Antimicrobial action of histone H2B in Escherichia coli: evidence for membrane translocation and DNA-binding of a histone H2B fragment after proteolytic cleavage by outer membrane proteinase T.

Previous studies have led to the isolation of histone H2B with antibacterial properties from an extract of the skin of the Schlegel's green tree frog Rhacophorus schlegelii and it is now demonstrated that the intact peptide is released into norepinephrine-stimulated skin secretions. In order to investigate the mechanism of action of this peptide, a maltose-binding protein (MBP)-fused histone H2B (MBP-H2B) conjugate was prepared and subjected to antimicrobial assay. The fusion protein showed bacteriostatic activity against Escherichia coli strain JCM5491 with a minimum inhibitory concentration of 11 microM. The lysate prepared from JCM5491 cells was capable of fragmenting MBP-H2B within the histone H2B region, but the lysate from the outer membrane proteinase T (OmpT) gene-deleted BL21(DE3) cells was not. FITC-labeled MBP-H2B (FITC-MBP-H2B) penetrated into the bacterial cell membrane of JCM5491 and ompT-transformed BL21(DE3) cells, but not into ompT-deleted BL21(DE3) cells. Gel retardation assay using MBP-H2B-deletion mutants indicated that MBP-H2B bound to DNA at a site within the N-terminal region of histone H2B. Consequently, it is proposed that the antimicrobial action of histone H2B involves, at least in part, penetration of an OmpT-produced N-terminal histone H2B fragment into the bacterial cell membrane with subsequent inhibition of cell functions.

Differential migratory properties of mouse, fish, and frog leukocytes treated with agonists of opioid receptors.

Zymosan-induced peritoneal inflammation was inhibited by morphine co-injection in mice and fish but not in anuran amphibians. In present experiments, an in vitro migration of mouse, goldfish, and frog leukocytes to L15 medium, control serum (S) or zymosan-activated serum (ZAS) was recorded following cell preincubation with L15 or with agonists of mu, delta, or kappa opioid receptors (morphine, deltorphine, or U-50,488H, respectively). In all species, migration of control leukocytes was in the order ZAS > S > L15. Pretreatment with morphine or deltorphine (but not with U-50,488H) enhanced leukocyte migration to L15 and S in each species, while it inhibited migration of mouse and fish (but not frog) leukocytes to ZAS, phenomena reversed by specific antagonists of mu and delta opioid receptors (CTOP or naltrindole, respectively). It seems that final effects of opioids on cell migration are dependent on a species-specific balance between up- and down-regulation of leukocyte migration resulted from interplay between receptors for opioids and chemotactic factors.

Immunohistochemical demonstration of FSH and LH in the pituitary of the developing frog, Rana esculenta.

The ontogenetic pattern of immunohistochemically detectable FSH beta and LH beta cells was investigated in the pars distalis of the pituitary of the frog, Rana esculenta. The appearance, distribution, and percentage of these cells were examined in tadpoles from soon after hatching to the end of metamorphosis and in juveniles. We used monoclonal antibodies against bullfrog FSH beta and LH beta for single staining, and either mouse anti-bullfrog LH beta + guinea pig anti-rat FSH beta or rabbit polyclonal anti-bullfrog LH beta + mouse monoclonal anti-bullfrog FSH beta for double staining. The first appearance of gonadotropes, immunopositive for FSH beta, was revealed in stage 26 tadpoles. In successive stages of development the percentage of FSH beta-positive cells increased progressively and significantly. The mean percentage of these in the pars distalis cells increased from 0.7% in stage 26 to nearly 10% during the metamorphic climax (stages 31-33). In juveniles, the mean percentage of FSH beta-positive cells increased more than twofold compared to the climax value. The appearance of LH beta-positive cells was first recorded during the climax, and the mean percentage of LH beta-positive cells in juveniles reached levels as high as 30% or more, exceeding the number of FSH beta-positive cells. In climax, all LH beta-positive cells stained with anti-FSH beta as well. In juveniles, however, up to 80% of gonadotropes demonstrated colocalization of FSH beta and LH beta staining. We argue that both gonadotropins may be synthesized in all gonadotropes, and a small number of cells immunoreactive to either of the two gonadotropins may simply indicate that at that particular moment the cell contained detectable amounts of only one form of gonadotropin. These observations are discussed in relation to the possible involvement of hypothalamic influence in the differentiation of gonadotropes of the pituitary.

Invertebrate and vertebrate immune cells express pro-opiomelanocortin (POMC) mRNA.

We show here that hemocytes and leukocytes with phagocytic activity from both invertebrates (Planorbarius corneus, Viviparus ater) and vertebrates (Carassius auratus, Rana esculenta) express pro-opiomelanocortin (POMC) mRNA, as assessed by in situ hybridization with a digoxigenin-labeled human DNA probe. These data are in accord with previous observations from our laboratories on the presence in these cells of POMC-derived peptides and strongly suggest that these molecules--highly conserved throughout evolution--play an important role in cell locomotion and phagocytosis. POMC mRNA was also detected in lymphocytes of R. esculenta, but not of C. auratus, suggesting that from anuran amphibians onwards lymphocytes also express this gene. This phenomenon could be related to the appearance of more than one immunoglobulin isotype in anurans.

The distribution of GABA-like-immunoreactive neurons in the brain of the newt, Triturus cristatus carnifex, and the green frog, Rana esculenta.

The distribution of gamma-aminobutyric acid (GABA) immunoreactivity was studied in the brain of two amphibian species (Triturus cristatus carnifex, Urodela; Rana esculenta, Anura) by employing a specific GABA antiserum. A noteworthy immunoreactive neuronal system was found in the telencephalic dorsal and medial pallium (primordium pallii dorsalis and primordium hippocampi) and in the olfactory bulbs. In the diencephalic habenular nuclei there was a rich GABAergic innervation, and immunoreactive neurons were observed in the dorsal thalamus. In the hypothalamus the GABA immunoreactivity was found in the preoptic area, the paraventricular organ and in the hypothalamo-hypophysial complex. In the preoptic area of the frog some GABA-immunoreactive CSF-contacting cells were shown. In the optic tectum immunolabeled neurons were present in all the cellular layers. A rich GABAergic innervation characterized both the fibrous layers of the tectum and the neuropil of the tegmentum and interpeduncular nucleus. In the cerebellum, in addition to the Purkinje cells showing a variable immunopositivity, some immunoreactive cells bodies appeared in the central grey. Abundant immunolabeled nerve fibers in the acoustico-lateral area and some immunopositive neurons in the region of the raphe nucleus were observed. In conclusion, the GABAergic central systems, well-developed in the amphibian species studied, were generally characterized by close similarities to the pattern described in mammals.

Comparison of reactions to skin grafts in green frogs: Rana lessonae cam., R. esculenta L. and R. ridibunda pall.

Adult, field-collected green frogs, Rana lessonae and R. ridibunda, and their interspecific hybrid R. esculenta were tested with skin grafts at 22 +/- 2 degrees C. Two types of reactions to grafts were observed. 1) Median survival time (MST) was about 25 days, which was observed in R. lessonae hosts in response to allogeneic stimuli and to skin grafts from R. esculenta, and R. esculenta hybrids given grafts from the parental species. 2) MST was equal to or longer than 30 days, which was observed in the case of grafts among R. esculenta, and in R. ridibunda hosts given grafts from R. esculenta and R. lessonae. It seems that a high histocompatibility allele polymorphism occurs in R. lessonae even within one population, whereas lower polymorphism and/or reduced immunological reactivity occurs in R. ridibunda.

Immunochemical investigations on water frog eggs: comparative studies on Rana ridibunda, Rana lessonae and their hybrid Rana esculenta.

Eggs of Rana ridibunda, Rana lessonae and their hybrid Rana esculenta were examined for their immunochemical properties. The reaction spectra obtained confirm the hybrid origin of Rana esculenta.

Macrophage disappearance reaction in Rana esculenta induced by specific antigen and rabbit lymphokines.

Macrophage disappearance reaction was induced by intraperitoneal injections of specific antigens in Rana esculenta sensitized by bovine gamma globulin and Mycobacterium bovis BCG. Rabbit lymphokines derived from concanavalin A stimulated blood lymphocytes injected intraperitoneally were able to induce macrophage disappearance reaction in normal Rana esculenta. This suggests that mammalian lymphokines are capable of acting in amphibia. Mammalian lymphokines have not an exclusive class-specificity in vertebrates.

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.