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PubMed2004

Phylogeny of caecilian amphibians (Gymnophiona) based on complete mitochondrial genomes and nuclear RAG1.

We determined the complete nucleotide sequence of the mitochondrial (mt) genome of five individual caecilians (Amphibia: Gymnophiona) representing five of the six recognized families: Rhinatrema bivittatum (Rhinatrematidae), Ichthyophis glutinosus (Ichthyophiidae), Uraeotyphlus cf. oxyurus (Uraeotyphlidae), Scolecomorphus vittatus (Scolecomorphidae), and Gegeneophis ramaswamii (Caeciliidae). The organization and size of these newly determined mitogenomes are similar to those previously reported for the caecilian Typhlonectes natans (Typhlonectidae), and for other vertebrates. Nucleotide sequences of the nuclear RAG1 gene were also determined for these six species of caecilians, and the salamander Mertensiella luschani atifi. RAG1 (both at the amino acid and nucleotide level) shows slower rates of evolution than almost all mt protein-coding genes (at the amino acid level). The new mt and nuclear sequences were compared with data for other amphibians and subjected to separate and combined phylogenetic analyses (Maximum Parsimony, Minimum Evolution, Maximum Likelihood, and Bayesian Inference). All analyses strongly support the monophyly of the three amphibian Orders. The Batrachia hypothesis (Gymnophiona, (Anura, Caudata) receives moderate or good support depending on the method of analysis. Within Gymnophiona, the optimal tree (Rhinatrema, (Ichthyophis, Uraeotyphlus), (Scolecomorphus, (Gegeneophis Typhlonectes) agrees with the most recent morphological and molecular studies. The sister group relationship between Rhinatrematidae and all other caecilians, that between Ichthyophiidae and Uraeotyphlidae, and the monophyly of the higher caecilians Scolecomorphidae+Caeciliidae+Typhlonectidae, are strongly supported, whereas the relationships among the higher caecilians are less unambiguously resolved. Analysis of RAG1 is affected by a spurious local rooting problem and associated low support that is ameliorated when outgroups are excluded. Comparisons of trees using the non-parametric Templeton, Kishino-Hasegawa, Approximately Unbiased, and Shimodaira-Hasegawa tests suggest that the latter may be too conservative.

Amphibians↗
PubMed2005

A PCR survey for posterior Hox genes in amphibians.

Hox genes encode transcription factors that play a key role in specifying the body plan in metazoans and are therefore essential in explaining patterns of evolutionary diversity. As an ancient tetrapod group with diverse limb types, amphibians are important for understanding the origin and diversification of limbs in land vertebrates. We conducted a PCR survey in two species of each amphibian order to identify Hox-9 to Hox-13, known to function in limb development. Fifteen distinct posterior Hox genes and one retro-pseudogene were identified, and the former confirm the existence of four Hox clusters in each amphibian order. Some genes expected to occur in all tetrapods, based on the posterior Hox complement of mammals, fishes and coelacanth, were not recovered from our survey, and may have been lost. Hoxd-12 is absent in frogs and possibly other amphibians. Considering its function in autopodial development, the loss of this gene may be related to the absence of the fifth finger in frogs and salamanders.

Amino Acid Sequence↗
PubMed2005

Phylogenetic relationships of Hynobius naevius (Amphibia: Caudata) as revealed by mitochondrial 12S and 16S rRNA genes.

Using mitochondrial 12S and 16S rRNA sequences, we investigated phylogenetic relationships among populations of the endemic Japanese salamander Hynobius naevius. Monophyly of this species was recovered only in the maximum parsimony tree and was unresolved in maximum likelihood and Bayesian trees. Instead the following four haplotype clades consistently emerged clearly: Clade 1 from northwestern Kyushu, Clade 2 from Chugoku and northeastern Kyushu, Clade 3 from western Shikoku and Kyushu, and Clade 4 from Chubu-Kinki and central-eastern Shikoku. Of these, Clades 1 and 2, and Clades 3 and 4, respectively, correspond to Groups A and B previously recognized from the analyses of allozyme data in this species, but monophyly of these groups was not strongly supported. Unlike the previous results, the western and eastern samples from Shikoku did not form a clade, and were grouped with Kyushu-B in Clade 3 and Chubu-Kinki in Clade 4, respectively. The reason for this conflict between mtDNA and allozyme results is unknown, but might be related to retention of ancestral mtDNA polymorphism in Shikoku populations. Nearly simultaneous divergence of as many as four lineages in wide-ranging H. naevius is inferred for the late Miocene-Pliocene history of this taxon.

Animals↗
PubMed2005

Extreme tadpoles: the morphology of the fossorial megophryid larva, Leptobrachella mjobergi.

The bizarre larvae of Leptobrachella mjobergi are fossorial and live in the gravel beds of small streams. These tadpoles are vermiform in body shape. Here we present details on their skeleton and musculature, particularly of the head. The entire cranium and its associated musculature are reconstructed in three dimensions from serial histological sections. The hyobranchial apparatus is highly reduced. The head of the L. mjobergi larva is more mobile than in other anuran species. This mobility can largely be ascribed to the exclusion of the notochord from the cranial base and an articulation of the foramen magnum floor with the atlas of the tadpole. The articulation is unique among anuran species, but design parallels can be drawn to salamanders and the articulation between atlas and axis in mammals. In L. mjobergi, the atlas forms an anterior dens that articulates with the basal plate in an accessory, third occipital articular face. The muscle arrangements deviate from the patterns found in other tadpoles: For instance, epaxial and ventral trunk muscles reach far forward onto the skull. The post-cranial skeleton of L. mjobergi is considerably longer than that of other anurans: it comprises a total of 35 vertebrae, including more than 20 post-sacral perichordal centra. Despite a number of features in cranial and axial morphology of L. mjobergi, which appear to be adaptations to its fossorial mode of life, the species clearly shares other features with its megophryid and pelobatid relatives.

Animals↗
PubMed1991

Comparison of the amino acid sequences of tissue-specific parvalbumins from chicken muscle and thymus and possible evolutionary significance.

Chicken leg muscle parvalbumin was digested with cyanogen bromide or trypsin or trypsin after citraconylation. Peptides isolated by reverse phase HPLC at pH 7.0 were subjected to acid hydrolysis and amino acid analysis and, in some cases, sequencing. The chicken muscle parvalbumin amino acid sequence has ca. 80% sequence identity with alpha-type parvalbumins from mammalian (rabbit, human and rat) muscle. By contrast, the chicken thymus parvalbumin ("avian thymic hormone") sequence is very similar to reptile (turtle, salamander and frog) muscle beta-type parvalbumins. We hypothesize that the evolutionary appearance of the warm-blooded reptiles was accompanied by recruitment of the beta parvalbumin isozyme for promotion of lymphocyte maturation.

Amino Acid Sequence↗
PubMed2002

Molecular mechanism of spontaneous pigment activation in retinal cones.

Spontaneous current and voltage fluctuations (dark noise) in the photoreceptor cells of the retina limit the ability of the visual system to detect dim light. We recorded the dark current noise of individual salamander L cones. Previous work showed that the dark noise in these cells arises from thermal activation of the visual pigment. From the temperature dependence of the rate of occurrence of elementary noise events, we found an Arrhenius activation energy E(a) of 25 +/- 7 kcal/mol (mean +/- SD). This E(a) is similar to that reported for the thermal isomerization of 11-cis retinal in solution, suggesting that the cone pigment noise results from isomerization of the retinal chromophore. E(a) for the cone noise is similar to that previously reported for the "photon-like" noise of rods, but the preexponential factor is five orders of magnitude higher. To test the hypothesis that thermal isomerization can only occur in molecules whose Schiff base linkage is unprotonated, we changed the pH of the solution bathing the cone outer segment. This had little effect on the rate of occurrence of elementary noise events. The rate was also unchanged when the cone was exposed to Ringer solution made up from heavy water, whose solvent isotope effect should reduce the probability, that the Schiff base nitrogen is naked.

Animals↗
PubMed2003

Role of hyperpolarization-activated currents for the intrinsic dynamics of isolated retinal neurons.

The intrinsic dynamics of bipolar cells and rod photoreceptors isolated from tiger salamanders were studied by a patch-clamp technique combined with estimation of effective impulse responses across a range of mean membrane voltages. An increase in external K(+) reduces the gain and speeds the response in bipolar cells near and below resting potential. High external K(+) enhances the inward rectification of membrane potential, an effect mediated by a fast, hyperpolarization-activated, inwardly rectifying potassium current (K(IR)). External Cs(+) suppresses the inward-rectifying effect of external K(+). The reversal potential of the current, estimated by a novel method from a family of impulse responses below resting potential, indicates a channel that is permeable predominantly to K(+). Its permeability to Na(+), estimated from Goldman-Hodgkin-Katz voltage equation, was negligible. Whereas the activation of the delayed-rectifier K(+) current causes bandpass behavior (i.e., undershoots in the impulse responses) in bipolar cells, activation of the K(IR) current does not. In contrast, a slow hyperpolarization-activated current (I(h)) in rod photoreceptors leads to pronounced, slow undershoots near resting potential. Differences in the kinetics and ion selectivity of hyperpolarization-activated currents in bipolar cells (K(IR)) and in rod photoreceptors (I(h)) confer different dynamical behavior onto the two types of neurons.

Animals↗
PubMed1970

The intracellular transport and distribution of cysteamine phosphate derivatives.

Radioautography and extractive techniques were used to analyze the transport of cysteamine phosphate and its derivatives in salamander oocytes. The quantitative relations among the processes involved - membrane permeation, enzymatic dephosphorylation, binding through mixed disulfide formation, and cytoplasmic diffusion - were elucidated. Within the detection limits, all of the intracellular material is present as dephosphorylated derivatives. Cytoplasmic diffusion is effectively slowed by binding (the "chromatographic" effect) and makes an appreciable contribution to cellular flux rates. As a consequence, one can observe by radioautography a cortical diffusion ring which spreads inward as a function of influx time, while also increasing in peak density because of the finite membrane permeability. Good agreement was found between the transport parameters determined by radioautography and those from influx data for the whole oocyte. The ratio of nuclear to cytoplasmic concentrations of the cysteamine phosphate derivatives at equilibrium is about 0.4. The nuclear membrane is, however, a negligible barrier to transport, and the asymmetry appears to arise primarily from the quantity and sulfhydryl content of the binding proteins in the two compartments.

Animals↗
PubMed1979

Reference phase analysis of free and bound intracellular solutes. I. Sodium and potassium in amphibian oocytes.

A method is described for the quantitative determination of free and bound solute concentrations in the cytoplasm of intact cells. The method includes (a) introduction of a gelatin gel reference phase (RP) into the cytoplasm; (b) diffusion of dissolved substances between cytoplasm and RP, (c) cell quenching to - 196 degrees C to prevent subsequent solute redistributions, (d) ultra-low temperature microdissection to isolate RP and cytoplasm samples, and (e) analysis of isolates for solute and water content. In normal oocytes of the salamander, Desmognathus ochrophaeus, free or RP Na+ and K+ are 21.0 +/- 1.1 and 128.8 +/- 2.4 mu eq/ml, respectively, and vary stoichiometrically in altered oocytes. Overall cytoplasmic concentrations are 75.2 +/- 2.7 mu eq Na+/ml and 88.6 +/- 1.5 mu eq K+/ml. Cytoplasmic chemical activities are 16.2 mu eq Na+/ml and 99.2 mu eq K+/ml, corresponding to activity coefficients of 0.22 and 1.12, respectively. The results demonstrate unambiguously that (a) oocytes actively transport Na+ and K+, and (b) cytoplasm has important binding properties which differentiate it from an ordinary aqueous solution. These cytoplasmic properties are investigated in the following paper.

Animals↗
PubMed1980

The location of olfactory receptor sites. Inferences from latency measurements.

Excitatory responses recorded from vertebrate olfactory sensory neurons are characterized by long latencies compared with those from other sensory receptors. Explanations which assume free access of the stimuli to receptor molecules presumably located on the olfactory cilia necessarily imply an intrinsic delay in the transduction mechanism. In contrast, the possibility of restricted or delayed access due to diffusion of the stimulus to molecular receptors located on the dendritic know or proximal portions of the cilia suggests transduction processes having time courses similar to those in other sensory systems. We show that the threshold stimulus concentrations and the latency of the excitatory response of the salamander can be predicted primarily on the basis of a diffusional delay and that the receptor molecules are well below the surface of the mucus. Examination of response latencies for other species reported in the literature support the generality of diffusional delay. The predicted location of molecular receptor sites is largely insensitive to assumptions based on the mode of clearance of the stimuli. Additional access restrictions are discussed but are shown to generate qualitatively different latency functions than does diffusion, suggesting that they exert only minor influences on latency and threshold characteristics.

Action Potentials↗
PubMed1989

Action potentials initiated by single channels opening in a small neuron (rat olfactory receptor).

Rat olfactory receptor neurons were enzymatically dissociated and studied with the cell-attached configuration of the patch-clamp technique. Biphasic current waveforms induced across the membrane patch by intracellular action potentials were observed in approximately 5% of cells studied. In one cell in particular, current injected by the opening of a single channel initiated an action potential in the remainder of the cell each time the channel opened. A conventional type of electrical model of the cell and patch allowed the accurate modeling of cell excitability. The same model was used to explain the shape of the action potential current waveforms induced across the patch. The analysis indicated that the whole cell resistance (Ro) was approximately 40 G omega and the membrane capacitance (Co) was close to the standard value of 1 microF.cm-2. In addition, the threshold potential change necessary to initiate an action potential (Vth) was approximately 13 mV and a minimum current injection of 1 pA was required to depolarize the cell to spike threshold. When the smaller size of mammalian receptors are taken into account, membrane electrical properties were found to be consistent with those of salamander cells investigated by others using whole-cell recording. The analysis also revealed possible errors in the determination of single-channel conductances and reversal potentials by cell-attached recording from small cells.

Action Potentials↗
PubMed1990

Transduction noise induced by 4-hydroxy retinals in rod photoreceptors.

New visual pigments were formed with 4-hydroxy retinals in isolated vertebrate rod photoreceptors by exposing bleached rods from the tiger salamander, Ambystoma tigrinum, to lipid vesicles containing the analogues. Formation of physiologically active pigment was demonstrated by the restoration of sensitivity and by a shift of approximately 50 nm in the peak of both the visual pigment absorptance spectrum and rod spectral sensitivity spectrum from approximately 520 to approximately 470 nm for 11-cis 4-hydroxy retinal. Membrane current recordings from the inner segments of isolated rods revealed excess fluctuations in membrane current after formation of the new pigment in bleached cells or after exposure of unbleached cells to flashes in the presence of the analogue. The excess current fluctuations are similar to the fluctuations elicited by steady light producing a few discrete responses per second, a rate approximately 100 times greater than the normal rate of spontaneous events in darkness. These results suggest that analogues of retinal can produce alterations in the frequency of production of discrete responses in darkness in rod photoreceptors.

Ambystoma↗
PubMed1996

Noise analysis of the glutamate-activated current in photoreceptors.

The glutamate-activated current in photoreceptors has been attributed both to a sodium/glutamate transporter and to a glutamate-activated chloride channel. We have further studied the glutamate-activated current in single, isolated photoreceptors from the tiger salamander using noise analysis on whole-cell patch-clamp recordings. In cones, the current is generated by chloride channels with a single-channel conductance of 0.7 pS and an open lifetime of 2.4 ms. The number of channels per cell is in the range of 10,000-20,000. Activation of the channels requires the presence of both glutamate and sodium. The single-channel conductance and the open lifetime of the channel are independent of the external concentration of glutamate and sodium. External glutamate and sodium affect only the opening rate of the channels. D,L-Threo-3-hydroxyaspartate (THA), a glutamate-transport blocker, is shown to be a partial agonist for the channel. The single-channel conductance is the same regardless of whether glutamate or THA is the ligand, but the open lifetime of the channel is only 0.8 ms with THA as ligand. The glutamate-activated current in rods has a similar single-channel conductance (0.74 pS) and open lifetime (3 ms). We propose a kinetic model, consistent with these results, to explain how a transporter can simultaneously act both as a sodium/glutamate-gated chloride channel and a glutamate/sodium cotransporter.

ATP-Binding Cassette Transporters↗
PubMed1996

Effect of changing temperature on the ionic permeation through the cyclic GMP-gated channel from vertebrate photoreceptors.

Native cGMP-gated channels were studied in rod outer segments of the larval tiger salamander Ambystoma tigrinum. The alpha subunit of the cGMP-gated channel from bovine rods, here referred to as the wild type (w.t.), and mutant channels were heterologously expressed in Xenopus laevis oocytes. These channels were studied in excised membrane patches in the inside-out configuration and were activated by the addition of 100 or 500 microM cGMP. The effect of temperature on the ionic permeation was studied. The macroscopic current flowing through the native channel at +100 mV had an activation energy of 35.8, 30, 31.8, 34.5, 41.3, and 22.4 kJ mol-1 in the presence of Li+, Na+, K+, Rb+, Cs+, and NH4+, respectively. The macroscopic current flowing through the w.t. channel at +100 mV had an activation energy of 45.2, 38.2, 37.5, 47.3, 49.4, and 38.9 kJ mol-1 in the presence of Li+, Na+, K+, Rb+, Cs+, and NH4+, respectively. The activation energy of the macroscopic current flowing through the native and w.t. channels did not vary significantly when the ionic concentration of the permeant ion was changed between 2.5 and 110 mM. The activation energy of the single-channel current of the w.t. channel at +100 mV was 40.4 and 33 kJ mol-1 for Na+ and NH4+, respectively. The reversal potential of biionic solutions changed significantly with temperature. These results can be used to obtain an estimate of the enthalpic and entropic contributions to the barrier of the Gibbs free energy experienced by an ion during its permeation through the open channel. These estimates indicate that the ionic permeation and selectivity of the cGMP-gated channel are controlled both by enthalpic and entropic factors and that the selectivity of the native channel for Li+ over Na+ is primarily caused by entropic effects.

Ambystoma↗
PubMed2000

Ultrastructural identification of synapses between mitral/tufted cell dendrites.

Asymmetrical, type 1 synapses between mitral and/or tufted (M/T) cell dendrites were observed in the glomerular layer (GL) and juxtaglomerular external plexiform layer (EPL) of salamander olfactory bulb sections. The dendrites had electron-lucent cytoplasm containing regularly-arrayed microtubules and spherical translucent vesicles. The vesicles were clustered against a thin pre-synaptic density that was aligned with a 17-20 nm-wide synaptic cleft and a thicker post-synaptic density. These dendrodendritic synapses could be a source of the delayed, prolonged excitation that originates from the GL/EPL. During spatiotemporal encoding of odor stimuli, they could amplify or synchronize M/T cell responses.

Ambystoma↗
PubMed1998

Calretinin-immunoreactive elements in the retina and optic tectum of the frog, Rana esculenta.

The frog retina contains numerous 28 kDa calbindin positive elements in its every layer. At the same time, parvalbumin has been observed only in a few elements in the visual system of amphibian species, whilst calretinin immunoreactivity could be detected in salamander retina and the optic tectum of tench. However, the presence and distribution of calretinin have been described to date neither in the retina nor in the other parts of the visual system of anurans. Therefore, the aim of this study is to describe the calretinin immunoreactive elements in the retina and the optic tectum of the frog and to establish whether or not the expression of this calcium-binding protein is transmitter-related and/or cell type specific in these parts of the central nervous system. In the retina, numerous bipolar cells showed calretinin immunoreactivity. The axon terminals of the bipolar cells branched in both the OFF and ON sublayers of the inner plexiform layer. The few labeled amacrine cells were larger than 10 microns in diameter. Over 50% of the cells in the ganglion cell layer contained calretinin. The labeled cells in the ganglion cell layer were of usually 16-22 microns in diameter, although a few smaller cells were also seen. Accordingly, many optic fibers were also labeled. In colocalization experiments, gamma-aminobutyric acid and calretinin were found in partially overlapping amacrine cell populations, cells with the former marker being much more numerous. At the same time, all the gamma-aminobutyric acid positive bipolar cells also contained calretinin. Most of the calretinin positive neurons in the ganglion cell layer however were only single-labeled. Axons of ganglion cells terminated in B, C and F sublayers of layer 9 in the optic tectum. Local tectal neuron populations in layer 4, 6, 8 and 9 were also labeled and a few calretinin positive cells were detected also in layer 2. Approximately 10% of the tectal cells were found to be immunoreactive for calretinin. Layer 4 and 6 cells were mostly large pear-shaped neurons while cells in the 8th layer were small pear-shaped and ganglion cells labeled too. Coexistence of gamma-aminobutyric acid and calretinin was characteristic in cells of the upper tectal layers while they were not detected in neurons of deep layers of the tectum. After monocular enucleation, contralateral to the removed eye, calretinin-immunoreactivity disappeared almost completely from F sublayer and became less pronounced in sublayers B and C after 90 days. Calretinin-immunoreactivity remained mostly unchanged in local tectal cells. The results show that, although its function remains undetermined, calretinin is the major EF-hand calcium-binding protein in the frog retina and optic tectum.

Animals↗
PubMed1995

Three-dimensional membrane crystals in amphibian cone outer segments: 2. Crystal type associated with the saddle point regions of cone disks.

In light-adapted, perfusion fixed retinas of the Congo eel salamander, Amphiuma, we have observed distinctive 3-D crystalline domains within the axial array of cone outer segment disks. These crystalline domains, each involving 2-12 disks, have been observed in the distal half of cone outer segment, and are associated with saddle point regions and immediately adjacent segments of the cone disk perimeter. In longitudinal sections, the crystals typically display an axially oriented array of cytoplasmic filaments with lateral spacings in the range of 12-13 nm. The width of the intradiskal compartment is expanded to 8-10 nm within the crystal, and approximates the width of the cytoplasmic compartment. In some sections, the cytoplasmic filaments are axially aligned with intradiskal filaments of similar length and width. In transverse sections, the projected lattice appears to be approximately rectangular, with unit cell dimensions of approximately 12 nm x 12.5 nm. In shape, orientation, dimension, location, projection symmetry and associated membrane spacing relationships, the cone outer segment crystal filaments share a strong resemblance with lattice filaments located along the perimeters and preincisures of rod disks. These similarities suggest that the cone crystal filaments may be related to the rim protein of rod disks. Lastly, the preferential association of these crystals with saddle point regions indirectly supports the hypothesis that reductions in COS disk area with apical displacement are accomplished by resorption of disk membrane components through the saddle points.

Animals↗
PubMed1987

Patterns of interdisk connections within the lamellar domains of retinal rod outer segment disks: observations relevant to the axial propagation of incisures.

In rod outer segments (ROSs) of the Congo eel salamander (Amphiuma), we have observed a system of cytoplasmic filaments that interconnect the lamellar domains of adjacent disks. In longitudinal sections, these filaments occur in pairs, spaced 18-20 nm apart. Each filament is 9-11 nm in length, and perpendicular to the disk membrane. Such filament pairs display extended axial alignment through the lamellar domains of successive disks, indicating the involvement of trans-membranous elements. In cross-sectional views, these filaments appear as a row of paired, punctate densities with a periodic spacing of 14-16 nm. Such views indicate that these filaments are organized on a two-dimensional crystalline lattice, oriented perpendicular to the disk lamellae. The linear interdisk densities of the terminal loop complex appear to be identical to these cytoplasmic filaments: both are equal in length, and both are organized as two-dimensional assemblies with equivalent orientations and lattice parameters. These shared features suggest a common subset of molecular components and lattice determinants. Where the paired filament assemblies intersect the disks, one observes a characteristic dilatation of the intradisk space. In these regions, the disk is 3-6 nm wider than adjacent lamellar regions, and tends to approximate the width of the terminal loop expansion along the disk perimeter. Within such dilatations, we have not been able to demonstrate crescentic densities corresponding to those found within the terminal loop region. Paired filament arrays are not randomly located within the lamellar domains, but lie parallel to the local disk perimeter. Both single and multiple arrays are commonly observed. Single arrays are recessed about 80 nm from the disk edge. When more than one array is present, the additional arrays lie parallel to the first and are spaced at intervals of about 70 nm. Thus, in three dimensions, the multiple arrays appear as regularly spaced sheets of filaments, parallel to the local disk perimeter and perpendicular to the lamellar domains. The characteristic interval between such filament arrays suggests the presence of 'spacer' molecules that organize the two-dimensional filament lattices in the third dimension. Such a spacing mechanism may also account for the minimum width observed for disk lobule segments defined by extended parallel incisures. The potential role of paired filament arrays in the axial propagation of incisures is discussed.

Animals↗