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D A Raftos

Publications and source records attributed to D A Raftos.

At least 19 recordsLinked to original sources
PubMed2002

A complement component C3-like protein from the tunicate, Styela plicata.

This study identifies a complement component C3-like protein in the solitary tunicate, Styela plicata. Three different polyclonal antibodies raised against C3 molecules from two species (humans and the tunicate, Halocynthia roretzi) were used to identify the C3-like protein in S. plicata hemolymph. The C3 cross-reactive protein is a 170kDa heterodimer composed of polypeptides (116 and 80kDa) that have molecular weights comparable to those of C3alpha and C3beta from other species. Amino acid sequencing and amino acid composition analysis confirmed that the C3-like protein from S. plicata is closely related to C3 from H. roretzi.

Amino Acid Sequence↗
PubMed2001

Effects of tributyltin and other metals on the phenoloxidase activating system of the tunicate, Styela plicata.

Toxic metals, such as tributyltin (TBT), contribute substantially to anthropogenic pollution in many estuarine environments. Animals that live in those environments, particularly invertebrate filter feeders like tunicates, are likely to be exposed to substantial metal contamination. This study investigates the effects of TBT and other metals on the phenoloxidase activity of the estuarine tunicate, Styela plicata, in an effort to identify a biochemical marker of metal pollution. Hemocytes harvested from S. plicata that were exposed to tributyltin or copper in aquaria had significantly enhanced phenoloxidase activities relative to non-exposed controls. This enhanced phenoloxidase activity could be explained by an increased frequency of morula cells, which contain high levels of phenoloxidase's proenzyme, prophenoloxidase. Unlike those from tunicates exposed to metals in aquaria, the phenoloxidase activities of hemocytes incubated with tributyltin in vitro were significantly reduced when compared with hemocytes cultured without tributyltin. The ability of tributyltin to decrease phenoloxidase activity in tissue culture may reflect its known inhibitory effects on calcium-dependent signaling systems such as those involved in the exocytosis of prophenoloxidase from morula cells.

Animals↗
PubMed2001

A C-type lectin from the tunicate, Styela plicata, that modulates cellular activity.

Previous studies have identified proteins from tunicates (invertebrate members of the Phylum Chordata) that have physicochemical and functional properties similar to those of the inflammatory cytokine, interleukin 1 (IL-1). Here we characterize one of those proteins from the tunicate, Styela plicata, that can stimulate tunicate and mammalian cell proliferation, activate phagocytosis, increase interleukin 2 (IL-2) secretion by mammalian peripheral blood mononuclear cells and enhance IL-2 receptor (IL-2R) expression by mammalian EL-4.IL-2 cells. Partial amino acid sequence data showed that the S. plicata protein resembles three C-type lectins (TC14, TC14-1 and TC14-2) from a closely related tunicate species, Polyandrocarpa misakiensis. Its similarity to carbohydrate recognition domains (CRDs) from P. misakiensis lectins suggests that the S. plicata protein modulates the activities of mammalian immunocompetent cells by interacting with carbohydrate moieties of glycosylated cell surface receptors.

Amino Acid Sequence↗
PubMed2000

Effects of metal-based environmental pollutants on tunicate hemocytes.

Tunicates are filter feeding marine invertebrates that are susceptible to environmental contamination by toxic metals and polyaromatic hydrocarbons. Recently, we have shown that tunicate immune reactions are profoundly affected by exposure to tributyltin (TBT) and copper, both of which are components of marine antifouling paints. This study tests the effects of those pollutants on the hemocytes of tunicates. Immunofluorescence labeling with an anti-hemocyte monoclonal antibody demonstrated that the antigenic structure of the circulating hemocyte population was substantially affected by TBT and copper. Antigen-positive hemocytes were also found to accumulate in the pharyngeal papillae of TBT-exposed tunicates. Histological analyses indicated that this cellular accumulation in pharyngeal papillae involved refractile vacuolated hemocytes. Refractile vacuolated cells from TBT-exposed tunicates also occurred at greater frequencies in the circulating hemolymph, and had altered morphologies, compared to cells from nontreated controls. These data confirm that exogenous metals can have profound effects on the hemocytes of tunicates.

Animals↗
PubMed2000

A collectin-like protein from tunicates.

Collectins are a sub-family of C-type lectins from mammals and birds that are characterized by their collagen-like domains. The mammalian collectin, mannose binding lectin, has attracted considerable interest because it can activate complement components via a lectin-mediated complement pathway that is independent of immunoglobulins. In this study, we have identified a calcium-dependent lectin from the invertebrate (tunicate), Styela plicata, that bears substantial similarities to mammalian collectins. The tunicate lectin, which was isolated by carbohydrate affinity chromatography, has a reduced apparent molecular mass of 43 kDa. The 43 kDa reduced polypeptide appeared as dimers, trimers and hexamers when analyzed by non-reducing and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while gel filtration suggested that the native form of the protein was a nonamer. Amino acid sequence and amino acid composition analysis revealed obvious similarities between the tunicate lectin and mammalian collectins, notably the inclusion of a collagenous domain and a short, cysteine bearing N-terminal domain. The identification of a collectin-like protein in an invertebrate such as S. plicata, which does not express immunoglobulin, indicates that lectin-mediated complement pathways may predate the origin of antibodies.

Amino Acid Sequence↗
PubMed1998

Chemotactic responses of tunicate (Urochordata, Ascidiacea) hemocytes in vitro.

A number of molecules were found to alter the motility of tunicate hemocytes. Bacterial lipopolysaccharide (LPS) significantly enhanced cell mobility relative to non-stimulated controls. Responses to LPS were not directional and so represented chemokinesis. In contrast, checkerboard analyses indicated that two tunicate hemolymph proteins, tunIL1-alpha and -beta, stimulated truly directional chemotaxis by hemocytes. The data suggest that tunIL1 proteins may contribute to defense by altering the localization of immunocompetent cells.

Animals↗
PubMed1994

A cell-surface opsonic receptor on leucocytes from the phylogenetically primitive vertebrate, Eptatretus stouti.

A humoral recognition molecule that is homologous to the mammalian complement components C3, C4 and C5 has recently been identified in the Pacific hagfish, Eptatretus stouti. One function of this complement-like protein (CLP) is to opsonize foreign material for phagocytosis by hagfish leucocytes. Here, we demonstrate that CLP's opsonic activity can be abrogated by pre-incubating phagocytes with an anti-hagfish leucocyte mAb (1B1). Moreover, antigen-activated CLP can block the binding of the 1B1 antibody to hagfish leucocytes. Flow cytometry and immunoprecipitation indicate that 1B1 recognizes a 105 kDa cell-surface, monomeric protein that is expressed exclusively on phagocytic hagfish leucocytes. It is concluded that this 105 kDa protein represents the cell surface receptor by which CLP mediates the phagocytosis of opsonized targets.

Animals↗
PubMed1993

Invertebrate cytokines. III: Invertebrate interleukin-1-like molecules stimulate phagocytosis by tunicate and echinoderm cells.

Phagocytosis is the predominant defense mechanism of invertebrates. Here we show that phagocytosis by echinoderm bladder amoebocytes and tunicate granular amoebocytes can be enhanced by invertebrate interleukin-1-like molecules. As little as 5 ng/ml of invertebrate interleukin-1 produced a significant stimulation of echinoderm and tunicate amoebocyte phagocytosis. Stimulation of phagocytosis by echinoderm interleukin-1-like molecules was inhibited by antisera to vertebrate interleukin-1. Invertebrate interleukin-1 also acted as an opsonin when preincubated with erythrocytes or yeast. In addition, the cellular mechanisms of invertebrate phagocytosis were studied using pharmacologic agents to inhibit echinoderm amoebocyte phagocytosis. The energy requirements and involvement of cellular cytoskeletal elements in phagocytosis by bladder amoebocytes were similar to those of mammalian macrophages. These results demonstrate a role for interleukin-1 in invertebrate host defense mechanisms.

Animals↗
PubMed1992

Hagfish humoral defense protein exhibits structural and functional homology with mammalian complement components.

A genomic clone and cDNA fragment encoding a portion of a humoral recognition molecule from the hagfish were isolated and sequenced. The serum protein has previously been described as having structural features that are immunoglobulin-like. Amino acid sequence obtained from the 77-kDa H1 heavy chain facilitated the isolation of a genomic clone containing at least two coding regions. Through use of primers derived from the genomic sequences, a 231-base-pair cDNA fragment was obtained by PCR from liver RNA. Comparison of the deduced 120-amino acid sequence from the N terminus of H1 with known protein sequences revealed substantial sequence similarity with the beta chain of the murine fourth complement component C4 and with the related third and fifth complement molecules C5 and C3 and the major histocompatibility complex-encoded sex-limited protein. Observation of structural and functional similarities associated with the sequence similarity indicate that these molecules share an evolutionary relationship: the polypeptide chain structure of hagfish complement-like protein (CLP) resembles that of C4; CLP contains a hidden thioester group on the 70-kDa chain; CLP binds to streptococcal cells and enhances the phagocytosis of yeast by hagfish leukocytes. These data suggest that CLP forms part of a non-clonally-derived complement-related humoral defense system in the hagfish.

Amino Acid Sequence↗
PubMed1992

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.

Animals↗
PubMed1992

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.

Animals↗
PubMed1992

Invertebrate cytokines II: release of interleukin-1-like molecules from tunicate hemocytes stimulated with zymosan.

Conditioned media and cell extracts from tunicate hemocytes that had been cultured with a variety of antigenic stimulants were tested for interleukin-1 (IL-1)-like activity. Media conditioned by hemocytes stimulated with zymosan significantly increased the proliferative and phagocytic activities of tunicate hemocytes. SDS-PAGE indicated that these biological activities were associated with the adaptive release of tunicate IL1-like (tunicate IL-1) molecules by stimulated hemocytes. The data suggest that tunicate IL1 molecules are expressed in response to selected antigenic stimuli. Such responses may form the basis for nonclonal, inducible immune reactions among phylogenetically primitive animals.

Animals↗
PubMed1991

Invertebrate cytokines: tunicate cell proliferation stimulated by an interleukin 1-like molecule.

Tunicate pharyngeal cells include lymphocyte-like cells and granular amoebocytes. They are involved in the specific allogeneic and phagocytic reactions of tunicates. Little is known about their regulation or control. A tunicate interleukin 1 (IL-1)-like fraction is shown to stimulate the proliferation of these cells in vitro. This fraction, designated tunicate IL-1 beta, was isolated from tunicate hemolymph by gel filtration and chromatofocusing chromatography. Mitogenic responses to tunicate IL-1 beta were dose dependent and could be eliminated rapidly by removing tunicate IL-1 beta from culture medium. A second tunicate hemolymph fraction had no effect on tunicate cell proliferation even though it exhibited IL-1-like activity in a mouse thymocyte proliferation assay. Phytohemagglutin did not act synergistically with either fraction. These data are discussed in terms of the function and evolution of IL-1-like molecules in invertebrates.

Animals↗
PubMed1991

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 naïve 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.

Animals↗
PubMed1991

Interleukin-2 and phytohaemagglutinin stimulate the proliferation of tunicate cells.

Proliferative responses of cells in tunicate pharyngeal explants to human interleukins and mitogenic lectins were tested. Increased tritiated-thymidine [( 3H]-TdR) uptake was detected among pharyngeal cells incubated with recombinant human interleukin-2 (IL-2), and phytohaemagglutinin-P (PHA-P). Responses to IL-2 were dose-dependent and affected lymphocyte-like cells. Enhanced proliferation was stimulated by IL-2 in the absence of co-stimulants and was not synergized by co-incubation with human interleukin-1 (IL-1) or PHA-P. Anti-IL-2 polyclonal antibody inhibited the stimulatory activity of recombinant human interleukin-2 (rhIL-2). Of three lectins tested (concanavalin-A [Con-A], pokeweed mitogen [PWM] and PHA-P), only PHA-P proved to be mitogenic. Con-A and PWM did not significantly increase proliferative activity even though both lectins were capable of binding pharyngeal cells as revealed by flow cytometry and fluorescence microscopy. Similarly, human IL-1 had no effect on [3H]-TdR uptake either alone or in combination with IL-2 and PHA-P. These data suggest that the functions of some interleukin-like cytokines have been conserved during evolution.

Animals↗