Purification and characterization of a humoral opsonin from the solitary urochordate Styela clava.

1. We have previously identified opsonic activity in the plasma of the solitary urochordate, Styela clava. 2. Here, we report the purification and further characterization of the opsonic molecule. 3. Two purification methods were employed. 4. Gel filtration yielded one strongly opsonic fraction that contained a single, electrophoretically-resolved protein. 5. Opsonic activity was dose-dependent and sensitive to tryptic digestion and heat denaturation. 6. SDS-PAGE and calibrated gel filtration indicated the opsonic protein was a 17.5 kDa monomer while isoelectrofocusing indicated a single pI of 7.0. 7. In an alternative procedure, a similar opsonic activity and protein were isolated by affinity purification using whole yeast cells.     

In vitro allogeneic cytotoxicity in the solitary urochordate Styela clava.

Hemocytes from the solitary urochordate Styela clava can effect allogeneic cytotoxicity in vitro. Spectrophotometric and microscopic quantification of eosin-y dye exclusion revealed significantly greater frequencies of cell death in allogeneic hemocyte cultures when compared to autogeneic controls. This cytotoxic response was characterized by 1) transient activity such that specific cytotoxicity could be detected for only 4 hours of culture though continued specific killing may have been obscured by spontaneous cell death; 2) a necessity for cellular interaction demonstrated by the elimination of allogeneic cytotoxicity in the absence of cell contact; 3) killing of multiple targets by effector cells due to high levels of response at low allogeneic ratios; 4) insensitivity to altered temperature; 5) increased cytotoxicity in the absence of autologous plasma; 6) an absence of xenogeneic reactivity; 7) the presence of three hierarchical levels (low, intermediate, and high) of response. These data reflect events involved in the recognition of allogeneic cellular determinants resulting in specific cytotoxicity effected by immunocompetent cells. Such in vitro recognition and cytotoxic recognition and cytotoxic reactivity may be responsible for adaptive reactions caused by histoincompatibility in solitary tunicates.     

Proliferation of lymphocyte-like cells from the solitary tunicate, Styela clava, in response to allogeneic stimuli.

Lymphocyte-like hemocytes (LLCs) of solitary tunicates proliferate in response to allogeneic stimuli. In vitro labeling of proliferative hemocytes from the solitary species Styela clava revealed significantly greater proliferative activity among individuals immunized with allogeneic tissue as opposed to autogeneically primed and na?ve animals. Enhanced proliferation was restricted to discrete crypts of dividing cells within the body wall of recipients. Here, increased proliferative activity was specifically associated with LLCs. These data support previous results which implicated LLC activity with immunological memory that is evident in allograft rejection. Hence, it is postulated that adaptive histoincompatibility responses in solitary tunicates depend upon the specific proliferation of immunocompetent cells.     

Protochordate immunity--II. Diverse hemolymph lectins in the solitary tunicate Styela clava

Hemolymph lectins (agglutinins) of the tunicate Styela clava were analyzed by agglutination, cross-absorption and carbohydrate-hemagglutination inhibition using several vertebrate erythrocytes. Lectin activity was heat labile, dependent on divalent cations and refractory to neuraminidase-treated erythrocytes. Four lectins with different carbohydrate specificities were found. Carbohydrate specificities included L-rhamnose, D-glucuronolactone, maltose, D-galactosamine, D-mannosamine, D-galactose, hyaluronic acid and bacterial lipopolysaccharide. Since S. clava lectins can be inhibited by carbohydrates found in the extracellular capsule or cell wall of most bacteria, we propose that the lectins may be part of the tunicate immuno-defense system.     

Endocrine Cells in the Oesophagus of the Ascidian Styela clava,a cytochemical and immunofluorescence study

Summary Immunocytochemical studies have demonstrated the occurrence of an insulin-immunoreactive cell type in the oesophageal epithelium of the Ascidian Styela clava. Staining with aldehyde fuchsin has demonstrated a number of similar small, triangular, cells located on the basement membrane, which may have an endocrine function. Argyrophilic cells have also been found, suggesting the presence of a second endocrine cell type. The absence of argentaffin cells has led us to believe that the cells so far observed do not produce biogenic amines such as 5-HT (5-Hydroxytryptamine). The nature of these cells is discussed with reference to endocrine-like cells found in the digestive tracts of other protochordates.Animals were collected by courtesy of the Admiralty Marine Trials Station, Portsmouth. This research was carried out during the tenure of an S.R.C. grant no. B/RG 82919 to one of us (M.C.T.). —The localisation of polypeptide hormones in the pharynx and gut of protochordates     

流式细胞术结合组织学方法对柄海鞘血细胞的分类

用流式细胞术研究柄海鞘(Styela clava)血细胞的分类,依据细胞大小及颗粒的复杂程度将血细胞分为5类:即类群R1-R5,各类群分别占血细胞总数的37.15%4±1.01%、15.85%4±2.91%、16.15%4±1.58%、23.65%±3.05%、5.87%±0.31%,上机血样品的密度约106个/ml.组织学方法依据细胞大小、所含颗粒的情况及细胞的染色特征将血细胞分为5类:成血细胞、透明细胞、嗜碱性颗粒细胞、嗜酸性颗粒细胞及吞噬细胞.对两种方法各自所分5类细胞的对应关系进行了讨论.     

Physiological energetics of the ascidian Styela clava in relation to body size and temperature.

The ascidian Styela clava is widely distributed in northern China and is thought to be important in the functioning of estuarine systems. This ascidian may experience highly variable physiological conditions, and its physiological responses to these are of interest considering its ecological role. This study presents data on the physiological parameters in relation to body size and temperature (12-28 degrees C) of S. clava. Respiration, excretion, feces, ingestion and absorption energy were positive related to size and its mass exponents (b-values) varied from 0.2930 to 0.7488. Respiration energy increased with increasing, but critical values were found at 20 degrees C for energy of ingestion and absorption, while 24 degrees C for energy in feces and excretion. At the range of 16-24 degrees C, the scope for growth, gross and net growth efficiencies of ascidians increased with increasing temperature. The energy budget equations of different sized ascidians were obtained at different temperatures. Excretion energy shared a minimal fraction in ingestion energy (1.30-2.47%), the ratios of feces energy and respiration energy to ingestion energy were 46.53-64.27% and 10.26-80.75%, respectively. The physiological data obtained in the present study indicated that S. clava could adjust its energy budget according to the environment and its physiological conditions to meet their nutritional and energetic demands. In the range of experimental temperature (12-28 degrees C), 16-20 degrees C was suitable for the rearing of S. clava to achieve optimum development, while deficient metabolic adjustment induced a negative scope for growth of S. clava at 28 degrees C.     

Physiological energetics of the ascidian Styela clava in relation to body size and temperature

The ascidian Styela clava is widely distributed in northern China and is thought to be important in the functioning of estuarine systems. This ascidian may experience highly variable physiological conditions, and its physiological responses to these are of interest considering its ecological role. This study presents data on the physiological parameters in relation to body size and temperature (12-28 degrees C) of S. clava. Respiration, excretion, feces, ingestion and absorption energy were positive related to size and its mass exponents (b-values) varied from 0.2930 to 0.7488. Respiration energy increased with increasing, but critical values were found at 20 degrees C for energy of ingestion and absorption, while 24 degrees C for energy in feces and excretion. At the range of 16-24 degrees C, the scope for growth, gross and net growth efficiencies of ascidians increased with increasing temperature. The energy budget equations of different sized ascidians were obtained at different temperatures. Excretion energy shared a minimal fraction in ingestion energy (1.30-2.47%), the ratios of feces energy and respiration energy to ingestion energy were 46.53-64.27% and 10.26-80.75%, respectively. The physiological data obtained in the present study indicated that S. clava could adjust its energy budget according to the environment and its physiological conditions to meet their nutritional and energetic demands. In the range of experimental temperature (12-28 degrees C), 16-20 degrees C was suitable for the rearing of S. clava to achieve optimum development, while deficient metabolic adjustment induced a negative scope for growth of S. clava at 28 degrees C.     

Design, synthesis and structure of an amphipathic peptide with pH-inducible haemolytic activity.

A synthetic, 26-residue peptide having a strong helix forming potential in the protonated state was designed to interact with lipid bilayers in a pH-dependent way. On the basis of this concept a cluster of four glutamic acid residues was inserted in the central region of the amphipathic peptide to promote helix destabilization by mutual charge repulsion at neutral pH. Protonation of these residues might then bring about both a pH-mediated change in hydrophobicity and conformation forming a membrane-active amphiphilic helix. The sequence GLGTLLTLLEFLLEELLEFLKRKRQQamide produced by the design strategy induced pH-triggered lysis of human erythrocytes. A molecular model correlating the lytic activity to the formation of transmembrane pores which were detected by electron microscopy in erythrocyte membranes is discussed. Circular dichroism studies indicated a self-association of the monomeric random coil form with increasing peptide concentration leading to the apparent induction of strong alpha-helix formation (approximately 100% helicity) in the fully aggregated state. However, no pH-dependent helix-random coil transition was observed, implying that interhelical hydrophobic and ionic interactions not only govern the self-association but also decisively influence the conformational stability of the peptide.     

Plicatamide,an antimicrobial octapeptide from Styela plicata hemocytes

Plicatamide (Phe-Phe-His-Leu-His-Phe-His-dc Delta DOPA), where dc Delta DOPA represents decarboxy-(E)-alpha,beta-dehydro-3,4-dihydroxyphenylalanine, is a potently antimicrobial octapeptide from the blood cells of the solitary tunicate, Styela plicata. Wild type and methicillin-resistant Staphylococcus aureus (MRSA) responded to plicatamide exposure with a massive potassium efflux that began within seconds. Soon thereafter, treated bacteria largely ceased consuming oxygen, and most became nonviable. Native plicatamide also formed cation-selective channels in model lipid bilayers composed of bacterial lipids. Methicillin-resistant S. aureus treated with plicatamide for 5 min contained prominent mesosomes as well as multiple, small dome-shaped surface protrusions that suggested the involvement of osmotic forces in its antimicrobial effects. To ascertain the contribution of the C-terminal dc Delta DOPA residue to antimicrobial activity, we synthesized several analogues of plicatamide that lacked it. One of these peptides, PL-101 (Phe-Phe-His-Leu-His-Phe-His-Tyr-amide), closely resembled native plicatamide in its antimicrobial activity and its ability to induce potassium efflux. Plicatamide was potently hemolytic for human red blood cells but did not lyse ovine erythrocytes. The small size, rapid action, and potent anti-staphylococcal activity of plicatamide and PL-101 make them intriguing subjects for future antimicrobial peptide design.     

Isolation and characterization of twelve polymorphic microsatellite markers for the invasive ascidian Styela clava (Tunicata)

The invasive solitary ascidian Styela clava has spread extensively along European coasts since its first occurrence in the early 1950s. In order to characterize spatial and temporal patterns of genetic change during its establishment and subsequent spread, we developed 12 species‐specific loci from an enriched microsatellite library. Polymorphism was explored in one native and two introduced populations (N = 31 ± 1). Number of alleles per locus varied from two to 13 (mean = 7). The average expected heterozygosity within populations ranged from 0.539 to 0.580.     

A cytochemical and immunofluorescence study of endocrine cells in the gut of the ascidian Styela clava

Summary Strong secretin-like immunofluorescence has been demonstrated in endocrine-like cells from the gastric epithelium of Styela. These cells also stain with lead haematoxylin and exhibit a brilliant formaldehyde-induced fluorescence, but do not show any other cytochemical features characteristic of the mammalian APUD series. Tests with antisera to glucagon, gastrin and somatostatin all proved negative. In the oesophagus tests with all four antisera proved negative. The significance of these results is discussed in relation to the phylogeny of vertebrate gastro-intestinal hormones.     

Structure-function analysis of tritrypticin, an antibacterial peptide of innate immune origin.

The structural requirements for the antibacterial activity of a pseudosymmetric 13-residue peptide, tritrypticin, were analyzed by combining pattern recognition in protein structures, the structure-activity knowledge-base, and circular dichroism. The structure-activity analysis, based on various deletion analogs, led to the identification of two minimal functional peptides, which by themselves exhibit adequate antibacterial activity against Escherichia coli and Salmonella typhimurium. The common features between these two peptides are that they both share an aromatic-proline-aromatic (ArProAr) sequence motif, and their sequences are retro with respect to one another. The pattern searches in protein structure data base using the ArProAr motif led to the identification of two distinct conformational clusters, namely polyproline type II and beta-turn, which correspond to the observed solution structures of the two minimal functional analogs. The role of different residues in structure and function of tritrypticin was delineated by analyzing antibacterial activity and circular dichroism spectra of various designed analogs. Three main results arise from this study. First, the ArProAr sequence motif in proteins has definitive conformational features associated with it. Second, the two minimal bioactive domains of tritrypticin have entirely different structures while having equivalent activities. Third, tritrypticin has a beta-turn conformation in solution, but the functionally relevant conformation of this gene-encoded peptide antibiotic may be an extended polyproline type II.     

Styela clava Herdman (Urochordata, Ascidiacea), a successful immigrant to North West Europe: ecology, propagation and chronology of spread

Since its first occurrence at Plymouth, southern England, in 1952 the East Asiatic ascidianStyela clava has spread to many localities along the coasts of the south and west British Isles, Ireland, northern France, Belgium, the Netherlands, Denmark and Germany. While some dispersal may occur by natural means, spreading over long distances is probably due to transfer along with oysters when relaid elsewhere. Transport while attached to the hulls of ships or driftingSargassum is also possible.Styela clava is a large, hardy and fast-growing species with a tough, leathery tunic, and has no recorded enemies or native analogues among the NW European ascidian fauna. At many sites it has established dense populations of 500–1000 specimens/m² and in some cases has nearly outcompeted some of the native ascidian species.     

Hepcidin is an antibacterial, stress-inducible peptide of the biliary system

Background/Aims

Hepcidin (gene name HAMP), an IL-6-inducible acute phase peptide with antimicrobial properties, is the key negative regulator of iron metabolism. Liver is the primary source of HAMP synthesis, but it is also produced by other tissues such as kidney or heart and is found in body fluids such as urine or cerebrospinal fluid. While the role of hepcidin in biliary system is unknown, a recent study demonstrated that conditional gp130-knockout mice display diminished hepcidin levels and increased rate of biliary infections.

Methods

Expression and localization of HAMP in biliary system was analyzed by real time RT-PCR, in-situ hybridization, immunostaining and –blotting, while prohepcidin levels in human bile were determined by ELISA.

Results

Hepcidin was detected in mouse/human gallbladder and bile duct epithelia. Biliary HAMP is stress-inducible, in that it is increased in biliary cell lines upon IL-6 stimulation and in gallbladder mucosa of patients with acute cholecystitis. Hepcidin is also present in the bile and elevated prohepcidin levels were observed in bile of primary sclerosing cholangitis (PSC) patients with concurrent bacterial cholangitis compared to PSC subjects without bacterial infection (median values 22.3 vs. 8.9; p = 0.03). In PSC-cholangitis subjects, bile prohepcidin levels positively correlated with C-reactive protein and bilirubin levels (r = 0.48 and r = 0.71, respectively). In vitro, hepcidin enhanced the antimicrobial capacity of human bile (p<0.05).

Conclusion

Hepcidin is a stress-inducible peptide of the biliary epithelia and a potential marker of biliary stress. In the bile, hepcidin may serve local functions such as protection from bacterial infections.     

Structure-activity relationship of an antibacterial peptide, maculatin 1.1, from the skin glands of the tree frog, Litoria genimaculata.

Maculatin 1.1 (Mac) is a cationic antibacterial peptide isolated from the dorsal glands of the tree frog, Litoria genimaculata, and has a sequence of GLFGVLAKVAAHVVPAIAEHF-NH2. A short peptide lacking the N-terminal two residues of Mac was reported to have no activity. To investigate the structure-activity relationship in detail, several analogs and related short peptides of Mac were synthesized. CD measurement showed that all the peptides took more or less an alpha-helical structure in the presence of anionic lipid vesicles. Analogs which are more basic than Mac had strong antibacterial and hemolytic activities, while short peptides lacking one or two terminal residues exhibited weak or no activity. Outer and inner membrane permeabilization activities of the peptides were also reduced with shortening of the peptide chain. These results indicate that the entire chain length of Mac is necessary for full activity, and the basicity of the peptides greatly affects the activity.     

Consequences of introducing a disulfide bond into an antibacterial and hemolytic peptide.

The effect of introducing a disulfide bridge between the N- and C-terminal ends on the structure and biological activities of the 13-residue linear peptide PKLLKTFLSKWIG(SPFK), which has both antibacterial and hemolytic activity, have been investigated. The terminal amino acids P and G in SPFK were replaced by cysteines to form a disulfide bridge. The linear peptides C(Acm)KLLKTFLSKWIC(Acm) and C(Acm) KLLKTFLSKWIC(Acm)-amide, where Acm is acetamidomethyl group, showed antibacterial activity but did not possess hemolytic activity unlike SPFK. Introduction of an S-S bridge resulted in enhanced hemolytic activity compared with SPFK. The hemolytic activity was particularly pronounced in the cyclic peptide CKLLKTFLSKWIC-amide. Circular dichroism studies indicate that the cyclic peptides tend to adopt distorted helical structures. The cyclic peptides also have a greater affinity for lipid vesicles, which could be the reason for the effective perturbation of the erythrocyte membrane.     

New antibacterial peptide derived from bovine hemoglobin

Peptic digestion of bovine hemoglobin at low degree of hydrolysis yields an intermediate peptide fraction exhibiting antibacterial activity against Micrococcus luteus A270, Listeria innocua, Escherichia coli and Salmonella enteritidis after separation by reversed-phase HPLC. From this fraction a pure peptide was isolated and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). This peptide correspond to the 107-136 fragment of the alpha chain of bovine hemoglobin. The minimum inhibitory concentrations (MIC) towards the four strains and its hemolytic activity towards bovine erythrocytes were determined. A MIC of 38 microM was reported against L. innocua and 76 microM for other various bacterial species. This peptide had no hemolytic activity up to 380 microM concentration.