Frog Erythrocytes Research Articles

Cryo-ET study from in vitro to in vivo revealed a general folding mode of chromatin with two-start helical architecture

The organization and dynamics of chromatin fiber play crucial roles in regulating DNA accessibility for gene expression. Here we combine cryoelectron tomography (cryo-ET), sub-volume averaging, and 3D segmentation to visualize the invitro and invivo chromatin fibers folding by linker histone. We discover that an increased nucleosome repeat length and prolonged fiber length do not change the two-start helical architecture in reconstituted chromatin of homogeneous composition. Additionally, an isolated chromatin fiber with heterogeneous composition was observed, which includes short-range regions compatible with two-start helix. Invivo, sub-volume averaging reveals similar subunits of two-start helical architecture in transcriptionally inactive chromatin in frog erythrocyte nuclei. Strikingly, unambiguous DNA trajectories that displayed a zigzag pattern universally between alternate N/N+2 nucleosomes were further determined by cryo-ET with voltage phase plate. Therefore, these structural similarities suggest a general folding mode of chromatin induced by linker histone, and heterogeneous compositions mainly affect local conformation rather than changing the overall architecture.

A new, widespread venomous mammal species: hemolytic activity of Sorex araneus venom is similar to that of Neomys fodiens venom

BackgroundVenom production has evolved independently many times in the animal kingdom, although it is rare among mammals. Venomous shrews produce toxins in their salivary glands and use their venoms to hunt and store prey. Thus far, the toxicity and composition of shrew venoms have been studied only in two shrew species: the northern short-tailed shrew, Blarina brevicauda, and the Eurasian water shrew, Neomys fodiens. Venom of N. fodiens has potent paralytic activity which enables hunting and storing prey in a comatose state. Here, we assayed the hemolytic effects of extracts from salivary glands of N. fodiens and the common shrew, Sorex araneus, in erythrocytes of Pelophylax sp. frogs. We identified toxins in shrew venom by high-performance liquid chromatography coupled to tandem mass spectrometry.ResultsOur results prove, confirming a suggestion made four centuries ago, that S. araneus is venomous. We also provide the first experimental evidence that shrew venoms produce potent hemolysis in frog erythrocytes. We found significant concentration-dependent effects of venoms of N. fodiens and S. araneus on hemolysis of red blood cells evaluated as hemoglobin release. Treatment of erythrocytes with N. fodiens venom at concentrations of 1.0 and 0.5 mg/ml and with S. araneus venom at concentration of 1.0 mg/ml caused an increased release of hemoglobin. Our findings confirm that hemolytic effects of N. fodiens venom are stronger than those produced by S. araneus venom. We identified four toxins in the venom of N. fodiens: proenkephalin, phospholipase A2 (PLA2), a disintegrin and metalloproteinase domain-containing protein (ADAM) and lysozyme C, as well as a non-toxic hyaluronidase. In the venom of S. araneus we found five toxins: proenkephalin, kallikrein 1-related peptidase, beta-defensin, ADAM and lysozyme C. PLA2 and ADAMs are likely to produce hemolysis in frog erythrocytes.ConclusionsOur results clearly show that shrew venoms possess hemolytic action that may allow them to hunt larger prey. Since a member of the numerous genus Sorex is venomous, it is likely that venom production among shrews and other eulipotyphlans may be more widespread than it has previously been assumed.

Specificity of Nuclear Size Scaling in Frog Erythrocytes.

In eukaryotes, the cell has the ability to modulate the size of the nucleus depending on the surrounding environment, to enable nuclear functions such as DNA replication and transcription. From previous analyses of nuclear size scaling in various cell types and species, it has been found that eukaryotic cells have a conserved scaling rule, in which the nuclear size correlates with both cell size and genomic content. However, there are few studies that have focused on a certain cell type and systematically analyzed the size scaling properties in individual species (intra-species) and among species (inter-species), and thus, the difference in the scaling rules among cell types and species is not well understood. In the present study, we analyzed the size scaling relationship among three parameters, nuclear size, cell size, and genomic content, in our measured datasets of terminally differentiated erythrocytes of five Anura frogs and collected datasets of different species classes from published papers. In the datasets of isolated erythrocytes from individual frogs, we found a very weak correlation between the measured nuclear and cell cross-sectional areas. Within the erythrocytes of individual species, the correlation of the nuclear area with the cell area showed a very low hypoallometric relationship, in which the relative nuclear size decreased when the cell size increased. These scaling trends in intra-species erythrocytes are not comparable to the known general correlation in other cell types. When comparing parameters across species, the nuclear areas correlated with both cell areas and genomic contents among the five frogs and the collected datasets in each species class. However, the contribution of genomic content to nuclear size determination was smaller than that of the cell area in all species classes. In particular, the estimated degree of the contribution of genomic content was greater in the amphibian class than in other classes. Together with our imaging analysis of structural components in nuclear membranes, we hypothesized that the observed specific features in nuclear size scaling are achieved by the weak interaction of the chromatin with the nuclear membrane seen in frog erythrocytes.

Multilocus Genotyping of Sympatric Hepatozoon Species Infecting the Blood of Ontario Ranid Frogs Reinforces Species Differentiation and Identifies an Unnamed Hepatozoon Species.

Intraerythrocytic gamonts of at least 2 named Hepatozoon species have been reported to infect the erythrocytes of ranid frogs in Ontario, Canada. Although gamonts of both species are morphometrically similar, the cytopathological changes that 1 of these species, Hepatozoon clamatae, causes to host erythrocytes, manifested by nuclear fragmentation, was used historically to distinguish this parasite from Hepatozoon catesbianae. Molecular characterization of these 2 Hepatozoon species has been equivocal in correlating genotype with gamont morphotype. Amplification and sequencing of multiple potential genotyping loci within the nuclear (18S ribosomal deoxyribonucleic acid [rDNA]; internal transcribed spacer 1), apicoplast (23S rDNA), and mitochondrial genomes (complete genomes, cytochrome c oxidase subunits I and III [COI and COIII], and cytochrome b) were conducted on Hepatozoon species that infect ranid frogs in Ontario. Sequence data were then used to evaluate the diversity of parasites present in these amphibian hosts and to assign genotypes to gamont morphotypes, if possible. Three distinct genotypes were identified at all loci; the data permitted the discovery of a third, formerly unrecognized Hepatozoon species in ranid frogs from Ontario. Although all genetic loci demonstrated differences between Hepatozoon species, mitochondrial COIII sequences were most suitable for genotypic differentiation of these parasites of frogs. Linking genotypes to gamont morphotypes proved impossible; genotypes identified as H. catesbianae and H. clamatae were found in infections with or without nuclear fragmentation of their host erythrocytes. This suggests that differentiating these species must rely on suitable genotyping methods for identification in the blood of their amphibian intermediate hosts.

Cytogenotoxicity evaluation of a heavy metal mixture, detected in a polluted urban wetland: Micronucleus and comet induction in the Indian green frog (Euphlyctis hexadactylus) erythrocytes and the Allium cepa bioassay

Heavy metal contamination in wetland ecosystems is a serious environmental and health concern. This study evaluated the cytogenotoxicity of a previously evidenced heavy metal contamination (Cd, Cr, Cu, Pb and Zn ∼5 ppm each) in a polluted urban wetland, the Bellanwila-Attidiya sanctuary (BAS) in Sri Lanka, using a battery of cytogenotoxic assays. Micronucleus and comet assays evaluated the genotoxicity in erythrocytes of a common amphibian, the Indian green frog (Euphlyctis hexadactylus), under natural metal exposure in the wetland, and in vitro exposure, respectively.The Allium cepa bioassay assessed the cytogenotoxicity of the heavy metal mixture and of the individual metals, under laboratory exposure. Although in vivo natural exposure showed no significant induction of micronuclei in frog erythrocytes (P > 0.1), a significant and dose dependent elevation of comets was evident with in vitro exposure to the metal mixture (P < 0.001). Field controls did not show significant impacts in the A. cepa bioassay, whereas individual exposure to heavy metals reported lower effects than their combined exposure under laboratory conditions; Pb2+was the most toxic metal, with the highest mitotic inhibition (Pb2+>Cd2+>Zn2+>Cr6 >Cu2+), mutagenic potential as evaluated in the percentage incidence of chromosomal aberrations (Pb2+> Zn2+> Cu2+> Cr6+> Cd2+) and cytotoxicity evaluated by the incidence of cell apoptosis and necrosis (Pb2+>Cr6+>Cu2+>Cd2+>Zn2+). Thus, the test battery of micronucleus, comet and A. cepa assays that reveal differential aspects of cytogenotoxicity may serve as a valuable tool in environmental monitoring, primarily to screen for complex environmental mixtures of heavy metals that may impact ecological health.

Leukocytic Indices and Micronucleus in Erythrocytes as Population Markers of the Immune Status of Pelophylax ridibundus (Pallas, 1771) (Amphibia: Ranidae) Living in Various Biotopic Conditions

Abstract—A differentiated assessment of blood cells and types of micronuclei in the erythrocytes of marsh frogs (Pelophylax ridibundus) living in reservoirs of the Nizhny Novgorod region was performed. The water reservoirs differ by origin, morphology, and chemical composition. The specific abiotic conditions (a peat bog) and anthropogenic load resulted in the most pronounced change in the leukocyte differential count, corresponding to the neutrophilic type of leukemoid reaction, and in an increased micronucleus fraction in the erythrocytes. A moderate positive correlation between the increase in the number of disintegrated micronuclei and the content (mg/L) of nitrites in the reservoir was found (r = 0.72, p = 0.0179). The complex action of high concentrations of technogenic chemical pollutants (copper, chromium, and oil products) caused the activation of erythropoiesis and humoral immunity, which was confirmed by the nature of the leukemoid reaction of a lymphatic type. The cellular and humoral types of immune reaction aimed at self–foreign molecule differentiation provided stability of the frog ontogenesis under anthropogenic pressure. Integral leukocyte indexes, such as the blood cell indicator, the neutrophil–lymphocyte ratio, the lymphocyte–granulocyte index, and the index of leukocyte shift, which reflect the level of the body’s general reactivity, can be considered as population markers of the immune status of amphibians.

Endemik Beyşehir kurbağası Pelophylax caralitanus (Arıkan,1988) populasyonlarında Comet Analizi ile DNA hasarın değerlendirilmesi

In study this, the level of DNA damage in three populations of endemic Beyşehir frog (Pelophylax caralitanus) inhabiting on Lakes of Karamık, Eber and Beyşehir with different anthropogenic pollution was assessed by using the DNA Comet Assay technique. Frog erythrocytes cells were used for the analysis. Seven adult individuals were collected from each biotope containing minimum 100 cells were analysed. While the results showing a significant increase in the DNA comet index in the contaminated zone [(Eber Lake= Idc=0.22±0.015), (p&lt;0.05)], the majority of the cells did not contain any DNA damage in the clean zone (Karamık Lake, Idc=0.08±0.001). The obtained data demonstrated t that the endemic Beyşehir frog could serve as a useful flag species in an indicator of anthropogenic impact on ecosystems.

Nuclear Abnormalities in Erythrocytes of Frogs from Wetlands and Croplands of Western Ghats Indicate Environmental Contaminations

Anuran amphibians are the biological models to assess the influence of environmental contamination. We conducted nuclear abnormality assessment and micronuclei test in erythrocytes of frogs to identify an early influence of environmental contaminations. In Western Ghats of India, farmers use different agrochemicals and obviously, the amphibian habitat is contaminated with combinations of many residues. Many frog species use these agro-ecosystem for breeding and to complete early life stage. In the present study, we used Indian skipper frog ( Euphlyctis cyanophlyctis (Anura: Ranidae)), a common inhabitant of water bodies in agro-ecosystems of Western Ghats for the assay. We collected the adult frogs from three different habitats; a) the rice paddy fields contaminated with agro-chemicals, b) uncontaminated marsh and c) a shallow water pool. We recorded micronucleus, blebbed, lobed, kidney bean shaped nucleus and nucleus with notches in erythro-cytes of these frogs. These nuclear abnormalities in erythrocytes are quantified. The incidences of occurrence of micronucleus ranged from 0 to 8 in 1000 analyzed cells. More than 80% of the frogs living in agro-chemical contaminated site showed these abnormalities. The appearance of nuclear abnormalities indicates that the frogs are affected by agro-chemicals.

Lethal and sub-lethal effects on the Asian common toad Duttaphrynus melanostictus from exposure to hexavalent chromium

Chromium discharged in industrial effluents frequently occurs as an environmental pollutant, but the lethal and sub-lethal effects the heavy metal might cause in animals exposed to it have been insufficiently investigated. Selecting the amphibian Duttaphrynus melanostictus, we carried out laboratory tests to investigate the effects of short and long term exposure to hexavalent chromium (Cr(VI)) in both tadpoles and adult toads. The concentrations used were 0.002, 0.02, 0.2, 1.0 and 2.0mg/L, the first three corresponding to field levels. In vitro exposures were also carried out using toad erythrocytes and Cr(VI) concentrations of 0.0015, 0.003, 0.015, 0.03, 0.15mg/L. Mortality, growth retardation, developmental delays and structural aberrations were noted in the metal-treated tadpoles, with increasing incidence corresponding to increase in Cr(VI) level and duration of exposure. Many of the sub-lethal effects were evident with long term exposure to environmentally relevant levels of the toxicant. Changes in selected blood parameters and erythrocyte morphometry were also detected in Cr(VI) exposed toads, indicating anaemic and leucopenic conditions. In the genotoxicity study, DNA damage indicated by comet assay and increased micronuclei frequency, occurred at the low Cr(VI) concentrations tested. The multiple deleterious effects of exposure to chromium signal the need for monitoring and controlling the discharge of chromium to the environment. The dose-dependency and genotoxic effects observed in this widely distributed Asian toad indicates its suitability for monitoring heavy metal pollution in aquatic systems.

The fatty acid composition of phospholipids and absorption spectra of lipid extracts from lamprey, frog, and rat erythrocytes

The work studies the content and fatty acid composition of phospholipids as well as the absorption spectra of lipid extracts from red blood cells of poikilothermal and homoiothermal animals at different evolutionary levels. The objects of study include two poikilothermal species, the river lamprey (Lampetra fluviatilis) that uses oxygen dissolved in water, and the common frog (Rana temporaria) that consumes oxygen both from water and from air. A homoithermal animal is the white rat (Rattus rattus) that inhabits the terrestrial-aerial environment. The animals are studied in winter and spring. The phospholipid content in lamprey blood plasma is found to be twice higher than that in its erythrocytes. In the frog and the rat, the ratio is reverse. Determination of the fatty acid lipid composition of red blood cell phospholipids suggests that membranes in the lamprey are denser than in the frog. As for the fatty acids in the erythrocyte fraction of rat blood, they appear to be less diverse, with a double prevalence of saturated acids over unsaturated ones and devoid of long chain (C22) ω3 fatty acids. All of this results in a lower degree of unsaturation and a denser packing of fatty acids in the membrane structures of rat erythrocytes. The mechanism of reversible binding of O2 molecules to hemoglobin in erythrocytes is discussed. Presumably, the mechanism of interaction between molecules of O2 and molecules of water prevents the exchange interaction of electrons of the hemoglobin iron atoms with an oxygen molecule. This is confirmed by our obtained absorption spectra, which show that in the lipid extract almost totally devoid of water the heme isolated from erythrocytes is converted to hemin.

Evaluation of DNA Damage in Eurasian Marsh Frogs (Pelophylax ridibundus) by Comet Assay for Determination of Possible Pollution in the Different Lakes in Central Anatolia, Turkey

In the present study, adult Eurasian marsh frogs, Pelophylax ridibundus, and water samples were collected from a reference lake and three water bodies in central Anatolia, Turkey, to evaluate the water for chemical pollutants and possible effects of pollutants on the DNA of frog erythrocytes by using a comet assay. The results for DNA damage parameters of the comet assay (total comet length, tail intensity, and olive tail moment) and their statistical analysis by ANOVA demonstrated that P. ridibundus and the comet assay together represent an useful approach for the early detection of polluted water bodies.

Factors determining the in vitro emergence of sexual stages of Hepatozoon clamatae from erythrocytes of the Green Frog (Rana clamitans)

Sexual reproduction of apicomplexan parasites in haematophagous arthropods requires that intracellular sexual stages of these protozoa escape vertebrate erythrocytes in the blood meal. Although cues that signal sexual stages of the human malaria parasite (Plasmodium falciparum Welch, 1897) to emerge from erythrocytes are well documented, such signals are poorly known for other blood-dwelling Apicomplexa. The objective of this comparative study was to investigate conditions required to induce in vitro emergence of sexual stages of Hepatozoon clamatae (Stebbins, 1905) from frog erythrocytes. Blood was drawn from Green Frogs (Rana clamitans Latreille in Sonnini de Manoncourt and Latreille, 1801 = Lithobates clamitans clamitans (Latreille in Sonnini de Manoncourt and Latreille, 1801)) infected with H. clamatae and treated with solutions of various concentrations of saline, pH, and xanthurenic acid at different temperatures. Hypertonic saline solutions of 200 and 222 mmol/L at pH 7.4 and 7.7 elicited emergence of nearly 100% of gamonts from frog erythrocytes, but at pH 8.0 resulted in decreased emergence. Solutions containing 1, 10, and 100 μmol/L xanthurenic acid increased gamont emergence at saline concentrations of 156, 178, and 244 mmol/L, but decreased emergence at 200 and 222 mmol/L. Gamont emergence increased as incubation temperatures increased from 18 to 26 °C. These results suggest that conditions necessary for emergence of sexual stages of bloodstream Apicomplexa from erythrocytes vary among genera.

Osmoregulatory reactions of frog erythrocytes under conditions of activation and blockade of Ca2+-channels

The kinetics of cell osmoregulatory reactions under conditions of activation and blockade of Ca2+-channels was studied on a model of frog polyfunctional nuclear erythrocyte. Both activation and blockade of Ca2+-channels has been established to promote swelling of nuclei and an increase of the nuclear-cytoplasmic ratios under conditions of hypotonic exposure. The osmoregulatory cell reactions after activation of Ca2+-channels are expressed as a decrease of the cell volume. The blockator of Ca2+-channels verapamil produces an alternated increase and decrease of the erythrocyte volume with time intervals of 30 and 60 s. The clearly expressed functional activity of the nuclear membrane in response to the hypotonic action under conditions of activation and blockade of Ca2+-channels indicates participation of Ca2+ ions in mechanisms of the nuclear-cytoplasmic transfer.

Two non-vesicular ATP release pathways in the mouse erythrocyte membrane

Erythrocytes are exceptionally suited for analysis of non-exocytotic release mechanisms of ATP, because these cells under physiological conditions lack vesicles. Previous studies have indicated, that Pannexin1 (Panx1) provides a key ATP permeation pathway in many cell types, including human and frog erythrocytes. Here we show that erythrocytes of Panx1−/− mice lend further support to this conclusion. However, ATP release, although attenuated, was still observed in Panx1−/− mouse erythrocytes. In contrast to Panx1+/+ cells, this release was not correlated with uptake of extracellularly applied dyes, was insensitive to Panx1 channel blockers, and was inhibited by dipyridamole and stimulated by iloprost. Thus, in erythrocytes, two independent pathways mediate the release of ATP. We also show that glyburide is a strong inhibitor of Panx1 channels.

Frogs, sentinels of DNA damage induced by pollution in Naples and the neighbouring Provinces

Many DNA mutation-based diseases recognised in Campania have recently been related to toxic substances in illegal dumping areas. We performed a comet assay on edible frog erythrocytes to evaluate DNA damage. Differences in genotoxic parameters were observed among populations. We show that severe DNA damage occurred in the north Campania where the emergence of environmental waste exploded recently. Although a similar magnitude of genotoxic damage was observed in some southern populations, it is attributable to a massive pesticide pollution related to intensive farming. The frog species analysed seems to be a good bioindicator for detecting genotoxic effects of chemical environmental hazards.

Experimental Transmission of Hepatozoon clamatae (Apicomplexa: Adeleida) to the Wood Frog, Rana sylvatica, and to the Mosquito Culex pipiens

Hepatozoon clamatae naturally infects the erythrocytes of green frogs (Rana clamitans), bullfrogs (Rana catesbeiana), and northern leopard frogs (Rana pipiens) in northeastern North America and uses the mosquito Culex territans as a definitive host. In this study, we show that the wood frog, Rana sylvatica, supports merogonic development, but not gamogonic development, of this protozoan parasite, and that the mosquito Culex pipiens serves as an experimental definitive host for sporogonic development. Two wood frogs were each force-fed Cx. territans, containing oocysts of H. clamatae in their Malpighian tubules, which had fed on blood of infected green frogs 30 days previously. Free merozoites were observed in 1 wood frog 35 days after inoculation, but intraerythrocytic gamonts were not observed. Fifteen Cx. pipiens were fed on a mixture of infected frog blood and physiological saline. Thirty days after blood feeding, 2 mosquitoes were infected with oocysts of H. clamatae, whereas the other 13 mosquitoes either were negative for infection or had died. The observed absence of gamogonic development of this parasite in wood frogs are discussed in light of previous records of host specificity of Hepatozoon species for their anuran hosts, and the importance of Cx. pipiens as an additional definitive host for H. clamatae.

Seasonal activity of frog erythrocytes by data of electrophoretic mobility

The peculiarities of frog erythrocyte electrophoretic mobility, coupled to the seasonal course of temperatures, have been studied. At the periods of anabiosis and of burst of hemopoiesis, in the vascular bed there increases the portion of functionally young erythrocytes (up to 22%) with increased values of the cell membrane surface charge. Preparation to winter is accompanied by a rise of the number of circulating functionally worn-down blood cells (up to 60%) with low values of the superficial charge and low mobility in electrical field. Use of the cell microelectrophoresis method of evaluation of seasonal activity of frog erythrocytes allows obtaining objective data about the cellular surface charge and its depending functional cell activity without submitting the erythrocytes to modifying actions.

Regulation of K–Cl cotransport in erythrocytes of frog Rana temporaria by commonly used protein kinase and protein phosphatase inhibitors

Recently (Agalakova and Gusev in J Comp Physiol 179:443-450, 2009), we demonstrated that the activity of K-Cl cotransport (KCC) in frog red blood cells is inhibited under stimulation of protein kinase C (PKC) with phorbol ester PMA (12-myristate-13-acetate). Present work was performed to uncover possible implication of protein kinases and protein phosphatases (PPs) in the regulation of baseline and volume-dependent KCC activity in these cells. K+ influx was estimated as 86Rb uptake by the cells in isotonic or hypotonic media in the presence of ouabain, K+ efflux was determined as the difference between K+ loss by the cells incubated in parallel in isotonic or hypotonic K(+)-free Cl(-)- and NO(3)(-)-media. Swelling of the cells in hypotonic medium was accompanied by approximately 50% activation of Cl-dependent K+ influx and efflux. Protein tyrosine kinase (PTK) inhibitor genistein (0.1 mM) stably and considerably (up to 89%) suppressed both baseline and volume-dependent KCC activity in each direction. Other PTK blockers (tyrphostin 23 and quercetin) had no influence on KCC activity in frog erythrocytes. PKC inhibitor chelerythrine (20 microM) and both PP inhibitors, fluoride (5 mM) and okadaic acid (1 microM), reduced KCC activity by 25-70%. Neither basal nor swelling-activated KCC in frog erythrocytes was affected by PKC inhibitor staurosporine (1 microM). Based on the previous and present results, we can suggest that the main role in the maintenance of basal and volume-dependent KCC activity in frog erythrocytes belongs to PTKs and PPs, whereas PKC is a negative regulator of this ion system.

Effects of phorbol 12-myristate 13-acetate on potassium transport in the red blood cells of frog Rana temporaria

Phorbol 12-myristate 13-acetate (PMA), a stimulator of PKC, was examined for its influence on K(+) ((86)Rb) influx in the frog erythrocyte. PMA, 0.1 microM, was found to accelerate ouabain-sensitive K(+) influx, which was suppressed by 73% with 1 mM amiloride, indicating secondary activation of the Na(+)-K(+)-pump due to stimulation of Na(+ )/H(+) exchange. PMA-induced stimulation of the sodium pump was completely inhibited with 1 microM staurosporine and by approximately 50% with 20 microM chelerythrine. In contrast to Na(+)-K(+)-pump, an activity of Cl(-)-dependent K(+) transport (K-Cl cotransport, KCC), calculated as the difference between K(+) influxes in Cl(-) and NO(3) (-)-media, was substantially decreased under the influence of PMA. Staurosporine fully restored the PMA-induced inhibition of KCC, whereas chelerythrine did not exert any influence. Osmotic swelling of the frog erythrocytes was accompanied by approximately twofold stimulation of KCC. Swelling-activated KCC was inhibited by approximately 50 and approximately 83% in the presence of PMA and genistein, respectively, but not chelerythrine. Exposure of the frog erythrocytes to 5 mM fluoride (F(-)) also reduced the KCC activity in isotonic and hypotonic media, with maximal suppression of K(+) influx in both media being observed upon simultaneous addition of PMA and F(-). Furosemide and [(dihydronindenyl)oxy] alkanoic acid inhibited the K(+) influx in both the media by approximately 50-60%. The results obtained show both the direct and indirect effects of PMA on the K(+) transport in frog erythrocytes and a complicated picture of KCC regulation in frog erythrocytes with involvement of PKC, tyrosine kinase and protein phosphatase.

PHYLOGENETIC ANALYSIS OF HEPATOZOON SPECIES (APICOMPLEXA: ADELEORINA) INFECTING FROGS OF NOVA SCOTIA, CANADA, DETERMINED BY ITS-1 SEQUENCES

Species of Hepatozoon are apicomplexan parasites infecting tetrapod vertebrates and hematophagous arthropods. Two species, Hepatozoon catesbianae and Hepatozoon clamatae, have been described inhabiting the erythrocytes of bullfrogs and green frogs. A number of characteristics typically used to distinguish between members of this genus are shared between these 2 species, prompting speculation as to whether or not these organisms are in fact distinct species. To test the species distinction, bullfrogs and green frogs were captured at various sites across Nova Scotia, blood samples were collected, and DNA was extracted from samples containing parasites. The internal transcribed spacer 1 (ITS-1) from geographically diverse samples of both species was amplified by PCR, sequenced, and analyzed. ITS-1 sequences from the 2 species revealed single-nucleotide polymorphisms at 6 sites. Phylogenetic analysis of these molecular data and cytopathological features place isolates of each species in separate monophyletic groups. Comparison of the ITS-1 sequences between isolates from Nova Scotia and Ontario revealed that ITS-1 sequences of H. catesbianae from a previous study were mischaracterized as being those of H. clamatae. Phylogenetic data based on molecular variation and cytopathological features from this study provide the strongest evidence to date supporting the distinction between these 2 species.