An orthologue of the host-defense protein psoriasin (S100A7) is expressed in frog skin.

Host-defense peptides and proteins are vital for first line protection against bacteria. Most host-defense peptides and proteins common in vertebrates have been studied primarily in mammals, while their orthologues in non-mammalian vertebrates received less attention. We found that the European Common Frog Rana temporaria expresses a protein in its skin that is evolutionarily related to the host-defense protein S100A7. This prompted us to test if the encoded protein, which is an important microbicidal protein in human skin, shows similar activity in frogs. The R. temporaria protein lacks the zinc-binding sites that are key to the antimicrobial activity of human S100A7 at neutral pH. However, despite being less potent, the R. temporaria protein does compromise bacterial membranes at low pH, similar to its human counterpart. We postulate that, while amphibian S100A7 likely serves other functions, the capacity to compromise bacterial cell membranes evolved early in tetrapod evolution.

[Regulatory interconnections of cyclooxigenase and inducible no-synthase in urinary bladder epithelial cells of the frog Rana temporaria under action of bacterial stimuli].

Earlier we have shown that in epithelial cells of the frog urinary bladder under action of bacterial lipopolysaccharides (LPS) there is activated expression of inducible NO-synthase (iNOS) and there is increased the NO production, which can play an important role in providing protective cell reactions from pathogens. The goal of the present work consisted in study of cyclooxigenase (cOG) products and mechanisms of their regulatory effect on expression of iNOS under action of LPS. In experiments on urinary bladder epithelial cells on the frog Rana temporaria it has been shown that incubation of the cells for 21 h with LPS leads to a rise in production of PGE2 and nitrites, stable NO metabolites. Inhibitor of iNOS 1400W decreased sharply production of nitrites, but did not affect the PGE2 level. Both the basal and the LPS-stimulated level of PGE2 and nitrites were inhibited in the presence of selective cOG inhibitors--SC-560 (cOG-1) and NS-398 (cOG-2). The IC50 value amounted to 90, 220, and 470 microM for NS-398, SC-560, and diclofenac (unspecific inhibitor of both isoforms), respectively. PGE2 and butaprost, the EP2-receptor agonist, but not agonists of EP1/EP3 or EP1 receptors, partially eliminated the inhibitory action of diclofenac on production of nitrites. Action of PGE2 was accompanied by an increase in the intracellular cAMP. Analysis of expression of iNOS mRNA in the epithelial cells incubated with LPS or LPS + inhibitor of cOG has shown the LPS-stimulated rise in expression of iNOS mRNA to decrease sharply in the presence of SC-560 or NS-398. Thus, the epithelial cells of the frog urinary bladder have the effectively functioning system of the congenital immune protection against bacterial pathogens, the most important component of this system being PGE2 and NO. Analysis of mechanisms of regulatory interactions of cOG and iNOS indicates that in this cell type the main regulators of iNOS expression and of the nitrogen oxide level are products of the cOG catalytic activity.

[Mast cells of lymph hearts during ontogenesis of frogs Rana temporaria].

Electron microscopic observations of the lymph hearts of tadpoles and yearling frogs of Rana temporaria showed that mast cells (MCs) were present not only between muscle fibers (population of resident MCs), but in the cavities of lymph heart (population of circulating MCs), too. There were some differences in the ultrastructure of the resident MCs at each studied stage of larval development. The first recognizable MCs were revealed in the lymph hearts at premetamorphosis (stages 39-41). MCs presented as mononuclear relatively small and slightly elongated cells with a few immature secretory granules and numerous free ribosomes, polysomes and short cisternae of rough endoplasmic reticulum (RER) in the cytoplasm. Chromatin of their nuclei was poorly condensed; the Golgi apparatus was moderately developed. At pro-metamorphosis (stages 44-45), we revealed MCs at different levels of their differentiation. Some MCs demonstrated an active process of granulogenesis in their cytoplasm. Among densely packed cytoplasmic organelles, immature secretory granules were closely associated with cisternae of RER and free ribosomes. Other MCs appeared as more differentiated cells. They were characterized by a predominantly heterochromatic nuclei and cytoplasm filled with polymorphic and heterogeneous granules. MCs also showed a reduction in the number of free ribosomes and cisternae of RER in the cytoplasm. On the contrary, the Golgi apparatus was well developed. Stacks of Golgi cisternae, detaching vacuoles, and progranules occupied the perinuclear region. The majority of the outlines above ultrastructural features of differentiated MCs were typical for MCs of yearling frogs. At metamorphic climax (stages 52-53), MCs often tightly contacted with macrophages. We did not reveal apoptotic MCs. However, some MCs exhibited morphological features typical for programmed necrosis-like death, which was characterized by mitochondria swelling, dilatation of cisternae of RER and nuclear envelope, plasma membrane rupture and subsequent loss of intracellular contents. Electron microscopical immunocytochemistry revealed the localization of atrial natriuretic peptide (ANP), substance S (SP) and heat shock protein (Hsp70) in the secretory granules of the resident and circulating MCs at different stages of tadpole development and in yearling frogs.

[GABA immunoreactivity in the main olfactory bulb of the frog Rana temporaria]. / GAMK-immunoreaktivnost' v osnovnoi oboniatel'noi lukovitse liagushkiRana temporaria.

Immunoreactivity for gamma-aminobutyric acid (GABA) was localized at the light microscopic level in the main olfactory bulb (MOB) of the frog, Rana temporaria. By means of free-floating peroxidase-antiperoxidase immunocytochemical technique, GABA was found in a large number of neurons in the granular cell layer, in a few small somata in the mitral cell layer and in two different types of cell somata in the glomerular layer. Individual GABA-immunopositive cells were found in the olfactory nerve layer. GABA immunostaining was also localized in cell processes and fiber fragments. There were many immunoreactive puncta in all layers of the MOB. GABA-positive punctate structures often outlined immunonegative cells in the mitral cell and glomerular layers. Rounded tightly packed groups of immunoreactive puncta were found only along ventral border of the glomerular layer. The results are discussed in comparison with data obtained on mammalian MOB in terms of MOB functional organization.

[Immunochemical approach to the identification of specific markers of the corneal epithelium in Rana temporaria]. / Immunokhimicheskii podkhod k identifikatsii spetsificheskikh markerov épiteliia rogovitsy Rana temporaria.

Biochemical and immunochemical studies of water soluble proteins in the corneal epithelium (CE) of adult frogs were carried out. Up to 30-40 protein fractions were found in the CE extracts using the methods of isoelectrofocusing and electrophoresis with SDS. The major part of the CE proteins is in the acid zone (pH 4.2-6.2) and is presented by three peptide fractions in the molecular weight range of 40-50 kD. The CE peptide spectrum is most close to that of epidermis and differs markedly from that of lens fibers. Up to 20 water-soluble antigens were revealed by rabbit antisera against the total extract, among them 10 antigens are immunologically similar with the blood serum proteins. Out of 10 tissue antigens, 6 are interorganic. Only two corneal antigens were not found in epidermis. One of them was found only in CE, retina and lens epithelium. Another antigen is present in considerable quantities only in liver, heart and lung. The synthesis of most tissue specific antigens and antigens immunologically similar with alpha- and beta-crystallins of R. temporaria was shown in CE by immunoautoradiography. The antigenic composition proved to be similar in CE, epidermis and lens epithelium.

Amphibian lymphokines: 2. Migration inhibition factor produced by antigenic and mitogenic stimulation of amphibian leucocytes.

Migration of Rana temporaria peritoneal exudate cells (PEC) was examined in vitro using both direct and indirect assay systems. After sensitization in vivo followed by in vitro challenge 7-21 days later with the appropriate sensitizing antigen, spleen cell culture supernatants were obtained which inhibited the normal in vitro migration of PEC from non-sensitized animals. Cultures of spleen cells with mitogen also gave rise to supernatants with migration inhibitory properties. Sephadex separation of supernatants showed that maximum inhibitory activity was present in the 27-50,000 MW range and, furthermore, that this inhibition was blocked by alpha-L-fucose, but not by beta-D-galactose. The inhibition did not appear to be species specific. The results indicate that following appropriate stimulation amphibian leucocytes produce a soluble, migration inhibition factor (MIF) with characteristics similar to those described for the mammalian lymphokine MIF.

Amphibian lymphokines: 1. Leucocyte chemotactic factors produced by amphibian spleen cells following antigenic and mitogenic challenge in vitro.

The production of soluble chemotactic factors by Rana temporaria leucocytes following in vitro stimulation with antigen or mitogen was examined. Stimulated spleen cells were found to produce a soluble factor with a molecular weight (MW) of between 16,000-27,000 daltons which was chemotactic for Rana peritoneal cells. Rana peritoneal cells also showed directional movement towards solutions of casein, which is a potent chemo-attractant for mammalian lymphoid cells.

Gorgoderina vitelliloba: interaction with frog leucocytes in vivo and in vitro.

Ultrastructural observations were made on the response in vivo of adult Rana temporaria to Gorgoderina vitelliloba and the interaction between host cells and the parasite in vitro. In both cases, the cellular attack on the fluke was most intense 21-23 days postinfection. Host leucocytes (believed to be eosinophils) lay flat against the surface of the parasite. Degranulation of their electron-dense, peroxidase-positive granules occurred and vacuoles formed. The outer plasmalemma of the tegument was breached and eosinophils migrated over the basal lamina, stripping the tegument from the surface of the fluke. The cellular response was so vigorous in vitro that the tegument was sometimes completely lost. In vivo, damage was slight, and the majority of the parasites successfully completed their migration to the bladder, where there was no further cellular response, and tegumental repair occurred. The fact that the migration of the fluke from the kidney to the bladder occurred at the same time as the peak of the cellular response may not be coincidental, and it is suggested that the cellular attack may initiate migration from the kidneys.

MHC zygosity in the frog, Rana temporaria.

The major histocompatibility complex (MHC) zygosity of the field-collected frogs, Rana temporaria, was detected by progeny testing. Groups of sibling tadpoles were grafted with intrafamilial tail-tip allografts and the ratio of rapidly rejected allografts to slowly rejected ones was estimated. Twenty-five percent of parental frogs appeared to be MHC homozygotes. Thus, MHC homozygosity in natural frog populations seems to be considerably higher than in wild mouse populations.

Ontogeny of transplantation immunity in the common frog, Rana temporaria L.

Viability of allografts exchanged between the field-collected individuals of the common frog, R. temporaria, was long in tadpoles grafted during and immediately after closing of operculum; median survival time (MST) was 26 and 18 days, respectively. This probably reflected the immaturity of the host immune system and temporary tolerance to weak transplantation antigens. Allograft viability was the shortest in tadpoles grafted at foot-paddle stage (MST, 11 days). It was independent from the origin and size of the grafts. Such rate of rejection might reflect a maximal immunological potential of the host and the absence of any suppressor factors in response to highly polymorphic frog transplantation antigens. A gradual prolongation of allograft viability was observed in animals grafted at final stages of metamorphosis, in froglets, and in sexually mature adults (MST: 13, 17, and 28 days, respectively). In particular age groups viability of allografts from sibling donors was longer and from nonsibling ones shorter than MST values cited above. Immunological memory of transplantation antigens did not disappear during the host metamorphosis, as MST (10 days) of second-set allografts in metamorphosing hosts sensitized during larval life was considerably shorter than the viability of the sensitizing grafts in the same age group. The ontogeny of the response to alloantigens reflecting the immunological potential and the appearance of self-tolerance can be realized in different ways, depending on a particular amphibian species.

[Study of the cytostatic action of the cytoplasm of oocytes and mature ova of the common frog and sturgeon (Acipenser stellatus)]. / Izuchenie istitostaticheskogo deistviia tsitoplazmy ootsitov i zrelykh iaits travianoi liagushki i sevriugi.

The presence of the cytostatic factor found by Masui and Markert (1971) in the cytoplasm of oocytes and eggs of Rana pipiens was verified in the cytoplasm and mature inactivated eggs of the common frog Rana temporaria and sevryuga Acipenser stellatus. The cytoplasm was injected to the embryos of the same species at different phases of the first cleavage division in the animal region of one of the two blastomeres. The injection of cytoplasm did not cause the arrest of cleavage divisions. Many embryos proceeded to gastrulation. The cytoplasm of maturing oocyte and mature egg in the common frog and sevryuga, unlike in R. pipiens, has no cytostatic effect.

Albumin evolution in frogs: a test of the evolutionary clock hypothesis.

Frogs are an ancient group compared to placental mammals. Yet, although there are about as many species of frogs as there are of mammals, zoologists consider that frogs have undergone only limited morphological divergence, while placental mammals have diversified greatly in morphology and way of life. The serum albumins of numerous frog species were compared by the quantitative microcomplement fixation technique. Frogs that are morphologically similar enough to merit taxonomic distinction at only the species level often exhibit differences in the serological properties of their albumins larger than those usually seen between mammals placed in distinct families or suborders. Thus, there seems to be a contrast between albumin evolution and evolution at the organismal level. The large differences between albumins among frogs can be explained by the hypothesis that albumin evolution has proceeded at the same rate in frogs as in mammals.