The evolution of new structures: clues from plant cytoskeletal genes.

How large numbers of genes were recruited simultaneously to build new organ structures is one of the greatest puzzles in evolutionary biology. Here, we present data suggesting that the vegetative and reproductive classes of actins and other cytoskeletal proteins arose concurrently with the macroevolutionary divergence of leaves and reproductive structures in the earliest land plants. That the cytoskeleton is essential for physically programming the development of organs and tissues is well established. Thus, we propose that this regulatory dichotomy represents an ancient landmark event in the global regulation of hundreds of higher-plant genes, an event that is linked to the macroevolution of plant vegetative and reproductive organs. The recent availability of sequence and expression data for large numbers of plant genes should make it possible to dissect this and other major macroevolutionary events.

Two-dimensional gel electrophoresis of membrane proteins from the R3327 prostate adenocarcinoma.

The Dunning rat prostate adenocarcinoma (R3327) is a reliable model that shares many similarities with the human tumor. Two sublines of the tumor, G and H, represent opposite extremes in histology and growth rate. Purified membrane fractions from G and H solid tumors were isolated by sucrose gradient. Tumor and normal prostate membrane proteins were labeled with 125I, incubated with G and H antisera, and precipitated by adsorption of antibody-antigen complexes to staphylococcal Protein A. Proteins were resolubilized and electrophoresed on two-dimensional gels, and the gels were autoradiographed. A total of eight labeled proteins were precipitated from the G and H tumors in the presence of G antisera. Of these, seven were homologous. One high-molecular-weight protein (Protein b) present on the G tumor was absent from the H tumor. The H tumor contained another high-molecular-weight protein (i) that was not found on the G tumor or on normal prostate. Normal prostate revealed a pattern similar to the G tumor except that Protein b appeared to be quantitatively reduced. Precipitation in the presence of H antisera showed similar patterns except that Protein b was not detected in the G tumor and was greatly reduced in the normal prostate. Therefore, despite variable growth characteristics, there were few changes in membrane proteins between the solid tumors and between the tumors and normal prostate. Iodination of surface proteins of cultured cells from normal prostate and the G and H sublines also showed a high degree of homology. No consistent differences between cultured cell lines were noted.

Members of the Arabidopsis actin gene family are widely dispersed in the genome.

Plant genomes are subjected to a variety of DNA turnover mechanisms that are thought to result in rapid expansion and presumable contraction of gene copy number. The evolutionary history of the 10 actin genes in Arabidopsis thaliana is well characterized and can be traced to the origin of vascular plant genomes. Knowledge about the genomic position of each actin gene may be the key to tracing landmark genomic duplication events that define plant families or genera and facilitate further mutant isolation. All 10 actin genes were mapped by following the segregation of cleaved amplified polymorphisms between two ecotypes and identifying actin gene locations among yeast artificial chromosomes. The Arabidopsis actin genes are widely dispersed on four different chromosomes (1, 2, 3, and 5). Even the members of three closely related and recently duplicated pairs of actin genes are unlinked. Several other cytoskeletal genes (profilins, tubulins) that might have evolved in concert with actins were also mapped, but showed few patterns consistent with that evolutionary history. Thus, the events that gave rise to the actin gene family have been obscured either by the duplication of very small genic fragments or by extensive rearrangement of the genome.

Detection of deleterious genotypes in multigenerational studies. I. Disruptions in individual Arabidopsis actin genes.

Plant actins are involved in numerous cytoskeletal processes effecting plant development, including cell division plane determination, cell elongation, and cell wall deposition. Arabidopsis thaliana has five ancient subclasses of actin with distinct patterns of spatial and temporal expression. To test their functional roles, we identified insertion mutants in three Arabidopsis actin genes, ACT2, ACT4, and ACT7, representing three subclasses. Adult plants homozygous for the act2-1, act4-1, and act7-1 mutant alleles appear to be robust, morphologically normal, and fully fertile. However, when grown as populations descended from a single heterozygous parent, all three mutant alleles were found at extremely low frequencies relative to the wild-type in the F2 generation. Thus, all three mutant alleles appear to be deleterious. The act2-1 mutant allele was found at normal frequencies in the F1, but at significantly lower frequencies than expected in the F2 and F3 generations. These data suggest that the homozygous act2-1/act2-1 mutant adult plants have a reduced fitness in the 2N sporophytic portion of the life cycle, consistent with the vegetative expression of ACT2. These data are interpreted in light of the extreme conservation of plant actin subclasses and genetic redundancy.

Structure and evolution of the actin gene family in Arabidopsis thaliana.

Higher plants contain families of actin-encoding genes that are divergent and differentially expressed. Progress in understanding the functions and evolution of plant actins has been hindered by the large size of the actin gene families. In this study, we characterized the structure and evolution of the actin gene family in Arabidopsis thaliana. DNA blot analyses with gene-specific probes suggested that all 10 of the Arabidopsis actin gene family members have been isolated and established that Arabidopsis has a much simpler actin gene family than other plants that have been examined. Phylogenetic analyses suggested that the Arabidopsis gene family contains at least two ancient classes of genes that diverged early in land plant evolution and may have separated vegetative from reproductive actins. Subsequent divergence produced a total of six distinct subclasses of actin, and five showed a distinct pattern of tissue specific expression. The concordance of expression patterns with the phylogenetic structure is discussed. These subclasses appear to be evolving independently, as no evidence of gene conversion was found. The Arabidopsis actin proteins have an unusually large number of nonconservative amino acid substitutions, which mapped to the surface of the actin molecule, and should effect protein-protein interactions.

IgM-mediated opsonization and cytotoxicity in the shark.

Two types of cytotoxic reactions have been observed using cells from the nurse shark: spontaneous cytotoxicity mediated by cells of the macrophage lineage and antibody-dependent killing carried out by a different effector cell population. Previous data showed that removal of phagocytic cells using iron particles abolished macrophage-mediated killing, but not antibody-dependent reactions. The current study used single cell assays and showed that the effector of antibody-driven reactions was the neutrophil. Surprisingly, the mechanism of killing was shown to be phagocytosis mediated by both 7S and 19S immunoglobulin M (IgM). Reactions proceeded with as little as 0.01 microg of purified 19S or 7S IgM and were complete within 4-6 h. In contrast, purified immunoglobulin did not adsorb to macrophages and had no effect on target cell binding or cytotoxicity. Pretreatment of cells with cytochalasin D abolished the phagocytic reaction, but not spontaneous cytotoxicity. These data show that antibody-mediated killing results from opsonization and phagocytosis; the mechanism of macrophage killing is currently unknown. In addition, these data show that the shark neutrophil, not the macrophage lineage, carries a receptor for Fc mu.

Inhibition of cell-mediated immune reactions by an aqueous extract of normal human skin.

An aqueous extract of normal human skin has been shown to contain an inhibitor of certain cell mediated immune reactions. In this report, the effect of the inhibitor on cell membrane markers and antibody dependent cellular cytotoxicity was determined. Significant diminution of E rosette formation was demonstrated using as little as 0.6 microgram of the skin fraction (p less than .02). Fc receptors for both IgG and IgM were reduced by 46-96% of controls in the presence of the skin inhibitor. On the other hand, no effect on the detection of the complement receptor or surface immunoglobulin was observed, indicating some specificity of binding. In addition, the antibody dependent cell-mediated cytotoxic reaction was inhibited on the skin extract. It was shown that the inhibitor interacted with the lymphocytes, not the antibody or target cells. No effect was detectable when the skin fraction was added after the interactions of effector cells, antibody, and target cells had occurred. This was in contrast to PHA-induced cytotoxicity which could be inhibited following the preincubation of the lymphocytes with the mitogen. Thus there appears to be 2 mechanisms by which the skin fraction interferes with cellular responses: inhibition of antibody binding to Fc receptors, and interference with a step in cellular activation following mitogen stimulation. Analysis of the extract showed the inhibitor was inactivated by trypsin, and did not contain sialic acid, 5'-nucleotidase of beta-N-acetylglucosaminidase, and thus was not associated with membrane or lysosomal enzymes.

Primed allogeneic reactions to leukocytes and epithelial cells in the bicolor damselfish.

The studies presented here describe allogeneic reactions in the bicolor damselfish, Pomacentrus partitus, a tropical marine teleost. Damselfish were immunized twice at 2-week intervals either by placement of reciprocal allografts or with primary cultures of epithelial cells. Two weeks after the second immunization, recipient splenocytes were tested for alloreactivity toward donor and third-party pronephros cells. For those animals immunized with cultured cells, reactivity toward donor and third-party epithelial cells was also examined. Unprimed animals responded to allogeneic pronephros between days 8 and 10 (28 degrees C). Fish immunized with epithelial cells showed accelerated mixed leukocyte reactions, with optimal responses occurring between days 4 and 6. Moreover, responses to the immunogen preceded responses to donor pronephros tissue by 2 days, occurring on day 2 or day 4. In addition, primed responses were of significantly greater magnitude than primary reactions. Reactivity toward third-party pronephros tissue paralleled responses of naive animals, indicating the specificity of response. No reactivity toward epithelial cells was observed in the absence of immunization, suggesting that accessory cell functions of damselfish pronephros and epithelial cells differ.

Shark spontaneous cytotoxicity: characterization of the regulatory cell.

Down regulation of shark macrophage-mediated spontaneous cytotoxicity is evident in vitro when animals are maintained at environmental temperatures greater than 26 degrees C. Previous work has shown that inhibition of spontaneous killing is mediated by viable, glass nonadherent, nonphagocytic cells which are sensitive to alterations in environmental temperature. The current report further characterizes the regulatory cell population. Glass nonadherent leukocytes were enriched for regulatory activity by density gradient centrifugation, and the majority of activity sedimented to 34% iso-osmotic Percoll. Two morphologically distinct cells are found in this fraction, lymphocyte-like cells and granulocytes. The 34% fraction was further separated by adherence to SIg-coated dishes, and the ability to inhibit spontaneous killing partitioned with the SIg- subset. In addition, fractionation of cells bearing Fc receptors (FcR) for shark Ig showed the regulator to be devoid of FcR. Sequential depletion of cells expressing SIg and FcR confirmed these data. Inhibition by allogeneic cells indicated that histocompatibility between the cytotoxic effector and the regulator is not a requirement for expression of activity. Thus, down regulation of spontaneous cytotoxicity is mediated by nonadherent, nonphagocytic, SIg-, FcR-cells which are not MHC restricted. The lymphoid or granulocytic lineage of the regulatory cell is discussed.

Damselfish with neurofibromatosis exhibit cytotoxicity towards retrovirus infected cells.

Lymphocytes from tumor-bearing damselfish are cytotoxic towards target cell lines derived from damselfish neurofibromatosis. These cell lines contain at least one retrovirus which appears to be related to the etiology of the disease. The current studies were designed to characterize the effectors of this cytotoxic reaction. Data presented here show that cells separated using an antibody (5C6.10.4) directed towards non-specific cytotoxic cells of catfish sequesters all antitumor activity in the 5C6.10.4 negative population. Thus, damselfish 5C6.10.4 positive cells bind to tumor targets, but do not contribute to target cell death. In contrast, 5C6.10.4 positive cells are cytotoxic towards xenogeneic erythrocytes. Cytotoxicity of splenocytes from animals inoculated with virus purified from the 88-503 cell line suggested that prior exposure to the retrovirus enhanced reactivity, especially towards 88-503. In addition, cytotoxicity was significantly greater in tumor homogenate injected animals that resisted tumor development for more than 5 months as compared to those that developed tumors quickly. Lastly, cytotoxic responsiveness towards primary cultures of mock and virus infected autologous and allogeneic cells implies that the cytotoxic effector is directed towards retrovirus infected cells.

Cell-mediated immune response of Chelonia mydas.

Blastogenic and cytotoxic responsiveness of peripheral blood leukocytes from the green turtle, Chelonia mydas were examined. Blastogenic responses were low level and showed considerable variation between animals. Blastogenesis in response to phytohemagglutinin and concanavalin A was observed through out all seasons. Responses to pokeweed mitogen and lipopolysaccharide were seasonal, appearing in spring. No blastogenic response to protein A or allogeneic leukocytes was observed. Cytotoxic responses to phytohemagglutinin and antibody-dependent cell-mediated cytotoxicity were of significant magnitude and, all animals responded in a similar manner. No spontaneous cytotoxic reactivity was observed.

Damselfish with neurofibromatosis exhibit cytotoxicity toward tumor targets.

Damselfish neurofibromatosis (DNF) is a malignant transmissible disease affecting Schwann cells, and is the only naturally occurring animal model of human neurofibromatosis type 1. The current study was designed to determine whether fish in the early stages of disease have measurable immune responses toward DNF tumor cells. Three DNF tumor cell lines were used as targets in standard 51Cr cytotoxic assays. In addition, Lutjanus griseus erythrocytes served as nonspecific targets, and concanavalon A (Con A) blasts from healthy animals served as normal target cells. Results of this study show that tumor-bearing damselfish have cells capable of destroying tumor targets but healthy animals display minimal, if any, reactivity toward the DNF tumor lines. The majority of antitumor activity resides in the spleen; the pronephros appears to contain the majority of nonspecific activity. Data also show that some of the effector cells are analogous to the nonspecific cytotoxic cells of catfish. No lysis of healthy damselfish targets was observed. Thus damselfish have cytotoxic cells capable of interacting with tumor targets, but in the majority of animals this response is not adequate to circumvent the process of tumorigenesis.

Fc receptor for shark IgM.

Fc receptors for shark IgM have been demonstrated on shark leukocytes. Measurement of receptor binding required treatment of leukocytes with Cytochalasin D to inhibit phagocytosis. EA rosetting assays were carried out using human erythrocytes coated with shark anti-human antibody. Binding to shark leukocytes was demonstrated to be specific to shark IgM in that affinity purified shark IgM and purified Fc5 mu fragments could block rosette formation, but not shark transferrin, bovine serum albumin or fetal bovine serum. The binding was shown to be saturable and reversible, characteristic of receptor-ligand interaction. Further, it was shown that affinity purified, radioiodinated IgM could also bind Cytochalasin D-treated shark leukocytes in a manner analogous to rosetting. We conclude that Fc receptors appeared early in evolution, and that previous difficulties in demonstrating the Fc mu receptor resulted from non-specific binding associated with phagocytosis.

Macrophage-like effector of spontaneous cytotoxicity from the shark.

The effector of spontaneous cytotoxicity from shark peripheral blood has been shown to be a macrophage-like cell. Effector cells are isolated by centrifugation over Lymphocyte Separation Medium, adherence to glass, Percoll density gradient centrifugation and adherence to fibronectin sequentially. Effector cells are adherent to glass, sediment to densities of 1.048-1.052 g/ml and are adherent to fibronectin. The isolated effectors represent less than 1% of the peripheral blood leukocytes, and exhibit potent cytotoxic capability, both spontaneous and in the presence of phytohemagglutinin. In addition, the activity of these cells is resistant to 3000 rads gamma irradiation. Although nurse sharks have natural antibody to trinitrophenol, spontaneously cytotoxic cells are incapable of killing trinitrophenol modified targets indicating that natural antibody is not required for reactivity, and that natural antibody and spontaneous effectors do not have the same repertoire. However, cold target inhibition studies showed that these effector cells can recognize four of five human lymphomyeloid targets. It is concluded that the spontaneous, extracellular killing by the macrophage-like effector most closely resembles that of activated mammalian tumoricidal macrophages with the exception that they do not appear to require in vitro activation.

Immune dysfunction in experimental versus naturally occurring neurofibromatosis in damselfish.

Damselfish neurofibromatosis [DNF], a neoplastic disease characterized by multiple, neurofibromas and malignant schwannomas, is currently the only naturally occurring animal model of human neurofibromatosis type-1. Previous studies of immune function in DNF affected fish indicated the potential for significant immunosuppression in advanced stages of the disease. The current study compares healthy animals with fish captured in the wild bearing spontaneous tumors and with animals bearing experimental tumors transmitted in the laboratory. In order to determine the effects of tumor burden on the immune capabilities of these animals, proliferative responses to mitogens and toward allogeneic cells were tested. The data presented here indicate that animals bearing advanced tumors of experimental origin are profoundly immunocompromised. Similarly, some spontaneous tumor-bearing animals are deficient in proliferative immune responses, and splenocytes from most animals fail to respond to mitogens. However, a proportion of animals with stage 5 spontaneous tumors retain immune reactivity, and are capable of alloreactions comparable to those of normal individuals when stimulated with cells from healthy [4/10, 40%] or other tumor-bearing [4/8, 50%] animals. The contributions of tumor size, distribution and cytokine production to the differential immune impairment are discussed.

Shark cytotoxic macrophages interact with target membrane amino groups.

The types of target structures recognized by cytotoxic macrophages have been described for various microorganisms, but have not been defined for tumor cells. Tumoricidal macrophages are selective in their destructive mechanisms, sparing normal cells while directing their lytic machinery toward neoplastic targets. The cytotoxic activity of macrophages from a primitive vertebrate, the nurse shark, closely resembles the activity of mammalian tumoricidal macrophages. Host defense mechanisms of these animals appear to rely on antigen nonspecific cellular effector systems, and it has been postulated that macrophage-mediated cytotoxicity plays a dominant role in protection during periods of decreased environmental temperatures when lymphocyte responses of poikilothermic vertebrates are compromised. Similar to mammalian tumoricidal macrophages shark macrophages display selective recognition of target cells. Previous studies showed that TNP modification of targets was protective, preventing recognition by the shark spontaneously cytotoxic macrophage. Additionally, it was shown that cytotoxic activity was inhibited in a dose dependent fashion by the addition of excess unlabeled targets. In the present study, similar inhibition experiments with hapten-modified targets have been used to determine the nature of the target structures recognized by the shark cytotoxic macrophage. Cold targets modified with haptens which react covalently with free amino groups on cell membranes, trinitrobenzene sulfonic acid (TNBS) and flourescein isothiocyanate (FITC), are not recognized by the cytotoxic macrophage. The relative amount of membrane bound TNP was correlated with inhibition of cytotoxicity. Conversely, target cells modified with sulfhydryl reacting reagents, N-iodoacetyl-N'-(5-sulfonic-1-naphthyl) ethylene diamine and dithionicotinic acid, are recognized similarly to untreated targets. Moreover, TNP-containing lipids, permitted to diffuse into target membranes without covalent binding, do not alter target recognition, indicating that TNP itself has no effect on macrophage:target interaction. From these data, it is concluded that the shark cytotoxic macrophage interacts with membrane bound amino, but not sulfhydryl groups. The ability to distinguish between membrane structures may have appeared early in evolution as a means of preserving self cells while retaining protective nonspecific cytotoxic mechanisms.