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Light-related changes in electron-dense material in photoreceptor synaptic clefts of the frog, Rana catesbeiana.

Lumps of electron-dense material were observed in synaptic clefts associated with all types of photoreceptors, in the vicinity of the synaptic ribbons, in the retinae of dark-adapted frogs. Frogs were reared under a cyclic illumination (light on at 8:00; light off at 20:00) and then exposed to one of two courses of dark adaptation: one started from 11:00 in the morning, and the other started from 20:00 in the evening. The synaptic clefts of red rods became wider at some places where spherical or polygonal lumps of dense material were accumulated. The frequency and sectional area of the lumps increased faster for the first hour in the regime starting from 20:00 than in the regime starting from 11:00, then they reached the similar saturation levels of about 0.6 (per ribbon) and 1.6 to 1.8 X 10(4) (nm2) in both the regimes. In green-rod synapses, plate-shaped lumps of dense material were present in synaptic clefts and interspaces between the processes of second-order neurons. In cone synapses at the end of about 1 h darkness, the frequency and area of the lumps reached maximum values of about 0.12 (per ribbon) and 9 X 10(3) (nm2) in the regime starting from 11:00 and, about 0.08 (per ribbon) and 4 X 10(3) (nm2) in the regime starting from 20:00. On exposure to light, the dense material abruptly disappeared from all types of photoreceptor synaptic clefts. Large dense-core vesicles, occasionally observed in light-adapted rod photoreceptor terminals, seem to participate in exocytosis of the dense material. The number of dense-core vesicles per synaptic ribbon in a terminal was about 0.55 at the end of 3 h light in the morning and about 1.28 at the end of 12 h light in the evening. The increased number of dense-core vesicles during the daytime may contribute to the faster accumulation of dense material in the synaptic clefts. Although the chemical identification or the functional significance of the electron-dense material remains unknown, it is interesting that this material showed a rise and fall in response to darkness and illumination. Also the fact that this material is clearly visible will be helpful for future analysis of frog photoreceptor synapses.

Animals↗

Synthesis and intracellular transport of proteins in the exocrine pancreas of the frog (Rana esculenta). II. An in vitro study of the transport process and the influence of temperature.

Frog pancreatic tissue was pulse-labelled in vitro with 3H-leucine and protein transport was studied in exocrine cells by electron microscope autoradiography. The proteins appeared to be synthesized in the RER and transported to the secretory granules along a similar route and with the same velocity as previously described under in vitro conditions. Evidence was obtained for the involvement of the vesicular and tubular elements at the periphery of the Golgi system in transferring protein from the RER to the Golgi cisternae. Kinetics of the release of newly synthesized proteins from the RER and their appearance in the condensing vacuoles are discussed and related to results reported from other tissues. The transport velocity in this poikilothermic system was studied in relation to the incubation temperature and compared with results reported from its mammalian counterpart. At temperatures between 20 and 30 degrees C intracellular protein transport occurs faster in the frog than in the Guinea pig pancreas. At higher temperature the transport process was severely disturbed in the frog.

Animals↗

The time course and frequency content of hydrodynamic events caused by moving fish, frogs, and crustaceans.

In the present study the time course and spectral-amplitude distribution of hydrodynamic flow fields caused by moving fish, frogs, and crustaceans were investigated with the aid of laser-Doppler-anemometry. In the vicinity of a hovering fish sinusoidal water movements can be recorded whose velocity spectra peak below 10 Hz. Single strokes during startle responses or during steady swimming of fish, frogs, and crustaceans cause short-lasting, low-frequency (less than 10 Hz), transient water movements. Low-frequency transients also occur if a frog approaches and passes a velocity-sensitive hydrodynamic sensor. In contrast, transient water movements caused by a rapidly struggling or startled fish or water motions measured in the wake of a slowly swimming (less than or equal to 47 cm/s) trout can be broadbanded, i.e., these water movements can contain frequency components up to at least 100 Hz. High-frequency hydrodynamic events can also be measured behind obstacles submerged in running water. The possible biological advantage of the ability to detect high-frequency hydroynamic events is discussed with respect to the natural occurrence of high frequencies and its potential role in orientation and predator-prey interactions of aquatic animals.

Animals↗

Unilateral pretectal microinjections of SR 95,531, a GABA A antagonist: effects on directional asymmetry of frog monocular OKN.

Monocular eye movements have been studied in frogs using the search coil technique before and after unilateral microinjection of SR 95,531, a GABA A antagonist, into the pretectal nuclei contralateral to the open eye. Before injection, monocular, horizontal optokinetic nystagmus (OKN) in frogs, as in other lower vertebrates, displays a directional asymmetry: the stimulation in the T-N (temporo-nasal) direction is more efficient in evoking OKN than is stimulation in the N-T (naso-temporal) direction. The N-T component is almost absent and displays only slow phases of very low speed. Unilateral SR 95,531 microinjection into the pretectum reversibly decreased the directional asymmetry of monocular horizontal OKN, by strongly increasing the N-T component slow phase velocity while the T-N slow phase velocity remained unchanged. These data show that SR 95,531 injected into the pretectum contralateral to the open eye reversibly decreased the inhibition upon the N-T component of monocular horizontal OKN, which suggests that a pretectal GABAergic system is involved in the directional asymmetry of monocular horizontal OKN in frogs.

Animals↗

Modulation of the activity of the transverse tubule Mg(2+)-ATPase from frog skeletal muscle by a monoclonal antibody in vitro.

We have established several hybridoma lines that produce monoclonal antibodies against transverse tubule (t-tubule) proteins from frog skeletal muscle. The specificity of these antibodies was characterized by ELISA and Western immunoblotting with purified t-tubule, sarcoplasmic reticulum and partially purified sarcolemmal membranes. One of the monoclonal antibodies (2/34.4) recognizes a band of 109,000 Da in immunoblots. When purified frog t-tubule vesicles were preincubated with this antibody we observed an increase in the rate of the Mg(2+)-ATPase enzyme (up to six fold) which was dependent on antibody concentration. Immunocytological experiments done on cryostat sections of frog muscle indicate that the antigen recognized by this antibody is localized mainly at the level of the t-tubules (I band) and to a lesser extent at the sarcolemma. These results indicate that monoclonal antibody 2/34.4 recognizes the t-tubule Mg(2+)-ATPase and modulates its activity. This antibody should be useful as a probe on studies designed to study the physiological function of the enzyme.

Animals↗

Histochemical and ultrastructural properties of myoid cells in the thymus of the frog.

Histochemical and ultrastructural properties of myoid cells in the thymus of the frog were investigated and compared with properties of skeletal muscle fibres. The histochemical reactions of phospholipids, phosphorylase, succinic dehydrogenase and adenosine triphosphatase activities in myoid cells were characterized by considerable variability. Individual myoid cells apparently possess different enzyme activities which correspond to different stages of development, maturity and degeneration of these cells. The mature mononucleated myoid cells have similar enzymatic properties to the fast muscle fibres of the frog. This finding has been extended by ultrastructural observations. Features, typical of fast muscle fibres of the frog, e.g. the presence of the M-line, straight and narrow Z-line and well developed triads were found in the majority of mature myoid cells.

Adenosine Triphosphatases↗

Evidence for chromosome number reduction and chromosomal homosequentiality in the 24-chromosome Korean frog Rana dybowskii and related species.

The karyotype of the Korean frog Rana dybowskii with its pattern of C-band heterochromatin distribution was numerically analyzed. There are 2n = 24 chromosomes in the karyotype representing a reduction in number from the typical 2n = 26 chromosome karyotype of Rana. The karyotype shows other evidence of reorganization relative to 26-chromosome species. The chromosomes grade smoothly in size from largest to smallest without the two size classes that are characteristic for 26-chromosome species. In contrast to many 26-chromosome species, there are few centromeric C-bands but many interstitial ones. C-bands for each homologous chromosome pair are distinctive. A prominent secondary constriction is located on one of the smallest chromosomes, chromosome 11, in a position similar to that seen in most 26-chromosome species. The karyotype of R. dybowskii is compared to those of other species of Rana known to have 2n = 24 chromosomes; it is most similar to that of R. chensinensis, less so that of R. ornativentris and less still to that of R. arvalis in terms of the positions of centromeres and secondary constrictions. C-bands as well as secondary constrictions in the karyotypes of these frogs show evidence of chromosomal homosequentiality. The process and possible consequences of chromosome number reduction from an ancestral 26-chromosome karyotype is also evident in the karyotypes of these closely allied palearctic frogs. Pericentric inversions followed by fusion of two small elements apparently produced a new chromosome, chromosome 6, occurring originally among northeast Asian populations.

Animals↗

Distribution of NCAM-180 and polysialic acid in the developing tectum mesencephali of the frog Discoglossus pictus and the salamander Pleurodeles waltl.

The 180 kDa component of the neural cell adhesion molecule (NCAM-180), total NCAM (NCAM-total) and the polysialic acid modification of NCAM (PSA) show similar temporal and spatial regulation in the developing tecta of Pleurodeles waltl (salamander) and Discoglossus pictus (frog). Whereas NCAM-total is found throughout the tectal tissue on neurons and glia, NCAM-180 is only found on non-proliferating neurons and in fiber layers. PSA is expressed by a subset of NCAM-180-positive cells. Western blots show that there is little polysialylated NCAM-140 in the developing amphibian tectum. Regions unstained for PSA and NCAM-180 correspond precisely to the growth zones of the tectum. NCAM-180 and PSA are not present in tecta of early larvae. Staining intensity is strongest at midlarval stages for both antigens. At metamorphosis, PSA is strongly downregulated, whereas NCAM-180 is downregulated in juvenile animals. Both antigens are still present in fiber layers of adult animals. In dissociated tissue culture of the frog tectum, NCAM-180 is not present on astrocytes, but on neuronal cells. Expression is enhanced at cell contact sites, suggesting that NCAM-180 is involved in cell contact stabilization. This study shows that general features of temporal and spatial regulation of NCAM isoforms and PSA are highly conserved in frog and salamander tecta, despite large differences in the rate of cell migration and the degree of lamination in these homologous brain regions.

Animals↗

Histology and lectin-binding patterns in the skin of the terrestrial horned frog Ceratophrys ornata.

The terrestrial horned frog, Ceratophrys ornata, lives on a wet substratum and absorbs water through the ventral epidermis; water is lost by evaporation from the dorsal skin. Thus, this species may be useful as a model for determining whether or not skin histology and lectin-binding patterns, indicative of glycoconjugates, are related to skin functions such as osmoregulation and water balance. With this in mind, a histological and lectin-histochemical study was carried out on dorsal and ventral skin of aquatic tadpoles and of a young terrestrial frog of C. ornata. Sections of skin were stained with various dyes to demonstrate general histological features and with two horseradish peroxidase (HRP)-conjugated lectins, Ulex europaeus agglutinin (UEA 1) and soybean agglutinin (SBA) which bind to specific terminal sugar residues of glycoconjugates, namely L-fucose and N-acetyl-D-galactosamine or D-galactose, respectively. In early stage tadpoles both lectin-binding patterns were similar in the bilaminar epidermis of dorsal and ventral skin (i.e., each lectin stained the apical cell layer). However, metamorphic changes resulted in a young frog with typical adult-type skin composed of a stratified squamous epidermis and three distinct types of glands containing glycoconjugates in their secretions. Strikingly different lectin-binding patterns were evident in the epidermis from dorsal and ventral regions of the body. The epidermis from the dorsal region was stained by both lectins; in contrast, that from the ventral region, although stained strongly by HRP-SBA, did not react with HRP-UEA 1, indicating that few, if any, fucose residues were present in the ventral epidermis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

The effects of gonadectomy and testosterone treatment on the Harderian gland of the green frog, Rana esculenta.

The effects of gonadectomy and testosterone treatment on the fine structure of the Harderian gland in male and female green frogs were investigated in different periods of the year. Gonadectomy, carried out when the glands are in the lowest secretory phase (September), causes degenerative changes consisting of a reduction of the rough endoplasmic reticulum, the appearance of autolysosomes, and an increase of nuclear heterochromatin. These effects can be prevented by testosterone treatment. No castration effects are found during the recovery (November) and enhancement (April-May) phases of secretory activity. The results suggest that the frog Harderian gland's sensitivity to testosterone changes during the annual cycle. The androgen dependence of the Harderian gland is correlated with the presence of androgen receptors in both male and female frogs.

Animals↗

Ultrastructural radioautographic analysis of neurogenesis in the hypothalamus of the adult frog, Rana temporaria, with special reference to physiological regeneration of the preoptic nucleus. II. Types of neuronal cells produced.

Light- and electron-microscopic radioautography was used to identify the newly formed neuronal cells in the hypothalamic preoptic area of the frog. Adult Rana temporaria that had been caught in May/June received repeated 3H-thymidine injections and were sacrificed 30 days later. Heavily labeled cells were found in 1-micron plastic coronal sections of the preoptic area and then analysed in electron-microscopic radioautographs of neighbouring thin sections. The cells were identified as newly generated by the presence of 3H-thymidine label over the nucleus. All frogs showed considerable numbers of new peptidergic neurosecretory cells, small conventional neurons, and glia in the preoptic area. Some new ependymally located cells contacting the cerebrospinal fluid displaying ultrastructural characteristics of monoaminergic cells were also revealed. We conclude that prominent ventricular neurogenesis normally exists in the intact adult frog hypothalamus. The birth of small hypothalamic neurons seems to represent a case of sustained growth leading to a net increase in neuron numbers without loss. Conversely, the birth of large peptidergic neurosecretory cells, in which the increased secretory activity often leads to natural death of some cells, is considered as a neuronal replacement phenomenon, referred to as physiological regeneration of the magnocellular preoptic nucleus. The possible significance of this phenomenon in adult Anamnia is discussed.

Animals↗

Immunoreactive Tamm-Horsfall protein in the kidney and skin of the frog Rana temporaria.

Tamm-Horsfall protein (THP) is the main protein in normal human urine, and is found in the thick limb of the Loop of Henle in human kidney, and in other mammalian species. The skin of the frog. Rana temporaria, has similar physiological properties to this mammalian kidney tissue. In the present study, an immunohistological method involving an antibody to human THP was used to investigate the distribution of this distinctive protein in frog kidney and skin, and to compare its distribution with that found in the kidney tubules of rat and rabbit. THP-positive material was detected in the distal renal tubules and nephric duct of frogs, and was also located in the superficial epidermis of skin. It is suggested that its presence in amphibian skin is consistent with the hypothesis that THP is an important component of tissues that absorb sodium and chloride ions, but remain impermeable to water.

Animals↗

MHC zygosity in the frog, Rana temporaria.

The major histocompatibility complex (MHC) zygosity of the field-collected frogs, Rana temporaria, was detected by progeny testing. Groups of sibling tadpoles were grafted with intrafamilial tail-tip allografts and the ratio of rapidly rejected allografts to slowly rejected ones was estimated. Twenty-five percent of parental frogs appeared to be MHC homozygotes. Thus, MHC homozygosity in natural frog populations seems to be considerably higher than in wild mouse populations.

Animals↗

Twin pulse facilitation in dependence on pulse duration and calcium concentration at motor nerve terminals of crayfish and frogs.

Phasic release from motor-nerve terminals of crayfish and frogs was elicited and recorded by means of a macro-patch-clamp electrode through which the terminal was depolarized in graded pulses. The tip of the electrode was perfused and the Ca concentration around the terminal, Cae, was controlled independent from that in the superfusion of the muscle, Cab. Release increased with pulse duration with a double-logarithmic slope of 5 to 9 in crayfish and frogs, which represents a form of "early facilitation" (Katz and Miledi 1968). In crayfish, this relation was shifted to longer pulse durations on lowering Cae, while in frogs, in addition, the saturation level of release was suppressed at low Cae. Responses to twin pulses with intervals of 7-10 ms showed facilitation, Fd. When pulse duration of the twin pulses was increased, starting from about 0.5 ms, Fd increased to a maximum, but declined for longer pulses which elicited release approaching the saturation range. On lowering Cae, the maximum of Fd, Fd, increased in amplitude and was shifted to larger pulse durations. Also reduction of Cab increased Fd. The effects of pulse duration and of Cae and Cab on Fd are predicted by the residual Ca theory of facilitation, if it is assumed that changes of Cae produce corresponding changes in Ca inflow during depolarization, and if the resting intracellular Ca concentration is influenced by the extracellular Ca concentration. The large values of early facilitation can not be explained by the residual Ca theory of facilitation and may indicate the action of another depolarization dependent factor which joins in the control of release.

Animals↗

Intracellular free Mg2+ concentration in skeletal muscle fibres of frog and crayfish.

The free intracellular Mg2+ concentration ([Mg2+]i) was investigated in frog sartorius and crayfish phasic and tonic skeletal muscle fibres, using a new Mg2(+)-sensitive microelectrode based on the ionophore ETH 5214 [Hu et al. (1989) Anal Chem 61:574-576]. In Ringer solution containing 0.5 mmol/l MgCl2, the mean [Mg2+]i of the frog muscle fibres was 1.3 mmol/l. In phasic crayfish muscle fibres, [Mg2+]i was about twice as high (mean 3.5 mmol/l) as in tonic fibres (mean 1.5 mmol/l), measured in van Harreveld solution containing 1.2 mmol/l MgCl2. Long-lasting (3-12 h) incubation of frog skeletal muscle fibres in Na(+)-free solution produced a reversible increase of [Mg2+]i by a factor of about 1.7. A tenfold rise of extracellular Mg2+ led to an increase in [Mg2+]i in the presence as well as in the absence of Na+. In these experiments, mean [Mg2+]i values of 3.2 mmol/l were never exceeded. Thus, [Mg2+]i remained at least 60 times lower than predicted from a passive distribution across the cell membrane. The results suggest the existence of a Na(+)-dependent and a Na(+)-independent Mg2+ extrusion mechanism, which is regulated by actual Mg2+ concentrations.

Animals↗

Regulation of resting ionic conductances in frog skeletal muscle.

The membrane electrical properties and resting ionic conductances of frog semitendinosus muscle fibres were studied in vitro at 25 degrees C with the two-micro-electrode cable technique, in the presence of an activator or inhibitor of protein kinase C (PKC) or in the presence of an activator of adenylate cyclase. The PKC activator, 4 beta-phorbol 12,13-dibutyrate (4 beta-PDB), reduced chloride conductance (GCl) at concentrations greater than 1 microM and did not affect potassium conductance (GK). At 150 microM, the maximum concentration of 4 beta-PDB tested, GCl was reduced by 42%. The "inactive" phorbol ester 4 alpha-phorbol 12,13-dibutyrate did not affect GCl or GK. The inhibitory effect of 4 beta-PDB on GCl was prevented by pretreatment of the muscle preparation with the PKC inhibitor staurosporine. The adenylate cyclase activator forskolin (1.5-8 microM) significantly increased the GK of the fibres, without affecting GCl. Thus, we conclude that frog skeletal muscle GCl, unlike rat muscle GCl, is relatively insensitive to activators of PKC. Moreover, in frog muscle, protein kinase A is a likely modulator of GK, but not GCl.

Adenylyl Cyclases↗

Inheritance and expression of a sex-linked enzyme in the frog, Rana clamitans.

The pattern of inheritance indicates that the gene for aconitase-1 is sex linked in the frog. Rana clamitans, and that the male is the heterogametic sex. Unlike mammals, both male and female frogs carry and express two alleles for this sex-linked gene. Therefore, the sex chromosomes in these frogs and probably others behave like an autosomal pair, with one homologue carrying a male-determining element.

Aconitate Hydratase↗

Immunocytochemical localization of tyrosine hydroxylase, dopamine-beta-hydroxylase and phenylethanolamine-N-methyltransferase in the adrenal glands of the frog and rat by a peroxidase-antiperoxidase method.

The subcellular localizations of tryrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT) in the adrenal glands of the frog and rat have been examined by a peroxidase-antiperoxidase (PAP) method. TH was localized in the ground substance of the adrenaline-containing cells and noradrenaline-containing cells, but not in the nucleus or in the mitochondria. TH was also located on the outside of the membrane of the chromaffin granules. DBH was observed only inside the granules. PNMT was found not only in the ground substance but also on the membrane of some adrenaline-containing granules. Cortical lipid cells of the frog adrenals did not show TH-, DBH-, and PNMT-reactions. The negative reactions to TH-, DBH-, and PNMT-antiserum exhibited by the summer cells of the frog adrenals prove that they belong to the cortical cells.

Adrenal Glands↗