Analyses of mRNA Expression Levels of Pituitary Hormones, Their Hypothalamic Regulating Factors, and Receptors Involved in Metamorphosis with Special Reference to the Summer and Winter Seasons.

Bullfrog (Rana catesbeiana) larvae inhabiting the main island of Japan overwinter as preclimax animals, whereas the larvae that reached climax in summer complete metamorphosis. We analyzed the mRNA expression levels of the adenohypophyseal hormones, hypothalamic hormones, and their receptors that are involved in controlling metamorphosis in tadpoles at various developmental stages available in summer and winter in order to understand the hormonal mechanism regulating metamorphosis progression. Corticotropin-releasing factor (CRF) and thyrotropin ß-subunit (TSHß) mRNA expression was enhanced as they reached the climax stage in metamorphosing summer tadpoles, although type 2 CRF receptor (CRFR2) mRNA levels demonstrated a tendency of elevation, indicating the activation of the hypothalamo-hypophyseal axis for stimulating the release of thyroid hormone in summer. Arginine vasotocin (AVT) mRNA levels were elevated as metamorphosis progressed, but mRNA expression levels were not synchronized with those of proopiomelanocortin (POMC) and V1b-type AVT receptor (V1bR). The elevation of mRNA levels of prolactin (PRL) 1A and type 3 thyrotropin-releasing hormone receptor (TRHR3), but not of thyrotropin-releasing hormone (TRH) precursor mRNA levels, was noted in climactic tadpoles, indicating that PRL mRNA levels are not simply dependent on the expression levels of TRH precursor mRNA. In the preclimactic larvae captured in winter, which are in metamorphic stasis, mRNA levels of pituitary hormones, hypothalamic factors, and their receptors remained low or at levels similar to those of the larvae captured in summer. These results indicate the relationship between the mRNA expression of metamorphosis-related factors and the seasonal progression/stasis of metamorphosis.

Molecular Cloning, Expression Analyses, and Physiological Roles of Cathelicidins in the Bursa of Fabricius of the Japanese Quail, Coturnix japonica.

Antimicrobial peptides (AMPs) act directly on pathogens and maintain the anti-inflammatory effects and activation of immunocompetent cells. Therefore, the activation of the immune system in poultry via the elevation of endogenous AMPs has been attempted. In this study, we focused on the host defense mechanisms in the bursa of Fabricius (BF) of Japanese quail, cloned the cDNA of cathelicidin (CATH)-1 to -3, and analyzed their expression sites. In situ hybridization experiments revealed the mRNA expression of the CATHs in the interfollicular epithelium surrounding the lumen of the quail BF, which suggests that each CATH may exert its antimicrobial action directly in the BF. The intravenous injection of bacterial lipoteichoic acid and lipopolysaccharide endotoxins into the quail promoted the mRNA expression of CATH-1 and CATH-3 in the BF. The addition of CATH-1 or CATH-2 at the time of the antigen injection into mice resulted in antiserum with high antibody titers. Ad libitum administration of butyrate, a short-chain fatty acid, in the drinking water induced an increase in CATH-2 mRNA expression in the BF under certain conditions. These results may improve the defense mechanisms of quail by stimulating CATH expression in the BF through their diet.

Hormonal and pheromonal studies on amphibians with special reference to metamorphosis and reproductive behavior.

In this article, we review studies which have been conducted to investigate the hormonal influence on metamorphosis in bullfrog (Rana catesbeiana) and Japanese toad (Bufo japonicus) larvae, in addition to studies conducted on the hormonal and pheromonal control of reproductive behavior in red-bellied newts (Cynops pyrrhogaster). Metamorphosis was studied with an emphasis on the roles of prolactin (PRL) and thyrotropin (TSH). The release of PRL was shown to be regulated by thyrotropin-releasing hormone (TRH) and that of TSH was evidenced to be regulated by corticotropin-releasing factor. The significance of the fact that the neuropeptide that controls the secretion of TSH is different from those encountered in mammals is discussed in consideration of the observation that the release of TRH, which stimulates the release of PRL, is enhanced when the animals are subjected to a cold temperature. Findings that were made by using melanin-rich cells of Bufo embryos and larvae, such as the determination of the origin of the adenohypophyseal primordium, identification of the pancreatic chitinase, and involvement of the rostral preoptic recess organ as the hypothalamic inhibitory center of α-melanocyte-stimulating hormone (α-MSH) secretion, are mentioned in this article. In addition, the involvement of hormones in eliciting courtship behavior in male red-bellied newts and the discovery of the peptide sex pheromones and hormonal control of their secretion are also discussed in the present article.

Cell proliferation and neurogenesis in the adult telencephalon of the newt Cynops pyrrhogaster.

Urodele amphibians have the ability to regenerate several organs, including the brain. For this reason, the research on neurogenesis in these species after ablation of some parts of the brain has markedly progressed. However, detailed information on the characteristics and fate of proliferated cells as well as the function of newly generated neurons under normal conditions is still limited. In this study, we focused on investigating the proliferative and neurogenic zones as well as the fate of proliferated cells in the adult brain of the Japanese red-bellied newt to clarify the significance of neurogenesis in adulthood. We found that the proximal region of the lateral ventricles in the telencephalon and the preoptic area in the diencephalon were the main sites for continuous cell proliferation in the adult brain. Furthermore, we characterized proliferative cells and analyzed neurogenesis through a combination of 5-ethynyl-2'-deoxyuridine (EdU) labeling and immunohistochemistry using antibodies against the stem cell marker Sox2 and neuronal marker NeuN. Twenty-four hours after EdU injection, most of the EdU-positive cells were Sox2-immunopositive, whereas, EdU-positive signals and NeuN-immunoreactivities were not colocalized. Two months after EdU injection, the colocalization ratio of EdU-positive signals with Sox2-immunoreactivities decreased to approximately 10%, whereas the ratio of colocalization of EdU-positive signals with NeuN-immunoreactivities increased to approximately 60%. Furthermore, a portion of the EdU-incorporated cells developed into γ-aminobutyric acid-producing cells, which are assumed to function as interneurons. On the basis of these results, the significance of newly generated neurons was discussed with special reference to their reproductive behavior.

Involvement of glucose in freeze tolerance in the Japanese tree frog Hyla japonica.

Glycerol and aquaporin 9 (aquaglyceroporin) are known to be involved in freeze tolerance in the Japanese tree frog Hyla japonica. However, the regulatory mechanisms of freeze tolerance in this species have not been fully elucidated. In the present study, we focused on the inter- and intracellular dynamics of glucose to analyze the role of glucose and glucose-related proteins such as transporter and metabolic enzymes in freeze tolerance. Serum glucose concentrations were compared among the frogs that were nonhibernating, hibernating, and thawed after freezing at -4°C for 6 hr. Serum concentrations of glucose in thawed frogs were significantly higher than those in hibernating and nonhibernating, active frogs. Periodic acid-Schiff staining showed that the accumulation of glycogen in the hepatocytes increased before hibernation and decreased after freezing and thawing. Quantitative RT-PCR analysis using the liver showed that, compared with active frogs, the type 2 glucose transporter gene (glut2) was upregulated in frozen frogs, the liver glycogen phosphorylase gene (pygl) was upregulated in frozen or thawed frogs, and the type 2 glycogen synthase gene (gys2) was upregulated in hibernating frogs. Immunohistochemistry of liver sections showed that, compared with nonhibernating frogs, Glut2 proteins were clearly increased most likely on the plasma membrane of hepatocytes in hibernating frogs and further increased by freezing, then decreased after thawing. These results suggest the possibility that glucose acts as a cryoprotectant in H. japonica.

Dual Roles of Extracellular Histone H3 in Host Defense: Its Differential Regions Responsible for Antimicrobial and Cytotoxic Properties and Their Modes of Action.

Extracellular histones play a dual role-antimicrobial and cytotoxic-in host defense. In this study, we evaluated the antimicrobial and cytotoxic activities of histone H3 and identified the responsible molecular regions for these properties. Broth microdilution assays indicated that histone H3 exhibits growth inhibitory activity against not only Gram-negative and -positive bacteria but also fungi. Observations under scanning electron microscopy (SEM) revealed that histone H3 induced morphological abnormalities on the cell surface of a wide range of reference pathogens. MTT assays and SEM observations indicated that histone H3 has strong cytotoxic and cell lytic effects on mammalian normal, immortal, and tumor cell lines. Assays using synthetic peptides corresponding to fragments 1-34 (H3DP1), 35-68 (H3DP2), 69-102 (H3DP3), and 103-135 (H3DP4) of histone H3 molecule demonstrated that its antimicrobial activity and cytotoxicity are elicited by the H3DP2 and H3DP3 protein regions, respectively. Enzyme-linked endotoxin binding assays indicated that histones H3 and H3DP1, H3DP2, and H3DP4, but not H3DP3, exhibited high affinities toward lipopolysaccharide and lipoteichoic acid. Our findings are expected to contribute to the development of new histone H3-based peptide antibiotics that are not cytotoxic.

Delayed Postnatal Growth and Anterior Pituitary Development in Growth-Retarded (grt) Female Mice.

Growth-retarded (grt) mice display primary congenital hypothyroidism due to the hyporesponsiveness of their thyroid glands to thyroid-stimulating hormone (TSH). We examined somatic growth, anterior pituitary development, and hormonal profiles in female grt mice and normal ones. Although growth in grt females was suppressed 2 weeks after birth, the measured growth parameters and organ weights gradually increased and finally reached close to the normal levels. Grt mice exhibited delayed eye and vaginal openings and remained in a state of persistent diestrus thereafter, plasma estrogen levels being lower than those in normal mice. Grt mice that received normal-donor thyroids showed accelerated growth and their body weights increased up to the sham-normal levels, indicating the importance of early thyroid hormone supplementation. In the anterior pituitary, there were fewer growth hormone (GH) and prolactin (PRL) cells in grt mice than in normal mice as examined at 12 weeks after birth, but the numbers of these cells did not differ from those in normal mice after 24 weeks. Grt mice had more TSH cells than normal mice until 48 weeks. Plasma GH levels in grt mice were lower than those in normal mice at 2 weeks, but did not differ substantially after 5 weeks. Compared with normal mice, grt mice had significantly lower plasma PRL and thyroxine levels, but notably higher TSH levels until 48 weeks. These findings indicate that thyroid hormone deficiency in grt mice causes delayed development and growth, and inappropriate development of GH, PRL and TSH cells, followed by the abnormal secretion of hormones by these pituitary cells.

Development of the hypothalamo-hypophyseal system in amphibians with special reference to metamorphosis.

In this review article, topics of the embryonic origin of the adenohypophysis and hypothalamus and the development of the hypothalamo-hypophyseal system for the completion of metamorphosis in amphibians are included. The primordium of the adenohypophysis as well as the primordium of the hypothalamus in amphibians is of neural origin as shown in other vertebrates, and both are closely associated with each other at the earliest stage of development. Metamorphosis progresses via the interaction of thyroid hormone and adrenal corticosteroids, of which secretion is enhanced by thyrotropin and corticotropin, respectively. However, unlike in mammals, the hypothalamic releasing factor for thyrotropin is not thyrotropin-releasing hormone (TRH), but corticotropin-releasing factor (CRF) and the major releasing factor for corticotropin is arginine vasotocin (AVT). Prolactin, the release of which is profoundly enhanced by TRH at the metamorphic climax, is another pituitary hormone involved in metamorphosis. Prolactin has a dual role: modulation of the metamorphic speed and the development of organs for adult life. The secretory activities of the pituitary cells containing the three above-mentioned pituitary hormones are elevated toward the metamorphic climax in parallel with the activities of the CRF, AVT, and TRH neurons.

Antimicrobial Property and Mode of Action of the Skin Peptides of the Sado Wrinkled Frog, Glandirana susurra, against Animal and Plant Pathogens.

The Sado wrinkled frog Glandirana susurra has recently been classified as a new frog species endemic to Sado Island, Japan. In this study, we cloned 12 cDNAs encoding the biosynthetic precursors for brevinin-2SSa-2SSd, esculentin-2SSa, ranatuerin-2SSa, brevinin-1SSa-1SSd, granuliberin-SSa, and bradykinin-SSa from the skin of G. susurra. Among these antimicrobial peptides, we focused on brevinin-2SSb, ranatuerin-2SSa, and granuliberin-SSa, using their synthetic replicates to examine their activities against different reference strains of pathogenic microorganisms that infect animals and plants. In broth microdilution assays, brevinin-2SSb displayed antimicrobial activities against animal pathogens Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, and Candida albicans and plant pathogens Xanthomonas oryzae pv. oryzae, Clavibacter michiganensis subsp. michiganensis, and Pyricularia oryzae. Ranatuerin-2SSa and granuliberin-SSa were active against C. albicans and C. michiganensis subsp. michiganensis, and granuliberin-SSa also was active against the other plant pathogenic microbes. Scanning electron microscopic observations demonstrated that brevinin-2SSb, ranatuerin-2SSa, and granuliberin-SSa induced morphological abnormalities on the cell surface in a wide range of the reference pathogens. To assess the bacterial-endotoxin-binding ability of the peptides, we developed an enzyme-linked endotoxin-binding assay system and demonstrated that brevinin-2SSb and ranatuerin-2SSa both exhibited high affinity to lipopolysaccharide and moderate affinity to lipoteichoic acid.

Some aspects of the hypothalamic and pituitary development, metamorphosis, and reproductive behavior as studied in amphibians.

In this review article, information about the development of the hypothalamo-hypophyseal axis, endocrine control of metamorphosis, and hormonal and pheromonal involvements in reproductive behavior in some amphibian species is assembled from the works conducted mainly by our research group. The hypothalamic and pituitary development was studied using Bufo embryos and larvae. The primordium of the epithelial hypophysis originates at the anterior neural ridge and migrates underneath the brain to form a Rathke's pouch-like structure. The hypothalamo-hypophyseal axis develops under the influence of thyroid hormone (TH). For the morphological and functional development of the median eminence, which is a key structure in the transport of regulatory hormones to the pituitary, contact of the adenohypophysis with the undeveloped median eminence is necessary. For the development of proopiomelanocortin-producing cells, contact of the pituitary primordium with the infundibulum is required. The significance of avascularization in terms of the function of the intermediate lobe of the pituitary was evidenced with transgenic Xenopus frogs expressing a vascular endothelial growth factor in melanotropes. Metamorphosis progresses via the interaction of TH, adrenal corticosteroids, and prolactin (PRL). We emphasize that PRL has a dual role: modulation of the speed of metamorphic changes and functional development of organs for adult life. A brief description about a novel type of PRL (1B) that was detected was made. A possible reason why the main hypothalamic factor that stimulates the release of thyrotropin is not thyrotropin-releasing hormone, but corticotropin-releasing factor is considered in light of the fact that amphibians are poikilotherms. As regards the reproductive behavior in amphibians, studies were focused on the courtship behavior of the newt, Cynops pyrrhogaster. Male newts exhibit a unique courtship behavior toward sexually developed conspecific females. Hormonal interactions eliciting this behavior and hormonal control of the courtship pheromone secretion are discussed on the basis of our experimental results.

A novel type of prolactin expressed in the bullfrog pituitary specifically during the larval period.

Prolactin (PRL) is one of the major hormones that control amphibian metamorphosis. Recently, a PRL (PRL1B) gene that is different from the known PRL (PRL1A) gene has been found in the genomes of several amphibian species. In order to ascertain whether the PRL1B gene is expressed in the bullfrog (Rana catesbeiana) pituitary, cloning of cDNA encoding PRL1B in the pituitary of the premetamorphic bullfrog tadpole was attempted. The bullfrog PRL1B amino acid sequence predicted from the obtained cDNA showed 62% identity with those of Xenopus PRL1Bs that have been presumed from the genome sequences, whereas the sequence identity between bullfrog PRL1A and PRL1B was 48%. A molecular phylogenetic tree showed that bullfrog PRL1B is most appropriately grouped with amphibian PRL1Bs. Quantitative PCR analysis revealed that the mRNA expression levels of bullfrog PRL1B in the pituitary were high during pre- and prometamorphosis, sharply declined at metamorphic climax and became undetectable after metamorphosis. In contrast, PRL1A mRNA levels were relatively low during pre- and prometamorphosis, rose at climax and remained high after metamorphosis. Immunohistochemical study using antibodies against partial peptides of PRL1A and PRL1B revealed that most of the PRL1A- and PRL1B-immunoreactive cells in the larval pituitary were distributed separately, but that some of the cells immunoreactive with both antibodies were also present. Western blot analysis with the larval pituitary extract indicated that PRL1B-immunoreactive band appeared at the position of molecular weight ca. 22.1 kDa and PRL1A-immunoreactive band at the position of ca. 22.8 kDa. The results obtained in this experiment suggest the possibility that PRL1B plays as-yet-unknown role(s) during the pre-climactic period of metamorphosis. This is the first report on the existence of PRL1B as a protein in the amphibian larval pituitary.

Possible involvement of thyrotropin-releasing hormone receptor 3 in the release of prolactin in the metamorphosing bullfrog larvae.

In amphibians, thyrotropin (TSH), corticotropin (ACTH) and prolactin (PRL) are regarded as the major pituitary hormones involved in metamorphosis, their releasing factors being corticotropin-releasing factor (CRF), arginine vasotocin (AVT), and thyrotropin-releasing hormone (TRH), respectively. It is also known that thyrotropes and corticotropes are equipped with CRF type-2 receptor and AVT V1b receptor, respectively. As for PRL cells, information about the type of receptor for TRH (TRHR) through which the action of TRH is mediated to induce the release of PRL is lacking. In order to fill this gap, an attempt was made to characterize the TRHR subtype existing in the PRL cells of the anterior pituitary gland of the bullfrog, Rana catesbeiana. We cloned cDNAs for three types of bullfrog TRHRs, namely TRHR1, TRHR2 and TRHR3, and confirmed that all of them are functional receptors for TRH by means of reporter gene assay. Analyses with semi-quantitative reverse transcription-PCR and in situ hybridization revealed that TRHR3 mRNA is expressed in the anterior lobe and that the signals reside mostly in the PRL cells. It was also noted that the expression levels of TRHR3 mRNA in the anterior pituitary as well as in the PRL cells of metamorphosing tadpoles elevate as metamorphosis progresses. Since the pattern of changes in TRHR3 mRNA levels in the larval pituitary is almost similar to that previously observed in the pituitary PRL mRNA and plasma PRL levels, we provide a view that TRHR3 mediates the action of TRH on the PRL cells to induce the release of PRL that is prerequisite for growth and metamorphosis in amphibians.

Molecular Cloning and Functional Characterization of Antimicrobial Peptides Brevinin-1ULf and Ulmin-1ULa in the Skin of the Newly Classified Ryukyu Brown Frog Rana ulma.

Antimicrobial peptides (AMPs) were previously isolated from the skin of the Ryukyu brown frog Rana okinavana. However, this species has recently been reclassified as two species, i.e., Rana kobai and Rana ulma. As a result, it was determined that AMPs isolated from R. okinavana were in fact products of R. kobai, but not of R. ulma. In the present study, we collected skin samples from the species R. ulma and cloned twelve cDNAs encoding AMP precursors for the acyclic brevinin-1ULa--1ULf, the temporin-ULa-ULc, ranatuerin-2ULa, japonicin-1ULa, and a novel peptide using reverse-transcription polymerase chain reaction techniques. The deduced amino acid sequence of the novel peptide had a high similarity to those of Rana chensinensis chensinin-1CEa--1CEc, which were cloned by Zhao et al. ( 2011 ), but had a low similarity with R. chensinensis chensinin-1, which was cloned by Shang et al. ( 2009 ). To avoid confusion with these two different chensinin-1 families, we termed our peptide ulmin-1. Among these peptides, we focused on two peptides, brevinin-1ULf and ulmin-1ULa, and examined the antimicrobial and cytotoxic activity of their synthetic replicates. In broth microdilution assays, growth inhibitory activities against Staphylococcus aureus, Bacillus cereus, and Candida albicans were detected for brevinin-1ULf but not for ulmin-1ULa, whereas scanning electron microscopic observations revealed that both peptides induce morphological abnormalities in these microbes. In addition, binding activity of ulmin-1ULa to the bacterial cell wall component lipoteichoic acid was higher than that of brevinin-1ULf. In contrast, hemolytic and cytotoxic activities of brevinin-1ULf were stronger than those of ulmin-1ULa.

Imorin: a sexual attractiveness pheromone in female red-bellied newts (Cynops pyrrhogaster).

The male red-bellied newt (Cynops pyrrhogaster) approaches the female's cloaca prior to performing any courtship behaviour, as if he is using some released substance to gauge whether she is sexually receptive. Therefore, we investigated whether such a female sexual attractiveness pheromone exists. We found that a tripeptide with amino acid sequence Ala-Glu-Phe is secreted by the ciliary cells in the epithelium of the proximal portion of the oviduct of sexually developed newts and confirmed that this is the major active substance in water in which sexually developed female newts have been kept. This substance only attracted sexually developed male newts and acted by stimulating the vomeronasal epithelial cells. This is the first female sexual attractiveness peptide pheromone to be identified in a vertebrate.

Arginine vasotocin is the major adrenocorticotropic hormone-releasing factor in the bullfrog Rana catesbeiana.

In a previous study, we showed that corticotropin-releasing factor (CRF) is the major thyroid-stimulating hormone (TSH)-releasing factor in the bullfrog (Rana catesbeiana) hypothalamus. Our findings prompted us to ascertain whether CRF or arginine vasotocin (AVT), a known adrenocorticotropic hormone (ACTH) secretagogue in several vertebrates, is the main stimulator of the release of ACTH from the bullfrog pituitary. Both the frog CRF and AVT stimulated the release of immunoassayable ACTH from dispersed anterior pituitary cells in vitro in a concentration-dependent manner. AVT, however, exhibited far more potent ACTH-releasing activity than CRF. Although CRF by itself weakly stimulated ACTH release, it acted synergistically with AVT to enhance the release of ACTH markedly. Mesotocin and AVT-related peptides such as hydrin 1 and hydrin 2 showed relatively weak ACTH-releasing activity. Subsequently, cDNAs encoding the bullfrog AVT V1a-type and V1b-type receptors were molecularly cloned. Reverse transcriptase-PCR using specific primers revealed that the anterior lobe of the pituitary predominantly expressed AVT V1b-type receptor mRNA but scarcely expressed AVT V1a-type receptor mRNA. Abundant signals for V1b-type receptor mRNA in the corticotropes were also detected by in situ hybridization. The results obtained by the experiments with the bullfrog pituitary indicate that AVT acts as the main ACTH-releasing factor through the AVT V1b-type receptor and that CRF acts synergistically with AVT to enhance the release of ACTH.

Possible hormonal interaction for eliciting courtship behavior in the male newt, Cynops pyrrhogaster.

Reproductive behavior in amphibians, as in other vertebrate animals, is under the control of multiple hormonal substances. Prolactin (PRL), arginine vasotocin (AVT), androgen, and 7α-hydroxypregnenolone (7α-OH PREG), four such substances with hormonal activity, are known to be involved in the expression of the tail vibration behavior which is the initial step of courtship performed by the male newt, Cynops pyrrhogaster. As current information on the interaction(s) between these hormones in terms of eliciting tail vibration behavior is limited, we have investigated whether the decline of expression of tail vibration behavior due to suppression of the activity of any one of these hormones can be restored by supplying any one of the other three hormones exogenously. Expression of the behavior was determined in terms of incidence (% of test animals exhibiting the behavior) and frequency (number of times that the behavior was repeated during the test period). Neither PRL nor androgen restored the decline in the incidence and frequency of the tail vibration behavior caused by the suppression of the activity of any one of other three hormones. AVT completely restored both the anti-PRL antibody-induced and flutamide (an androgen receptor antagonist)-induced, but not ketoconazole (an inhibitor of the steroidogenic CYP enzymes)-induced decline in the incidence and frequency of the tail vibration behavior. The neurosteroid, 7α-OH PREG, failed to restore flutamide-induced decline in the incidence and frequency of the behavior. However, it was able to restore both anti-PRL antibody-induced and AVT receptor antagonist-induced decline in the incidence, but not in the frequency of the behavior. In another experiment designed to see the activity of hormones enhancing the frequency of the tail vibration behavior, AVT was revealed to be more potent than 7α-OH PREG. The role of each hormonal substance in determining the expression of the tail vibration behavior was discussed based on the results.

Bacterial toxin-inducible gene expression of cathelicidin-B1 in the chicken bursal lymphoma-derived cell line DT40: functional characterization of cathelicidin-B1.

Chicken cathelicidin-B1 (chCATH-B1) is a major host defense peptide of the chicken bursa of Fabricius (BF). To investigate the mechanisms of chCATH-B1 gene expression in the BF, we focused on the DT40 cell line derived from chicken bursal lymphoma as a model for analysis. A cDNA encoding chCATH-B1 precursor was cloned from DT40 cells. The nucleotide sequence of the cDNA was identical with that of the BF chCATH-B1. A broth dilution analysis showed that the synthetic chCATH-B1 exhibited a significant defensive activity against both Escherichia coli and Staphylococcus aureus. A scanning microscopic analysis demonstrated that chCATH-B1 inhibited bacterial growth through membrane destruction with formation of blebs and spheroplasts. Limulus amoebocyte lysate assay and electromobility shift assay results revealed that chCATH-B1 bound to lipopolysaccharide (LPS) and lipoteichoic acid (LTA), which are the surface substances of the E. coli and S. aureus cell, respectively. A chemotactic assay results revealed that chCATH-B1 showed mouse-derived P-815 mastocytoma migrating activity dose-dependently but with a higher concentration, resulting in a loss of the activity. A semi-quantitative real-time RT-PCR analysis revealed that LPS stimulated chCATH-B1 gene expression in a dose-dependent manner and that the LPS-inducible chCATH-B1 gene expression was inhibited by the administration of dexamethasone. The chCATH-B1 mRNA levels in DT40 cells were also increased by the administration of bacterial LTA. The results indicate that bacterial toxins induce chCATH-B1 gene expression in the chicken BF and the peptide expressed in the organ would act against pathogenic microorganisms not only directly but also indirectly by attracting mast cells.

Expression of G proteins in the olfactory receptor neurons of the newt Cynops pyrrhogaster: their unique projection into the olfactory bulbs.

We analyzed the expression of G protein α subunits and the axonal projection into the brain in the olfactory system of the semiaquatic newt Cynops pyrrhogaster by immunostaining with antibodies against Gαolf and Gαo , by in situ hybridization using probes for Gαolf , Gαo , and Gαi2 , and by neuronal tracing with DiI and DiA. The main olfactory epithelium (OE) consists of two parts, the ventral OE and dorsal OE. In the ventral OE, the Gαolf - and Gαo -expressing neurons are located in the apical and basal zone of the OE, respectively. This zonal expression was similar to that of the OE in the middle cavity of the fully aquatic toad Xenopus laevis. However, the Gαolf - and Gαo -expressing neurons in the newt ventral OE project their axons toward the main olfactory bulb (MOB) and the accessory olfactory bulb (AOB), respectively, whereas in Xenopus, the axons of both neurons project solely toward the MOB. In the dorsal OE of the newt, as in the principal cavity of Xenopus, the majority of the neurons express Gαolf and extend their axons into the MOB. In the vomeronasal organ (VNO), the neurons mostly express Gαo . These neurons and quite a few Gαolf -expressing neurons project their axons toward the AOB. This feature is similar to that in the terrestrial toad Bufo japonicus and is different from that in Xenopus, in which VNO neurons express solely Gαo , although their axons invariably project toward the AOB. We discuss the findings in the light of diversification and evolution of the vertebrate olfactory system.

Roles of arginine vasotocin receptors in the brain and pituitary of submammalian vertebrates.

This chapter reviews the functions of arginine vasotocin (AVT) and its receptors in the central nervous system (CNS) of primarily submammalian vertebrates. The V1a-type receptor, which is widely distributed in the CNS of birds, amphibians, and fish, is one of the most important receptors involved in the expression of social and reproductive behaviors. In mammals, the V1b receptor of arginine vasopressin, an AVT ortholog, is assumed to be involved in aggression, social memory, and stress responses. The distribution of the V1b-type receptor in the brain of submammalian vertebrates has only been reported in an amphibian species, and its putative functions are discussed in this review. The functions of V2-type receptor in the CNS are still unclear. Recent phylogenetical and pharmacological analyses have revealed that the avian VT1 receptor can be categorized as a V2b-type receptor. The distribution of this newly categorized VT1 receptor in the brain of avian species should contribute to our knowledge of the possible roles of the V2b-type receptor in the CNS of other nonmammalian vertebrates. The functions of AVT in the amphibian and avian pituitaries are also discussed, focusing on the V1b- and V1a-type receptors.

Effect of neuropeptide Y on food intake in bullfrog larvae.

Neuropeptide Y (NPY) is a potent orexigenic neuropeptide implicated in appetite regulation in mammals. However, except for teleost fish such as the goldfish and zebrafish, the involvement of NPY in the regulation of feeding in non-mammalian vertebrates has not been well studied. Anuran amphibian larvae feed and grow during the pre- and pro-metamorphic stages, but, thereafter they stop feeding as the metamorphic climax approaches. Therefore, orexigenic factors seem to play important roles in pre- and pro-metamorphic larvae. We investigated the role of NPY in food intake using bullfrog larvae including pre- and pro-metamorphic stages, and examined the effect of feeding status on the expression level of the NPY transcript in the hypothalamus. NPY mRNA levels in hypothalamus specimens obtained from larvae that had been fasted for 3 days were higher than those in larvae that had been fed normally. We then investigated the effect of intracerebroventricular (ICV) administration of NPY on food intake in the larvae. Cumulative food intake was significantly increased by ICV administration of NPY (5 and 10 pmol/g body weight, BW) during a 15-min observation period. The NPY-induced orexigenic action (10 pmol/g BW) was blocked by treatment with a NPY Y1 receptor antagonist, BIBP-3226 (100 pmol/g BW). These results indicate that NPY acts as an orexigenic factor in bullfrog larvae.