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At least 865 records · Page 48Linked to original sources
PubMed1994

Cloning of cDNAs for fructose 6-phosphate 2-kinase/fructose 2,6-bisphosphatase from frog skeletal muscle and liver, and their expression in skeletal muscle.

Frog (Rana catesbeiana) skeletal muscle (M-type) and liver (L-type) cDNAs of fructose 6-phosphate 2-kinase/fructose 2,6-bisphosphatase were isolated from lambda gt10 phage cDNA library. The full-length L-type cDNA (1829 bp) encodes a 469 amino acids subunit (M(r) 54,800), while the M-type cDNA (1792 bp) encodes 455 amino acids (M(r) 52,901). The amino acid sequence of the M-type isozyme is identical to that of the L-type isozyme except for the N-terminus. The N-terminal 30 amino acids of the L-type isozyme are replaced by an unique sequence of 16 amino acids in the M-type isozyme. Both L- and M-type cDNAs were detected also in a lambda gt10 phage library of skeletal muscle. Relative amount of the M- and L-type mRNAs is skeletal muscle was determined by the reverse transcription-polymerase chain reaction method. The M/L mRNA ratio in frog skeletal muscle shows seasonal variations, being 0.56/1 in early summer and 5.3/1 in winter. These results suggest that there is a seasonal change in the isozyme composition and that the glycolysis in frog skeletal muscle may be regulated by type of the isozyme synthesized.

Amino Acid Sequence↗
PubMed1998

A highly active microsomal glutathione transferase from frog (Xenopus laevis) liver that is not activated by N-ethylmaleimide.

Microsomal glutathione transferase has hitherto only been purified from mammalian species. N-ethylmaleimide and trypsin activation (discriminating features of this enzyme) has only been observed in microsomes from mammals. In this paper we describe the first isolation and characterization of a non-mammalian microsomal glutathione transferase from frog (Xenopus laevis) liver. This protein has a molecular weight similar to that of the mammalian enzyme (approximately 17 kDa), but cannot be activated by N-ethylmaleimide or trypsin. In fact the enzyme is rapidly inactivated by this sulfhydryl reagent and protease. It thus appears that N-ethylmaleimide activation is not an obligatory property of microsomal glutathione transferase. The frog liver microsomal glutathione transferase has one of the highest specific activities towards the second substrate 1-chloro-2,4-dinitrobenzene (CDNB) (200 mumol/min mg) obtained with any glutathione transferase and accounts for the high activity found in frog liver microsomes. The kcat/K(m) for glutathione and CDNB are 0.017 and 1.1 x 10(6) M-1 s-1, respectively. The enzyme also functions as a glutathione peroxidase (dilinoleoyl phosphatidylcholine hydroperoxide is reduced (5.2 mumol/min mg)). It is now evident that a highly active microsomal glutathione transferase, with a molecular weight similar to that of the mammalian enzymes also exists in a non-mammal species.

Animals↗
PubMed2000

Kassinatuerin-1: a peptide with broad-spectrum antimicrobial activity isolated from the skin of the hyperoliid frog, Kassina senegalensis.

Kassinatuerin-1 (GFMKYIGPLI(10)PHAVKAISDL(20)I.NH(2)) was isolated in high yield (75 nmol/g) from an extract of the skin of a Hyperoliid frog, the African running frog Kassina senegalensis and its sequence was confirmed by total synthesis. The peptide inhibited growth of the gram-negative bacterium Escherichia coli (minimum inhibitory concentration, MIC = 4 microM), the gram-positive bacterium Staphylococcus aureus (MIC = 8 microM), and the yeast Candida albicans (MIC = 70 microM). A structurally related peptide, kassinatuerin-2 (FIQYLAPLI(10)PHAVKAISDL(20)I.NH(2)) was also isolated in high yield (96 nmol/g) from the extract but was devoid of antimicrobial activity against these microrganisms. Kassinatuerin-1 may be classified with other linear, cationic antimicrobial peptides that can potentially adopt an amphipathic alpha-helical conformation but it contains almost no amino acid sequence identity with previously characterized bioactive peptides from frog skin.

Amino Acid Sequence↗
PubMed2000

A protease inhibitor of the Kunitz family from skin secretions of the tomato frog, Dyscophus guineti (Microhylidae).

Norepinephrine-stimulated skin secretions of the tomato frog, Dyscophus guineti, contained a trypsin inhibitor whose primary structure was established as: SPAEVCF LPK(10) ESGLCRARAL(20) RYYYDRGDGK(30) CEEFIYGGCG(40) GNGNNY KSLL(50) TCKISCE. This amino acid sequence identifies the peptide as a member of the Kunitz/bovine pancreatic trypsin inhibitor (BPTI) family and demonstrates that selective evolutionary pressure has acted to conserve those domains in the molecule (corresponding to positions 12-18 and 34-39 in BPTI) that interact with trypsin. Extracellular proteases produced by pathogenic microorganisms play important roles in facilitating invasion of the host and broad spectrum antimicrobial activity of BPTI has been described. Cationic, amphipathic alpha-helical antimicrobial peptides of the magainin type, important in the defense strategy of several species of frog, were not detected in the skin secretions. We speculate, therefore, that synthesis of a proteinase inhibitor in the skin of the tomato frog may be a component of an alternative strategy of this animal to defend itself against microorganisms.

Adrenergic alpha-Agonists↗
PubMed1997

De novo protein biosynthesis responses to water stresses in wood frogs: freeze-thaw and dehydration-rehydration.

Protein biosynthesis responses occurring during the postthaw (after 12 h freezing at -1.4 degrees C), dehydration (to 27 or 40% of total body water lost), or rehydration (after the loss of 40% of body water) were monitored in tissues of spring-collected wood frogs (Rana sylvatica) after intraperitoneal injection of 35S-labeled methionine + cysteine. All six organs tested accumulated radiolabeled amino acids and organs of both thawing and rehydrating frogs held at 3-5 degrees C showed a linear increase in amino acid incorporation into the acid-precipitable protein fraction over time. By contrast, dehydrating animals showed little or no increment in protein bound radioactivity over the course of the stress, a result that may be indicative of metabolic suppression in organs when dehydration became severe. Isoelectrofocusing (IEF) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to characterize the proteins synthesized by liver under each experimental state. IEF revealed both new peaks of 35S-labeled proteins and enhanced labeling of others in extracts from experimental animals, compared with controls. In particular, new synthesis of proteins with isoelectric points of about 6.0 was prominent and labeled proteins in this IEF peak persisted at 5, 10, or 24 h postinjection, becoming proportionally more important over time. SDS-PAGE analysis of the p(I) 6.0 peaks from thawed, dehydrated, and rehydrated frogs revealed the presence of one major low molecular weight protein in each case with molecular masses of 15, 13, and 21 kDa, respectively. These data indicate that the biochemical adaptations supporting freeze tolerance and dehydration tolerance in anurans include the stress-induced biosynthesis of a suite of proteins including the novel synthesis of selected specific proteins. These proteins may represent stress-related (or shock) proteins or may have specific roles in metabolic adaptation in each state such as in water and ionic balance or cell volume regulation.

Animals↗
PubMed1997

In vitro metabolic competence of the frog retina: effects of glucose and oxygen deprivation.

The metabolic competence and histological integrity of the frog retina in vitro were evaluated as a function of the presence/absence of exogenous glucose and of oxygen tension. Dark- and light-adapted frog neural retinas were incubated for 1-8 hr at 23 degrees C in a modified Ringer's-bicarbonate medium under aerobic and anaerobic conditions, in the presence and absence of 10 mM glucose. Control retinas (+glucose, aerobic conditions) maintained ATP levels comparable to those of freshly excised tissue (ave. 17 nmol mg protein-1), produced minimal lactate (ave. 0.12 mumol mg protein-1 hr-1), and exhibited normal histology. In the absence of any exogenous carbon source, retinas incubated aerobically maintained ATP levels, produced lactate, incorporated [3H]acetate into nonsaponifiable lipids, and exhibited histology comparable to controls. In the presence of 1 mM iodoacetate, aerobic ATP levels declined markedly, with or without exogenous glucose. Under anaerobic conditions with glucose present, lactate production increased ca. 8.5-fold, while ATP levels were maintained at control levels, demonstrating a marked Pasteur effect; under these conditions, retinas exhibited only moderate histopathological changes. However, in the absence of both glucose and oxygen, ATP levels declined precipitously, with concomitant massive cytological deterioration. No major differences in the biochemical measurements or histological appearance were observed as a function of light adaptation. These results demonstrate the remarkable resilience of the frog retina to anoxia and hypoglycemic stress. Aerobically, with or without exogenous glucose, ATP production and de novo lipid synthesis are maintained, apparently by recruitment of an endogenous carbohydrate substrate (e.g., glycogen).

Adenosine Triphosphate↗
PubMed1994

Seasonal changes in plasma growth hormone and prolactin concentrations of the frog Rana esculenta.

Plasma growth hormone (GH), prolactin (PRL), and vitellogenin (VTG) concentrations were determined during the annual reproductive cycle of the frog Rana esculenta. Plasma GH and PRL were measured using a RIA that employed purified bullfrog (Rana catesbeiana) GH and PRL as standards and radioligand, and their respective antibodies. Using ELISA, plasma VTG titers were related to ovarian weight. GH, PRL, and VTG displayed different trends related to season and sex. In male frogs the GH and PRL trends have been found parallel, showing the highest concentrations (35 and 85 ng/ml, respectively) during the winter months. In the female frogs, the GH trend behaved differently from that in males; in fact, plasma GH changed during the annual reproductive cycle, showing two main peaks occurring during the reproductive period and the autumn ovarian recrudescence that are well correlated with the vitellogenesis as well as with the main changes in ovarian weight.

Animals↗
PubMed1994

Circulating thyroglobulin in tadpoles and adult frogs of Rana catesbeiana.

Using purified thyroglobulin (TG) of Rana catesbeiana and its antisera, a specific double-antibody radioimmunoassay (RIA) was developed. Serum TG content was measured in tadpoles, froglets, and adult frogs by RIA. Serum TG increased during pre- and prometamorphosis and reached a peak of 476.9 +/- 29.1 ng/ml at the climax. Thereafter the amount of circulating TG decreased slowly toward the end of metamorphosis, and it was at the lowest level (154.1 +/- 18.4 ng/ml) in froglets. Serum TG in adult frogs was 267.3 +/- 31.7 ng/ml. The serum TG in tadpoles and adult frogs was identified by immunoblotting as only one component corresponding to the purified TG. The circulating TG level was much higher than that in mammalian species and chickens reported so far. These results seem to show that the release of TG is a preferential function in the bullfrog thyroid gland.

Aging↗
PubMed1994

Regeneration of the testicular interstitial compartment after ethane dimethane sulfonate treatment in the hypophysectomized frog Rana esculenta: independence of pituitary control.

The effects of ethane dimethane sulfonate (EDS) on the testes of hypophysectomized frogs (Rana esculenta) were investigated by light and electron microscopy. Initial signs of interstitial cell damage were observed in EDS- and EDS plus pituitary homogenate (PH)-treated animals 5 days after a single injection of EDS (100 mg/Kg body weight). The germinal compartment in these two groups appeared disorganized adjacent to the damaged interstitial tissue only in the EDS-treated animals, and by Day 8, spermatogenesis seemed to be affected in the EDS + PH-treated frogs in which Leydig cells had disappeared in some areas. On Day 28, regeneration of the interstitial tissue was complete and spermatogenesis was restored to normal. These data suggest that, in hypophysectomized frogs, the regeneration of the interstitial compartment is independent of pituitary activity and that the lack of interstitial cells activates the production of local factors responsible for the differentiation and proliferation of new Leydig cells. It is concluded that in addition to gonadotropins, the intratesticular environment is fundamental in the maintenance and regulation of testicular structure and function.

Androgens↗
PubMed1995

Effect of thyroid state on cardiac electrical activity of the frog Rana esculenta.

The effects of altered thyroid states on the heart rate and ventricular electrophysiological properties of the frog were examined. Hypothyroidism was induced by a 10-day treatment with propylthiouracil and produced decreased serum-free and total triiodothyronine levels below detectable concentrations. Hyperthyroidism, elicited by a 5-day treatment with triiodothyronine, was associated with increased serum thyroid hormone levels. The hypothyroid state was associated with a significantly decreased heart rate measured in vivo and an increased duration of the action potential recorded in vitro from ventricular fibers. Hyperthyroidism was associated with an increased heart rate and a decreased ventricular action potential duration (APD). The dependence of APD on temperature was affected by thyroid status. An increase from 25 to 30 degrees barely shortened the repolarization phase in hyperthyroids, minimally (13.3%) shortened that in euthyroids, and greatly (43.7%) shortened that in hypothyroids; the APD was similar in euthyroid and hypothyroid frogs. The shortening of the repolarization phase, by increased stimulation frequency, was also greater for hypothyroid frogs. In this case, however, the differences in APD among groups remained significant at all the frequencies tested.

Action Potentials↗
PubMed1997

Coexistence of alpha1 and beta adrenergic receptors in the liver of the frog Rana esculenta, the toad Bufo bufo, the lizard Podarcis sicula campestris, and the turtle Pseudemys picta elegans.

In mammals and birds the characteristics of alpha1 adrenergic receptors and their biological role in liver metabolism have been clearly described, although the predominance of receptor subtypes varies with species. In contrast, the actual presence of hepatic alpha1 adrenergic receptors in fish, amphibians, and reptiles has been questioned. Only recently has their existence been demonstrated in some fish species and also in the wood frog Rana sylvatica. The present study assessed the presence of alpha1 adrenergic binding sites on hepatic membranes of frogs, toads, lizards, and turtles using the specific alpha1 adrenergic receptor antagonist [3H]prazosin; for comparison, the binding of the specific beta adrenergic receptor antagonist [3H]CGP-12177A was evaluated in the same preparations. alpha1 Adrenergic receptors are indeed present in the liver of the ectotherms examined. Specific binding is saturable, reversible, and linear as a function of tissue concentration. The binding data indicated the presence of two classes of binding sites displaying high and low affinities with Kds in the nanomolar and micromolar ranges, respectively. The present study provides the first evidence for the presence of alpha1 adrenergic receptors in the liver of toad, lizard, and turtle while confirming their existence in another species of frog, Rana esculenta.

Adrenergic alpha-Antagonists↗
PubMed2000

Evidence for a mate-attracting chemosignal in the dwarf African clawed frog Hymenochirus.

Many male frogs and toads possess sexually dimorphic skin glands (breeding glands). However, in most anuran species, the functional significance of the glands is unknown. Here we show that the breeding glands of male dwarf African clawed frogs (Hymenochirus sp. ) release a mate-attractant chemosignal. The mate-attractant activity was assessed using a two-choice aquatic Y-maze. Female Hymenochirus were allowed to choose between different treatment waters (e.g., plain water and water housing males) in the upstream arms of the maze, and the females' movements were monitored by computer-linked motion sensors. Females showed a positive chemotaxis to water housing males and to water containing homogenized breeding glands. Females showed no reaction to water housing conspecific females or to water housing breeding gland-ablated males. Additional choice tests demonstrated that females were more attracted to water housing males than to water housing females and to water containing homogenized breeding glands than to water housing breeding gland-ablated males. Males in the maze showed no response to water housing either females or other males, indicating that the attractant is specific for females and is therefore neither a species aggregation signal nor a food-related attractant. These results represent the first experimental demonstration of a mate-attractant function for anuran breeding glands. Because many anuran species possess breeding glands, these results suggest that pheromonal communication could be more widespread among frogs and toads than previously believed.

Animals↗
PubMed1994

Pachymatisma johnstonii extract opens ATP-sensitive potassium channels in cardiac myocytes of the frog heart.

The effects of a proteic extract (PachyTX), isolated from the sponge Pachymatisma johnstonii, were studied respectively with intracellular microelectrode and the "whole cell" patch clamp techniques on fibers and myocytes isolated from the sinoatrial region of frog heart. PachyTX significantly and reversibly shortened the action potential duration and decreased the stimulated peak contraction. Under voltage clamp, PachyTX increased, in a dose-dependent manner, a tetraethylammonium-insensitive but glibenclamide-inhibited outward current. This current failed to develop when adenosine 5'-triphosphate (ATP) was intracellularly applied to the cell by diffusion through the patch electrode. PachyTX mimics the effect of the ATP-sensitive K channel opener SR 44866 on frog atrial action potential and peak contraction. From this evidence, it can be concluded that PachyTX is a new natural potent opener of ATP-sensitive K channels in frog atrial heart muscle and that the opening of these channels is associated with the inhibition of the contraction of cardiac muscle.

Adenosine Triphosphate↗
PubMed1994

Instability of frog virus 3 mRNA in productively infected cells.

Cloned DNA restriction fragments encoding representative frog virus 3 messages were used as probes to assess the stability of viral transcripts in infected fathead minnow cells. Analysis of Northern blot hybridization profiles confirmed earlier findings and revealed that in infected cells the steady-state level of representative frog virus 3 (FV3) messages increased throughout the replication cycle. However, when actinomycin D was added at 4 hr after infection to block the synthesis of new transcripts, viral messages were observed to turn over rapidly, with half-lives of approximately 2 hr. These results indicate that viral transcripts were not preferentially stabilized in FV3-infected cells and suggest that the high steady-state level of viral messages present at late times after infection was due to viral transcription outpacing message degradation. Moreover, the instability of viral messages challenges the suggestion that the terminal dyad symmetry (hairpin structure) observed in all frog virus 3 messages sequenced to date plays a role in transcript stability.

Animals↗
PubMed1982

Calmodulin-dependent regulation of calcium-activated outward current in frog atrial membrane.

The steady-state outward current underlying the inward-going rectification in frog atrial fibers has been studied by the double sucrose gap technique. Similar to the case in sheep Purkinje fibers (18,19), the inward rectifying potassium channels in frog atrium are blocked by cesium (5 mM) and activated by increasing cytosolic calcium concentration (replacing 30% of the Na by sucrose). It was shown that trifluoperazine (5 x 10(-6) M), a widely used blocker of the Ca-calmodulin complex, inhibits the inward rectifying potassium channels. Diphenylhydantoin (5 x 10(-6) M), a putative inhibitor of Ca-calmodulin-mediated membrane phosphorylation produces a similar action. The inhibitory action of both blockers is not manifested after preliminary Cs+ (5 mM) exposure. The data obtained suggest that calcium activates the inward rectifying potassium channels in frog atrial fibers via Ca-calmodulin regulatory phosphorylation of the membrane proteins.

Animals↗
PubMed1994

Cytochemical localization of adenylate cyclase in the sodium-transporting epithelium isolated from frog skin.

A modified cytochemical technique with 5'-adenylylimidodiphosphate as substrate, was used to examine the distribution of adenylate cyclase in cells comprising the transepithelial Na+ transport pathway in isolated frog skin epithelium. Particular attention was paid to the effects of fixation on the activity and localization of adenylate cyclase. Fixation in glutaraldehyde alone or in combination with paraformaldehyde reduced the amount of reaction product, while better results were obtained using unfixed tissues. Optimum results were obtained following stimulation of adenylate cyclase with forskolin and in the presence of specific metabolic inhibitors. Adenylate cyclase was localized in the basolateral membranes of the principal cells which constitute a functional syncytium for Na+ transport and was absent from the apical membranes of the outermost granulosum cells. This distribution is consistent with the transepithelial Na+ transport model and defines the functional morphology of the cells involved in Na+ transport across frog skin. The results are compatible with the process of Na+ re-absorption across other epithelial cells, verifying that frog skin is a convenient model-tissue to study Na+ transport mechanisms. Adenylate cyclase was also found in membranes of the mitochondria-rich cells, a minor and parallel Na+ transporting pathway.

Adenylyl Cyclases↗
PubMed1981

Fine structure of intramembranous particle aggregates in ADH-treated frog urinary bladder and skin: influence of glutaraldehyde and N-ethyl maleimide.

The fine structure of ADH-induced intramembrane particle aggregates has been studied in different tissues and under different experimental conditions. Particle aggregates similar to those previously observed in the amphibian urinary bladder and in the mammalian collecting duct were also found in the frog skin, another ADH target tissue. In the frog urinary bladder, typical aggregates were observed in the absence of glutaraldehyde fixation. Two experimental approaches were used 1) the absence of both fixative and cryoprotectant treatments and b) the absence of only glutaraldehyde treatment. In the latter case the reversal of hydrosmotic action was prevented by exposing the preparations to N-ethyl maleimide. In specimens of frog urinary bladder conventionally fixed with glutaraldehyde, two fracture levels could be observed in the aggregates, suggesting that the aggregated particles span an appreciable part of the membrane thickness.

Aldehydes↗
PubMed1984

Ultrastructure of muscle fibers and cells synthesizing DNA in lymph hearts of developing frogs and chick embryos.

The ultrastructure of striated muscle fibers and 3H-thymidine (3HTdr)-labeled cells adjacent to them in the lymph hearts of larvae of Rana temporaria, yearling frogs, and 9- to 13-day-old chick embryos was studied by use of electron-microscopic autoradiography. A comparatively high level of differentiation of lymph-heart muscle fibers was observed not only in yearling frogs but also in larvae. Myosatellites occurred in all stages of development. No mitoses were found in muscle fibers. In 9- to 13-day-old chick embryos the myofibers of lymph hearts were somewhat less differentiated than those of the larvae and yearling frogs. Differentiating sarcomeres were often seen in the sarcoplasm of myofibers of chick embryos. The analysis of the ultrastructure of 3HTdr-incorporating cells shows that 2-4 h after the single 3HTdr administration only mononucleated cells devoid of myofilaments are commonly labeled both in tadpoles and chick embryos. When fixation is postponed by 48-70 h, myonuclei frequently become labeled. Thus, the data obtained support the evidence that proliferation and differentiation processes in the developing muscle tissue of the lymph heart of both species studied are mutually exclusive, similar to the situation in differentiating vertebrate skeletal muscle.

Animals↗