Actin isoforms in amphioxus Branchiostoma lanceolatum.

Actin is a highly conserved cytoskeletal protein that is ubiquitous in all eukaryotes. Little is known about actin expression in amphioxus, the closest living relative of the vertebrates. In the present study, involving Western blotting and indirect immunofluorescence, we report the characterization and localization of various actin isoforms in amphioxus (Branchiostoma lanceolatum) tissues. Three antibodies against vertebrate actins were used: a polyclonal antibody recognizing beta-cytoplasmic actin (anti-beta actin), a monoclonal antibody against sarcomeric actins (anti-alphaSR-1), and a monoclonal antibody specific for alpha-smooth actin (anti-alphaSM-1). Western blot analysis of amphioxus extracts immunodecorated with these antibodies showed a 43-kDa-positive band co-migrating with respective controls. The amphioxus isoactin expression patterns recognized by these antibodies were similar to those of vertebrates, i.e., anti-beta actin showed positive staining mainly in non-muscle cells, anti-alphaSR-1 labelled dorsolateral myotomal muscles, and anti-alphaSM-1 stained ventral muscles. These results demonstrate that at least two muscle actins are present in amphioxus, suggesting that muscle actin gene duplication events began before vertebrate divergence from the amphioxus lineage.

The effects of copper on actin and fibronectin organization in Mytilus galloprovincialis haemocytes.

The effects of copper on actin and fibronectin organization in Mytilus galloprovincialis haemocytes were studied. The Cu2+ exposure of mussels caused severe perturbations in haemocyte actin and fibronectin organization with respect to non-exposed organisms. Cytoskeletal actin was analysed by indirect immunofluorescence, using an antitotal actin monoclonal antibody, and by rhodamine-conjugated phalloidin. The majority of haemocytes from Cu(2+)-exposed mussels displayed a round morphology, with short and blunt filopodia; they lacked the polarized phenotype which was typical in control samples. The cytoskeleton alteration, more evident after phalloidin staining, resulted in the disappearance of filamentous actin. The actin cortical meshwork also appeared disorganized. The cytoskeletal morphology studied by transmission electron microscopy after negative staining of Triton X-100-treated haemocytes confirmed these observations. The structural organization of actin when analysed by Western blotting showed a larger number of Triton-soluble actin pools in treated mussel haemocytes. Fibronectin was studied by indirect immunofluorescence using a polyclonal antiserum directed against mussel fibronectin. In treated mussels, fibronectin appeared to be strongly disorganized and its levels decreased in both haemocytes and haemolymph. The mechanism(s) of the copper-induced alterations on actin and fibronectin organization in mussel immunocytes is discussed.

Characterization and immunocytochemical localization of actin and fibronectin in haemocytes of the mussel Mytilus galloprovincialis.

Cell-extracellular matrix interactions are recognized to be important for human leucocyte functions, including chemotaxis and phagocytosis. These activities depend on a reorganization of the microfilament actin (F-actin) promoted by fibronectin, one of the major components of extracellular matrices. Although invertebrate haemocytes are, in many aspects, similar to the human granulocyte-monocyte-macrophage cell lineage, actin and fibronectin have not been well studied in these cells. Consequently, the characterization and structural organization of actin and fibronectin in mussel (Mytilus galloprovincialis) haemocytes was investigated using Western blotting analysis, indirect immunofluorescence and immunoelectron microscopy. Actin was immunocharacterized by an anti-total actin monoclonal antibody. Fibronectin was immunocharacterized by an autologous polyclonal antiserum directed against the protein of mussel haemolymph. Actin was mainly localized along the peripheral cytoplasm of the haemocyte. The distribution of the F-actin microfilaments was assayed with Rhodamine-labelled phalloidin. F-actin was associated mainly with stress-fibres of spreading haemocytes and with microspikes at the adhesion sites. The labelling by the anti-fibronectin antiserum of the haemocyte rough endoplasmic reticulum vesicles, revealed by immunoelectron microscopy, suggests that these cells are involved in fibronectin biosynthesis. Gold particles were also present along the outer surfaces of the cell plasma membrane and its protrusions. Mussel fibronectin was localized immunohistochemically at the adhesion sites and in the extracellular matrix fibrils. The relationships between fibronectin and the actin cytoskeleton in Mytilus galloprovincialis haemocytes are discussed.

Expression of alpha SM actin in terrestrial ectothermic vertebrates.

alpha-Smooth muscle (alpha SM) actin of endothermic vertebrates is selectively recognized by the monoclonal antibody anti-alpha SM-1. Immunoreactivity to this antibody has been shown to be localized in the NH2-terminal sequence Ac-EEED (Chaponnier et al. 1994). Among terrestrial ectothermic vertebrates, two amphibian (Triturus vulgaris, Rana esculenta) and three reptilian species (Pseudemys scripta elegans, Natrix natrix, Podarcis sicula) were screened to investigate if their vascular and visceral smooth muscles were stained by anti-alpha SM-1. In all the specimens tested, Western-blot analysis of tissue extracts immunodecorated with anti-alpha SM-1 revealed a single polypeptide chain having the same electrophoretic mobility as bovine alpha SM actin. The binding to amphibian and reptilian tissue extracts was inhibited by the synthetic peptide Ac-EEED, but not Ac-DEED, as occurs in mammals. alpha SM actin expression was found in vascular and visceral smooth muscle cells of the species tested. The media of small and large blood vessels was labelled by anti-alpha SM-1. In the stomach and intestine the outer longitudinal and inner circular layers of the muscularis and of the muscularis mucosae were stained. In addition, myofibroblasts of the subepithelial layer were labelled. A more restricted expression of this isoactin was detected in turtle (P. scripta elegans) visceral smooth muscle cells, which may be related to the involvement of the digestive system in respiratory activity. These data suggest that in vertebrate evolution alpha SM actin arose earlier than previously proposed.

Factor VIII inhibitor prior to and during secondary acute nonlymphocytic leukemia in a patient with cured Hodgkin's disease.

We report a 54-year-old patient with Hodgkin's disease who achieved a complete remission after combined modality treatment. Three years later the patient developed a severe hemorrhagic syndrome, concomitant with the onset of a factor VIII inhibitor in plasma. The control of very proteiform bleedings was extremely difficult, even with plasmaphereses, as well as with immunosuppressive and substitutive therapies. Two years later, a secondary acute nonlymphocytic leukemia (ANLL) was diagnosed. Two courses of chemotherapy with fludarabine, cytosine arabinoside and G-CSF (FLAG) were able to obtain a complete remission. Hemorrhagic complications were mainly linked to thrombocytopenia and continued until recovery of thrombopoiesis. Factor VIII inhibitor levels and related clinical symptoms decreased progressively. In conclusion, we suggest that FLAG succeeded in inhibiting an abnormal lymphoid clone responsible for factor VIII inhibitor production, suggesting a possible role for intensive chemotherapy in similar situations, which are often refractory to conventional immunosuppressive and depletive therapy.

Actin expression in some Platyhelminthe species.

Actin expression in some Platyhelminthe species was demonstrated by western-blotting and immunocytochemical analysis using two distinct anti-actin antibodies: the anti-total actin that reacts against all actin isoforms of higher vertebrates and the anti-alpha SM-1 that recognizes the alpha-smooth muscle (alpha SM) isotype of endothermic vertebrates (Skalli et al., 1986). Western-blotting experiments showed that all species tested, including some free-living Platyhelminthes (Tricladida and Rhabdocoela) and the parasitic Fasciola hepatica, were stained by anti-total actin antibody while only Dugesidae and Dendrocoelidae showed a positive immunoreactivity against anti-alpha SM-1. These results were confirmed by cytochemical immunolocalization using both avidin biotin conjugated peroxidase reaction on paraffin sections, and immunogold staining on Lowicryl 4KM embedded specimens. Our findings may contribute to the understanding of Platyhelminthes phylogeny.

Purification and some properties of a Mg(2+)-activated acid phosphatase from rat testis.

1. The acid phosphatase (AcPase, EC 3.1.3.2) IV from rat testicular tissue was purified to apparent homogeneity. 2. The enzyme displays a native molecular weight of 70 kDa determined on gel permeation chromatography on a Sephadex G-100 column and 68 kDa using linear 5-20% sucrose density gradient centrifugation. The subunit molecular weight on SDS-PAGE analysis is 67 kDa, suggesting that the enzyme is a monomeric protein. 3. The enzyme does not bind to Concanavaline A-Sepharose 4B column, indicating that it is not a glycoprotein. 4. The rat testis AcPase IV is a metal activated enzyme in which Mg2+ is the metal activating agent with a Ka = 0.88 x 10(-3) M. The Michaelis constant for p-nitrophenylphosphate, in the presence of saturating concentrations of Mg2+ ions, is 0.23 x 10(-3) M. 5. The enzyme preferentially hydrolyzes p-nitrophenylphosphate, phenylphosphate and ATP.