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The cerebral giant cell (CGC) is known to play a crucial role in the regulation of feeding response in the pond snail, Lymnaea stagnalis. However, the mechanisms of signal transduction from the CGC to the following buccal motor neurons are not clear. In the present study, we examined whether cyclic AMP (cAMP)-dependent protein kinase (PKA) contributes to enhancement of a monosynaptic connection between the presynaptic CGC and the postsynaptic buccal motor neuron 1 (B1 cell). Injection of cAMP into the CGC or inhibition of phosphodiesterase by isobutylmethylxanthine in the CGC increased the amplitude of excitatory postsynaptic potential (EPSP) in the B1 cell, whereas no changes were detected in the electrical properties of the CGC. The synaptic enhancement in the B1 cell was completely blocked by inhibition of PKA in the CGC but did not require a de novo protein synthesis due to a PKA phosphorylation. The increase in the EPSP amplitude of B1 cell was associated with the increase in the amount of serotonin release from the CGC. These results hence provided the physiological evidence of the direct regulation of a synaptic enhancement by PKA in the CNS of L. stagnalis, indicating the completely different mechanism from that in the well-studied siphon- and gill-withdrawal reflex in Aplysia.
The distribution of serotonin-immunoreactive (5HT-IR) neurons was studied in the brain of a primitive bony fish, Polypterus senegalus. The diencephalon contained a prominent 5HT-IR cell group (G2) consisting mainly of liquor-contacting neurons in the hypothalamic periventricular wall including the paraventricular organ. Their ependymofugal processes arborized profusely and gave rise to dense networks of varicose fibers in the hypothalamus. Other groups of 5HT-IR cells were situated in the preoptic area including the periventricular preoptic nucleus (G1) and in the dorsal thalamus (G3). In the brain stem, 5HT-IR cells were found in the medial part, mainly in the superior raphe nucleus (G4). No 5HT-IR cells were demonstrated in other regions of the brain. The cell groups referred to as G1 and G4 in the present study seem to be new components comprising the serotonergic systems in the Polypterus brain.
We identified electrophysiologically cardioinhibitory neurons in the central nervous system of the isopod crustacean Ligia exotica. A pair of the cardioinhibitory neurons are located at the ventral side in the anterior region of the 1st thoracic ganglion. Intracellular injection of neurobiotin into the cardioinhibitory neurons revealed that the each neuron sends the peripheral axon to the heart via a contralateral nerve root of its own ganglion. In the central nervous system, each of the neurons has arborization in the 1st thoracic and subesophageal ganglia and sends the longitudinal processes through the ipsilateral connective toward the circumesophageal connective and down to the 7th thoracic ganglion.
The olfactory center (procerebral lobe; PC lobe) of the terrestrial slug, Limax marginatus, shows oscillatory behavior with a frequency of about 1 Hz and an activity wave that propagates from the apical to distal regions of the PC surface. To study the dynamic properties of this oscillatory network, serotonin, glutamate and acetylcholine were applied to the PC lobe. Serotonin and acetylcholine increased the frequency of PC oscillation and decreased the velocity of wave propagation. The effect of serotonin on the frequency was long-lasting and there was a delay before it caused a decrease in the wave propagation velocity. In contrast, the effect of acetylcholine on the frequency was phasic, and no delay was observed. Glutamate first decreased, then increased, the frequency. However, specific changes in the wave propagation velocity were not observed. From these experimental results, it is suggested that the oscillatory neural network of PC lobe has a potential to represent odor information as a series of spatially and temporally distributed ensembles of coherent firing neurons.
In Drosophila elegans, copulation duration is shorter in the brown morph (about 11 min) than in the black morph (25–35 min). In this study, it appeared that sperm transfer also occurred at an earlier stage of copulation in the brown morph than in the black morph and both sexes were responsible for the control of sperm transfer. In the HK (brown morph) strain, number of eggs produced per female decreased to some extent when copulation was interrupted 3 min after the start of copulation. In the OH (black morph) strain, the rate of sperm transfer was low when copulation was interrupted 3 or 5 min after the start of copulation. Thus, shortened (3–5 min) copulation was disadvantageous in this species. However, adaptive significance of prolonged (>10 min) copulation of the black morph was not apparent in the present experiments. Females of the HK strain recovered receptivity earlier than those of the OH strain irrespective of male-type in the previous mating: i.e., the former will mate more times in their lives than the latter. The adaptive significance of multiple mating was discussed.
C. elegans becomes habituated to repetitive mechanical stimuli. We compared the habituated states induced by three types of mechanical stimuli: touch on the head (head-touch), touch on the anterior body (body-touch), and mechanical tapping of the Petri dish, all of which evoke backward movement. The habituation patterns were similar, but differed in retention period and/or the rate of recovery. We found a hierarchy between the habituated states induced by the three types of mechanical stimuli in the decreasing order of head-touch, body-touch, and tap stimulus. Evidence is presented that the hierarchy is brought out by the magnitude of stimuli rather than by independent neural pathways.
Variegated patterns on the caudal fin are a common and popular trait in guppy strains commercially cultured in Singapore. Gene control of this highly variable mosaic pattern of black spots and patches of different shapes and sizes on a brightly colored tail fin was elucidated by reciprocal crosses between the Green Variegated (GV) strain and wild-type (WT) stock. F1 progenies were produced by single-pair crossing between GV and WT, while the F2 generation was obtained from full-sib mating between F1 males and F1 females. Data for the F1 and F2 generations were segregated according to phenotypes and sex, and tested by chi-square analyses. Inheritance of variegated tail patterns appears to be determined by a single locus on the X- and Y-chromosomes. Genotypes of males and females of the GV strain are proposed to be X VarYVar and XVarXVar, respectively. The allele for variegated tail patterning, Var, is dominant over that of the wild-type, Var, which does not exhibit these patterns. Recombination frequency between the Var locus and sex-determining region (SdR) in male guppies was estimated to be about 1.9% (map distance ≈1.9 map units). The segregation and mode of inheritance of the Var gene are illustrated by genetic models.
ABP-280 cross-links actin filaments and connects membrane glycoproteins and actin filaments in mammalian blood cells. We isolated cDNA clones for ABP-280 from hydra by screening a cDNA expression library with an antiserum against a cell membrane-enriched crude fraction. The amino acid sequences deduced from the cDNA clones showed significant similarity to human ABP-280, including intramolecular repetition of a unique sequence motif, suggesting similar tertiary structure and molecular activity. Monospecific antibodies were purified from the antiserum, by affinity to fusion proteins produced from the cDNA clones, and used for immunohistochemistry. Specific staining was detected at epithelial cell-cell boundaries in the hydra. The signals were restricted to the subapico-lateral regions corresponding to the locations of septate junctions. These results suggest that the identified molecule is a possible component of the septate junction, and presumably connects membrane cell adhesion molecules to actin filaments in the epithelial cells of hydra. This is the first report describing the subcellular distribution of ABP-280-related molecules in epithelial cells.
Blastomeres of the 16-cell stage embryos of the sea urchin, Hemicentrotus pulcherrimus, were separated by an elutriator. By differential display, several RNA species that are enriched in micromeres are detected and their cDNA was cloned. One of the cloned cDNA encodes mt 12S rRNA. cDNA for mt 16S rRNA was also cloned from the cDNA library of unfertilized eggs. Two mt rRNAs contain poly(A) tails in their 3′ ends. Both mt rRNAs distribute asymmetrically along a vegetal-animal axis of the 16-cell embryos and are enriched in micromeres, and this is also confirmed by whole mount in situ hybridization as well as electron microscopic in situ hybridization. As development proceeds, these mt rRNAs become more enriched in small micromeres. Results of electron microscopical in situ hybridization reveal both mt rRNAs localize extramitochondrially. Though at present we have no evidence on the role of the extramitochondrial mt rRNAs in sea urchin development, it is speculated considering roles of extramitochondrial mt 16S rRNA in Drosophila development that extramitochondrial mt rRNA may be implicated in development of sea urchin embryos.
Formation of embryonic axis is an essential step for animal development but its mechanism is not well understood. For axial determination in the fish embryos, participation of the yolk syncytial layer (YSL; a unique multinucleated structure formed in the yolk cell) has been shown. To investigate relationship between the YSL and axial specification, we examined whether or not expressions of the mesodermal marker genes no tail (Brachyury) or goosecoid are dependent on the YSL in explants isolated from the medaka embryos. The results of whole-mount in situ hybridization showed that these genes were expressed in the explants irrespective of the YSL, indicating that activation of these genes is a separate process from YSL-dependent axis formation.
Chaetognaths are bilateral animals totally symmetrical on both sides of their body. To elucidate the manner in which cell fates are established, single blastomeres of the two-cell stage embryos of the benthic arrow worm, Paraspadella gotoi, were injected with a fluorescent lineage-tracing dye. The distribution of labels was observed in the hatchlings by using a confocal laser scanning microscope. A total of four different labeling patterns was observed: 1) the dorsal epidermis, the right half of the ventral epidermis, and the right half of the dorsal longitudinal muscles (DR pattern); 2) the dorsal epidermis, the left half of the ventral epidermis, and the left half of the dorsal longitudinal muscles (DL pattern); 3) the right half of the ventral epidermis, the ventral longitudinal muscles, the right half of the dorsal body muscles, and the primordial germ cells (PGCs) (VR pattern); and 4) the left half of the ventral epidermis, the ventral longitudinal muscles, the left half of the dorsal body muscles, and the PGCs (VL pattern). Thus, one blastomere of the two-cell stage largely contributes the dorsal epidermis and the other contributes the ventral longitudinal muscles and the PGCs. Bilateral halves of the dorsal longitudinal muscles and the ventral epidermis were labeled as complementary pairs. These labeling patterns indicate that the first cleavage plane runs oblique to the bilateral and dorsoventral axes. In addition, the occurrence of complementary “DL and VR” and “DR and VL” labeling patterns indicates that the first cleavage plane bears one of two different angular relationships relative to the future body axes.
We isolated and characterized two distinct cDNAs for mammalian gonadotropin-releasing hormone (mGnRH) and chicken GnRH-II (cGnRH-II) precursors from the Japanese eel by rapid amplification of cDNA ends. Each GnRH precursors were composed of a signal peptide, a GnRH decapeptide, a processing site and a GnRH-associated peptide. Northern blot and reverse transcription-polymerase chain reaction analysis revealed that the mGnRH precursor gene is expressed in all tissues tested including the brain, pituitary, eye, olfactory epithelium, ovary, testis, liver, kidney, spleen, heart, gill, intestine, pancreas, muscle, skin, fin and peripheral blood leukocyte. In contrast, the cGnRH-II precursor gene expression was detected only in the brain, pituitary, olfactory epithelium, ovary and testis. These findings suggest unknown physiological function(s) for mGnRH besides the well-documented role in the pituitary gonadotropin synthesis and release. The eel mGnRH and cGnRH-II precursors have high amino acid homologies with seabream GnRH (sbGnRH) precursors of the Perciforms and cGnRH-II precursors of other teleosts, respectively. Phylogenetic analysis showed the existence of three distinct evolutionary arms of GnRHs; multiple GnRH forms (mGnRH, guinea pig GnRH, chicken GnRH-I, sbGnRH and catfish GnRH (cfGnRH)) on the first, cGnRH-II on the second, and salmon GnRH (sGnRH) on the third arm. This analysis suggests that mGnRH progenitor has undergone sequence divergence to give rise to sbGnRH and cfGnRH, whereas sGnRH represents a separate evolutionary line.
Angiotensin II at pharmacological doses induces drinking in various vertebrate species. However, its role in physiological regulation of drinking remains to be determined. Eel angiotensin II was infused slowly into freshwater eels to examine whether sustained, physiological increases in its plasma level, as observed after transfer to seawater, stimulate drinking. Hourly infusion was initiated with 0.9% NaCl, followed by increasing concentrations of angiotensin II (0.1–10 pmol/kg/min), and ended with only saline again for 2 hr.
Angiotensin II infusion at a rate of 0.1 pmol/kg/min significantly induced drinking; neither plasma angiotensin II concentration nor plasma atrial natriuretic peptide (ANP), Na concentrations or arterial pressure were significantly altered. The dipsogenic effect of angiotensin II was dose-dependent, and enhanced drinking disappeared quickly after infusate was switched back to saline. Blood pressure and plasma ANP concentration increased dose-dependently at more than 1 pmol/kg/min, and both parameters declined to their initial levels after return to normal saline infusion. By contrast, plasma Na concentration was decreased by more than 1 pmol/kg/min of angiotensin II.
These results show that drinking is induced by angiotensin II within physiological range in water-replete freshwater eels. At higher doses, enhanced drinking occurs despite hypertension, hyponatremia and increased plasma ANP, all of which are inhibitory to elicitation of drinking. Thus, it is likely that angiotensin II could be a physiological stimulus for drinking in the eel.
To know whether or not molluscs are capable of recognizing tissue alloantigens, dorsal skin-allografts were exchanged between adult terrestrial slug, Incilaria fruhstorferi. We succeeded for the first time in orthotopic transplantation of allografts and observed chronic rejection of allografts. During the first two weeks after transplantation (WAT), in all grafts, both foreign (allo-) and self (auto-), many macrophages infiltrated from the host toward the grafts. This phenomenon is seemed to heal wounds. In the case of autografts, many macrophages observed in the grafted site until 8 weeks, whereas at 4 WAT, grafted tissues such as muscle fibers and mucous cells begun to regenerate slowly and the regeneration of these cells had been over at 20 WAT. However, in the case of allografts, regenerative phenomena were not observed, rather than muscle fibers had been actively attacked by macrophages. Numerous macrophages which phagocytosed cell debris were observed in host connective tissues during this experiment. These observations strongly suggest that an allorecognition system is present in molluscs, and in the case of terrestrial slugs dorsal skin transplantation is a useful assay system for analyses of immunological incompatibility.
Metallic ion-mediated interactions between chromosomal proteins and DNA during mammalian spermatogenesis are poorly understood. In search of specific proteins related to this process, we fractionated soluble proteins from rat testis using Ca2 and DNA in low ionic strength solution. A testis specific 16 kDa protein that sedimented in the presence of both Ca2 and DNA was identified as PERF 15 (Swiss-Prot ID: TLBP_RAT) based on amino acid sequence and immunochemical analysis. PERF 15 is the major germ cell protein of the rat perforatorium, but its role during spermatogenesis is unknown. Indirect immunofluorescence showed that PERF 15 was present in the nucleus from spermatocyte to middle spermatid stage. In late-pachytene spermatocytes, PERF 15 colocalized with DNA. In addition, PERF 15 contained phosphotyrosine like other fatty acid binding proteins with high similarity. These results suggest that PERF 15 specifically associates with germ cell chromatin and that its function may be regulated by Ca2 and tyrosine phosphorylation.
Silver sea bream (Sparus sarba) were adapted to a hyposmotic environment of 6‰ for 5 or 21 day periods. Hyposmotic adaptation did not significantly alter serum Na, Cl– or muscle moisture content. After 5 days in 6‰, chloride cell (CC) apical and fractional areas increased with no alteration in CC numbers. Elevated CC apical and fractional area was coupled with an increase in CC numbers after 21 days in 6‰. In fish adapted to 6‰, an increase was found in the area of CC cytoplasm occupied by mitochondria. Branchial Na-K-ATPase decreased after 21 days in 6‰, resulting in an uncoupled “typical” correlative relationship between this enzyme and CC numbers. Kidney Na-K-ATPase activity elevated after 5 days in 6‰ but was not significantly elevated after 21 days. In gill and kidney tissue, alterations were found in the activity of key metabolic enzymes after 5 days acclimation to 6‰, with few differences occurring after 21 days. Serum cortisol levels were unaltered by low salinity acclimation suggesting that an increase in the number of CCs found in 6‰-adapted S. sarba did not occur as a result of hypercortisolemia. Based on the present evidence, it seems possible that alterations in the form and function of the branchial epithelium may play an important role in the ability of S. sarba to acclimate to low salinity conditions, with renal assistance occurring during short term exposure. The response of S. sarba to low salinity adaptation appears to differ from the generally accepted teleostean model of response and offers insight into the as yet undefined hyperosmoregulatory strategies of estuarine marine migrant fish.
We found many isoforms of membrane-bound guanylyl cyclase (membrane GC) in the gonads of several echinoid species including Hemicentrotus pulcherrimus (six isoforms), Glyptocidaris crenularis (four isoforms), Clypeaster japonicus (three isoforms), Diadema setosum (three isoforms), and Brissus agassizii (three isoforms). In addition to these membrane GC isoforms, several putative soluble GC isoforms were also found, one in H. pulcherrimus, two in G. crenularis, and three in D. setosum. Other echinoderm species also possess many GC isoforms in their gonads: the starfish, Asterina pectinifera, has five membrane GC isoforms; the brittle star, Ophioplocus japonicus, possesses six membrane and two putative soluble GC isoforms; and the sea cucumber, Stichopus japonicus, contains five membrane and two putative soluble GC isoforms. We also obtained the full-length cDNA sequence of some of these isoforms, one for sperm-activating peptide (SAP) receptor-type GC, and the other for a homolog of the vertebrate natriuretic peptide receptors/membrane GCs, and analyzed their phylogenetic relationship among various invertebrate and vertebrate GC isoforms.
The family Cidaridae of the order Cidaroida from Japanese waters includes four common echinoid species belonging to four different genera: Stereocidaris japonica, Eucidaris metularia, Prionocidaris baculosa, and Phyllacanthus dubius. Phylogenetic relationship among the four species were investigated by allozyme analysis. From the allozyme variation in 18 genetic loci, Nei's genetic distances between species were calculated. The genetic distances were higher than those observed between confamilial genera in many other echinoids, but comparable to those between different families. The result suggests that the four cidarids diverged in earlier time from one another and generally have older evolutionary origin than members of families of the orders Echinoida and Diadematoida. A molecular phylogenetic tree for the four cidarids indicated the following: (1) S. japonica and Pr. baculosa are the most closely related to each other and diverged later. (2) E. metularia is more closely related to the cluster of S. japonica and Pr. baculosa than Ph. dubius. (4) Ph. dubius is the most distant among four species and diverged first. These allozyme results are discussed through the comparison with other non-molecular evidence.
Phylogenetic relationships among 12 species of the genus Draco were inferred from 779 base pairs of mitochondrial 12S and 16S rRNA genes and allozymes for 20 presumptive loci. Results indicated the presence of at least four distinct lineages within the genus. The first lineage consists of D. volans and D. cornutus, whereas the second only of D. lineatus, which exhibits a great genetic divergence between two subspecies. The third is monotypic with D. dussumieri, the only species distributed in southern India. The fourth included all the remaining species. The third and fourth lineages are supposed to exclusively share a common ancestor. It is likely that the common ancestor of whole Draco originally diverged into three groups, the ancestors of the first, second, and third and fourth lineages, by vicariance. In the fourth lineage, D. blanfordii, D. haematopogon, D. melanopogon, D. obscurus and D. taeniopterus are likely to be exclusively close to each other. The resultant phylogenetic tree contradicts the dichotomous relationships previously hypothesized on the basis of morphological characters.
It is almost impossible to discriminate each region's black Echinometra based on appearance, such as the color of spines, milled ring, and skin around the peristome. However, it is possible to divide them into several groups based on such characteristics as the spicules of the gonads and tubefeet, the porepairs, the shape of the sperm, and the pedicellaria. The percentage of spicules of the gonads and tubefeet depends on the region the individuals inhabit. The five pore-pair percentage of Guamanian individuals is very low, and this is significant compared to the individuals of the other regions. The sperm morphology of Guamanian and Hawaiian individuals is significantly more compact than that of Mauritian, Indonesian, Okinawan, and Boninian individuals, while the difference between the Guamanian and Hawaiian individuals is also significant. The tridentate pedicellaria of the Hawaiian individuals are longer than those of the other regions' individuals, and the difference is significant. The length differences of the ophiocephalous pedicellaria and trifiliate pedicellaria are significant depending on the individuals of the region, and it is possible to discriminate individuals of each region into several groups. Guamanian and Hawaiian individuals are separated into different groups based on all these characteristics, and it is also possible to discriminate these two groups from the other regions' individuals by several characteristics. Therefore, black Echinometra individuals living in the Indo-West Pacific are divided into three groups, i.e., Mauritian, Indonesian, Okinawan, Boninian individuals; Guamanian individals; and Hawaiian individuals. The number of pore-pairs and morphology of the sperm in the Okinawan Echinometra species complex reflect species level differences, and it is suggested that cross-fertilizations between black Echinometra from Okinawa and Guam as well as Okinawa and Hawaii were not successful. This indicates that the three groupings based on these characteristics reflect a species level difference. In other words, the black Echinometra inhabiting the Indo-West Pacific are a species complex, or a cryptic species composed of at least three species.
The endostyle is a special organ in the pharynx of urochordates, cephalochordates and cyclostomes. During evolution of the primitive chordates, the endostyle was organized in their common ancestor(s) with a shift to internal feeding for extracting suspended food from the water. In addition, the endostyle has an iodine-concentrating activity and is therefore thought to be functionally homologous to the vertebrate thyroid gland. Human TITF1 and mouse TITF1 are members of the Nkx-2.1/TTF-1 gene subfamily, which encode an NK-2 type homeodomain transcription factor. The genes are expressed in the thyroid gland and are essential for thyroid-specific structural gene expression. In the present study, we isolated cDNA clones for ascidian homologs of TITF1 from Halocynthia roretzi and Ciona intestinalis, and examined whether the genes are expressed in the ascidian endostyle. Results clearly indicated that both the H. roretzi homolog Hrtitf1 and the C. intestinalis homolog Cititf1 are expressed specifically in the endostyle. The present finding therefore provide molecular evidence for the functional relationship between the ascidian endostyle and vertebrate thyroid gland. However, the genes are expressed in the supporting element regions but not in the putative iodine-concentrating regions of the endostyle.