Frog Liver Research Articles

DDTs in rice frogs (Rana limnocharis) from an agricultural site, South China: Tissue distribution, biomagnification, and potential toxic effects assessment

Contamination with agricultural pesticides such as dichlorodiphenyltrichloroethane (DDT) and its metabolites, dichlorodiphenyldichloroethylene (DDE) and dichlorodiphenyldichloroethane (DDD), is among several proposed stressors contributing to the global declines in amphibian populations and species biodiversity. These chemicals were examined in insects and in the muscle, liver, and eggs of rice frogs (Rana limnocharis) from the paddy fields of an agricultural site in South China. The ΣDDT (sum of DDT, DDE, and DDD) concentrations ranged from 154 to 915, 195 to 1,400, and 165 to 1,930 ng/g lipid weight in the muscle, liver, and eggs, respectively. All the DDTs (DDT, DDE, and DDD) showed higher affinity for the liver relative to muscle tissue and can be maternally transferred to eggs in female frogs. The average biomagnification factors for DDTs ranged from 1.6 to 1.9 and 1.5 to 2.9 in female and male frogs, respectively, providing clear evidence of their biomagnification from insects to frogs. Compared with the reported DDT levels demonstrated to have toxic effects on frogs, DDTs in the present frogs are unlikely to constitute an immediate health risk. However, the adverse impacts of high DDT residues in eggs on the hatching success and their potential toxicity to the newly metamorphosed larval frogs should be assessed further.

Seasonal Variation in the Hepatoproteome of the Dehydration- and Freeze-Tolerant Wood Frog, Rana sylvatica

Winter’s advent invokes physiological adjustments that permit temperate ectotherms to cope with stresses such as food shortage, water deprivation, hypoxia, and hypothermia. We used liquid chromatography (LC) in combination with tandem mass spectrometry (MS/MS) quantitative isobaric (iTRAQ™) peptide mapping to assess variation in the abundance of hepatic proteins in summer- and winter-acclimatized wood frogs (Rana sylvatica), a northerly-distributed species that tolerates extreme dehydration and tissue freezing during hibernation. Thirty-three unique proteins exhibited strong seasonal lability. Livers of winter frogs had relatively high levels of proteins involved in cytoprotection, including heat-shock proteins and an antioxidant, and a reduced abundance of proteins involved in cell proliferation, protein synthesis, and mitochondrial function. They also exhibited altered levels of certain metabolic enzymes that participate in the biochemical reorganization associated with aphagia and reliance on energy reserves, as well as the freezing mobilization and post-thaw recovery of glucose, an important cryoprotective solute in freezing adaptation.

Effects of omethoate on certain oxidative biomarkers in various tissues of frogs (Rana ridibunda) at acute exposure

The study aimed mainly to assess effects of omethoate on certain oxidative stress biomarkers in various tissues of frogs (Rana ridibunda). Biomarkers selected for stress monitoring were malondialdehyde (MDA) and antioxidant defense system (ADS) constituent such as reduced glutathione (GSH) level, glutathione peroxidase (GSH-Px), glutathione-S-transferase (GST) and superoxide dismutase (SOD) activities in the liver, brain, heart and kidney tissues of frogs exposed to 10 and 20 ppm dosages of omethoate for 24, 48, 72 and 96 hours. Results showed that the administrations of omethoate promote MDA content in the tissues of the frogs treated with both dosages of omethoate. With regard to the ADS, GSH-Px, GST, SOD activities and GSH levels decreased after 24, 48, 72 and 96 hours in the tissues of treatment groups compared with controls. Collective results demonstrated that the exposures to omethoate of frogs induced an increase in MDA joined with fluctuated ADS. This may reflect the potential role of these parameters as useful biomarkers for assessment of aquatic pollution.

Acute effects of fenthion on certain oxidative stress biomarkers in various tissues of frogs (Rana ridibunda)

This study was aimed mainly to assess the effects of fenthion on certain oxidative stress biomarkers in various tissues of frogs (Rana ridibunda). Biomarkers selected for stress monitoring were malondialdehyde (MDA) and antioxidant defense system (ADS) such as reduced glutathione (GSH) level, glutathione peroxidase (GSH-Px), glutathione-S-transferase (GST), and superoxide dismutase (SOD) activities in the liver, kidney, heart, and brain of frogs exposed to 10 and 20 ppm dosages of fenthion for 24, 48, 72, and 96 h. The results demonstrate an increase in MDA levels in selected tissues following exposure to both concentrations of fenthion. The ADS, GSH-Px, GST, SOD activities and GSH levels also fluctuated after 24, 48, 72, and 96 h in all the treatment groups compared with controls. From the evidence obtained here, it is concluded that the exposure of frogs to fenthion induced an increase in MDA combined with fluctuated ADS. This may reflect the potential role of these parameters as useful biomarkers for oxidative stress in amphibian species.

Regulation of cell cycle components during exposure to anoxia or dehydration stress in the wood frog, Rana sylvatica

The wood frog (Rana sylvatica) exhibits a well-developed natural anoxia and dehydration tolerance. The degree of stress tolerance depends on numerous biochemical adaptations, including stress-induced hypometabolism that helps to preserve long-term viability by reducing ATP demand. We hypothesized that the mechanisms involved in cell cycle control could act to aid in the establishment of the hypometabolic state required for stress survival. Selected proteins involved in the proliferation of cells were evaluated using immunoblotting in liver and skeletal muscle of wood frogs comparing controls with animals subjected to either 24-hr anoxia exposure under a nitrogen gas atmosphere or dehydration to 40% of total body water lost (all at 5°C). Levels of cyclins (type A, B, D, and E) decreased significantly under both stresses in liver and skeletal muscle. Similar reductions were seen for Cyclin-dependant kinases (Cdk) types 2, 4, and 6 in both liver and skeletal muscle; however, an increase in the relative amount of phosphorylated inactive p-Cdk (Thr14/Tyr15) was observed in liver under both stresses. Levels of positive regulators of Cdk activity (Cdc25 type A and C) were significantly reduced in both tissues under both stresses, whereas negative regulators of Cdk activity (p16(INK4a) and p27(KIP1) ) increased significantly in liver under both anoxia and dehydration stress (but not in muscle). This study provides the first report of differential regulation of cell cycle components in an anoxia and dehydration tolerant vertebrate, the wood frog, suggesting that cell cycle suppression is an active part of stress resistance and life extension in hypometabolic states.

Sialic acid complement of indigenous Nigerian lizards and frogs

The sialic acid complement of some selected organs of indigenous Nigerian Agama agama lizards and frogs, namely, heart, brain, liver, lungs and kidney, was determined using the thiobarbituric acid assay method and it was observed that the mean sialic acid concentration was highest in the kidney of female frogs (9.80 ± 1.20 mg/ml) followed by the liver of male frogs (9.0 ± 1.0 mg/ml) and least in the liver of female frogs (0.50 ± 0.080 mg/ml). Apart from establishing the baseline data on the sialic acid complement of the aforementioned organs in the above-mentioned species, this study also provides good information on the best organs for sialic acid isolation and preparation of sialic acid standard for research. Results from the present study suggest that lower vertebrates offer a more appealing alternative for sialic acid isolation, for the preparation of sialic acid standards, in the place of calves and this advantage should be exploited. It is concluded that the sialic acid complement in the organs of interest investigated in this study should be characterized to document their nomenclature and configuration in indigenous Nigerian A. agama lizards and frogs.

The Siberian tree frog (Rana amurensis Bulenger, 1886) in Yakutia

The paper studies the distribution, life style, propagation, nutrition, helminthes invasion, seasonal fluctuations of hematologic parameters, content of ascorbic acid in the organs and glycogen in the liver of the Siberian tree frog.

Phenotypic characterization and phylogenetic analysis of a virulent Bacillus cereus strain from the tiger frog, Hoplobatrachus rugulosus Wiegmann.

Bacillus cereus is a common opportunistic human pathogen well known as a food contaminant. In recent years, tiger frog farming has been undertaken in China and many other countries to meet human food habits. However, there is no definitive data to indicate the contamination of potentially pathogenic B. cereus strains to fresh cultured tiger frogs. In this study, a virulent B. cereus strain W10 with hemolytic activity was first isolated from the liver of cultured tiger frogs. It was initially identified based on the spore-forming characterization and phenotypic characterization using API identification kits and its taxonomic position, was further determined by nucleotide blast search in NCBI website and phylogenetic analysis. The constructed phylogenetic tree using neighbor-joining method further showed that strain W10 was closely related to B. cereus strain F46 (GenBank accession no. EF203906), which was isolated from the contaminated food. To further confirm the virulence and potential danger of strain W10, its LD50 value was tested to be 5.22×104 cfu/g, which indicated that strain W10 had strong potential virulence to healthy tiger frogs. Pathological changes were observed in liver and body surface of the challenged tiger frogs. Thus, as a potential pathogen of a zoonotic disease, the distribution of the virulent B. cereus strain, may be a threat to the safety of tiger frog food and more importance should be attached to the virulent B. cereus strain in tiger frog farming. Key words: Tiger frog, Bacillus cereus, phenotypic characterization, phylogenetic analysis.

Mycobacterium liflandiiOutbreak in a Research Colony ofXenopus (Silurana) tropicalisFrogs

A research colony of Xenopus (Silurana) tropicalis frogs presented with nodular and ulcerative skin lesions. Additional consistent gross findings included splenomegaly with multiple tan-yellow nodular foci in the spleen and liver of diseased frogs. Copious acid-fast positive bacteria were present in touch impression smears of spleen, skin, and livers of diseased frogs. Histologically, necrotizing and granulomatous dermatitis, splenitis, and hepatitis with numerous acid-fast bacilli were consistently present, indicative of systemic mycobacteriosis. Infrequently, granulomatous inflammation was noted in the lungs, pancreas, coelomic membranes, and rarely reproductive organs. Ultrastructurally, both extracellular bacilli and intracellular bacilli within macrophages were identified. Frogs in the affected room were systematically depopulated, and control measures were initiated. Cultured mycobacteria from affected organs were identified and genetically characterized as Mycobacterium liflandii by polymerase chain reaction amplification of the enoyl reductase domain and specific variable numbers of tandem repeats. In recent years, M. liflandii has had a devastating impact on research frog colonies throughout the United States. This detailed report with ultrastructural description of M. liflandii aids in further understanding of this serious disease in frogs.

Regulation of glucose-6-phosphate dehydrogenase by reversible phosphorylation in liver of a freeze tolerant frog

Glucose-6-phosphate dehydrogenase (G6PDH) and the pentose phosphate pathway play a key role in reductive biosynthesis and antioxidant defense, while diverting glucose from other cellular functions. G6PDH was isolated from liver of the wood frog, Rana sylvatica, a freeze tolerant species that uses glucose as a cryoprotectant. Analysis of kinetic parameters (K(m) and V(max)) of G6PDH showed a significant increase in K(m) G6P (from 98.2 ± 3.8 to 121 ± 5.3 μM) and K(m) NADP(+) (from 65.5 ± 2.3 to 89.1 ± 4.8 μM) in frogs following freezing exposure, indicating lower affinity for G6PDH substrates in this state. Subsequent analyses indicated that differential phosphorylation of G6PDH between the two states was responsible for the altered kinetic properties. Thus, two differentially charged forms of G6PDH were resolved by DEAE ion-exchange chromatography and, compared with controls, the proportion of G6PDH activity in peak I decreased and in peak II increased in liver from frozen frogs. G6PDH in peak I had a K(m) G6P of 94.1 ± 1.1 μM and K(m) NADP(+) of 61.2 ± 3.5 μM, whereas Peak II G6PDH showed higher values (K(m) G6P was 172 ± 4.3 μM, K(m) NADP(+) was 98.2 ± 3.3 μM). G6PDH from each peak was incubated with ions and second messengers to stimulate the actions of protein kinases with results indicating that G6PDH can be phosphorylated by protein kinase G, protein kinase C, AMP-activated protein kinase, or calmodulin-dependent protein kinase. The data indicate that in control frogs, G6PDH is in a high phosphate form and displays a high substrate affinity, whereas in frozen frogs G6PDH is less phosphorylated, with lower substrate affinity.

Insight into the interaction sites between fatty acid binding proteins and their ligands

Fatty acid binding proteins (FABPs), are evolutionarily conserved small cytoplasmic proteins that occur in many tissue-specific types. One of their primary functions is to facilitate the clearance of the cytoplasmic matrix from free fatty acids and of other detergent-like compounds. Crystallographic studies of FABP proteins have revealed a well defined binding site located deep inside their beta-clam structure that is hardly exposed to the bulk solution. However, NMR measurements revealed that, when the protein is equilibrated with its ligands, residues that are clearly located on the outer surface of the protein do interact with the ligand. To clarify this apparent contradiction we applied molecular dynamics simulations to follow the initial steps associated with the FABP-fatty acid interaction using, as a model, the interaction of toad liver basic FABP, or chicken liver bile acid binding protein, with a physiological concentration of palmitate ions. The simulations (approximately 200 ns of accumulated time) show that fatty acid molecules interact, unevenly, with various loci on the protein surface, with the favored regions being the portal and the anti-portal domains. Random encounters with palmitate at these regions led to lasting adsorption to the surface, while encounters at the outer surface of the beta-clam were transient. Therefore, we suggest that the protein surface is capable of sequestering free fatty acids from solution, where brief encounters evolve into adsorbed states, which later mature by migration of the ligand into a more specific binding site.

MicroRNA regulation below zero: Differential expression of miRNA-21 and miRNA-16 during freezing in wood frogs

Natural freeze tolerance depends on numerous biochemical adaptations that address the multiple stresses imposed on cells by freezing and preserves viability by suppressing energy-expensive cell functions in the frozen state. We hypothesized that microRNAs, small non-coding RNA transcripts that bind to mRNA, could act to establish rapid biological controls that aid the reorganization of metabolic priorities for freezing survival. Selected microRNA species (miR-16 and miR-21) were evaluated using RT-PCR in liver and skeletal muscle of wood frogs (Rana sylvatica) comparing controls (5°C acclimated) with animals frozen for 24h at −3°C. Levels of miR-21 increased significantly during freezing by 1.5-fold and 1.3-fold in liver and skeletal muscle, respectively. MiR-16 transcripts also rose significantly by 1.5-fold in liver of frozen frogs but fell by 50% in skeletal muscle. Protein levels of Dicer, a type III RNase that is responsible for mature microRNA processing in the cytoplasm, were unchanged in liver and decreased significantly by 50% in muscle. This data provides the first report of differential regulation of microRNA species in a freeze tolerant vertebrate and suggest a mechanism for rapid, yet reversible, gene silencing when animals transition into the frozen state.

Residues of Polybrominated Diphenyl Ethers in Frogs (Rana limnocharis) from a Contaminated Site, South China: Tissue Distribution, Biomagnification, and Maternal Transfer

Environmental pollutants are suspected to be a cause of global declines in amphibian populations, but few data are available on the bioaccumulation of polybrominated diphenyl ethers (PBDEs) in amphibians. To examine the tissue distribution, biomagnification potential, and maternal transfer of PBDEs in frogs, eighteen PBDE congeners were measured in the muscle, liver, and egg tissues of rice frogs (Rana limnocharis) and insects collected from an electronic waste (e-waste) recycling site in South China. PBDE levels in the frogs ranged from 0.63 to 11.6, 4.57 to 56.2, and 10.7 to 125 ng/g wet wt in the muscles, livers, and eggs, respectively. The frogs exhibited a unique congener profile, compared to those in aquatic and terrestrial species, with BDEs 99, 153, 183, 209, and 47 as the dominant congeners, intermediating between aquatic and terrestrial species. Most of the PBDE congeners in general showed higher affinity to liver than to muscle tissue. Except for BDEs 28, 47, 66, 138, and 206, the average biomagnification factors (BMFs) for all PBDE congeners were greater than 1.0, providing clear evidence of their biomagnification from insects to frogs. A parabolic relationship between log BMFs and bromine atom numbers or log Kow of PBDEs was observed, with the maximum BMF values for PBDEs with 6 bromine atoms (or at a log K(ow) of approximately 8.0). Relatively higher levels of 3-MeO-BDE 47 were found in male frogs, suggesting that male frogs in the present study might have higher metabolic capacity for PBDEs compared to female frogs. The ratio of levels in egg/female liver, indicating mother-to-egg transfer capacity, increased with increasing bromine atom numbers up to 7 and then declined as the bromine atom numbers rose. This indicated that the physicochemical properties of the congeners (e.g., K(ow), molecular sizes, and structures), resulting in different affinities to transport proteins, might impact their maternal transfer in frogs.

Goussia Labbé, 1896 (Apicomplexa, Eimeriorina) in Amphibia: Diversity, Biology, Molecular Phylogeny and Comments on the Status of the Genus

We provide new data on morphology, host specificity, life history, pathology and phylogeny of the coccidian genus Goussia in European anurans. Divergence in the SSU rDNA sequences (3-4%) of three Goussia isolates from three anuran hosts suggests that the isolates represent three distinct species. The isolate from Pelophylax kl. esculentus was determined as Goussia neglecta. The isolates from Rana dalmatina and Rana temporaria are considered conspecific and are, on the basis of host specificity, light microscopical, ultrastructural, and molecular phylogenetic data, described as a new species, Goussia noelleri. The new Goussia species from Bufo bufo remains unnamed. During the host's metamorphosis the infection disappears, however, oocysts are retained in the liver of post-metamorphic frogs. Molecular phylogenetic analysis showed that anuran-host Goussia form a well-supported monophyletic clade, which together with a clade represented by piscine Goussia metchnikovi, constitute basal lineages of the Eimeriidae. The two lineages show polytomy, likely reflecting undersampling of the basal eimeriid taxa. Goussia janae represents a distinct lineage, sister to the clade containing the other eimeriorinid taxa, suggesting a paraphyly of the genus Goussia. The identity of G. neglecta, the status of the genus Goussia, the presence of cryptic species in anuran-host Goussia and their ecological peculiarities are discussed.

Cold acclimation-induced up-regulation of the ribosomal protein L7 gene in the freeze tolerant wood frog, Rana sylvatica

Natural freezing survival by the wood frog, Rana sylvatica, involves multiple organ-specific changes in gene expression. The present study used differential display PCR to find cold-responsive genes in wood frog skin. A cDNA was retrieved from skin that was in higher amounts in cold- versus warm-acclimated frogs. The cDNA was used to probe a wood frog liver cDNA library and retrieve a long sequence that, after the further application of 5′RACE, was shown to encode the full sequence of the ribosomal large subunit protein 7 (RPL7) (GenBank accession number AF175983). Wood frog RPL7 contained 246 amino acids and shared 90% identity with Xenopus laevis RPL7, 82–83% with chicken and zebrafish homologues, and 79% with mammalian RPL7. Multiple binding domains found in human RPL7 showed differing degrees of conservation in the frog protein. Transcript levels of rpl7 were elevated up to 4-fold in skin of cold-acclimated frogs as compared with warm-acclimated animals. Organ-specific responses by rpl7 transcripts also occurred when frogs were given survivable freezing exposures. Transcripts rose by 1.8–3.3 fold in brain and skeletal muscle during freezing but were unaffected in central organs such as liver and heart. Up-regulation of rpl7 also occurred in brain of anoxia-exposed frogs and RPL7 protein levels increased strongly in heart under both freezing and dehydration stresses. Cold- and freezing-responsive up-regulation of the rpl7 gene and RPL7 protein in selected organs suggests that targeted changes in selected ribosomal proteins may be an integral part of natural freeze tolerance.

Aspecto bioquímico em fígados de Rana catesbeiana (SHAW, 1802) submetida a diferentes dietas

Com o objetivo de adequar uma dieta, rãs-touro (Rana catesbeiana) foram submetidas a diferentes dietas e analisadas, posteriormente, as substâncias bioquímicas de seu fígado. As dietas experimentais, com base em alimentos contendo proteínas/lipídios, apresentaram 40% de proteína bruta (PB) e foram suplementadas com diferentes quantidades de óleo de soja (OS) (tratamento 1: 6% de OS; tratamento 2: 9% OS, e tratamento 3: 12% OS). A dietacontrole foi um alimento comercial com 40% PB e 4% de extrato etéreo. Os animais foram avaliados depois de 30,60 e 90 dias do início da alimentação. Retiram-se extratos do fígado e quantificados em relação às proteínas, aos fenóis, à enzima peroxidase e ao glicogênio. Os animais que tiveram maior concentração de proteínas e glicogênio foram submetidos à alimentação com 9% de OS e 40% PB. Os mesmos animais apresentaram decréscimo da atividade de peroxidase e de concentração de fenóis, que podem ser os indicadores necessários para demonstrar a melhor dieta.

Functional evolution of the vitamin D and pregnane X receptors

BackgroundThe vitamin D receptor (VDR) and pregnane X receptor (PXR) are nuclear hormone receptors of the NR1I subfamily that show contrasting patterns of cross-species variation. VDR and PXR are thought to have arisen from duplication of an ancestral gene, evident now as a single gene in the genome of the chordate invertebrate Ciona intestinalis (sea squirt). VDR genes have been detected in a wide range of vertebrates including jawless fish. To date, PXR genes have not been found in cartilaginous fish. In this study, the ligand selectivities of VDRs were compared in detail across a range of vertebrate species and compared with those of the Ciona VDR/PXR. In addition, several assays were used to search for evidence of PXR-mediated hepatic effects in three model non-mammalian species: sea lamprey (Petromyzon marinus), zebrafish (Danio rerio), and African clawed frog (Xenopus laevis).ResultsHuman, mouse, frog, zebrafish, and lamprey VDRs were found to have similar ligand selectivities for vitamin D derivatives. In contrast, using cultured primary hepatocytes, only zebrafish showed evidence of PXR-mediated induction of enzyme expression, with increases in testosterone 6β-hydroxylation activity (a measure of cytochrome P450 3A activity in other species) and flurbiprofen 4-hydroxylation activity (measure of cytochrome P450 2C activity) following exposure to known PXR activators. A separate assay in vivo using zebrafish demonstrated increased hepatic transcription of another PXR target, multidrug resistance gene (ABCB5), following injection of the major zebrafish bile salt, 5α-cyprinol 27-sulfate. The PXR target function, testosterone hydroxylation, was detected in frog and sea lamprey primary hepatocytes, but was not inducible in these two species by a wide range of PXR activators in other animals. Analysis of the sea lamprey draft genome also did not show evidence of a PXR gene.ConclusionOur results show tight conservation of ligand selectivity of VDRs across vertebrate species from Agnatha to mammals. Using a functional approach, we demonstrate classic PXR-mediated effects in zebrafish, but not in sea lamprey or African clawed frog liver cells. Using a genomic approach, we failed to find evidence of a PXR gene in lamprey, suggesting that VDR may be the original NR1I gene.

Immunohistochemical Demonstration of Ranavirus Antigen in the Tissues of Infected Frogs (Rana temporaria) with Systemic Haemorrhagic or Cutaneous Ulcerative Disease

Ranavirus disease emerged as a cause of annually recurring epidemic mortality of common frogs (Rana temporaria) in Britain in the late-1980s. Affected frogs present with a peracute disease characterized by systemic haemorrhage, or with a chronic disease characterized by skin ulceration, but no internal gross lesions. Common toads (Bufo bufo) have also been found with haemorrhagic ranavirus disease. In order to investigate possible differences in the pathogenesis of ranavirus infection for each main disease syndrome, we studied a range of tissues from both naturally and experimentally infected frogs using anti-ranavirus immunohistochemistry. Ranavirus was located in a variety of cells, including fibrocytes, epithelial cells, lymphocytes, hepatocytes and melano-macrophages, but fewer tissues were infected in frogs with the skin ulcerative syndrome than in frogs with systemic haemorrhagic disease. Specifically, and in contrast to frogs with haemorrhagic syndrome, there was no labelling for viral antigen in the splenic lymphocytes, pancreas or gastrointestinal epithelium in frogs with ulcerative syndrome. Intracytoplasmic virus inclusions were seen in the liver, kidney, pancreas and stomach of frogs with systemic haemorrhagic disease, but not in frogs with the ulcerative syndrome. Immunohistochemical labelling of selected tissues from an affected toad demonstrated ranavirus antigen in the skin and viscera. This technique demonstrates that, in comparison to ranavirus ulcerative syndrome, the haemorrhagic form of ranavirus disease is associated with virus infection of a wider range of internal organs and identifies the infection of certain tissues, such as the spleen, which might be important in the pathogenesis of the haemorrhagic disease.

Comparison of Metal Bioavailability in Frogs from Urban and Rural Sites of Western Ukraine

The seasonal and spatial fluctuations of heavy metals in the liver of the frog Rana ridibunda from a river in Western Ukraine were investigated. Liver weight was seen to increase from spring to summer/autumn, most probably as a result of accumulation of metabolites, especially fat and glycogen. The concentrations of the metals found in the liver of the frog was in the order: Fe>Cu approximately Zn>Mn>Cd. For most metals, the highest concentration was recorded in the frogs inhabiting the urban site. The highest level of Cu in the liver was observed in the spring, in the agricultural site, while the highest level of other metals was observed in the summer. The most probable explanation for the high concentration of Cu in the rural site was that in this wetland there were discharge effluents from fungicides with Cu in their formula. Compared to other metals, the bioavailability of Cu was approximately 1000 times higher. The high concentration of Fe rather reflects its fluctuation in the water. Despite its very low concentration in the water (below the limit of detection), Cd was detected in the liver of frogs inhabiting both sites. This is an indication that tissues accumulate Cd, despite the very low concentration detected in the water. This may be an indication of intermittent exposure of frogs to Cd and possibly of other heavy metals.

Molecular dynamics simulations of palmitate entry into the hydrophobic pocket of the fatty acid binding protein

The entry of substrate into the active site is the first event in any enzymatic reaction. However, due to the short time interval between the encounter and the formation of the stable complex, the detailed steps are experimentally unobserved. In the present study, we report a molecular dynamics simulation of the encounter between palmitate molecule and the Toad Liver fatty acid binding protein, ending with the formation of a stable complex resemblance in structure of other proteins of this family. The forces operating on the system leading to the formation of the tight complex are discussed.