Hepatic oxidative stress and neurotoxicity in Pelophylax kl. esculentus frogs: Influence of long-term exposure to a cyanobacterial bloom.

Although the long-term exposure of aquatic organisms to cyanobacterial blooms is a regular occurrence in the environment, the prooxidant and neurotoxic effects of such conditions are still insufficiently investigated in situ. We examined the temporal dynamics of the biochemical parameters in the liver of Pelophylax kl. esculentus frogs that inhabit the northern (N) side of Lake Ludas (Serbia) with microcystins (MCs) produced in a cyanobacterial bloom over three summer months. The obtained data were compared with data on frogs that live on the southern (S), MC-free side of the same lake. Our results showed that the MC-producing bloom induced oxidative damage to proteins and lipids, observed as a decrease in the concentration of protein -SH groups and increased lipid peroxidation (LPO) in the liver of N frogs in comparison to S frogs. Glutathione (GSH) played a key role in the transient defense against the MC-induced development of LPO. The low glutathione peroxidase (GPx) activity detected in all groups of frogs from the N site was crucial for the observed prooxidant consequences. The bloom impaired cholinergic homeostasis as a result of a decrease in ChE activity. A delayed neurotoxic effect in relation to the prooxidant outcomes was observed. Our results also showed that even though the integrated biomarker response (IBR) of the antioxidant biomarkers increased during exposure, the individual biochemical parameters did not exhibit a well-defined time-dependent pattern because of specific adaptation dynamics and/or additional effects of the physicochemical parameters of the water. This comprehensive environmental ecotoxicological evaluation of the cyanobacterial bloom-induced biochemical alterations in the liver of frogs provides a new basis for further investigations of the prolonged, real-life ecotoxicity of the blooms.

Biochemical parameters in skin and muscle of Pelophylax kl. esculentus frogs: Influence of a cyanobacterial bloom in situ.

There is little information in scientific literature as to how conditions created by a microcystin (MC) producing cyanobacterial bloom affect the oxidant/antioxidant, biotransformation and neurotoxicity parameters in adult frogs in situ. We investigated biochemical parameters in the skin and muscle of Pelophylax kl. esculentus from Lake Ludas (Serbia) by comparing frogs that live on the northern bloom side (BS) of the lake with those that inhabit the southern no-bloom side (NBS). A higher protein carbonylation level and lower antioxidant defense system capability in the skin of frogs living in conditions of the cyanobacterial bloom were observed. Inhibition of glutathione-dependent machinery was the major mechanism responsible for the induction of cyanobacterial bloom-mediated oxidative stress in frog skin. On the other hand, the detected higher ability of muscle to overcome bloom prooxidant toxicity was linked to a higher efficiency of the biotransformation system through glutathione-S-transferase activity and/or was the consequence of indirect exposure of the tissue to the bloom. Our results have also revealed that the cyanobacterial bloom conditions induced the cholinergic neurotransmitter system in both tissues. This study provides a better understanding of the ecotoxicological impact of the MC producing cyanobacterial bloom on frogs in situ. However, further investigations of the complex mechanism involved in cyanobacterial bloom toxicity in real environmental conditions are required.

Oxidative stress in Pelophylax esculentus complex frogs in the wild during transition from aquatic to terrestrial life.

During life, anuran individuals undergo drastic changes in the course of transition from aquatic to terrestrial habitat, when they are faced with metabolically demanding processes (growth, responses to developmental pressures), which result in increased production of reactive oxygen species (ROS), signaling molecules involved in development that can induce oxidative damage and stress. This situation can be further complicated by environmental influences. The aim of this study was to investigate oxidative stress parameters in naturally developing Pelophylax esculentus complex frogs during four developmental periods: premetamorphosis, prometamorphosis, metamorphic climax and juvenile stage, in order to examine changes in the response of the antioxidative system (AOS) and oxidative damage during the transition from aquatic to terrestrial life. Results show that ontogenetic shifts in anurans are accompanied by different levels of damage and AOS responses, which vary from the increased first-line enzymatic activities during the early period of development (premetamorphosis), through increased changes in the non-enzymatic complement during the metamorphic climax, to changes in both the enzymatic and non-enzymatic components observed in juvenile individuals. Premetamorphic individuals and individuals in metamorphosis displayed higher levels of lipid peroxidation, indicating that direct exposure to the environment for the first time and the modulation of organs are the most susceptible stages for oxidative damage. On the other hand, lower oxidative damage in juveniles points to the ability of their AOS to efficiently respond to challenges of the terrestrial environment. This study highlights the importance of ROS and the AOS of anurans in response to different developmental and/or environmental pressures that individuals face.

Identification and pharmaceutical evaluation of novel frog skin-derived serine proteinase inhibitor peptide-PE-BBI (Pelophylax esculentus Bowman-Birk inhibitor) for the potential treatment of cancer.

Amphibian venom-derived peptides have high potential in the field of anticancer drug discovery. We have isolated a novel Bowman-Birk proteinase inhibitor (BBI)-type peptide from the skin secretion of Pelophylax esculentus (PE) named PE-BBI, and evaluated its bio-functions and anti-cancer activity in vitro. PE-BBI is a heptadecapeptide with C-terminal amidation. The mRNA sequence and primary structure of PE-BBI were identified using RT-PCR and LC/MS, respectively. A trypsin inhibitory assay was used to characterize the serine proteinase inhibitory activity of synthetic PE-BBI. PE-BBI's myotropic activity was analyzed using isolated rat bladder and rat-tail artery smooth muscle tissues, and the anti-cancer ability of PE-BBI using human colorectal cancer cells. PE-BBI's mechanism of action was investigated using Discovery studio software. PE-BBI showed trypsin inhibitory activity (Ki = 310 ± 72 nM), strong myotropic activity, and cytotoxicity that were specific to cancer cells, and no side effect to normal epithelial cells. The docking stimulation showed that PE-BBI had high affinity to several members of human kallikrein related peptidase (KLK) family. This finding helps to enrich our understanding of BBI peptides' mode of action. Moreover, the data presented here validates frog secretions as sources of potential novel proteinase inhibitors for cancer treatment.

The programmed DNA elimination and formation of micronuclei in germ line cells of the natural hybridogenetic water frog Pelophylax esculentus.

DNA elimination is a radical form of gene silencing and occurs both in somatic and germ cells. The programmed DNA elimination occurs during gametogenesis in interspecies hybrids that reproduce by hybridogenesis (stick insects, fishes, and amphibians) and concerns removal of whole genomes of one of the parental species and production of clonal gametes propagating the genome of the other species. The cellular mechanisms differ considerably in hybridogenetic insects and fishes but remains unknown in edible frogs Pelophylax esculentus, natural hybrids between Pelophylax lessonae and Pelophylax ridibundus. Here we report DNA elimination mechanism in early developing gonads of diploid and triploid hybrid frogs, studied by TEM, immunofluorescence, and cytochemistry. In gonocytes of both sexes (primary oogonia and prespermatogonia), micronuclei emerge as detached nuclear buds formed during interphase. We found depletion of nuclear pore complexes in micronuclear membrane and chromatin inactivation via heterochromatinization followed by degradation of micronuclei by autophagy. Micronuclei formation does not lead to apoptotic cell death showing that genome elimination is a physiological process. Chromatin elimination via micronuclei in P. esculentus is unique among hybridogenetic animals and contributes to broadening the knowledge about reproductive modes in animals.

Identification and target-modifications of temporin-PE: A novel antimicrobial peptide in the defensive skin secretions of the edible frog, Pelophylax kl. esculentus.

A potent natural antimicrobial peptide named temporin-PE was identified and encoded from the skin secretions of Pelophylax kl. esculentus via "shotgun" cloning and LC-MS/MS fragmentation analysis. Target-modifications were carried out to further enhance the antimicrobial and anti-proliferative bioactivities, whilst decreasing the hemolytic effect. A range of bioassays demonstrated that replacing a proline with a tyrosine residue resulted in a loss of the bioactivity against Gram-negative bacteria, but dramatically improved the hemolytic and anti-proliferative activity, indicating the FLP- motif influences the hemolytic activity of temporins. Moreover, the coupling of TAT to the peptide dramatically improved its antimicrobial activity, indicating coupling TAT to these peptides could be considered as a potential tool to improve their antimicrobial activity. Overall, we have shown that targeted modifications of this natural antimicrobial peptide can adjust its bioactivities to help its development as an antibiotic or anti-proliferative agent.

Oxidative stress parameters in two Pelophylax esculentus complex frogs during pre- and post-hibernation: Arousal vs heavy metals.

In spring, frogs from temperate regions are faced with arousal-induced oxidative stress and exposure to various xenobiotics from the environment. The question is whether pollutants can significantly modify the antioxidative defense system (AOS) response of hibernators during recovery from hibernation. If this assumption is true, we would then expect different patterns of seasonal variations in the AOS between individuals exposed to different levels of pollution. To examine this assumption, we determined the relationship between seasonal variations of accumulated metals and AOS parameters in the skin and muscle of two frog species from the Pelophylax esculentus complex (P. ridibundus and P. esculentus) inhabiting two localities (the Danube-Tisza-Danube canal and the Ponjavica River) with different levels of pollution during pre- and post-hibernation periods, respectively autumn and spring. Our results showed that even though there were differences in the concentrations of accumulated metals and AOS parameters between localities and species, the frogs displayed almost the same patterns of AOS variations during seasons, with a higher AOS response observed in spring. The parameters SH groups, GSH, GR and SOD had been contributed most rather than others. Our findings indicate that oxidative stress during the post-hibernation period was mainly caused by the organisms' recovery from hibernation, as the result of natural selection acting on the AOS, and that the accumulated metals did not significantly modify the AOS response. The present study provides new insight into the biological and physiological cellular responses of frogs to arousal stress.

Identification of Miscellaneous Peptides from the Skin Secretion of the European Edible Frog, Pelophylax kl. Esculentus.

The chemical compounds synthesised and secreted from the dermal glands of amphibian have diverse bioactivities that play key roles in the hosts' innate immune system and in causing diverse pharmacological effects in predators that may ingest the defensive skin secretions. As new biotechnological methods have developed, increasing numbers of novel peptides with novel activities have been discovered from this source of natural compounds. In this study, a number of defensive skin secretion peptide sequences were obtained from the European edible frog, P. kl. esculentus, using a 'shotgun' cloning technique developed previously within our laboratory. Some of these sequences have been previously reported but had either obtained from other species or were isolated using different methods. Two new skin peptides are described here for the first time. Esculentin-2c and Brevinin-2Tbe belong to the Esculentin-2 and Brevinin-2 families, respectively, and both are very similar to their respective analogues but with a few amino acid differences. Further, [Asn-3, Lys-6, Phe-13] 3-14-bombesin isolated previously from the skin of the marsh frog, Rana ridibunda, was identified here in the skin of P. kl. esculentus. Studies such as this can provide a rapid elucidation of peptide and corresponding DNA sequences from unstudied species of frogs and can rapidly provide a basis for related scientific studies such as those involved in systematic or the evolution of a large diverse gene family and usage by biomedical researchers as a source of potential novel drug leads or pharmacological agents.

Anandamide acts via kisspeptin in the regulation of testicular activity of the frog, Pelophylax esculentus.

In the frog Pelophylax esculentus, the endocannabinoid anandamide (AEA) modulates Gonadotropin Releasing Hormone (GnRH) system in vitro and down-regulates steroidogenic enzymes in vivo. Thus, male frogs were injected with AEA ± SR141716A, a cannabinoid receptor 1 (CB1) antagonist, to evaluate possible effects on GnRH and Kiss1/Gpr54 systems, gonadotropin receptors and steroid levels. In frog diencephalons, AEA negatively affected both GnRH and Kiss1/Gpr54 systems. In testis, AEA induced the expression of gonadotropin receptors, cb1, gnrh2 and gnrhr3 meanwhile reducing gnrhr2 mRNA and Kiss1/Gpr54 proteins. Furthermore, aromatase (Cyp19) expression increased in parallel to testosterone decrease and estradiol increase. In vitro treatment of testis with AEA revealed direct effects on Cyp19 and induced the expression of the AEA-degrading enzyme Faah. Lastly, AEA effects on Faah were counteracted by the antiestrogen ICI182780, indicating estradiol mediated effect. In conclusion, for the first time we show in a vertebrate that AEA regulates testicular activity through kisspeptin system.

Immunolocalization of the TASK2 Potassium Channel in Frog Kidney.

TASK2 (K2P5. 1, KCNK5) is a two-pore domain K⁺ channel belonging to the TALK subgroup of the K2P family of proteins. TASK2 expression has been reported in a variety of cells and tissues ranging from kidney to immune cells and including specific neurons, its proposed functions spanning from involvement in the regulation of cell volume to control of excitability. The purpose of this study was to determine the tubule location ofthe TASK2 K⁺ channel protein in frog kidney applying polyclonal antibody against the carboxyl terminus of human TASK2 (KCNK5) protein. Immunohistochemical analysis revealed that TASK2 is expressed on distal tubules and proximal epithelial cells. TASK2 is strongly expressed predominantly on the luminal part ofthe proximal epithelial cells and slightly cytoplasmatic staining is expressed. Distal tubules showed diffuse cytoplasmatic staining as well as slight staining on the apical parts ofthe cells. These findings suggest that the TASK2 K⁺ channel has cell-specific roles in renal potassium ion transport.

Neuroanatomical organization of the brain gonadotropin-inhibitory hormone and gonadotropin-releasing hormone systems in the frog Pelophylax esculentus.

Growing evidence suggests that gonadotropin-inhibitory hormone (GnIH) may play a key role in mediating vertebrate reproduction. GnIH inhibits gonadotropin synthesis and release by decreasing the activity of gonadotropin-releasing hormone (GnRH) neurons as well as by directly regulating gonadotropin secretion from the pituitary. Whereas the presence of GnIH has been widely investigated in various classes of vertebrates, there are very few immunohistochemical reports focusing on GnIH in amphibians. The aim of this study was to assess the presence and neuroanatomical distribution of GnIH-like immunoreactivity in the brain of the anuran amphibian Pelophylax (Rana) esculentus (esculenta) and to explore any potential anatomical relationship with mammalian GnRH-immunoreactive (mGnRH-ir) elements. The GnIH-like immunoreactive (GnIH-ir) system constitutes two distinct subpopulations in the telencephalon and diencephalon, with the highest number of immunoreactive cells located in the preoptic and suprachiasmatic areas. GnIH-ir neurons were also observed in the medial septum, the anterior commissure, the dorsal hypothalamus, the periventricular nucleus of the hypothalamus, and the posterior tuberculum. Scattered GnIH-ir fibers were present in all major subdivisions of the brain but only occasionally in the median eminence. mGnRH-ir neurons were distributed in the mediobasal telencephalon, the medial septal area, and the anterior preoptic area. Double-label immunohistochemistry revealed that the GnRH and GnIH systems coexist and have overlapping distributions at the level of the anterior preoptic area. Some GnIH-ir fibers were in close proximity to mGnRH-ir cell bodies. Our results suggest that both the neuroanatomy and the functional regulation of GnRH release are conserved properties of the hypothalamic GnIH-ir system among vertebrate species.

Kisspeptin drives germ cell progression in the anuran amphibian Pelophylax esculentus: a study carried out in ex vivo testes.

Kisspeptin, via Gpr54 receptor, regulates puberty onset in most vertebrates. Thus, the direct involvement of kisspeptin activity in testis physiology was investigated in the anuran amphibian, Pelophylax esculentus. In this vertebrate gpr54 mRNA has been localized in both interstitial compartment and spermatogonia (SPG), whereas SPG proliferation requires the cooperation between estradiol and testicular Gonadotropin releasing hormone (Gnrh). In the pre-reproductive period, dose response curve to assess the effects of Kisspeptin-10 (Kp-10) was carried out in vitro (dose range: 10(-9)-10(-6)M; incubation times: 1 and 4h); proliferative activity and germ cell progression were evaluated by expression analysis of proliferating cell nuclear antigen (pcna), estrogen receptor beta (erß), Gnrh system (gnrh1, gnrh2, gnrhr1, r2, r3) and by the count of empty, mitotic and meiotic tubules. All selected markers were up regulated at 4h Kp-10 incubation. Histological analysis also proved the increase of mitotic activity and the progression of spermatogenesis. Besides Kp-10 modulation of testicular Gnrh system, in vitro treatment with 17ß-estradiol (10(-6)M) ± the antagonist ICI182-780 (10(-5)M) revealed gnrh2 and gnrhr3 estrogen dependent expression. In the reproductive period, testes were incubated for 1 and 4h with Kp-10 (10(-7)M) or Kp-10 (10(-7)M)+kisspeptin antagonist [Kp-234 (10(-6)M)]. Results obtained in the pre-reproductive period were confirmed and Kp-234 completely counteracted Kp-10 effects. In conclusion, Kp-10 modulated the expression of pcna, erß, gnrhs and gnrhrs, inducing the progression of the spermatogenesis.

Hypothalamus-pituitary axis: an obligatory target for endocannabinoids to inhibit steroidogenesis in frog testis.

Endocannabinoids - primarily anandamide (AEA) and 2-arachidonoylglycerol (2-AG) - are lipophilic molecules that bind to cannabinoid receptors (CB1 and CB2). They affect neuroendocrine activity inhibiting gonadotropin releasing hormone (GnRH) secretion and testosterone production in rodents, through a molecular mechanism supposed to be hypothalamus dependent. In order to investigate such a role, we choose the seasonal breeder, the anuran amphibian Rana esculenta, an experimental model in which components of the endocannabinoid system have been characterized. In February, at the onset of a new spermatogenetic wave, we carried out in vitro incubations of frog testis with AEA, at 10(-9)M dose. Such a treatment had no effect on the expression of cytochrome P450 17alpha hydroxylase/17,20 lyase (cyp17) nor 3-ß-hydroxysteroid dehydrogenase/Δ-5-4 isomerase (3ß-HSD), key enzymes of steroidogenesis. To understand whether or not the functionality of the hypothalamus-pituitary axis could be essential to support the role of endocannabinoids in steroidogenesis, frogs were injected with AEA, at 10(-8)M dose. Differently from in vitro experiment, the in vivo administration of AEA reduced the expression of cyp17 and 3ß-HSD. Whereas the effect on 3ß-HSD was counteracted by SR141716A (Rimonabant) - a selective antagonist of CB1, thus indicating a CB1 dependent modulation - the effect on cyp17 was not, suggesting a possible involvement of receptors other than CB1, probably the type-1 vanilloid receptor (TRPV1), since AEA works as an endocannabinoid and an endovanilloid as well. In conclusion our results indicate that endocannabinoids, via CB1, inhibit the expression of 3ß-HSD in frog testis travelling along the hypothalamus-pituitary axis.

Role of cutaneous surface fluid in frog osmoregulation.

The study investigated whether evaporative water loss (EWL) in frogs stems from water diffusing through the skin or fluid secreted by mucous glands. Osmolality of cutaneous surface fluid (CSF) of Rana esculenta (Pelophylax kl. esculentus) subjected to isoproterenol or 30°C-34°C was 191±9.3 and 181±7.5 mosm/kg, respectively, as compared to lymph osmolality of, 249±10 mosm/kg. Cation concentrations of CSF were likewise independent of pre-treatment with averages of, [Na(+)]=65.5±5.1 and [K(+)]=14.9±1.6 mmol/L, and lymph concentrations of 116 mmol Na(+)/L and 5.1 mmol K(+)/L. The relatively high [K(+)] confirms that CSF is produced by submucosal glands. Since the chemical energy of water of CSF was always higher than that of body fluids, diffusion of water would be from CSF to the interstitial fluid and not in the opposite direction. It is concluded that volume and composition of CSF are regulated by subepidermal exocrine gland secretion balanced by EWL into the atmosphere and ion reuptake by the epidermal epithelium. Previously discovered regulatory mechanisms of epithelial ion absorption, hitherto not ascribed a body function, fit well with a role in regulating turnover of CSF. As a regulated external physiological compartment, CSF would be of importance for the immune defenses that amphibians employ in protecting their skin.

Kisspeptin receptor, GPR54, as a candidate for the regulation of testicular activity in the frog Rana esculenta.

Kisspeptins, acting via GPR54, are new players in the control of reproductive axis. They have the ability to communicate with GnRH neurons sending environmental, metabolic, and gonadal signals, with the induction of GnRH and LH secretion as final effect. At present, the physiological significance of kisspeptin signaling in the gonad is poorly investigated. We cloned GPR54 receptor from the anuran amphibian Rana esculenta testis and investigated its expression in several tissues (brain, spinal cord, ovary, muscle, and kidney). In particular, the expression analysis was carried out in pituitary and testis during the annual sexual cycle. Pituitary and testicular GPR54 mRNA increased at the end of the winter stasis (February) and reached high levels during the breeding season (April). The analysis of GPR54 expression in testis was reinforced by in situ hybridization that revealed GPR54 presence in the interstitial compartment and in proliferating germ cells. Testicular GPR54 expression in February and in June was indicated to be estradiol dependent. Furthermore, in February, kisspeptin-10 (Kp-10) induced the testicular expression of both GPR54 and estrogen receptor alpha (ERalpha) in a dose-dependent manner. Conversely, in March, Kp-10 had a biphasic effect on the expression of ERalpha, being inhibitory at short (1 h) and stimulatory at longer (4 h) incubation time. In conclusion, our results demonstrate that frog testis expresses GPR54 in an estradiol-dependent manner and that Kp-10 modulates the testicular expression of ERalpha; thus, the kisspeptin/GPR54 system might be locally involved in the regulation of estrogen-dependent testicular functions such as germ cell proliferation and steroidogenesis.

Thyroid hormone receptor-ß gene expression in the brain of the frog Pelophylax esculentus: seasonal, hormonal and temperature regulation.

Thyroid hormone receptor-ß (trß) cDNA was identified in the adult of Pelophylax esculentus (previously: Rana esculenta), a seasonally breeding species, in order to detect spatial brain trß expression, its levels through the seasons and in response to 6-n-propyl-2-thiouracil, T(4) and T(3) administrations as well as to thermal manipulations. The deduced amino acid sequence of P. esculentus trß showed a high similarity to the homologous of other vertebrates. By in situ hybridization we found trß mRNA signal in the anterior preoptic nucleus, the habenulae, the hypothalamic-pituitary region and the ependyma. Brain trß transcript levels varied through the seasons, and they were well correlated with brain T(4) levels but only partially with T(3) levels. Experimentally-induced hypothyroidism decreased brain trß expression. The administration of exogenous thyroid hormones increased brain trß expression, with T(4) appearing more potent than T(3). The experiments of thermal manipulations further strengthen the hypothesis that T(4) is more effective than T(3) in brain trß regulation. This study also shows that, as in other vertebrates, deiodinase enzymes could modulate trß expression via thyroid hormone regulation.

Steroidogenic gene expression following D-aspartate treatment in frog testis.

Previous studies have provided evidence that D-Asp plays a role in steroid-mediated reproductive biology in amphibians, reptiles, birds and mammals. To examine the molecular involvement of D-Asp on steroidogenic pathway regulation, we analysed the expression of StAR, P450 aromatase and 5αRed2 mRNAs in Pelophylax esculentus testis, either in relation to the reproductive cycle or D-Asp treatment. Basal StAR mRNA levels, as well as D-Asp and testosterone concentrations, were higher in reproductive than in post-reproductive frogs. D-Asp treatment increased StAR mRNA expression and immunolocalisation in both the reproductive and post-reproductive periods. In control testis, aromatase mRNA levels were higher in the post-reproductive period, but following D-Asp administration, they increased only in the reproductive period. The level of 5αRed2 mRNA was higher in reproductive frogs than in post-reproductive frogs, and it increased after D-Asp treatment only in the post-reproductive phase. Our results suggest that, in P. esculentus testis, D-Asp increases StAR mRNA in both periods, and P450 aromatase and 5αRed2 mRNAs at different points during the reproductive cycle.

First evidence of a cDNA encoding for a melatonin receptor (mel 1b) in brain, retina, and testis of Pelophylax esculentus.

Melatonin, nocturnally secreted by the pineal gland, regulates a variety of physiological functions, including reproduction. Here, we investigated the evidence of melatonin binding sites in frog tissue (brain, retina, and testis) through saturation and competition binding experiments. In the frog, Pelophylax esculentus, our results confirm the presence of a single class of melatonin-specific binding sites in the brain and retina, but not in the testis. Further experiments have been done using biomolecular approaches (PCR analysis). Here, we report the isolation of a cDNA encoding for a melatonin receptor type (mel 1b) from brain, retina, and testis of the P. esculentus. PCR analysis revealed that melatonin expression is higher in the brain and retina, whereas it is lower in the testis. The presence of a melatonin receptor transcript in the frog testis corroborates our previous results obtained in in vitro experiments that suggest that melatonin might act directly in male vertebrate gonads, and indicates that the frog testis may be a suitable model to verify the role of indolamine in testicular activity.

Anandamide modulates the expression of GnRH-II and GnRHRs in frog, Rana esculenta, diencephalon.

In the hypothalamus, endocannabinoids affect neuroendocrine activity by means of Gonadotropin-Releasing-Hormone-I (GnRH-I) inhibition. Since most vertebrates, human included, possess at least two GnRH molecular forms, the aim of this work was to investigate the effect of endocannabinoids on GnRH molecular forms other than GnRH-I and on GnRHRs. Thus, we cloned GnRH precursors as well as GnRH receptors (GnRHR-I, GnRHR-II, GnRHR-III) from the diencephalons of the anuran amphibian, Rana esculenta. GnRH-II expression was evaluated in pituitary, whole brain, spinal cord, hindbrain, midbrain and forebrain during the annual sexual cycle. Then, in post-reproductive period (May), GnRH-I, GnRH-II and GnRHRs expression was evaluated by quantitative real time (qPCR) after incubation of diencephalons with the endocannabinoid anandamide (AEA). AEA significantly decreased GnRH-I and GnRH-II expression, up regulated GnRHR-I and GnRHR-II mRNA and it had no effect upon GnRHR-III expression. These effects were counteracted by SR141716A (Rimonabant), a selective antagonist of type I cannabinoid receptor (CB1). In conclusion our results demonstrate a CB1 receptor dependent modulation of GnRH system expression rate (both ligands and receptors) in frog diencephalons. In particular, we show that AEA, besides GnRH-I, also acts on GnRH-II expression.

Solution structure of antimicrobial peptide esculentin-1c from skin secretion of Rana esculenta.

Granular glands in the skins of frogs synthesize and secrete a remarkably diverse range of peptides capable of antimicrobial activity. These anuran skin antimicrobial peptides are commonly hydrophobic, cationic and form an amphipathic α-helix in a membrane mimetic solution. Recently, they have been considered as useful target molecules for developing new antibiotics drugs. Esculentin-1c is a 46-amino acid residue peptide isolated from skin secretions of the European frog, Rana esculenta. It displays the most potent antimicrobial activity among bioactive molecules. Esculentin-1c has the longest amino acids among all antimicrobial peptides. The present study solved the solution structure of esculentin-1c in TFE/water by NMR, for the first time. We conclude that this peptide is comprised of three α-helices with each helix showing amphipathic characteristics, which seems to be a key part for permeating into bacterial membranes, thus presenting antimicrobial activity.