Plasma sex steroid and thyroid hormones profile in male water frogs of the Rana esculenta complex from agricultural and pristine areas.

Some chemical compounds used in intensive agriculture have been found to induce estrogenic effects; therefore a histological analysis of the testes and an evaluation of plasma levels of sex steroid, thyroid hormones, and vitellogenin were carried out in adult male water frogs of two coexisting taxa (Rana lessonae and the hemiclonal hybrid Rana esculenta) sampled in agricultural and pristine areas. Differences in seasonal profiles of hormones were found in water frogs living in the agricultural area where the presence of endocrine disrupting compounds was suspected on the basis of a previous study. In R. esculenta, sampled in the pristine area, high androgen levels were found in May; the opposite trend was found for R. esculenta sampled in agricultural areas in which the highest androgen levels were found in September, significantly lower compared with those found in R. esculenta sampled in the pristine area. Low androgen levels were also recorded in R. lessonae males sampled both in pristine and agricultural areas, while the highest levels were found in September. Regarding the trend of estradiol-17beta, an increase of this hormone was found in July both in esculenta and lessonae sampled in the agricultural area, and in the same month an estradiol-17beta peak, even though lower, was also found both in esculenta and lessonae males captured in the pristine area; detectable vitellogenin was found neither in males captured in the agricultural area, nor in those sampled in the pristine one. Moreover, while no significant changes of thyroid hormones were found either in the esculenta or lessonae males sampled in the pristine area, increased T3 and T4 titers were found in July in both esculenta and lessonae captured in the agricultural area. Morphological differences of the testes in males of parental species captured in the agricultural area were also observed. These findings indicate alterations in endocrine and reproductive function in frogs in the agricultural area, that could suggest the presence of endocrine disrupting compounds.

Parasitism by Dermocystidium ranae in a population of Rana esculenta complex in Central Italy and description of Amphibiocystidium n. gen.

We report the enigmatic parasite Dermocystidium ranae in a green frog population (Solomeo, Umbria, Italy) of the Rana esculenta complex, consisting of the parental species R. lessonae (L) and hybrid form R. esculenta (E). In this population a rapid 50% decline of the parental form L was observed. Large dermal U-shaped cysts of D. ranae were found primarily on the ventral aspect of infected individuals, with a significantly higher incidence of infection in the parental species compared to the clonal hybrid. In each form, however, there was little pathological change associated with infection, and the cause of the recent declines of R. lessonae at this site remains unknown. In this paper we present the first ultrastructural description of an amphibian Dermocystidium sp. and we review the taxonomy of Dermocystidium, Dermosporidium and Dermomycoides spp. from amphibians. We conclude that Dermosporidium multigranulare Broz & Kulda, 1954 is synonymous with Dermocystidium ranae Guyénot & Naville, 1922 and, due to lack of sufficient differences between genera and significant dissimilarities with fish Dermocystidium spp., the 3 amphibian genera are synonymous. We propose that they should be designated to a new genus, Amphibiocystidium n. gen., and Dermocystidium retained for those species parasitic in fish.

Multiple isoform recovery (MIR)-PCR: a simple method for the isolation of related mRNA isoforms.

We present a rapid and efficient method for the detection of related transcripts with different expression levels. This approach combines the rapid amplification of cDNA ends (RACE) method with a cDNA subtractive technique. The strategy is based on successive subtractions of prevalent isoforms resulting in enrichment of less expressed transcripts. For each subtraction, a biotinylated primer specific for the prevalent isoform is hybridized on the total cDNA and the hybrid is retained on a streptavidin affinity column. The unbound cDNA serves as a template for subsequent isoform identification. To illustrate its application we describe the isolation of three new actin cDNA isoforms in the freshwater planarian Dugesia (S) polychroa.

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.

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.

The mammalian anti-alpha-smooth muscle actin monoclonal antibody recognizes an alpha-actin-like protein in planaria (Dugesia lugubris s.l.).

The presence of an alpha-smooth muscle (alpha-sm) actin-like protein in planaria (Dugesia lugubris s.l.) is reported. The protein shows a 42 kDa molecular weight determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis and is specifically recognized by the mammalian anti alpha-sm actin monoclonal antibody. When a planarian is induced to regenerate by head amputation, the immunostaining of the alpha-sm actin-like molecule becomes important in the area of growing blastema, reaching a maximum between 70-120 hours after injury. Conventional electron microscopy at the 4-day-regeneration stage shows that blastema-forming cells are a homogeneous population whose morphological features resemble those of migrating mesenchyme-like cells; only the myoblasts show a recognizable phenotype. The immunocytochemical localization of alpha-sm actin-like molecule by immunoperoxidase (light microscopy) and immunogold stains (electron microscopy) was carried out on both intact and injured worms. The antigen was localized mainly at the basal portion of the epidermal cells and in the undifferentiated mesenchyme-like cells. Myoblasts, but not differentiated myofibers, were also labelled by this antibody. The results indicate that in the lower Eumetazoan planarians, as well as in vertebrates, the alpha-sm actin can be considered to be a marker for myoid differentiation. The suggestion that alpha-sm actin can be used as a marker for mesenchyme-like cells in vertebrates and in invertebrates is also discussed.

Acid phosphatases in mammalian tissues. Evidence for the existence of a 57 kDa Zn(2+)-dependent acid phosphatase form.

1. A comparative study of multiple forms of acid phosphatase (AcPase) in various organs of mammals was carried out. 2. These studies indicated that the high-molecular weight AcPase is preferentially expressed by tissues which undergo cell proliferation such as epithelial tissues; on the contrary, the low-molecular weight enzyme seems to be characteristic of highly differentiated tissues such as nervous, muscle and blood erythrocytes. 3. The existence of a new AcPase activated by Zn2+ ions was observed in all tissues studied with the exception of erythrocytes. 4. The enzyme shows a molecular weight of 57 kDa, is insensitive to NaF, hydrolyzes p-nitro-phenylphosphate and o-c-phenylphosphate; ATP, a-naphthyl-phosphate and beta-glycerolphosphate are also dephosphorylated.

Characterization and fine-structural localization of actin- and fibronectin-like proteins in planaria (Dugesia lugubris s.l.).

Actin- and fibronectin-like proteins were characterized in the planarian, Dugesia lugubris s.l., by sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblotting analysis using antisera to vertebrate actin and fibronectin. These antisera recognized protein bands of 42 kDa and 220 kDa, respectively. In addition, the immunohistochemical distribution of both actin- and fibronectin-like material was examined by using immuno-electron microscopy. Actin-like protein was localized in myofibrils in various differentiation stages, and in the peripheral cytoplasm and lamellipodia of cells that were migrating. The fibronectin-like component was associated with the extracellular matrix in the fibrillar structures and with the surface of the migrating cells. Our data suggest that similar cellular and molecular mechanisms are involved in cell-matrix interactions and in the morphogenesis of living organisms at different evolutionary levels.

Acid phosphatases in the frog (Rana esculenta) skeletal muscle. Purification and some properties of the low molecular weight enzyme.

1. The presence of high-Mr and low-Mr acid phosphatases [orthophosphoric-monoester phosphohydrolase, (acid optimum), EC 3.1.3.2] in the skeletal muscle of frog Rana esculenta was reported. 2. The subcellular localization and some characteristics of both enzymes were also described. 3. The low-Mr AcPase was purified to homogeneity. The enzyme did not absorb on Concanavalin A-Sepharose 4B indicating that this was not a glycoprotein. 4. The enzyme is homogeneous on polyacrylamide gel electrophoresis and moves as a single band of Mr 13.7 +/- 0.8 kDa in the presence of sodium dodecyl sulphate. 5. The Mr of the native enzyme was 14.0 +/- 1.1 kDa as determined by gel filtration on a Sephadex G-100 column. The isoelectric point was 6.02. 6. The enzyme was strongly inhibited by 1 mM Ag+, Hg2+, Sn2+ and Cu2+ while other cations both at 10(-2) and 10(-3) M showed little or no effect. 7. The enzyme was insensitive to NaF and tartrate but was strongly deactivated by formaldehyde, PMB, Iodoacetamide and Triton X-100. Phosphate was a competitive inhibitor (k1 = 0.83 mM). 8. The best substrate for the enzyme was p-nitrophenylphosphate but phenylphosphate, flavin mononucleotide and o-P-tyrosine were also hydrolyzed, though at different rates. 9. The enzyme activity was enhanced in the presence of methanol, ethanol, acetone and glycerol indicating a phosphotransferase activity.

Purification and subcellular localization of Zn-dependent acid p-nitrophenylphosphatase in frog liver and comparison with other vertebrates.

Zn2(+)-dependent acid p-nitrophenylphosphatase (Zn-AcPase) from liver of Rana esculenta was purified to homogeneity. The purified enzyme moves as a single electrophoretic band at pH 8.3 in 7.5% acrylamide and was coincident with the enzyme activity. The enzyme has a molecular weight of 102,000 +/- 5,000D and is a dimer with two apparently similar polypeptide chains of 48,000 +/- 3,000D as determined by sodium dodecylsulfate gel electrophoresis. Zn-AcPase from frog liver requires Zn2+ ions for catalytic activity; other bivalent cations have little or no effect. The enzyme with a pI of 7.07 does not appear to be a glycoprotein and was associated with the soluble fraction after liver cell fractionation. The biochemical and molecular properties of frog liver Zn-AcPase were compared with that of the enzyme partially purified from carp (Cyprinus carpio), pike (Esox lucius), and rat (Rattus norvegicus).

Functional aspects of calcium transport in sarcoplasmic reticulum vesicles derived from frog skeletal muscle treated with saponin.

To study the physiological aspects of the excitation-contraction cycle, saponin (10-100 micrograms ml-1) was used as a skinning agent on muscle and sarcotubular vesicles derived from fast muscles (sartorius and tibialis anterior) of Rana esculenta. The vesicles showed similar Ca2+-ATPase activity and similar protein profiles carried out by SDS-PAGE. Calcium transport in untreated vesicles and those treated with different concentrations of saponin seemed to have the same quantitative and qualitative parameters if the saponin was used in a range between 10 and 50 micrograms ml-1. Our results confirm that saponin may be considered to be a valid skinning agent for the external membranes of fast skeletal muscles.

Acid phosphatases from liver of Rana esculenta. Subcellular localization and partial characterization of multiple forms.

1. Four acid phosphatases (AcPase I, II, III and IV) were found in the liver of the frog Rana esculenta. 2. AcPases I, II, III, and IV were associated with the microsomal, mitochondrial-lysosomal, nuclear and soluble fractions respectively and showed apparent molecular weights of about 240,000, 110,000, 38,000 and 17,000. 3. All the enzymes show acid pH optima, and similar Km values for p-nitrophenylphosphate. 4. AcPases I, II, and III hydrolyze most of the common phosphate esters whereas AcPase IV hydrolyzes effectively only p-nitrophenylphosphate, phenylphosphate and flavine mononucleotide. 5. AcPases I and II are inhibited by NaF and tartrate. AcPases III and IV are tartrate resistant. 6. Temperature inhibits AcPases I, II, IV, whereas it activates AcPase III.

The phorbol ester TPA dramatically inhibits planarian regeneration.

The effect of phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) on head regeneration in decapitated planarians (Dugesia lugubris s.1.) has been studied. TPA-treatment soon after head amputation dramatically inhibited the regenerative process. Ultrastructural analysis revealed that the migration of fixed parenchymal cells (FPCs) to the wound area was strongly activated by TPA. FPCs interacted with various types of cells inducing lysis and phagocyting cell debris. The resulting fluid was removed through diaphanous protrusions appearing at the level of the wound zone. Moreover the close association of FPCs with neoblastlike cell clusters in the parenchyma indicated their possible role in the modulation of neoblast migration.

Immunoelectron microscopic localization of a fibronectin-like molecule in Dugesia lugubris s.l.

Fibronectin (FN)-like protein has been localized by immunoelectron microscopy in the extracellular matrix (ECM) of planaria Dugesia lugubris s.l. The immunolabeling was present in both intercellular spaces of epidermal cells and the basement membrane, however the amount and distribution of gold particles seemed to be substantially different. FN-like material increased markedly during the passage of migrating cells through the basement membrane from the parenchyma to the epidermis. Gold particles were often found at cell-matrix contacts. Our result suggest that FN-like molecules detected in planarian ECM may be involved not only in cell adhesion but also in promoting cell migration and in regulating the epidermal cell turnover.

Immunoelectron microscopic localization of actin in migrating cells during planarian wound healing.

Wound repair in planarians is mainly characterized by two cell-migratory events involving the epidermis adjacent to the wound and its basement membrane. The first event is the migration of epidermal cells to cover the wound surface; the second one is the migration of newly differentiating replacement epidermal cells from the parenchyma to the epidermis. In addition to these events, migration of fixed parenchymal cells is observed during wound healing. All migrating cells were characterized by the presence of actin, as shown by the results obtained by means of indirect immunolocalization with fluorescent and electron microscopy. Migrating cells were heavily labeled with gold particles, which clustered at the level of cell-matrix and cell-cell contacts.