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At least 379 records · Page 21Linked to original sources
PubMed2006

Antimicrobial peptides from the skin of the Japanese mountain brown frog Rana ornativentris: evidence for polymorphism among preprotemporin mRNAs.

A previous study led to the isolation of antimicrobial peptides belonging to the temporin and brevinin-2 families from a pooled extract of the skin of adult specimens of the Japanese mountain brown frog Rana ornativentris Werner 1903. In order to ascertain whether individual frogs expressed the full complement of temporin genes, we individually cloned cDNAs encoding the temporin precursors from total RNA extracted from the skins of 12 frogs by RT-PCR using a set of preprotemporin-specific primers. All the specimens examined contained mRNAs directing the synthesis of the novel, but inactive, temporin-1Oe (ILPLLGNLLNGLL x NH2). Nucleotide sequence analysis revealed marked polymorphism among individual frogs. Twenty-seven distinct preprotemporin-1Oe mRNAs were identified that contained synonymous substitutions in the antimicrobial peptide region and both synonymous and non-synonymous substitutions in the signal peptide and intervening sequence regions. Up to eight preprotemporin-1Oe mRNA variants were found within a single frog. In addition, several cDNAs encoding preprotemporin-1Oa and -1Ob and a single cDNA encoding preprotemporin-1Oc were characterized. Peptidomic analysis of norepinephrine-stimulated skin secretions revealed the presence of temporin-1Oe, temporin-1Of (SLILKGLASIAKLF x NH2), temporin-1Og (FLSSLLSKVVSLFT x NH2), four members of the ranatuerin-2 family and one member of the palustrin-2 family in addition to previously characterized temporin and brevinin-2 peptides.

Amino Acid Sequence↗
PubMed2004

Spermatogenesis and plasma testosterone levels in Western Australian burrowing desert frogs, Cyclorana platycephala, Cyclorana maini, and Neobatrachus sutor, during aestivation.

Changes in testis size, histological status, and plasma levels of testosterone were monitored for males of three species of Western Australian desert frogs, Cyclorana maini, Cyclorana platycephala, and Neobatrachus sutor during aestivation. The frogs were induced to burrow and form cocoons soon after their capture and then disinterred at intervals in order to monitor changes in reproductive activity of the testes. All stages of spermatogenesis were evident in active frogs, which were collected a few days following rain from breeding choruses. Relative testis mass declined gradually in all species during the first 7 months of aestivation and then increased significantly at 16-19 months in the two species for which extended data were available (C. maini and N. sutor). A decrease in the number of sperm bundles 2-4 months after cocooning was associated with an initial increase in the number of free spermatazoa in all three species, which then returned to the levels seen in active animals after 7 months. Increases in the number of primary and secondary spermatogonia were most evident in C. platycephala after 4-7 months of aestivation, but early stages of spermatocytogenesis were evident in all species after 7 months of aestivation, especially in individuals that contained neither sperm bundles nor mature spermatazoa. Changes in plasma testosterone levels correlated significantly with variations in the diameter of the seminiferous tubules and the GSI, suggesting that this hormone plays a major role in controlling testicular recrudescence in aestivating, cocooned, desert frogs. Data from this study show that, in the absence of any external cues, testicular recrudescence is evident after approximately one year of aestivation in desert frogs which prepares them to breed again, once rain falls.

Animals↗
PubMed2004

Expression of natriuretic peptides, nitric oxide synthase, and guanylate cyclase activity in frog mesonephros during the annual cycle.

Natriuretic peptides (NPs), a family of structurally related hormones and nitric oxide (NO), generated by nitric oxide synthase (NOS), are believed to be involved in the regulation of fluid balance and sodium homeostasis. Differential expression and regulation of these factors depend on both physiological and pathological conditions. Both NPs and NO act in target organs through the activation of guanylate cyclase (GC) and the generation of guanosine 3',5'-cyclic monophosphate (cGMP), which is considered a common messenger for the action of these factors. The present study was designed to investigate--by histochemical methods--the expression of some NPs (proANP and ANP) and isoforms of NOS (neuronal NOS, nNOS, and inducible NOS, iNOS) in the mesonephros of Rana esculenta in different periods of the year including hibernation, to evaluate possible seasonal changes in their expression. We also studied the enzyme activity of NOS-related nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) and of GC. The experiments were performed on pieces of kidney of R. esculenta collected in their natural environment during active and hibernating life. The study was carried out using immunohistochemical techniques to demonstrate proANP, ANP, and some NOS isoforms. Antigen capture by enzyme linked immunosorbent assay (ELISA) was also performed to determine the presence of NPs in the frog kidney extract. Enzyme histochemistry was used to demonstrate the NOS-related NADPHd activity at light microscopy; GC activity was visualized at the electron microscope, using cerium as capture agent. The application of the immunohistochemical techniques demonstrated that frog mesonephros tubules express different patterns of distribution and/or expression of ANP and NOS during the annual cycle. Comparing the results obtained on active and hibernating frogs has provided interesting data; the NOS/NADPHd and GC activities showed some variations as well. Furthermore, the presence of NPs in the frog kidney extract was evidenced by dose-dependent response in the ELISA. The data suggest that both ANP and NO are intra-renal paracrine and/or autocrine factors which may modulate the adaptations of frog renal functions to seasonal changes through the action of the cGMP generated from GC activity.

Animals↗
PubMed2004

Expression of P450 aromatase protein in developing and in sex-reversed gonads of the XX/XY type of the frog Rana rugosa.

Gonadal differentiation in some species of amphibians is sensitive to steroids. The phenotypic sex of XX/XY-type frogs such as Rana rugosa can be reversed from female to male by injection of testosterone into tadpoles, but little is known about the molecular mechanism of this sex reversal. To elucidate the mechanism of the sex differentiation, we examined the role of P450 aromatase (P450arom), an enzyme that converts testosterone to estrogen, during gonadal differentiation of amphibians. In this study, we first cloned a P450arom cDNA homolog of the frog R. rugosa and analyzed by RT-PCR its expression profile in developing and in female-to-male sex-reversed gonads. P450arom expression was observed in the gonad of tadpoles during ovarian differentiation and became much stronger in the developing ovary in which only immature oocytes were observed. However, its expression declined significantly in the ovary of frogs 2 months after metamorphosis, when oocytes were growing; and it was no longer seen in adult ovaries. By RT-PCR, we also examined the expression of P450arom and SF-1 (steroidogenic factor-1; the orphan nuclear receptor) in the female-to-male sex-reversed gonad. The level of P450arom mRNA was high in the ovary, but it declined rapidly after the injection of testosterone. In contrast, no change in the SF-1 (also known as Ad4BP) expression was observed. Moreover, to identify the type(s) of cells expressing P450arom protein, we performed immunostaining with an antibody against frog P450arom protein. Cells giving positive signals were observed around oocytes in the ovary of frogs 1 month after metamorphosis. They were identified as follicle cells by both light and electron microscopy. The results, taken together, indicate that P450arom protein is synthesized in follicle cells and that P450arom is very much involved in ovarian differentiation in R. rugosa.

Amino Acid Sequence↗
PubMed1990

Characterization of the cDNA encoding proopiomelanocortin in the frog Rana ridibunda.

In the amphibian pars intermedia, secretion of proopiomelanocortin (POMC)-derived peptides is controlled by multiple factors including classical neurotransmitters and neuropeptides. To pursue questions concerning the regulation of POMC gene expression in Rana ridibunda, we have isolated and characterized a full-length cDNA for frog POMC. A cDNA clone isolated from a frog pituitary library contains an open-reading frame of 780-bp that predicts a 260 amino acid POMC protein. The structure of frog POMC demonstrates considerable amino acid sequence similarity with POMC from other species. In particular, the sequence of alpha-melanotropin (alpha-MSH) is identical in frog and all mammalian species studied so far, while adrenocorticotropin (ACTH) and beta-endorphin exhibit 79% and 84% homology with their human counterpart. Frog POMC contains only one potential asparagine-linked N-glycosylation signal (Asn-Ser-Thr) within the gamma-MSH domain. The alpha-MSH sequence is C-terminally flanked by the Gly-Lys-Lys amidation signal while the joining peptide is not amidate.

Amino Acid Sequence↗
PubMed1999

Functional studies of a glucagon receptor isolated from frog Rana tigrina rugulosa: implications on the molecular evolution of glucagon receptors in vertebrates.

In this report, the first amphibian glucagon receptor (GluR) cDNA was characterized from the liver of the frog Rana tigrina rugulosa. Functional expression of the frog GluR in CHO and COS-7 cells showed a high specificity of the receptor towards human glucagon with an EC(50) value of 0.8+/-0.5 nM. The binding of radioiodinated human glucagon to GluR was displaced in a dose-dependent manner only with human glucagon and its antagonist (des-His(1)-[Nle(9)-Ala(11)-Ala(16)]) with IC(50) values of 12.0+/-3. 0 and 7.8+/-1.0 nM, respectively. The frog GluR did not display any affinity towards fish and human GLP-1s, and towards glucagon peptides derived from two species of teleost fishes (goldfish, zebrafish). These fish glucagons contain substitutions in several key residues that were previously shown to be critical for the binding of human glucagon to its receptor. By RT-PCR, mRNA transcripts of frog GluR were located in the liver, brain, small intestine and colon. These results demonstrate a conservation of the functional characteristics of the GluRs in frog and mammalian species and provide a framework for a better understanding of the molecular evolution of the GluR and its physiological function in vertebrates.

Amino Acid Sequence↗
PubMed2000

Anticholinergic effects in frogs in a Morris water maze analog.

We determined the effect of two doses of the centrally acting anticholinergic drug, atropine sulfate (AS), on the performance of female Northern Leopard frogs (Rana pipiens) in a visual cue analog of the Morris water task. Untreated frogs learned the visually cued task, while frogs treated with 150 mg/kg AS were significantly slower than controls in learning to escape warm water by finding a visible platform, and there was a dose-dependent response, with frogs treated with 50 mg/kg AS performing midway between the higher dose and control frogs. These results suggest that the general role of the cholinergic system in learning is important in amphibians, and that this role is evolutionarily conserved across vertebrate species.

Analysis of Variance↗
PubMed1997

Absence of tetrodotoxins in a captive-raised riparian frog, Atelopus varius.

Bufonid frogs of the genus Atelopus contain two classes of skin toxins, namely the steroidal bufadienolides and the water-soluble tetrodotoxins. Frogs of the Panamanian species Atelopus varius have now been raised in captivity and levels in skin extracts of bufadienolides and of tetrodotoxin-like compounds assessed, using inhibition of [3H]ouabain binding and inhibition of [3H]saxitoxin binding, respectively. Levels of ouabain equivalents, corresponding to bufadienolides, were comparable to those found in wild-caught frogs from the same population in Panama, while tetrodotoxin-like activity was undetectable. The results strongly implicate environmental factors, perhaps symbiotic microorganisms, in the genesis of tetrodotoxins in the skin of frogs of the genus Atelopus, while indicating that the frog itself produces the skin bufadienolides.

Amphibian Venoms↗
PubMed1997

Absence of skin alkaloids in captive-raised Madagascan mantelline frogs (Mantella) and sequestration of dietary alkaloids.

Mantelline frogs of the genus Mantella contain a variety of pumiliotoxin, allopumiliotoxin and homopumiliotoxin alkaloids in their skin. Pyrrolizidines, indolizidines and quinolizidines are also present. In contrast, captive-raised frogs (Mantella aurantiaca) have no alkaloids detectable in skin extracts. Frogs fed alkaloid-dusted fruit flies accumulate alkaloids into their skin. Thus, these mantelline frogs, like the neotropical dendrobatid frogs, appear dependent on dietary sources for their skin alkaloids and have the requisite alkaloid-sequestering system(s).

Alkaloids↗
PubMed1997

Differential regulation of the mitochondrial ADP/ATP translocase gene in wood frogs under freezing stress.

The gene Aat coding for ADP/ATP translocase (AAT) was cloned from liver of the freeze-tolerant wood frog, Rana sylvatica, via differential screening of a cDNA library from liver of frozen frogs and using probes from control versus frozen frogs. Sequence analysis showed that clone pBfFR07 bearing the AAT cDNA contained a 1318-bp insert with one full-length open reading frame. The deduced amino acid sequence included 317 residues, with 81-86% identities to mammalian AAT. A 1750-nt transcript from the Aat gene was detected using pBfFR07 probe and a putative frog AAT of over 30 kDa was visualized by immunoblotting using a polyclonal antibody raised against chicken AAT. Analysis of liver samples from a time course of freezing showed a maximal 4.5-fold increase in mRNA after 8 h with AAT protein peaking in 24-h frozen frogs. Freezing also induced Aat expression in bladder and lung. In liver, mRNA expression also responded positively to anoxia stress but not to experimental dehydration of the animals. These results suggest that AAT induction during freezing may be stimulated by the ischemia that develops when plasma freezes; changes in AAT may contribute to stabilizing energetics in mitochondrial versus cytosolic pools over freeze/thaw cycles.

Amino Acid Sequence↗
PubMed2000

Purification and characterization of antimicrobial peptides from the skin of the North American green frog Rana clamitans.

Ten peptides with differential growth-inhibitory activity against the gram-positive bacterium, Staphylococcus aureus, the gram-negative bacterium, Escherichia coli, and the yeast Candida albicans were isolated from an extract of the skin of a North American frog, the green frog Rana clamitans. Ranatuerin-1C (SMLSVLKNLGKVGLGLVACKINKQC), ranalexin-1Ca (FLGGLMKAFPALICAVTKKC), ranalexin-1Cb (FLGGLMKAFPAIICAVTKKC), ranatuerin-2Ca (GLFLDTLKGAAKDVAGKLLEGLKCKIAGC KP), and ranatuerin-2Cb (GLFLDTLKGLAGKLLQGLKCIKAGCKP), are members of three previously characterized families of antimicrobial peptides, first identified in the North American bullfrog Rana catesbeiana. In addition, five structurally related peptides (temporin-1Ca, -1Cb, -1Cc, -1Cd, and -1Ce), comprising 13 amino acid residues and containing a C-terminally alpha-amidated residue, belong to the temporin family first identified in the European common frog Rana temporaria. Peptides belonging to the brevinin-1, brevinin-2, esculentin-1, and esculentin-2 families, previously isolated from the skins of Asian and European Ranid frogs, were not identified in the extract. The data support the hypothesis that the distribution and amino acid sequences of the skin antimicrobial peptides are valuable tools in the identification and classification of Ranid frogs.

Amino Acid Sequence↗
PubMed1997

Effects of urea on M4-lactate dehydrogenase from elasmobranchs and urea-accumulating Australian desert frogs.

We measured the effect of urea on M4-lactate dehydrogenase (M4-LDH) from elasmobranchs and Australian desert frogs (urea accumulators) and from two animals that do not accumulate urea, the axolotl and the rabbit. An analysis of the effect of urea on the Kd(NADH), V, V/K(m(prr)) and V/K(m(NADH)) shows that in all cases the major effect of urea was on the binding of pyruvate, which fits with data in the literature that show that urea acts as a competitive inhibitor of LDH. The characteristics of the elasmobranch enzymes are consistent with a proposed adaptation model, but the situation for the enzymes from the aestivating frogs is equivocal. Urea (400 mM) had less effect on the K(m(prr)) of M4-LDH from the urea accumulators than it did on the non-accumulators, suggesting a general adaptation and that the enzyme produced by the aestivating frogs (urea accumulators) is kinetically different from that of non-aestivating frogs (non-accumulators). A new approach is used to characterize the overall pattern of adaptation to urea. The pattern is similar in an enzyme from an elasmobranch and an aestivating frog despite the temporary presence of urea in the latter and the phylogenetic difference between these animals.

Ambystoma↗
PubMedDate not supplied

The frog as a unique vertebrate model for studying the rhombomeric organization of functionally identified hindbrain neurons.

The segmental organization of cranial nerve efferent, vestibular, and precerebellar neurons of larval ranid frogs is essentially retained in adult frogs, indicating the absence of any substantial postembryonic longitudinal migration of hindbrain neurons in this group. Comparison of the rhombomeric organization of vestibulomotor and branchiomotor pathways across different species suggests that the frog hindbrain blueprint is common to most vertebrates. The unique segmental stability seen in frogs can be used to create a quantitative stereotactic map of the adult brain that mirrors the embryonic and larval rhombomeric framework. Such a map allows the large number of physiologically identified hindbrain neurons in adult frogs to be linked with their underlying genetic specification. Transgenic reporters and antisense knockdown of gene activities suspected of having necessary functions in patterning neurons within specific rhombomeres may allow direct testing of the proposed map.

Animals↗
PubMed2000

Heart rate response to gentle handling of frog and lizard.

Heart rate was counted telemetrically in lizards (Iguana iguana) and frogs (Rana catesbeiana and Rana pipiens) to estimate their response to gentle 1-min handling. The animals were kept at steady body temperatures of ca. 28 degrees C (lizards), and 24 degrees C (frogs). Handling increased the heart rate of lizards from ca. 70 to 110 beats per min immediately during and after handling and this tachycardia decreased in ca. 10 min. Similar handling did not modify significantly the frogs' heart rates. Although the absence of a response to mild stress is not synonymous with the absence of emotion, the absence of handling-tachycardia in frogs and its presence in lizards (as well as in mammals and birds), together with the emotional fever in mammals, birds, and reptiles, but not frogs or fish as reported in the literature, might suggest that 'emotional' response to stress emerged in phylogeny between amphibians and reptiles.

Journal Article↗
PubMed2000

Two isoforms of FTZ-F1 messenger RNA: molecular cloning and their expression in the frog testis.

FTZ-F1, a member of the orphan nuclear receptors, is a transcriptional factor regulating the expression of the fushi tarazu gene in Drosophila (Lavorgna et al., 1991. Science 252, 848-851). Previously, we cloned a frog homologue of FTZ-F1 (rrFTZ-F1alpha; GenBank Accession No. AB035498). In this study, we isolated the rrFTZ-F1beta cDNA encoding a protein of 469 amino acids. Then, expressions of two types (alpha and beta) of rrFTZ-F1 mRNAs were examined during development of embryos and gonads in the frog Rana rugosa. They were expressed in the embryo at stage 12. Expressions of both the alpha and beta mRNAs became stronger in the testis of frogs at stage XXV and were most prominent in that of frogs 2months after metamorphosis. In the former testis, spermatogonia were the only germ cells in the seminiferous tubules, whilst postmeiotic cells were observed in the latter testis. Expression of the typealpha mRNA was more prominent. In addition, we cloned the regions with either exon I or II of the rrFTZ-F1 gene. Genomic structure analysis revealed that rrFTZ-F1beta is a partial exon I-truncated variant of rrFTZ-F1alpha. The results suggest that rrFTZ-F1alpha and -beta are expressed from the same gene by alternative splicing and that they may play an important role(s) in differentiation of premeiotic germ cells in the testis of the frog R. rugosa.

Alternative Splicing↗
PubMed2001

Expression of Dax-1 during gonadal development of the frog.

Dax-1, a member of the nuclear hormone receptor superfamily of transcription factors, is known to be involved in gonadal development in mammals. To date, Dax-1 has only been isolated in reptiles, birds and mammals. The expression of Dax-1 is down-regulated in the developing testis, but persists in the ovary of mice (Swain et al., Nat. Genet. 12 (1996) 404) and chicken (Smith et al., J. Mol. Endocrinol. 24 (2000) 23). Curiously, there is no sex difference in the expression patterns of Dax-1 in the American alligator (Western et al., Gene 241 (2000) 223). To understand its role(s) in gonadal development in vertebrates, molecular cloning of Dax-1 in amphibians is required. In this study, we cloned an amphibian Dax-1 homologue of the frog Rana rugosa and examined its expression profile during gonadal development. Cloned Dax-1 cDNA encoded a protein of 287 amino acids. Unlike mammalians that possess the three and one half repeat elements representing the putative DNA binding domain in the predicted sequence of Dax-1 protein, the frog had a single poorly conserved copy of the repeat unit. By RT-PCR analysis, the Dax-1 mRNA was detected in the liver and pancreas, but not in the testis and ovary of adult frogs. However, Dax-1 expression was seen first in the embryo at stage 12 and became stronger in tadpoles until stage X. The Dax-1 was transcribed in the testis stronger than in the ovary of frogs at stage XXV (just after completion of metamorphosis). In the gonad of frogs 2 months after metamorphosis (at this stage postmeiotic cells can be seen in the seminiferous tubules), the Dax-1 was expressed only in males. In addition, the Dax-1 transcription declined gradually as ovarian development proceeded, but its expression was down-regulated and then up-regulated rapidly when female-to-male sex reversal was caused by administration of testosterone into female tadpoles. Taken together, the results suggest that the Dax-1 may be closely involved in testicular development of amphibians.

Amino Acid Sequence↗
PubMed1999

Potassium currents in auditory hair cells of the frog basilar papilla.

The whole-cell patch-clamp technique was used to identify and characterize ionic currents in isolated hair cells of the leopard frog basilar papilla (BP). This end organ is responsible for encoding the upper limits of a frog's spectral sensitivity (1.25-2.0 kHz in the leopard frog). Isolated BP hair cells are the smallest hair cells in the frog auditory system, with spherical cell bodies typically less than 20 microm in diameter and exhibiting whole-cell capacitances of 4-7 pF. Hair cell zero-current resting potentials (Vz) varied around a mean of -65 mV. All hair cells possessed a non-inactivating, voltage-dependent calcium current (I(Ca)) that activates above a threshold of -55 mV. Similarly all hair cells possessed a rapidly activating, outward, calcium-dependent potassium current (I(K)(Ca)). Most hair cells also possessed a slowly activating, outward, voltage-dependent potassium current (I(K)), which is approximately 80% inactive at the hair cell Vz, and a fast-activating, inward-rectifying potassium current (I(K1)) which actively contributes to setting Vz. In a small subset of cells I(K) was replaced by a fast-inactivating, voltage-dependent potassium current (I(A)), which strongly resembled the A-current observed in hair cells of the frog sacculus and amphibian papilla. Most cells have very similar ionic currents, suggesting that the BP consists largely of one homogeneous population of hair cells. The kinetic properties of the ionic currents present (in particular the very slow I(K)) argue against electrical tuning, a specialized spectral filtering mechanism reported in the hair cells of birds, reptiles, and amphibians, as a contributor to frequency selectivity of this organ. Instead BP hair cells reflect a generalized strategy for the encoding of high-frequency auditory information in a primitive, mechanically tuned, terrestrial vertebrate auditory organ.

Animals↗
PubMed2002

First evidence of prothymosin alpha in a non-mammalian vertebrate and its involvement in the spermatogenesis of the frog Rana esculenta.

A cDNA clone encoding for a Prothymosin alpha (Prot-alpha) has been isolated and characterized from the testis of the frog Rana esculenta. Frog Prothymosin alpha (fProt-alpha) predicted a 109 amino acid protein with a high homology to the mammalian Prot-alpha. fProt-alpha contains 28 aspartic and 25 glutamic acid residues and presents the typical basic KKQK amino acid sequence in the close carboxyl terminal region. Northern blot analysis revealed that fProt-alpha is highly expressed in the testis. A different expression of fProt-alpha transcript was found during the frog reproductive cycle with a peak in September/October in concomitance with germ cell maturation, strongly suggesting a role for this protein in the testicular activity. In situ hybridization evidenced that the only germ cells expressing fProt-alpha are the primary and secondary spermatocytes; in addition, the hybridization signal was stronger in the October testis. Taken together, our findings indicate that fProt-alpha might contribute to the efficiency of frog spermatogenesis with a role during the meiosis. This study is the first report on the isolation and characterization of a Prot-alpha in a non-mammalian vertebrate. In addition, our results indicate that the testis of the frog R. esculenta may be a useful model to increase the knowledge concerning the physiological role of Prot-alpha in vertebrates.

Amino Acid Sequence↗