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1.
A lipopolysaccharide- and beta-1,3-glucan-binding protein (LGBP) was isolated and characterized from blood cells (hemocytes) of the freshwater crayfish Pacifastacus leniusculus. The LGBP was purified by chromatography on Blue-Sepharose and phenyl-Sepharose, followed by Sephacryl S-200. The LGBP has a molecular mass of 36 kDa and 40 kDa on 10% SDS-polyacrylamide gel electrophoresis under reducing and nonreducing conditions, respectively. The calculated mass of LGBP is 39,492 Da, which corresponds to the native size of LGBP; the estimated pI of the mature LGBP is 5.80. LGBP has binding activity to lipopolysaccharides as well as to beta-1,3-glucans such as laminarin and curdlan, but peptidoglycan could not bind to LGBP. Cloning and sequencing of LGBP showed significant homology with several putative Gram-negative bacteria-binding proteins and beta-1, 3-glucanases. Interestingly, LGBP also has a structure and functions similar to those of the coelomic cytolytic factor-1, a lipopolysaccharide- and glucan-binding protein from the earthworm Eisenia foetida. To evaluate the involvement of LGBP in the prophenoloxidase (proPO) activating system, a polyclonal antibody against LGBP was made and used for the inhibition of phenoloxidase (PO) activity triggered by the beta-1,3-glucan laminarin in the hemocyte lysate of crayfish. The PO activity was blocked completely by the anti-LGBP antibody. Moreover, the PO activity could be recovered by the addition of purified LGBP. These results suggest that the 36-kDa LGBP plays a role in the activation of the proPO activating system in crayfish and thus seems to play an important role in the innate immune system of crayfish.  相似文献   

2.
The Down syndrome cell adhesion molecule, also known as Dscam, is a member of the immunoglobulin super family. Dscam plays an essential function in neuronal wiring and appears to be involved in innate immune reactions in insects. The deduced amino acid sequence of Dscam in the crustacean Pacifastacus leniusculus (PlDscam), encodes 9(Ig)-4(FNIII)-(Ig)-2(FNIII)-TM and it has variable regions in the N-terminal half of Ig2 and Ig3 and the complete Ig7 and in the transmembrane domain. The cytoplasmic tail can generate multiple isoforms. PlDscam can generate more than 22,000 different unique isoforms. Bacteria and LPS injection enhanced the expression of PlDscam, but no response in expression occurred after a white spot syndrome virus (WSSV) infection or injection with peptidoglycans. Furthermore, PlDscam silencing did not have any effect on the replication of the WSSV. Bacterial specific isoforms of PlDscam were shown to have a specific binding property to each tested bacteria, E. coli or S. aureus. The bacteria specific isoforms of PlDscam were shown to be associated with bacterial clearance and phagocytosis in crayfish.  相似文献   

3.
An antibacterial peptide with 16 amino acid residues was found in plasma of the freshwater crayfish, Pacifastacus leniusculus. This peptide, designated astacidin 1, was purified by cation-exchange column chromatography and reverse-phase high performance liquid chromatography. Astacidin 1 has a broad range of antibacterial activity, and it inhibits growth of both Gram-positive and Gram-negative bacteria. The primary sequence of astacidin 1 was FKVQNQHGQVVKIFHH-COOH. The molecular mass was 1945.2 Da, and no carbohydrate-linked amino acid residues could be found by mass spectrometry. A synthetic astacidin 1 resulted in similar activity as the authentic astacidin 1 against Gram-positive bacteria, whereas it had less or no activity against Gram-negative bacteria. Three amino-terminal-truncated synthetic peptides were made; they all showed low activity, suggesting that the amino-terminal part of astacidin 1 contributes to the antibacterial activity. The structure of astacidin 1 based on the CD results showed that it has a beta-sheet structure in citric acid buffer at pH 4, 6, and 8. Cloning of astacidin 1 shows that it is the carboxyl-terminal part of crayfish hemocyanin and that astacidin 1 is produced by a proteolytic cleavage from hemocyanin under acidic conditions. The processing and release of astacidin 1 from hemocyanin is enhanced when crayfish are injected with lipopolysaccharide or glucan.  相似文献   

4.
Sperm from the crayfish, Pacifastacus leniusculus, resemble other reptantian sperm in that they are composed of an acrosome, subacrosomal region, nucleus, membrane lamellar complex, and spikes which radiate from the nuclear compartment. The acrosome (PAS positive vesicle) can be subdivided into three regions: the apical cap, crystalline inner acrosomal material, and outer acrosomal material which is homogeneous except for a peripheral electron dense band. The nucleus contains uncondensed chromatin and bundles of microtubules which project into the spikes. The orientation of the microtubule bundles relative to the nuclear envelope near the base of the subacrosomal region suggests that the nuclear envelope may function in the organization of the spike microtubules.  相似文献   

5.
  • 1 Experiments were designed to determine the relative importance of chemical and visual stimuli in eliciting predator avoidance behaviour in juvenile freshwater crayfish Pacifastacus leniusculus (Dana).
  • 2 Crayfish placed in visual and/or chemical contact with one of two predators exhibited marked avoidance behaviour, spending less time walking and climbing and more time within shelters.
  • 3 The combined effects of both visual and chemical stimuli increased crayfish shelter use and reduced walking and climbing activity to a greater degree than either stimulus when presented alone.
  • 4 Crayfish exhibited avoidance behaviour in response to chemical stimuli during periods of light and darkness. Visual detection of predators elicited avoidance behaviour during the day.
  • 5 It is suggested that the behavioural response of P. leniusculus to chemical stimuli reduces the likelihood of being detected by visual predators, and that chemical stimuli lower the response threshold for avoidance behaviour in crayfish reacting to visual stimuli. The adaptiviry of using chemical cues to detect predators is emphasized.
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6.
1. The American signal crayfish Pacifastacus leniusculus, an invasive species widely introduced throughout Europe, is a major threat to native European crayfish species and is causing increasing concern because of its wide impact on aquatic ecosystems. 2. Whilst various control and management methods have been proposed, very little is known about the factors influencing dispersal and movements of signal crayfish. 3. Sixty‐four adult signal crayfish (carapace length 31.9–63.8 mm) were radiotagged in upland rivers in northern England, during four periods. Tracking was carried out at two sites, a low‐density establishing population and a high‐density established population. Tracking was carried out at both sites concurrently during midsummer (June to August 2002), during late summer (August to September 2001) at the low‐density population site and during autumn to winter (October to February 2000/01) at the high‐density population site. 4. Maximum movement occurred during midsummer. Temperature appeared to be a major factor influencing the timing and extent of movements between tracking periods. 5. The frequency distribution of the maximum distance moved upstream and downstream by radiotagged crayfish showed an inverse power relationship. The median maximal upstream and downstream distances moved were 13.5 m (range 0–283 m) and 15 m (range 0–417 m), respectively. There was a significant difference between the distributions of upstream and downstream ranges, with greater distances moved downstream. 6. All downstream movements made by crayfish appeared to be active movements and not the result of passive movement during periods of high discharge. There was no apparent influence of size, sex or density on the amount of movement recorded. 7. The study provides important information on the spatial and temporal behaviour of introduced crayfish in upland lotic systems. In contrast to lowland rivers, our results suggest that flow or gradient may influence the invasive potential of signal crayfish in an upstream direction in upland rivers.  相似文献   

7.
The signal freshwater crayfish Pacifastacus leniusculus was found to be susceptible to infection with white spot syndrome virus (WSSV). Histopathological observations of various tissues of virus-injected crayfish showed similar symptoms to those from WSSV-infected penaeid shrimp, but no appearance of white spots on the cuticle or reddish body colour were observed, although these are the prominent gross signs of white spot disease in shrimp. A gene probe for detecting WSSV was developed in order to detect the virus in affected cells and tissues using in situ hybridisation. Strong signals were observed in cells of virus-injected crayfish, but not in control-injected crayfish. The number of granular haemocytes in virus-injected crayfish was significantly higher than in sham-injected and non-injected crayfish from Days 5 to 8 (p < or = 0.05) and Days 3 to 8 (p < 0.01) post-injection, respectively. The proportion of granular haemocytes in virus-injected crayfish was also significantly higher than in sham-injected controls from Days 3 to 8 (p < 0.01). These results indicate that WSSV has a significant effect on the proportion of different haemocyte types in the freshwater crayfish.  相似文献   

8.
The expression of both swimmeret and postural motor patterns in crayfish (Pacifastacus leniusculus) were affected by stimulation of a second root of a thoracic ganglion. The response of the swimmeret system depended on the state of the postural system. In most cases, the response of the swimmeret system outlasted the stimulus.Stimulation of a thoracic second root also elicited coordinated responses from the postural system, that outlasted the stimulus. In different preparations, either the flexor excitor motor neurones or the extensor excitor motor neurones were excited by this stimulation. In every case, excitation of one set of motor neurones was accompanied by inhibition of that group's functional antagonists.This stimulation seemed to coordinate the activity of both systems; when stimulation inhibited the flexor motor neurones, then the extensor motor neurones and the swimmeret system were excited. When stimulation excited the flexor motor neurones, then the extensor motor neurones and the swimmeret system were inhibited.Two classes of interneurones that responded to stimulation of a thoracic second root were encountered in the first abdominal ganglion. These interneurones could be the pathway that coordinates the response of the postural and swimmeret systems to stimulation of a thoracic second root.Abbreviations TSR thoracic second root - epsp excitatory post-synaptic potential - ipsp inhibitory post-synaptic potential - EJP excitatory jonctional potential - PS power-stroke - RS return-stroke - INT interneurone - N1 first segmental nerve - N2 second segmental nerve - N3 third segmental nerve - A1 abdominal ganglion 1  相似文献   

9.
A trypsin was purified from the hepatopancreas of snakehead (Channa argus) by ammonium sulfate fractionation and a series of column chromatographies including DEAE-Sepharose, Sephacryl S-200 HR and Hi-Trap Capto-Q. The molecular mass of the purified trypsin was about 22 kDa, as estimated by SDS-PAGE. The optimum pH and temperature of the purified trypsin were 9.0 and 40 °C, respectively. The trypsin was stable in the pH range of 7.5-9.5 and below 45 °C. The enzymatic activity was strongly inhibited by serine proteinase inhibitors, such as MBTI, Pefabloc SC, PMSF, LBTI and benzamidine. Peptide mass fingerprinting (PMF) of the purified protein obtained 2 peptide fragments with 25 amino acid residues and were 100% identical to the trypsinogen from pufferfish (Takifugu rubripes). The activation energy (Ea) of this enzyme was 24.65 kJ·M− 1. Apparent Km was 1.02 μM and kcat was 148 S− 1 for fluorogenic substrate Boc-Phe-Ser-Arg-MCA. A trypsinogen gene encoding 247 amino acid residues was further cloned on the basis of the sequence obtained from PMF and the conserved site peptide of trypsinogen together with 5′-RACE and 3′-RACE. The deduced amino acid sequence contains a signal peptide of 15 residues and an activation peptide of 9 amino acid residues with a mature protein of 223 residues. The catalytic triad His-64, Asp-107, Ser-201 and 12 Cys residues which may form 6 disulfide bonds were conserved. Compared with the PMF data, only 2 amino acid residues difference were identified, suggesting the cloned trypsinogen is quite possibly the precursor of the purified trypsin.  相似文献   

10.
The plasma of the crayfish Pacifastacus leniusculus contains a protein which is able to bind to laminarin (a soluble beta-1,3-glucan) and which has been isolated by two independent methods, affinity precipitation with a beta-1,3-glucan or immunoaffinity chromatography. The purified beta-1,3-glucan binding protein was homogenous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is a monomeric glycoprotein with a molecular mass of approximately 100,000 Da and an isoelectric point of approximately 5.0. Amino acid analysis showed a very high similarity with the amino acid composition of beta-1,3-glucan binding proteins recently purified from two insects, the cockroach Blaberus craniifer and the silkworm Bombyx mori. The N-terminal amino acid sequence was determined to be: H2N-Asp-Ala-Gly-X-Ala-Ser-Leu-Val-Thr-Asn-Phe-Asn-Ser-Ala-Lys-Leu-X-X-Ly s--- Using monospecific rabbit polyclonal antibodies, the presence of this protein has also been shown within the blood cells. The purified beta-1,3-glucan binding protein did not show any peptidase or phenoloxidase activity but was able to enhance the activation of hemocyte-derived peptidase and prophenoloxidase only in the presence of the beta-1,3-glucan, laminarin, whereas mannan, dextran (alpha-glucan), or cellulose (beta-1,4-glucan) incubated with the beta-1,3-glucan binding protein had no effect on these enzyme activities. The beta-1,3-glucan binding protein could only be affinity-precipitated from crayfish plasma by the beta-1,3-glucans laminarin or curdlan (an insoluble beta-1,3-glucan), while mannan or dextran did not bind to the beta-1,3-glucan binding protein. No hemagglutinating activity of the purified beta-1,3-glucan binding protein could be detected.  相似文献   

11.
1. Two glutathione peroxidases (GSH-Px), D1 and D2, were partially purified from the hepatopancreas of common carp Cyprinus carpio by ion-exchange, gel filtration, and hydrophobic chromatography. The enzymatic properties of those enzymes were examined. 2. The D1 and D2 differed in following enzymatic properties. The pH optima of D1 and D2 were 8.0 and 9.0, respectively. 3. When stored at pH 4.0 for 36 hr, D1 kept 70% of maximal activity while only 30% of that was shown in D2. 4. D1 and D2 lost activity by 50% when incubated at 55 degrees C and 65 degrees C for 10 min, respectively.  相似文献   

12.
A receptor for the 100 kDa beta-1,3-glucan-binding protein [Duvic, B. and S?derh?ll, K. (1990) J. Biol. Chem. 265, 9327-9332] has been purified from hemocyte membranes of the crayfish Pacifastacus leniusculus. The purification was achieved by DEAE-cellulose chromatography of detergent-solubilized membranes. The receptor had an apparent molecular mass of 350 kDa when subjected to native polyacrylamide-gel electrophoresis and was composed of two non-covalently associated subunits of about 230 kDa and 90 kDa, as judged by SDS/polyacrylamide-gel electrophoresis or two-dimensional electrophoresis. The receptor could only bind the beta-1,3-glucan-binding protein if this protein had previously reacted with a beta-1,3-glucan, laminarin, and the binding site was located on the 230 kDa subunit. The binding of laminarin-treated beta-1,3-glucan-binding protein to its receptor was a saturable process and binding data indicated a single high-affinity-binding site with a Kd of 0.35 +/- 0.15 microM as determined by Scatchard analysis. The receptor had a requirement for divalent cations and a pH optimum of 6.5 for binding the laminarin-treated beta-1,3-glucan-binding protein. Laminarin, as well as oligosaccharides such as D-glucose, sialic acid, N-acetyl glucosamine or methyl-alpha-D-mannoside, could not affect the binding of the beta-1,3-glucan-binding protein to its receptor.  相似文献   

13.
Jens Lübeck  Jürgen Soll 《Planta》1995,196(4):668-673
Nucleoside diphosphate kinase (NDPK; EC 2.7.4.6) was enriched 1900-fold from purified pea (Pisum sativum L. cv. Golf.) chloroplasts. The active enzyme preparation contained two polypeptides of apparent molecular weight 18.5 kDa and 17.4kDa. Both proteins were enzymatically active and were recognized by an antiserum raised against NDPK from spinach chloroplasts, suggesting the existence of two isoforms in pea chloroplasts. The N-terminal protein sequence data were obtained for both polypeptides and compared with the nucleotide sequence of a cDNA clone isolated from a pea cDNA library. The analysis revealed that the two NDPK forms are encoded for by one mRNA, indicating that the lower-molecular-weight form could represent a proteolytic breakdown product of the 18.5-kDa NDPK. The pea chloroplastic NDPK is made as a larger precursor protein which is imported into chloroplasts. The NDPK precursor is then processed by the stromal processing peptidase to yield the 18.5-kDa form.Abbreviations NDPK nucleoside diphosphate kinase - preNDPK precursor NDPK - ps-NDPK cDNA coding for Pisum sativum NDPK II We thank Dr. Schmidt, University Göttingen, Germany, for doing the protein sequencing. This work was supported in part by grants from the Deutsche Forschungsgemeinschaft.  相似文献   

14.
Cytokinins are degraded by cytokinin oxidases (CKOs) which catalyse cleavage of the N6-(isopent-2-enyl)-side chain resulting in formation of adenine-type compounds. CKO activity has been recorded in many plants and is thought to play a key role in controlling cytokinin levels in plants. Several partially purified CKOs have been characterised but no genes have been isolated yet. CKO activity is known to be inhibited by phenylureas, cytokinin agonists. We used 1-(2-azido-6-chloropyrid-4-yl)-3-(4-[3H])phenylurea ([3H]-azidoCPPU) to photolabel a glycosylated CKO from maize kernels. This enabled us to purify the enzyme. Peptide sequences were determined and the corresponding cDNA was cloned. The deduced amino acid sequence shares homology domains with FAD-dependent oxidases. An original assay based on transient expression of the enzyme in moss protoplasts allowed the functionality of the recombinant enzyme to be demonstrated.  相似文献   

15.
Alpha 2-Macroglobulin (alpha 2 M) was isolated from plasma of the freshwater crayfish, Pacifastacus leniusculus, using ultracentrifugation, ion-exchange chromatography and gel filtration techniques. The Pacifastacus alpha 2 M molecule (P alpha 2 M) was radio-actively labeled in the thiol ester structure with iodo [14C]acetic acid in the presence of methylamine. After reduction and carboxymethylation of the protein, it was digested with trypsin. A 14C-labeled tryptic peptide was sequenced and contained an amino acid sequence very similar to other known thiol ester sequences from human alpha 2 M and related proteins. The N-terminal sequence of P alpha 2 M was related to that recently determined for lobster alpha 2 M [(1987) J. Biol. Chem. 262, 14606-14611].  相似文献   

16.
Chloroplast and cytosolic triosephosphate isomerases from spinach were separated and purified to homogeneity. Both enzymes were partially sequenced by Edman degradation. Using degenerate primers designed against the amino acid sequences, a homologous probe for the chloroplast enzyme was amplified and used to isolate several full-size cDNA clones. Chloroplast triosephosphate isomerase is encoded by a single gene in spinach. Analysis of the chloroplast cDNA sequence in the context of its homologues from eukaryotes and eubacteria reveals that the gene arose through duplication of its pre-existing nuclear counterpart for the cytosolic enzyme during plant evolution.Abbreviations TPI triosephosphate isomerase - PEG polyethylene glycol - cp plastid - c cytosolic - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - PVP polyvinylpyrrolidone - PCR polymerase chain reaction - PGK 3-phosphoglycerate kinase  相似文献   

17.
An immunocytochemical technique with the use of three different antibodies raised against serotonin was applied to localize the immunoreactive neurons in the central nervous system of the crayfish, Pacifastacus leniusculus. Immunoreactive neurons were found in three optic ganglia (medulla externa, interna and terminalis). They appeared in three layers of the medulla externa and interna. The medulla terminalis displayed three prominent groups of immunoreactive perikarya and mainly marginal immunoreactive fibres. Immunoreactive areas of the brain comprised the protocerebral bridge, central body, paracentral lobes and two loci in the anterior portion of the protocerebrum, i.e., the terminal areas for immunoreactive fibres from the optic centres. The olfactory lobes showed a specific immunoreactive pattern. In addition, diffusely and sparsely distributed immunoreactive fibres were found throughout the brain. The immunoreactive neurons are largely localized in the same areas of the central nervous system as the catecholaminergic neurons although some distinct differences occur.  相似文献   

18.
A trypsin inhibitor from Dimorphandra mollis seeds was isolated to apparent homogeneity by a combination of ammonium sulfate precipitation, gel filtration, ion-exchange and affinity chromatographic techniques. SDS-PAGE analysis gave an apparent molecular weight of 20 kDa, and isoelectric focusing analysis demonstrated the presence of three isoforms. The partial N-terminal amino acid sequence of the purified protein showed a high degree of homology with various members of the Kunitz family of inhibitors. This inhibitor, which inhibited trypsin activity with a Ki of 5.3 x 10(-10) M, is formed by a single polypeptide chain with an arginine residue in the reactive site.  相似文献   

19.
Hepatopancreas of king crab Paralithodes camtschatica produces a metalloprotease, which belongs to the astacin family, as cDNA cloning and sequencing showed. The metalloprotease has been purified chromatographically to apparent homogeneity. The purification factor was 16 and activity recovery was 20%. pH and temperature optimum have been determined. In its properties (molecular weight, pI, metal content) the metalloprotease is close to crayfish astacin. However, analysis of the enzyme sequences revealed differences, which account for differences in substrate specificities and imply a different activation mechanism.  相似文献   

20.
A cDNA encoding a protein resembling masquerade, a serine proteinase homologue expressed during embryogenesis, larval, and pupal development in Drosophila melanogaster, was identified in hemocytes of the adult freshwater crayfish, Pacifastacus leniusculus. The crayfish protein is similar to Drosophila masquerade in the following aspects: (a) overall sequence of the serine proteinase domain, such as the position of three putative disulfide bridges, glycine in the place of the catalytic serine residue, and the presence of a substrate-lining pocket typical for trypsins; (b) the presence of several copies of a disulfide-knotted motif in the putative propeptide. This masquerade-like protein is cleaved into a 27-kDa fragment, which could be detected by immunoblot analysis using an affinity-purified antibody against a synthetic peptide in the C-terminal domain of the protein. The 27-kDa protein could be immunoaffinity-purified from hemocyte lysate supernatant and exhibited cell adhesion activity in vitro, indicating that the C-terminal domain of the crayfish masquerade-like protein mediates cell adhesion.  相似文献   

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