Neuro-immunohistochemical and molecular gene expression variations during hibernation and activity phases between Rana mascareniensis and Rana ridibunda.

Low temperatures and the lack of food during the winter lead the marsh frog Rana ridibunda and the grass frog Rana mascareniensis to hibernate in order to survive. The present study aimed to investigate the cytoarchitecture of brain sub-regions affected by the thermal cycle's fluctuations during the hibernation and activity period, besides the regional distribution quantitative expression of Na(+)/K(+)-ATPase and Pax6 transcriptional factor, the molecular gene expressions of some heat shock proteins, uncoupling protein, and metallothionein. The two frog species were isolated from the field during summer and hibernation time in winter. During hibernation it was notable the destitution of degenerated, pyknotic and vasogenic neurons in different brain areas with high rate nearby the pallium. The immunohistochemical expression of Na+/ K+-ATPase and Pax 6 is decreased during hibernation in different brain sub-regions in the two species suggesting their tendency for energy conservation strategy during hibernation. Additionally, RT-qPCR recorded the up regulation of a number of heat shock protein genes during hibernation with sharing increase between two species for hsp90 besides and the non-significant expression in summer and hibernation periods for hsp47 for both species. Moreover, uncoupling protein (ucp1and ucp2) and metallothionein genes in olfactory bulb were with significant up regulation during the hibernation suggesting that these proteins possibly have a protective effect against reactive oxygen species ROS. So, brain adaptations to low temperature play a crucial role in coordinating stress responses. The present study shed light on the importance of the olfactory bulb in the thermoregulation and sensation of temperature elevations during the hibernation period and defended by the expression of heat shock proteins and uncoupling proteins preventing the cellular damage and proteins misfolding. Neuronal energy production and regeneration activities among amphibians are markedly reduced with decreasing body temperature.

Sensitivity of Quails (Coturnix coturnix), Siskins (Carduelis spinus), and Frogs (Rana ridibunda) to West Nile Virus.

The level of viremia and features of the course of experimental infection caused by West Nile virus were studied in two species of migratory birds, siskins Сarduelis spinus and quails Coturnix coturnix, and in one species of amphibians, frogs Rana ridibunda. In quails, the virus caused a fatal disease; histological analysis revealed pathological changes in the heart, kidneys, liver, and brain stem. In siskins and frogs, virus antigen was detected in cloacal smears despite the absence of clinical manifestations, the level of viremia was sufficient to infect insect vectors during bloodsucking. These findings suggest that siskins and frogs can be potential reservoirs of West Nile virus and play a role in its circulation.

Molecular determinants of protein reabsorption in the amphibian kidneys.

Participation of molecular determinants of endocytosis in the processes of glomerular filtration and tubular reabsorption of albumin and lysozyme in the mesonephros of grass frogs (Rana temporaria L.), lake frogs (Rana ridibunda P.), and newts (Triturus vulgaris L.) is investigated. In all studied species, the constitutive expression of endocytic receptors in proximal tubule (PT) cells is established using immunofluorescence microscopy and immunoblotting. The certain stages of lysozyme and albumin endocytosis involving megalin/LRP2, cubilin, clathrin and protein Rab11 are detailed, and the central role of ligand-induced megalin/LRP2 activity in this process is shown. Increased ligand-induced expression for clathrin and Rab11was also found. In grass frogs, the different patterns of endocytic receptors and both absorbed proteins in the initial parts of proximal tubules suggest the proximo-distal specialization of absorptive processes along these tubule segments, similar to this in more complex mammalian nephrons. This data, as well as the revealed peculiarities of ligand-receptor interactions during intracellular trafficking of proteins prove that megalin is mainly involved in the absorption of lysozyme. At the same time, albumin absorption is mediated by both receptors, or cubilin contributes the most. The detection of endocytic receptor in glomerular structural elements in frogs and newts suggests the participation of filtration barrier components in endocytosis of filterable proteins. The results represent a new contribution to the study of the fundamental mechanisms of renal protein uptake in the amphibian mesonephros as a more primitive kidney compared to mammalian metanephros.

Capture and return of sexual genomes by hybridogenetic frogs provide clonal genome enrichment in a sexual species.

Hybridogenesis is a reproductive tool for sexual parasitism. Hybridogenetic hybrids use gametes from their sexual host for their own reproduction, but sexual species gain no benefit from such matings as their genome is later eliminated. Here, we examine the presence of sexual parasitism in water frogs through crossing experiments and genome-wide data. We specifically focus on the famous Central-European populations where Pelophylax esculentus males (hybrids of P. ridibundus and P. lessonae) live with P. ridibundus. We identified a system where the hybrids commonly produce two types of clonal gametes (hybrid amphispermy). The haploid lessonae genome is clonally inherited from generation to generation and assures the maintenance of hybrids through a process, in which lessonae sperm fertilize P. ridibundus eggs. The haploid ridibundus genome in hybrids received from P. ridibundus a generation ago, is perpetuated as clonal ridibundus sperm and used to fertilize P. ridibundus eggs, yielding female P. ridibundus progeny. These results imply animal reproduction in which hybridogenetic taxa are not only sexual parasites, but also participate in the formation of a sexual taxon in a remarkable way. This occurs through a process by which sexual gametes are being captured, converted to clones, and returned to sexual populations in one generation.

Effects of HCN channel blockade on the intensity­response function of electroretinographic ON and OFF responses in dark adapted frogs.

Hyperpolarization­activated and cyclic nucleotide­gated (HCN) channels are well expressed in the vertebrate retina. Their role in formation of electroretinographic (ERG) responses to stimulus onset (b­wave) and stimulus offset (d­wave) are largely unknown. In this study we investigated the effects of pharmacological blockade of HCN channels (with ZD7288 or ivabradine) on the ERG b­ and d­waves in dark adapted frog eyecup preparations. Initially, the dose­response relationship of ZD7288 effects on the b­ and d­waves was investigated. Afterwards, the effects of 75 µM ZD7288 on the stimulus ­ response function of the ERG b­ and d­waves were explored over a wide intensity range (10 log units). Finally, the effects of 30 µM ivabradine on the same function were studied. Perfusion with 75 µM ZD7288 did not change the absolute and relative sensitivity of the ERG ON and OFF responses. It caused an enhancement of the d­wave amplitude at all suprathreshold stimulus intensities, while the b­wave amplitude was slightly enhanced only in the range of higher intensities. As a result of the greater blocker effect on the OFF response amplitude, the b/d amplitude ratio was significantly decreased over the whole intensity range. ZD7288 caused a prolongation of the b­wave half­width duration, but a shortening of the d­wave half­width duration at higher intensities. Similar results were obtained when 30 µM ivabradine was used for HCN channel blockade. Our results clearly demonstrate that the blockade of retinal HCN channels changes the balance between the ON and OFF responses in the distal frog retina. This ON/OFF imbalance may be one of the causes for visual disturbances reported in ivabradine treated patients.

Helminth Species and Infracommunities in Frogs Pelophylax ridibundus and P. esculentus (Amphibia: Ranidae) in Northern Ukraine.

PURPOSE: The study aims to characterise and compare the helminth assemblages and helminth infracommunities in the marsh frog, Pelophylax ridibundus and the edible frog, P. esculentus collected in the northern part of Ukraine. METHODS: Occurrence and abundance of the helminths were analysed by calculating the prevalence, intensity, and mean abundance of infection; similarities between the infracommunities were estimated by the Bray-Curtis index and visualised using nMDS plots. Dissimilarities were estimated using the ANOSIM and SIMPER routines. RESULTS: In total, 27 helminth species were found in 143 frogs. Pelophylax ridibundus (n = 86) harboured 20 species of helminths, 24 species were found in P. esculentus (n = 57), and 17 species were shared by the two hosts. Oswaldocruzia bialata and larval Strigea sp. were absent in P. ridibundus, while they reached the prevalence of 30% and 10%, respectively, in P. esculentus. Cosmocerca ornata, Diplodiscus subclavatus, Opisthioglyphe ranae, and Codonocephalus urniger had significantly larger prevalence in P. ridibundus, whereas Haematoloechus asper was found to be more prevalent in P. esculentus. Acanthocephalus ranae, Icosiella neglecta, Haematoloechus variegatus, Pleurogenes claviger, Pleurogenoides medians, and Prosotocus confusus were equally common in both hosts. Helminth infracommunities in the two hosts had identical species richness (1-10 species, 4 on average); abundance was significantly higher in P. ridibundus. CONCLUSIONS: Helminth assemblages of the two hosts in northern Ukraine are rather similar; however, small but significant differences were found in their species composition, parameters of infection in some species, and structure of helminth infracommunities.

Rejection of the biophoton hypothesis on the origin of photoreceptor dark noise.

Rod photoreceptors of the vertebrate retina produce, in darkness, spontaneous discrete current waves virtually identical to responses to single photons. The waves comprise an irreducible source of noise (discrete dark noise) that may limit the threshold sensitivity of vision. The waves obviously originate from acts of random activation of single rhodopsin molecules. Until recently, it was generally accepted that the activation occurs due to the rhodopsin thermal motion. Yet, a few years ago it was proposed that rhodopsin molecules are activated not by heat but rather by real photons generated within the retina by chemiluminescence. Using a high-sensitive photomultiplier, we measured intensities of biophoton emission from isolated retinas and eyecups of frogs (Rana ridibunda) and fish (sterlet, Acipenser ruthenus). Retinal samples were placed in a perfusion chamber and emitted photons collected by a high-aperture quartz lens. The collected light was sent to the photomultiplier cathode through a rotating chopper so that a long-lasting synchronous accumulation of the light signal was possible. The absolute intensity of bio-emission was estimated by the response of the measuring system to a calibrated light source. The intensity of the source, in turn, was quantified by measuring rhodopsin bleaching with single-rod microspectrophotometry. We also measured the frequency of discrete dark waves in rods of the two species with suction pipette recordings. Expressed as the rate constant of rhodopsin activation, it was 1.2 × 10-11/s in frogs and 7.6 × 10-11/s in sterlets. Approximately two thirds of retinal samples of each species produced reliably measurable biophoton emissions. However, its intensity was ≥100 times lower than necessary to produce the discrete dark noise. We argue that this is just a lower estimate of the discrepancy between the hypothesis and experiment. We conclude that the biophoton hypothesis on the origin of discrete dark noise in photoreceptors must be rejected.

Mortality of embryos, developmental disorders and changes in biochemical parameters in marsh frog (Rana ridibunda) tadpoles exposed to the water-soluble fraction of Kazakhstan crude oil and O-Xylene.

The effects of different concentrations of water-soluble fraction of crude oil (WSFO) from the Zhanazhol oil field (Aktobe region, Kazakhstan) and compared to o-xylene, prevalent in this oil, on growth and development of marsh frog (Rana ridibunda) were assessed. In subchronic experiments (7 d), a dose-related increase in mortality and incidence of deformities in embryos were observed. In chronic experiments (60 d; starting from the Gosner stage 26), a dose-dependent decrease in body weight, size and developmental delay by 3-4 stages were also detected. In addition, the content of lipid hyperoxide (LHO) and malondialdehyde (MDA), as well as activities of superoxide dismutase (SOD) and catalase (CAT) enzymes in liver of the tadpoles were determined at the end of chronic experiment. Exposure to 0.5 mg/L or 1.5 mg/L WSFO elevated the content of LHO by 76% and 86%, and MDA by 47% and 58% but decreased SOD activity by 26% and 49%, and CAT by 35% and 46%, respectively. A less pronounced adverse effect was found after chronic exposure to the same concentrations of o-xylene. In tadpole liver exposed to o-xylene levels of LHO was increased by 40% and 51%, MDA by 11% and 29%, while the activity of SOD was lowered by 18% and 41%, and CAT - by 13% and 37% in the 0.5 mg/L and 1.5 mg/L treatment groups, respectively. Data demonstrated the embryotoxic and teratogenic effects attributed to WSFO and o-xylene exposure which may involve oxidative stress mechanisms.

Toxic effect of acetamiprid on Rana ridibunda sciatic nerve (electrophysiological and histopathological potential).

In this study, the effects of a neonicotinoid insecticide acetamiprid on the sciatic nerve of Rana ridibunda were investigated by using electrophysiological and histological methods. A total of 35 preparations of sciatic nerve isolated from 35 frogs (Nervus ischiadicus) were used in the experiments. Experiments were designed as four different dose groups (n = 8 per group). Acetamiprid solutions of 1 (group 1), 10 (group 2), 100 (group 3), and 1000 µM (group 4) were applied to the nerves in dose groups. In each group, action potentials were recorded before application of acetamiprid which served as control data. The extracellular action potentials were recorded for each group of 30th, 60th, 90th and 120th min of application time. Action potential amplitude and area were measured from recordings. Histological evaluation was performed by transmission electron microscopy. In electrophysiological examination, all doses in which acetamiprid applied have shown the effect from the 30th min and suppressed the sciatic nerve action potential. Acetamiprid significantly reduced the amplitude at the rate of 78-96% and the area at the rate of 79-98% (p < 0.05). In electron microscopic examination, the control nerves were in normal appearance. Disorganization, irregularity, dense ovoid body formation, fragmentation of the myelin sheath, and loss on some axoplasm of the nerves in the dose group have been observed. Our findings showed that acetamiprid can cause neuropathic changes in sciatic nerve at all applied doses. These results indicate that acetamiprid as other insecticides can have harmful effects on non-target organisms.

EThcD Discrimination of Isomeric Leucine/Isoleucine Residues in Sequencing of the Intact Skin Frog Peptides with Intramolecular Disulfide Bond.

Our scientific interests involve de novo sequencing of non-tryptic natural amphibian skin peptides including those with intramolecular S-S bond by means of exclusively mass spectrometry. Reliable discrimination of the isomeric leucine/isoleucine residues during peptide sequencing by means of mass spectrometry represents a bottleneck in the workflow for complete automation of the primary structure elucidation of these compounds. MS3 is capable of solving the problem. Earlier we demonstrated the advanced efficiency of ETD-HCD method to discriminate Leu/Ile in individual peptides by consecutive application of ETD to the polyprotonated peptides followed by HCD applied to the manually selected primary z-ions with the targeted isomeric residues at their N-termini and registration of the characteristic w-ions. Later this approach was extended to deal with several (4-7) broad band mass ranges, without special isolation of the primary z-ions. The present paper demonstrates an advanced version of this method when EThcD is applied in the whole mass range to a complex mixture of natural non-tryptic peptides without their separation and intermediate isolation of the targeted z-ions. The proposed EThcD method showed over 81% efficiency for the large natural peptides with intact disulfide ring, while the interfering process of radical site migration is suppressed. Due to higher speed and sensitivity, the proposed EThcD approach facilitates the analytical procedure and allows for the automation of the entire experiment and data processing. Moreover, in some cases it gives a chance to establish the nature of the residues in the intact intramolecular disulfide loops. Graphical Abstract ᅟ.

Presynaptic serotonin 5-HT1B/D receptor-mediated inhibition of glycinergic transmission to the frog spinal motoneurons.

Endogenous monoamine 5-hydroxytryptamine (5-HT, serotonin) is a phylogenetically ancient neurotransmitter present in vertebrates. The functions of 5-HT in central nervous system are intensively studied; however, the presynaptic effects of 5-HT in frog spinal motoneurons are practically unexplored. We have previously shown that 5-HT decreases the frequency of glycinergic miniature inhibitory postsynaptic potentials (mIPSPs), but does not affect the frequency of GABAergic mIPSPs and increases the frequency of glutamatergic postsynaptic potentials. In the present study, using pharmacological methods and intracellular recordings in motoneurons from an adult frog's isolated spinal cord, we aimed to identify the 5-HT receptor subtype responsible for inhibiting the release of glycine. Аn agonist of 5-HT1A and 5-HT7 receptors, 8-OH-DPAT, and a selective agonist of 5-HT2 receptors, α-Ме-5-НТ, did not show any significant effect on inhibitory transmission, indicating that 5-HT1A, 5-HT2, and 5-HT7 receptors are not involved in the modulation of glycine release in the adult frog spinal cord. An agonist of 5-HT1B/D receptors sumatriptan decreased the frequency (but not the amplitude) of glycinergic mIPSPs similar to 5-HT. An antagonist of 5-HT1,2 receptors, methysergide, abolished the effect of sumatriptan. Together our results suggest that 5-HT inhibits the release of glycine by activation of 5-HT1B/D receptors.

Dopamine D2 receptor blockade differentially affects the light­adapted turtle and frog electroretinogram.

The effects of dopamine D2-class receptor blockade by sulpiride on the electroretinographic (ERG) b-wave (ON response) and d-wave (OFF response) were investigated in light-adapted turtle (Trachemys scripta elegans) and frog (Rana ridibunda) eyecups. For turtle ERG, sulpiride (240 µM) produced an amplitude increase of the b- and d-waves, while the 40 µM and 120 µM of sulpiride were ineffective. Alternatively, for frog ERG, a well-developed and dose-dependent b- and d-wave amplitude decrease was obtained with 40 µM and 240 µM sulpiride. In both species, 240 µM sulpiride significantly affected the maximal voltage range of the ERG responses without altering their relative sensitivity (determined by the semi-saturation point). The absolute sensitivity of the ON and OFF responses (evaluated by threshold estimation) was not significantly altered for turtle ERG, but it was decreased for frog ERG. The time characteristics of the ERG responses were unchanged in both species. Our results show important differences between dopamine D2-class receptor-mediated pathways in turtle and frog retina (revealed by ERG).

Purinergic modulation of frog electroretinographic responses: The role of the ionotropic receptor P2X7.

The contribution of the purinergic receptors P2X7 (P2X7Rs) to the electroretinographic (ERG) responses was studied by testing the effects of the selective P2X7R antagonist A438079 and the selective P2X7R agonist Bz-ATP on the electroretinograms obtained in perfused frog (Rana ridibunda) eyecup preparations under a variety of stimulation conditions. The P2X7R blockade by 200 µM A438079 diminished the amplitude of the photoreceptor components: the a-wave and the pharmacologically isolated mass receptor potential. In the pure rod-driven and pure cone-driven responses, the amplitude of the postreceptoral ON (b-wave) and OFF (d-wave) components was also diminished. The OFF responses were affected to a greater extent compared to the ON responses. In the mixed rod- and cone-driven responses, obtained in the mesopic intensity range, the b-wave amplitude was increased, while the d-wave amplitude was decreased. The amplitude of the oscillatory potentials was diminished. The relative amplitude changes produced by the P2X7R blockade were greater in the dark-adapted compared to the light-adapted eyes. The application of 100 µM Bz-ATP produced small effects opposite to those of the antagonist, while a prolonged (>20 min) treatment with 1 mM Bz-ATP resulted in a significant amplitude reduction or even abolishment of b- and d-waves. Our results show that endogenous ATP through its P2X7Rs exerts significant, mostly potentiating effects on the ERG photoreceptor and postreceptoral responses. There is a clear ON/OFF asymmetry of the effects on the ERG postreceptoral responses favoring OFF responses: they are always strongly potentiated, while the ON responses are either less potentiated (in the rod-driven and most of the cone-driven responses) or even inhibited (in the mixed rod- and cone-driven responses). The overstimulation of P2X7Rs can produce acute pathological changes, that is, a decrease or abolishment of the ERG responses.

Origins of the phototransduction delay as inferred from stochastic and deterministic simulation of the amplification cascade.

PURPOSE: To identify steps of the phototransduction cascade responsible for the delay of the photoresponse. METHODS: Electrical responses of fish (Carassius) cones and Rana ridibunda frog rods and cones were recorded with a suction pipette technique and as an aspartate-isolated mass receptor potential from isolated perfused retinas. Special attention was paid to sufficiently high temporal resolution (1-ms flash, 700 Hz amplification bandpass). Stochastic simulation of the activation steps from photon absorption to the formation of catalytically active phosphodiesterase (PDE) was performed. In addition, a deterministic mathematical model was fit to the experimental responses. The model included a detailed description of the activation steps of the cascade that enabled identification of the role of individual transduction stages in shaping the initial part of the response. RESULTS: We found that the apparent delay of the photoresponse gets shorter with increasing stimulus intensity and reaches an asymptotic value of approximately 3 ms in cones and greater than or equal to 10 ms in rods. The result seems paradoxical since it is suggested that the delay occurs in the chain of steps from photon absorption to the formation of active transducin (T*) which in cones is, on average, slower than in rods. Stochastic simulation shows that actually the steps from photon absorption to T* may not contribute perceptibly to the delay. Instead, the delay occurs at the stage that couples the cycle of repetitive activation of T by rhodopsin (R*) with the activation of PDE. These steps include formation of T* (= T α GTP) out of T αßγ GTP released from the activation cycle and the subsequent interaction of T* with PDE. This poses a problem. The duration of an average cycle of activation of T in rods is approximately 5 ms and is determined by the frequency of collisions between R* and T in the photoreceptor membrane. The frequency is roughly proportional to the surface packing density of T in the membrane. As the packing density of PDE is approximately 12 times lower than that of T, it could be expected that the rate of the T*-PDE interaction were an order of magnitude slower than that of R* and T. As modeling shows, this is the case in rods. However, the delay in cones is approximately 3 ms which could be achieved only at a T*-PDE interaction time of less than or equal to 5 ms. This means that either the frequency of the collisions of T* and PDE, or the efficiency of collisions, or both in cones are approximately ten times higher than in rods. This may be a challenge to the present model of the molecular organization of the photoreceptor membrane. CONCLUSIONS: The delay of the photoresponse is mainly set by the rate of interaction of T* with PDE. In cones, the delay is shorter than in rods and, moreover, shorter than the duration of the cycle of repetitive activation of T by R*. This poses a problem for the present model of diffusion interaction of phototransduction proteins in the photoreceptor membrane.

Elevated cAMP improves signal-to-noise ratio in amphibian rod photoreceptors.

The absolute sensitivity of vertebrate retinas is set by a background noise, called dark noise, which originates from several different cell types and is generated by different molecular mechanisms. The major share of dark noise is produced by photoreceptors and consists of two components, discrete and continuous. Discrete noise is generated by spontaneous thermal activations of visual pigment. These events are undistinguishable from real single-photon responses (SPRs) and might be considered an equivalent of the signal. Continuous noise is produced by spontaneous fluctuations of the catalytic activity of the cGMP phosphodiesterase. This masks both SPR and spontaneous SPR-like responses. Circadian rhythms affect photoreceptors, among other systems by periodically increasing intracellular cAMP levels ([cAMP]in), which increases the size and changes the shape of SPRs. Here, we show that forskolin, a tool that increases [cAMP]in, affects the magnitude and frequency spectrum of the continuous and discrete components of dark noise in photoreceptors. By changing both components of rod signaling, the signal and the noise, cAMP is able to increase the photoreceptor signal-to-noise ratio by twofold. We propose that this results in a substantial improvement of signal detection, without compromising noise rejection, at the rod bipolar cell synapse.

Muscarinic cholinoreceptors (M1-, M2-, M3- and M4-type) modulate the acetylcholine secretion in the frog neuromuscular junction.

Muscarinic cholinoreceptors regulate the neurosecretion process in vertebrate neuromuscular junctions. The diversity of muscarinic effects on acetylcholine (ACh) secretion may be attributed to the different muscarinic subtypes involved in this process. In the present study, the location of five muscarinic receptor subtypes (M1, M2, M3, M4 and M5) on the motor nerve terminals of frog cutaneous pectoris muscle was shown using specific polyclonal antibodies. The modulatory roles of these receptors were investigated via assessment of the effects of muscarine and specific muscarinic antagonists on the quantal content of endplate currents (EPCs) and the time course of secretion, which was estimated from the distribution of "real" synaptic delays of EPCs recorded in a low Ca2+/high Mg2+ solution. The agonist muscarine decreased the EPC quantal content and synchronized the release process. The depressing action of muscarine on the EPC quantal content was abolished only by pretreatment of the preparation with the M3 blockers 4-DAMP (1,1-Dimethyl-4-diphenylacetoxypiperidinium iodide) and J 104129 fumarate ((αR)-α-Cyclopentyl-α-hydroxy-N-[1-(4-methyl-3-pentenyl)-4-piperidinyl]benzeneacetamide fumarate). Moreover, antagonists of the M1, M2, M3 and M4 receptors per se diminished the intensity of secretion, which suggests a putative up-regulation of the release by endogenous ACh.

An EThcD-Based Method for Discrimination of Leucine and Isoleucine Residues in Tryptic Peptides.

An EThcD-based approach for the reliable discrimination of isomeric leucine and isoleucine residues in peptide de novo sequencing procedure has been proposed. A multistage fragmentation of peptide ions was performed with Orbitrap Elite mass spectrometer in electrospray ionization mode. At the first stage, z-ions were produced by ETD or ETcaD fragmentation of doubly or triply charged peptide precursor ions. These primary ions were further fragmented by HCD with broad-band ion isolation, and the resulting w-ions showed different mass for leucine and isoleucine residues. The procedure did not require manual isolation of specific z-ions prior to HCD stage. Forty-three tryptic peptides (3 to 27 residues) obtained by trypsinolysis of human serum albumin (HSA) and gp188 protein were analyzed. To demonstrate a proper solution for radical site migration problem, three non-tryptic peptides were also analyzed. A total of 93 leucine and isoleucine residues were considered and 83 of them were correctly identified. The developed approach can be a reasonable substitution for additional Edman degradation procedure, which is still used in peptide sequencing for leucine and isoleucine discrimination. Graphical Abstract ᅟ.

Morphological and ultrastructural characteristics of Myxobolus ridibundae n. sp. (Myxosporea: Bivalvulida) infecting the testicular tissue of the marsh frog Rana ridibunda (Amphibia: Ranidae) in Egypt.

Myxozoans are one of the most economically important groups of protozoan parasites causing many serious diseases of their hosts. In the present study, a total of 60 live adult male specimens of the marsh frog Rana ridibunda have been randomly captured during the period of January-December 2015 in different areas at Kafr El-Sheikh Governorate, Egypt and were examined for infection by myxosporidian parasites. A total of 48 (80.0 %) out of 60 frog specimens were found to be infected with Myxobolus species. Parasitic infection was restricted to the testicular tissue of the examined frogs. Macroscopic cysts (plasmodia) which heavily infested different parts of the testes were recovered. Morphological and ultrastructural characteristics of these myxosporidian species were carried out using light and transmission electron microscopy. Plasmodia measured 0.16-0.53 (0.34 ± 0.01) mm in diameter. Mature spores appeared oval in frontal view, measuring 8.9-11.5 (9.6 ± 0.1) µm in length and 7.5-9.1 (8.4 ± 0.1) µm in width containing 5-6 turns of polar filaments. Morphometric characterization revealed that the very small size of the present Myxobolus species was the most distinctive feature that separates them from all previously described Myxobolus species. Ultrastructural analysis showed that the plasmodia are surrounded by a plasma membrane with numerous pinocytotic protrusions extending toward the host cell. The generative cells and the different developmental stages are arranged at the periphery of the plasmodia, while immature and mature spores are centrally located. Sporogenesis, capsulogenesis, valvogenesis, and spore maturation of the present parasite are also described. The present species is described as Myxobolus ridibundae and represents a new species.

Metabotropic and ionotropic glutamate receptors mediate the modulation of acetylcholine release at the frog neuromuscular junction.

There is some evidence that glutamate (Glu) acts as a signaling molecule at vertebrate neuromuscular junctions where acetylcholine (ACh) serves as a neurotransmitter. In this study, performed on the cutaneous pectoris muscle of the frog Rana ridibunda, Glu receptor mechanisms that modulate ACh release processes were analyzed. Electrophysiological experiments showed that Glu reduces both spontaneous and evoked quantal secretion of ACh and synchronizes its release in response to electrical stimulation. Quisqualate, an agonist of ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptors and metabotropic Group I mGlu receptors, also exerted Glu-like inhibitory effects on the secretion of ACh but had no effect on the kinetics of quantal release. Quisqualate's inhibitory effect did not occur when a blocker of Group I mGlu receptors (LY 367385) or an inhibitor of phospholipase C (U73122) was present. An increase in the degree of synchrony of ACh quantal release, such as that produced by Glu, was obtained after application of N-methyl-D-aspartic acid (NMDA). The presence of Group I mGlu and NMDA receptors in the neuromuscular synapse was confirmed by immunocytochemistry. Thus, the data suggest that both metabotropic Group I mGlu receptors and ionotropic NMDA receptors are present at the neuromuscular synapse of amphibians, and that the activation of these receptors initiates different mechanisms for the regulation of ACh release from motor nerve terminals. © 2016 Wiley Periodicals, Inc.

Presynaptic nicotinic cholinoreceptors modulate velocity of the action potential propagation along the motor nerve endings at a high-frequency synaptic activity.

Experiments on frog neuromuscular junctions have demonstrated that asynchrony of the acetylcholine quantal release forming the multi-quantal evoked response at high-frequency synaptic activity is caused, in particular, by a decrease in velocity of the action potential propagation along the non-myelinated nerve endings, which is mediated by activation of the α7 and α4ß4 nicotinic cholinoreceptors.