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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.

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.

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&ndash, 2SSd, esculentin-2SSa, ranatuerin-2SSa, brevinin-1SSa&ndash, 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.

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.

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.

The Harderian gland (HG) is an orbital gland found in many terrestrial vertebrates that possess a nictitating membrane. Using reverse-transcription polymerase chain reaction (RT-PCR), we cloned five cDNAs encoding antimicrobial peptide (AMP)-homologs, catesbeianalectin, ranacyclin-CBa, ranatuerin-1CBa, ranatuerin-2CBa, and ranatuerin-2CBb, from the bullfrog HG total RNA. Of these, catesbeianalectin has not been thoroughly studied in terms of its biological activities. We examined antimicrobial activities of the synthetic replicate of catesbeianalectin and its putative unprocessed precursor, catesbeianalectin-GK. Both peptides showed slight but significant growth inhibitory activity against the Gram-negative bacterium Escherichia coli. Subsequently, we tested catesbeianalectin and catesbeianalectin-GK for mast cell degranulation activity as a criterion of the release of N-acetyl-β-D-glucosaminidase from the mouse-derived mastocytoma cell line P-815, followed by the standard MTT assay to assess cell survival and recovery after peptide treatment. We found that catesbeianalectin and catesbeianalectin-GK invariably exhibited mast cell degranulation activity without cytotoxic effects. Hemagglutination assay revealed the presence of lectin-like activity in both catesbeianalectin and catesbeianalectin-GK. Our findings strongly suggest that these multifunctional host defense peptides in the amphibian HG are involved in innate immunodefense of the eye of the host against pathogenic environmental microorganisms.

The Harderian gland is an orbital gland found in many tetrapod species that possess a nictitating membrane. While the main role of the Harderian gland is lubrication of the eyeballs, numerous other functions are attributed to this gland. In amphibians, mast cells have been detected in the Harderian gland, suggesting that the gland is involved in the host's system of innate immunity defending against microbial invasions. Using reverse-transcription polymerase chain reaction, we cloned from the bullfrog Harderian gland total RNA preparations, cDNAs encoding biosynthetic precursors for the antimicrobial peptides temporin-CBa (FLPIASLLGKYL-NH 2 ), previously isolated from an extract of bullfrog skin, and chensirin-2CBa (IIPLPLGYFAKKP) that contained the amino acid substitution Thr 13 → Pro compared with chensirin-2 from the Chinese brown frog, Rana chensinensis. By means of in situ hybridization using digoxigenin-labeled cRNA probes for preprotemporin-CBa and preprochensirin-2CBa, we have demonstrated for the first time in an amphibian the presence of mRNAs encoding these two precursors in the cytoplasm of the glandular cells in the bullfrog Harderian gland.

Pacific herring Clupea pallasii and Japanese anchovy Engraulis japonicus, which belong to the same order Clupeiformes, spawn different types of eggs: demersal adherent eggs and pelagic eggs, respectively. We cloned three cDNAs for Pacific herring hatching enzyme and five for Japanese anchovy. Each of them was divided into two groups (group A and B) by phylogenetic analysis. They were expressed specifically in hatching gland cells (HGCs), which differentiated from the pillow and migrated to the edge of the head in both species. HGCs of Japanese anchovy stopped migration at that place, whereas those of Pacific herring continued to migrate dorsally and distributed widely all over the head region. During evolution, the program for the HGC migration would be varied to adapt to different hatching timing. Analysis of the gene expression revealed that Pacific herring embryos synthesized a large amount of hatching enzyme when compared with Japanese anchovy. Chorion of Pacific herring embryo was about 7.5 times thicker than that of Japanese anchovy embryo. Thus, the difference in their gene expression levels between two species is correlated with the difference in the thickness of chorion. These results suggest that the hatching system of each fish adapted to its respective hatching environment. Finally, hatching enzyme genes were cloned from each genomic DNA. The exon–intron structure of group B genes basically conserved that of the ancestral gene, whereas group A genes lost one intron. Several gene-specific changes of the exon–intron structure owing to nucleotide insertion and/or duplication were found in Japanese anchovy genes. J. Exp. Zool. (Mol. Dev. Evol.) 312B:95–107, 2009. © 2008 Wiley-Liss, Inc.

Antimicrobial peptides (AMPs) are important components of the host innate defense system against pathogenic microbial invasion in many organisms. In the present study, we cloned by RT-PCR a cDNA from total RNA prepared from the skin of the Japanese brown frog Rana japonica. The cDNA directs the synthesis of a novel, non-C-terminally alpha-amidated peptide composed of 21 amino acid residues (FLGSLIGAAIPAIKQLLGLKK). The putative peptide showed limited sequence similarity to atypical acyclic brevinin-1OK family AMPs originally isolated from the skin of the Ryukyu brown frog (R. okinavana), which lacks the COOH-terminal cyclic domain commonly observed in typical brevinin-1 groups, but that contains a C-terminally alpha-amidated residue. Although it is unclear whether the peptide, designated brevinin-1Ja, is produced in R. japonica skin, a synthetic replicate of the peptide showed differential growth-inhibiting activity against the Gram-positive bacterium Staphylococcus aureus and Gram-negative bacterium Escherichia coli (minimal inhibitory concentrations: 15 microM and 119 microM, respectively), and produced cell death of mammalian COS7 cells (LD50=28 microM).

An extract of the skin of the Japanese tree frog, Hyla japonica Gunther, 1859 (Anura: Hylidae) did not inhibit the growth of the bacteria Escherichia coli or Staphylococcus aureus, but contained a protein that was strongly hemolytic against human erythrocytes. The protein was purified to near homogeneity by reverse-phase HPLC, and its N-terminal amino acid sequence (SGRGKGGKGL…) identified it as histone H4. The complete primary structure of the 102-amino-acid-residue histone H4 was determined by a combination of molecular cloning of genomic and complementary DNAs encoding the protein. The molecular mass of the purified histone H4 determined by electrospray mass spectrometry was 71 ± 2 Daltons greater than that predicted from the deduced amino acid sequence of the protein. The + 71 mass units is consistent with the proposal that the protein isolated from the skin was post-translationally modified by addition of one acetyl and two methyl groups. The stem-loop structure at the 3′ flanking region of the H. japonica histone H4 gene, which acts as a transcription termination signal, contained a nucleotide sequence (5′-GGCTCTCCTCAGAGCC-3′) with unusual structural features not seen in other histone genes.

Complementary DNAs encoding two major osteoclastic markers, tartrate-resistant acid phosphatase (TRAP) and cathepsin K (Cath K) were cloned from the scales of a teleost, the goldfish. This is the first report of the full coding sequence of TRAP and Cath K molecules in fish. In the goldfish scale both TRAP and Cath K mRNAs were expressed in the multinucleate osteoclasts, which showed large numbers of mitochondria and lysosomes, and a well developed ruffled border. These characteristic features of osteoclasts in the scales are similar to those in mammals. Most teleosts use the scale as an internal calcium reservoir during the reproductive season. The expression of TRAP and Cath K mRNAs in the scale significantly increased in April, which is a reproductive season, compared with that in October, a non-reproductive season. Thus, both of these molecular markers should be useful for the study of osteoclasts in the teleost scale.

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

Peptidomic analysis of an extract of the skin of the stream brown frog Rana sakuraii Matsui and Matsui, 1990 led to the isolation of a C-terminally α-amidated peptide (VR-23; VIGSILGALASGLPTLISWIKNR·NH 2 ) with broad-spectrum antimicrobial activity that shows structural similarity to the bee venom peptide, melittin together with two peptides belonging to the temporin family (temporin-1SKa; FLPVILPVIGKLLNGIL·NH 2 and temporin-1SKb; FLPVILPVIGKLLSGIL·NH 2 ), and peptides whose primary structures identified them as belonging to the brevinin-2 (2 peptides) and ranatuerin-2 (1 peptide) families. Using a forward primer that was designed from a conserved region of the 5′-untranslated regions of Rana temporaria preprotemporins in a 3′-RACE procedure, a cDNA clone encoding preprotemporin-1SKa was prepared from R. sakuraii skin total RNA. Further preprotemporin cDNAs encoding temporin-1SKc (AVDLAKIANIAN KVLSSL F·NH 2 ) and temporin-1SKd (FLPMLAKLLSGFL·NH 2 ) were obtained by RT-PCR. Unexpectedly, the 3′-RACE procedure using the same primer led to amplification of a cDNA encoding a preprobradykinin whose signal peptide region was identical to that of preprotemporin-1SKa except for the substitution Ser 18 → Asn. R. sakuraii bradykinin ([Arg 0 ,Leu 1 ,Thr 6 ,Trp 8 ] BK) was 28-fold less potent than mammalian BK in effecting B2 receptor-mediated relaxation of mouse trachea and the des[Arg 0 ] derivative was only a weak partial agonist. The evolutionary history of the Japanese brown frogs is incompletely understood but a comparison of the primary structures of the R. sakuraii dermal peptides with those of Tago's brown frog Rana tagoi provides evidence for a close phylogenetic relationship between these species .

The temporins are a family of hydrophobic, C-terminally alpha-amidated antimicrobial peptides that are synthesized in the skins of a wide range of species of frogs belonging to the genus Rana. In the present study, we investigated using RT-PCR the expression of preprotemporin mRNAs in extradermal tissues of Tago's brown frog Rana tagoi. cDNAs encoding temporin-1TGa (FLPILGKLLS(10)GIL.NH2), previously isolated from an extract of the skin of R. tagoi skin, were amplified and cloned from the stomach, liver, kidney, skeletal muscle. However, a net insertion of 10 nucleotides resulted in the presence of a premature stop codon in the open reading frame that was not present in the corresponding region of preprotemporin-1TGa from skin. The preprotemporin cDNA obtained from small intestine contained an additional 12 nucleotide insertion in the region that encodes the temporin sequence so that a novel peptide (FLPVILPVIG(10)KLLSGIL.NH2), termed temporin-1TGc, is specified. This cDNA also contained a premature stop codon in the open reading frame. Although it is unclear whether temporin-1TGc is produced in R. tagoi tissues, a synthetic replicate of the peptide of was biologically active, inhibiting the growth of Staphylococcus aureus (minimal inhibitory concentration = 37.5 microM) and producing hemolysis of human erythrocytes (LD50 = 50 microM).

Using RT-PCR, two cDNAs encoding preprotemporins were cloned from a total RNA preparation of the skin of Tago's brown frog Rana tagoi. Preprotemporin-1TGa cDNA directs the synthesis of temporin-1TGa (FLPILGKLLSGIL.NH2) previously isolated from R. tagoi skin. Preprotemporin-1TGb cDNA directs the synthesis of a novel 16-amino-acid-residue peptide (AVDLAKIANKVLSSLF.NH2) that, atypically for members of the temporin family, inhibits the growth of Gram-negative bacteria more effectively than Gram-positive bacteria. Preprotemporin-1TGa mRNA and preprotemporin-1TGb mRNA were not detected in skin prior to the onset of metamorphosis (stage 35) but the levels of the transcripts increased markedly during metamorphosis reaching a maximum at stage 38. Exposure of adult animals to 10(-8) M triiodothyronine (T3) for 72 h enhanced expression of the preprotemporin-1TGb gene (approximately threefold) but did not significantly change the level of expression of the preprotemporin-1TGa gene. Exposure of the animals to 10(-8) M T3 and 10(-6) M bisphenol A, an endocrine disrupting chemical that potently inhibits the action of thyroid hormones (THs), reduced expression of the preprotemporin-1TGb gene by 10-fold and the preprotemporin-1TGa gene by threefold. We propose that T3-stimulated synthesis of antimicrobial peptides is important in protecting the animal against microorganisms, particularly at metamorphosis and during skin moulting, but environmental pollutants can inhibit peptide synthesis and render the animal susceptible to invasion by pathogens.

Osteogenesis in the teleost was morphologically observed using regenerating scales of goldfish. Histological observations indicated that osteoblasts around the regenerating scales on days 7 to 10 were greater in size and number than those at other stages. Therefore, further experiments were carried out to examine the activity of osteoblasts in the regenerating period. To quantify their osteoblastic activities, scales on the left side of the body were taken, and the regenerating scales were then used to measure the activities of alkaline phosphatase (ALP), a marker of osteoblasts, on days 7, 10, and 15. The ontogenic scales on the right side of the body were also collected and used to measure ALP activity on the same days. Osteoblasts at all stages of regenerating scales were more active than those in the remaining ontogenic scales. The regenerating scales on day 10 had the highest activity. Furthermore, we found that estrogen receptor (ER) mRNA was expressed in the regenerating scales because estrogen participates in osteoblastic growth and differentiation in mammals. Therefore, using a scale culture system reported previously, the estrogenic response was examined in the ontogenic and regenerating scales on day 10. The reactivity was much higher in regenerating scales, although estrogen treatment significantly activated the osteoblastic activities in both scales. We are the first to demonstrate that ER is expressed in regenerating scales and that estrogen participates in osteogenesis as it does in mammalian bone. Our findings strongly suggest that regenerating scales can be used as a model of osteogenesis in vertebrates.

Effects of bisphenol A (BPA) on embryonic and larval development were investigated. In Xenopus laevis blastulae treated with 2.5-3.0 x 10(-5) M BPA or with 10(-5) M 17beta estradiol (E2), malformation of the head region, scoliosis (curved vertebrate), and suppression of organogenesis were observed. In addition, 10(-5)-10(-4) M BPA blocked tri-iodothyronine (T3)-inducible resorption of the tail segments from premetamorphic (stage 52-54) larvae in vitro. When stage 52 tadpoles were immersed in 1.0-2.5 x 10(-5) M BPA, deceleration of both spontaneous and thyroxin (T4)-induced metamorphic changes occurred. Furthermore, BPA suppressed thyroid hormone receptor (TR) beta gene expression both in vivo and in vitro. Thus, we concluded that BPA at the concentrations examined affects both embryonic development and larval metamorphosis.

Six peptides with antimicrobial activity were isolated from an extract of freeze-dried skin of the Japanese mountain brown frog Rana ornativentris. Two structurally related peptides (brevinin-20a GLFNVFKGALKTAGKHVAGSLLNQLKCKVSGGC, 11 nmol/g dried tissue, and brevinin-20b GIFNVFKGALKTAGKHVAGSLLNQLKCKVSGEC, 170 nmol/g) belong to the brevinin-2 family, previously identified in Asian and European, but not North American, Ranid frogs. Four peptides (temporin-10a FLPLLASLFSRLL.NH2, 13 nmol/g; temporin-10b FLPLIGKILGTI L.NH2, 350 nmol/g; temporin-10c FLPLLASLFSRLF.NH2, 14 nmol/g; and temporin-10d FLPLLASLFSGLF.NH2, 8 nmol/g) are members of the temporin family first identified in the European common frog Rana temporaria but also found in the skins of North American Ranids. The brevinin-2 peptides showed broad-spectrum activity against the gram-positive bacterium, Staphylococcus aureus, the gram-negative bacterium, Escherichia coli and the yeast Candida albicans, whereas the temporins showed potent activity only against S. aureus. The brevinins and temporins belong to the class of cationic antimicrobial peptides that adopt an amphipathic alpha-helical conformation but it is significant that temporin-10d, which lacks a basic amino acid residue, is still active against S. aureus (minimum inhibitory concentration=13 microM compared with 2 microM for temporin-10a). This suggests that strong electrostatic interaction between the peptide and the negatively charged phospholipids of the cell membrane is not an absolute prerequisite for antimicrobial activity.

Adrenal corticoids accelerate metamorphosis of amphibians by potentiating the action of thyroid hormone. Adrenal corticoid secretion is considered to be controlled mainly by adrenocorticotropic hormone generated from proopiomelanocortin (POMC) in the anterior lobe of the pituitary. In order to assess the changes in POMC mRNA levels during metamorphosis, a cDNA for POMC was isolated from a cDNA library constructed from bullfrog (Rana catesbeiana) pituitary polyadenylated RNA. Northern blot analysis using the POMC cDNA as a probe revealed that POMC mRNA levels in the anterior lobe were relatively low during premetamorphosis, rose during prometamorphosis, reached the maximum at the end of prometamorphosis and remained very high during climax. The POMC mRNA levels of the intermediate lobe, where α-melanophore-stimulating hormone is generated from POMC, were also determined in metamorphosing tadpoles. The POMC mRNA levels of the intermediate lobe increased as metamorphosis progressed and were maximal ...

Labeling and immunoprecipitation experiments confirmed that three proopiomelanocortin (POMC)-derived peptides, the N-terminal peptide of POMC (NPP), joining peptide (JP) and adrenocorticotropic hormone (ACTH), were released by the bullfrog (Rana catesbeiana) anterior pituitary. The effects of these three peptides on luteinizing hormone (LH) release by bullfrog dispersed anterior pituitary cells were studied. NPP and ACTH, but not JP, enhanced LH release concentration-dependently. Approximately 6 hr elapsed before the gonadotrophs responded to NPP and ACTH by releasing LH, whereas their response to human GnRH (hGnRH) was faster, suggesting that the modes of action of these two peptides and hGnRH differ. These results raise the possibility that NPP and ACTH act as paracrine factors in the bullfrog pituitary to enhance LH release either directly or indirectly.

In an attempt to explain the contrasting patterns of expression of Xenopus thyroid hormone (xTR) and retinoid X (xRXR) receptor genes and to extend our understanding of the role of heterodimerization of these receptors during amphibian metamorphosis, we have investigated the response to their respective ligands of cells in which xTR and xRXR were overexpressed. Results obtained with two separate approaches are now described. In the first, 3,3′,5-triiodothyronine (T 3 ) was found to strongly upregulate xTR β mRNA in XTC-2 cells, but not of xTR α or xRXR α mRNAs, while xRXR γ transcripts could not be detected. 9- cis -retinoic acid (9- cis -RA) did not substantially influence the expression of any of these four receptor genes. When transcription from three different thyroid response elements (TREs) (a palindromic TREpal, an inverted repeat +6 [F2] and a direct repeat +4 [DR+4] as present in the promoter of xTR β gene) was measured in XTC-2 cells in which xTR β and xRXR α were overexpressed, only T 3 upregulated transcription while 9- cis -RA, alone or together with T 3 , was ineffective. 9- cis -RA however enhanced transcription from an RXR responsive element (RXR-RE). The second approach involved overexpression of xTR β and xRXR α in premetamorphic Xenopus tadpole tail muscle followed by measuring the response of the tails to T 3 in organ culture. After validating the microinjection/culture procedure histochemically, we found that T 3 enhanced transcription from the xTR β DR+4 TRE in tails in which xTR β was overexpressed but the overexpression of xRXR α failed to modify this response. It is concluded that in both XTC-2 cells and tadpole tails, overexpressed xRXR fails to modify the enhanced transcriptional response of endogenous and overexpressed xTR β to T 3 and that exogenous 9- cis -RA is ineffective.

We previously reported the activities and modes of action of arginine (Arg)-rich histones H3 and H4 against Gram-negative bacteria. In the present study, we investigated the properties of the Arg-rich histones against Gram-positive bacteria in comparison with those of lysine (Lys)-rich histone H2B. In a standard microdilution assay, calf thymus histones H2B, H3, and H4 showed growth inhibitory activity against Staphylococcus aureus with minimum effective concentration values of 4.0, 4.0, and 5.6 μM, respectively. Laser confocal microscopic analyses revealed that both the Arg-rich and Lys-rich histones associated with the surface of S. aureus. However, while the morphology of S. aureus treated with histone H2B appeared intact, those treated with the histones H3 and H4 closely resembled each other, and the cells were blurred. Electrophoretic mobility shift assay results revealed these histones have binding affinity to lipoteichoic acid (LTA), one of major cell surface components of Gram-positive bacteria. Scanning electron microscopic analyses demonstrated that while histone H2B elicited no obvious changes in cell morphology, histones H3 and H4 disrupted the cell membrane structure with bleb formation in a manner similar to general antimicrobial peptides. Consequently, our results suggest that bacterial cell surface LTA initially attracts both the Arg- and Lys-rich histones, but the modes of antimicrobial action of these histones are different; the former involves cell membrane disruption and the latter involves the cell integrity disruption.

The direct effects of a pair of tilapia prolactins (tPRL177 and tPRL188) and of ovine prolactin (oPRL) on cultured xanthophores and erythrophores isolated from some fish species were examined. Cultured xanthophores of the Nile tilapia, paradise goby, and medaka responded to tPRL177 and oPRL at concentrations of less than 100 nM by pigment dispersion, whereas tPRL188 had an effect only at concentrations of 10 μM or more. Swordtail erythrophores also responded to prolactin even in winter. It is likely, therefore, that in fish species possessing many xanthophores or erythrophores in their skin throughout the year, these cells respond to prolactin all the time. In fall and/or winter, prolactin had little or no effect on xanthophores isolated from the dark chub and rose bitterling or on erythrophores from the tilapia and paradise goby. However, it was shown that xanthophores of the rose bitterling and erythrophores of the tilapia responded better to prolactin in spring and/or summer as compared with fall and/or winter. Such seasonal change in the responsiveness of the cells to prolactin implies the involvement of the hormone in nuptial coloration and an augmentation of red and/or yellow skin color by pigment dispersion in the spawning season. Prolactin-induced pigment dispersion within erythrophores isolated from the red region of tail fins of the rose bitterling assuming nuptial coloration also lends support to this supposition. © 1996 Wiley-Liss, Inc.

The precocious induction of amphibian metamorphosis is an ideal system for analyzing the developmental action of TH, while the hormonal activation of tadpole tail regression offers the further advantage of studying programmed cell death. One of the striking features of thyroid hormone (TH)-induced tail regression (as with morphogenetic responses of all tadpole tissues) in Xenopus is the rapid autoinduction of TRbeta gene, but it is not known how TH would affect the expression of the genes encoding TR's heterodimeric partner, retinoid X receptor (RXR). Here we first show that the synthetic glucocorticoid dexamethasone (Dex) potentiates and prolactin (PRL) suppresses, 3,3',5-triiodothyronine (T3)-induced regression of pre-metamorphic Xenopus tadpole tails in organ culture. T3 strongly upregulated (11-35-fold) the concentration of Xenopus TRbeta (xTRbeta) mRNA in these cultures while downregulating by 50% that of Xenopus RXRgamma (xRXRgamma) mRNA in the same samples of tail RNA. DEX and PRL enhanced or diminished the T3-regulated expression of these two transcripts, respectively, which parallels their other effects in whole tadpoles or cultured tails. The contrasting effects of the three hormones on the steady-state levels of xTRbeta and XRXRgamma mRNAs were time- and dose-dependent. T3 and DEX also strongly upregulated the transcription of xTRbeta gene transfected into Xenopus XTC-2 cells but PRL failed to prevent this autoinduction. The actions of these three hormones involved in amphibian metamorphosis, as judged by the expression of xTRbeta and xRXRgamma genes, reveal a new facet of hormonal interplay underlying their developmental actions.

There is growing evidence of the antimicrobial properties of histones and histone-derived peptides; however, most of them are specific to lysine (Lys)-rich histones (H1, H2A, and H2B). In the present study, we focused on arginine (Arg)-rich histones (H3 and H4) and investigated their antimicrobial properties in comparison with those of histone H2B. In a standard microdilution assay, calf thymus histones H2B, H3, and H4 showed growth inhibitory activity against the bacterial outer membrane protease T (OmpT) gene-expressing Escherichia coli strain JCM5491 with calculated 50% growth inhibitory concentrations of 3.8, 10, and 12.7 μM, respectively. A lysate prepared from the JCM5491 cells was capable of strongly, moderately, and slightly fragmenting histones H2B, H3, and H4, respectively. While the lysate prepared from the cells of the ompT-deleted E. coli strain BL21(DE3) did not digest these histones, the ompT-transformed BL21(DE3), termed BL21/OmpT(+), cell lysate digested the histones more strongly than the JCM5491 cell lysate. Laser confocal and scanning electron microscopic analyses demonstrated that while histone H2B penetrated the cell membrane of JCM5491 or BL21/OmpT(+) cells, histones H3 and H4 remained on the cell surface and subsequently disrupted the cell membrane structure with bleb formation in a manner similar to general antimicrobial peptides. The BL21(DE3) cells treated with each histone showed no bleb formation, but cell integrity was affected and the cell surface was corrugated. Consequently, it is suggested that OmpT is involved in the antimicrobial properties of Arg- and Lys-rich histones and that the modes of antimicrobial action of these histones are different.

Using a combination of reverse-transcription polymerase chain reaction and the 5'- and/or 3'-rapid amplification of cDNA ends, we cloned, from a Japanese brown frog (Rana japonica) skin total RNA preparation, cDNAs encoding biosynthetic precursors for the antimicrobial peptides (AMPs) japonicin-1Ja (FFPIGVFCKIFKTC), japonicin-2Ja (FGLPMLSILPKALCILLKRKC), and temporin-1Ja (ILPLVGNLLNDLL.NH2). These peptides were previously isolated from an extract of R. japonica skin. The present study is the first report to describe the molecular cloning of the cDNA encoding a japonicin-2 family peptide. The nucleotide and deduced amino acid sequence analyses revealed that the hypothetical precursor protein of japonicin-2Ja, as well as japonicin-1Ja and temporin-1Ja, is organized similarly to those of typical amphibian AMP precursors, with a highly conserved signal peptide, a relatively well conserved intervening sequence, and a hypervariable AMP mature region. Antimicrobial assays for synthetic replicates of cyclic and linear japonicin-2Ja revealed that the intramolecular disulfide bond is necessary for activity. A semi-quantitative analysis by real-time RTPCR using TaqMan probes revealed that the relative values of preprojaponicin-2Ja mRNA expression levels in the skin, skeletal muscle of hind leg, kidney, testis, small intestine, and stomach total RNA sample specimens in adult R. japonica were 6.5×10(5), 9.6, 2.0, 1.6, 1.6, and 1.0, respectively. The presence of preprojaponicin-2Ja mRNAs in the cytoplasm of glandular cells in R. japonica dorsal skin glands was demonstrated by means of in situ hybridization using digoxigenin-labeled cRNA probes for the precursor.

Previous studies led to the isolation from skin extracts of Oki Tago's brown frog, Rana tagoi okiensis of five antimicrobial peptides belonging to the brevinin-1 (brevinin-1TOa), temporin (temporin-TOa and -TOb), and ranatuerin-2 (ranatuerin-2TOa and -2TOb) families, and bradykinin (BK) identical to mammalian BK. Using the reverse-transcription polymerase chain reaction (RT-PCR), we have now cloned from skin total RNA preparations cDNAs encoding biosynthetic precursors of brevinin-1TOa and brevinin-1TOb (containing the substitution Gly(1)-->Val), temporin-TOa and -TOb, and ranatuerin-2TOa and -2TOb. In addition, three cDNA clones encoding preprobradykinins were obtained that contained either one, two, or three tandem repeats of the sequence of BK followed by the sequence of [Thr(6)]-BK. In tissue expression analyses, preprobrevinin-1, preprotemporin, and preproranatuerin-2 gene transcripts were detected at higher levels in brain compared with peripheral tissues (heart, small intestine, kidney, liver lung, skeletal muscle, stomach, and testis). RT-PCR of brain RNA resulted in the amplification of cDNAs encoding ranatuerin-2TOc and ranatuerin-2TOd that contained the amino acid substitutions Lys(6)-->Arg and Ala(14)-->Thr, respectively compared with ranatuerin-2TOb. cDNAs encoding preprobrevinin-1TOa and preprotemporin-TOa were amplified from brain RNA as well as a second preprotemporin cDNA that contained a 10-nucleotide insertion that introduced a frame shift resulting in a premature stop codon. A cDNA encoding a novel peptide, DK25 (DVNDLKNLCAKTHNLLPMCAMFGKK) was amplified from brain RNA but neither DK25 nor its putative post-translationally modified form, DF22-amide (DVNDLKNLCAKTHNLLPMCAMF.NH(2)) displayed antimicrobial or hemolytic activities.

Antimicrobial peptides (AMPs) play an important role in the host's innate defence system in many organisms. Amphibian skin is expected to be a particularly rich source of novel AMPs. In amphibians, AMPs are produced from precursor proteins via specific cleavage by processing enzymes. While the nucleotide sequences of the AMP coding region in precursors are hypervariable, those of other regions, including the 5(')- and 3(')-untranslated regions (UTRs), are highly or relatively conserved in different precursors. Such nucleotide sequence conservation suggests an efficient strategy for molecular cloning of the antimicrobial peptide genes by 3(')-rapid amplification of cDNA ends (3(')-RACE) and reverse transcriptase polymerase chain reaction (RT-PCR) methods using specific primers. With this strategy in mind we have established an efficient protocol suitable for amplification of multiple cDNAs encoding amphibian AMP precursor proteins.

Antimicrobial peptides (AMPs) play an important role in the host's innate defence system in many organisms. Amphibian skin is expected to be a particularly rich source of novel AMPs. In amphibians, AMPs are produced from precursor proteins via specific cleavage by processing enzymes. While the nucleotide sequences of the AMP coding region in precursors are hypervariable, those of other regions, including the 5(')- and 3(')-untranslated regions (UTRs), are highly or relatively conserved in different precursors. Such nucleotide sequence conservation suggests an efficient strategy for molecular cloning of the antimicrobial peptide genes by 3(')-rapid amplification of cDNA ends (3(')-RACE) and reverse transcriptase polymerase chain reaction (RT-PCR) methods using specific primers. With this strategy in mind we have established an efficient protocol suitable for amplification of multiple cDNAs encoding amphibian AMP precursor proteins.

Previous studies led to the isolation of antimicrobial peptides (AMPs) of the brevinin-2, palustrin-2, and ranatuerin-2 families from skin extracts and/or skin secretions of the Japanese mountain brown frog, Rana ornativentris. In the present study, we cloned cDNAs encoding the precursors of brevinin-2Oc, palustrin-2Oa, and ranatuerin-2Ob and -2Oe from skin total RNA preparations from adult R. ornativentris and established a semi-quantitative RT-PCR system to measure the concentrations of these mRNAs. The levels of preprobrevinin-2 and preproranatuerin-2 mRNAs in the skin specimens of developing R. ornativentris larva were detectable only at stages later than the onset of metamorphosis and reached peaks at the stage of metamorphic climax. In contrast, prepropalustrin-2 mRNA was detected prior to the onset of metamorphosis and levels peaked at stages earlier than those of the other two mRNAs. In adult animals, preprobrevinin-2 and preproranatuerin-2 gene transcripts were detected at low levels in the small intestine and skeletal muscle but not in the stomach, liver, or kidney, whereas prepropalustrin-2 gene transcripts were detected at relatively high concentrations in all tissues examined. These results indicate that the expression of amphibian AMP genes is correlated with metamorphosis but is subjected to differential regulation.

There is growing evidence to suggest that proopiomelanocortin (POMC), a precursor of adrenocorticotropin and alpha-melanocyte-stimulating hormone (alpha-MSH), also exists in extrapituitary organs, including immune organs. We investigated the presence of the POMC signaling system in the avian-specific primary immune organ the bursa of Fabricius (BF) of the adult quail. Immunohistochemical staining revealed the presence of cells showing immunopositive reaction with anti-alpha-MSH antibody in the quail specimens. RT-PCR analysis revealed the expression of POMC, prohormone convertases (PC1/3 and PC2), and three melanocortin receptor subtype (MC1R, MC4R, and MC5R) mRNAs in total RNA specimens of the BF. These findings demonstrate that in the quail BF, just as in the pituitary, immunopositive alpha-MSH substances may be produced via specific cleavages of POMC by the sequential actions of PC1/3 and PC2. The observation of MCR expression within the BF suggests that the alpha-MSH substances may exert paracrine actions within the BF.

Cationic peptides with the propensity to adopt an amphipathic alpha-helical conformation in a membrane-mimetic environment are synthesized in the skin of many species of frogs. These peptides frequently display potent cytolytic activities against a range of pathogenic bacteria and fungi, consistent with the hypothesis that they play a role in host defense. However, the importance of the peptides in the survival strategy of the animal is not clearly understood. At this time, antimicrobial peptides have been identified in the skin of frogs from species belonging to the Bombinatoridae, Hylidae, Hyperoliidae, Leiopelmatidae, Leptodactylidae, Myobatrachidae, Pipidae, and Ranidae families, but several well-studied species from the Bufonidae, Ceratophryidae, Dicroglossidae, Microhylidae, Pelobatidae, Pyxicephalidae, Rhacophoridae, and Scaphiopodidae families do not appear to synthesize these peptides. Although cytolytic activity against the chytrid fungus Batrachochytrium dendrobatidis, responsible for anuran population declines worldwide, has been demonstrated in vitro, the ability of frog skin antimicrobial peptides to protect the animal in the wild appears to be limited. While the production of dermal cytolytic peptides may offer definite evolutionary advantage to anurans, their precise biological function, for example during metamorphosis, may need to be re-evaluated.

Antimicrobial peptides (AMPs), which are widely distributed in various organisms, comprise part of the host innate defense system to kill or damage bacterial and fungal pathogens. Amphibian skin is known to produce various AMPs, and is used as a source material in attempts to identify novel therapeutic AMPs. More than one hundred frog AMPs have been identified to date. In our previous study, we isolated histone H2B with antibacterial properties from the skin of the Schlegel's green tree frog Rhacophorus schlegelii. Although antimicrobial histone H2B has not been obtained from the skin of any species other than R. schlegelii, histones and histone-derived fragments with antimicrobial activities have been found in some specific cells of a diverse range of organisms from shrimps to humans. At least a portion of these fragments are known to be produced from "precursor histones" via specific cleavage by endogenous proteases. These antimicrobial histones and the fragments that act as physiological barriers of cells have a variety of antimicrobial actions and functions, including bacterial cell membrane permeabilization, penetration into the membrane followed by binding to bacterial DNA and/or RNA, binding to bacterial lipopolysaccharide (LPS) in the membrane, neutralizing the toxicity of bacterial LPS, and entrapping pathogens as a component of neutrophil extracellular traps (NETs). This review discusses the literature regarding the isolation, antimicrobial properties, and modes of action of antimicrobial histones and fragmented histones along with a brief introduction of typical amphibian skin AMPs.

Previous studies have led to the isolation of histone H2B with antibacterial properties from an extract of the skin of the Schlegel's green tree frog Rhacophorus schlegelii and it is now demonstrated that the intact peptide is released into norepinephrine-stimulated skin secretions. In order to investigate the mechanism of action of this peptide, a maltose-binding protein (MBP)-fused histone H2B (MBP-H2B) conjugate was prepared and subjected to antimicrobial assay. The fusion protein showed bacteriostatic activity against Escherichia coli strain JCM5491 with a minimum inhibitory concentration of 11 microM. The lysate prepared from JCM5491 cells was capable of fragmenting MBP-H2B within the histone H2B region, but the lysate from the outer membrane proteinase T (OmpT) gene-deleted BL21(DE3) cells was not. FITC-labeled MBP-H2B (FITC-MBP-H2B) penetrated into the bacterial cell membrane of JCM5491 and ompT-transformed BL21(DE3) cells, but not into ompT-deleted BL21(DE3) cells. Gel retardation assay using MBP-H2B-deletion mutants indicated that MBP-H2B bound to DNA at a site within the N-terminal region of histone H2B. Consequently, it is proposed that the antimicrobial action of histone H2B involves, at least in part, penetration of an OmpT-produced N-terminal histone H2B fragment into the bacterial cell membrane with subsequent inhibition of cell functions.

Certain proteins insoluble in aqueous salt solutions are difficult to separate from impurities by immunoaffinity techniques, even when the proteins are solubilized with denaturants due to interference of the antigen-antibody reaction. Representative examples of such proteins are the ubiquitin-protein conjugates that accumulate in neuronal tissues of neurodegenerative diseases, the hallmark of such disorders. In this study, we developed a novel sample preparation method comprising two successive steps: Sodium dodecyl sulfate (SDS) removal from the SDS-containing extracts and renaturation of the denatured proteins. The application of this method was tested on ubiquitin-protein conjugates in the brains of Niemann-Pick type C disease mouse and in heat-shocked K562 erythroleukemia cells. The ubiquitin-protein conjugates in both cases are insoluble in Tris-buffered saline but soluble in 2% SDS. The SDS-solubilized fractions prepared from each of the samples were further pretreated by the method mentioned above, and the ubiquitin-protein conjugates were efficiently immunoprecipitated with the anti-ubiquitin antibody from them. This method was also applied successfully to the immunoprecipitation of flotillin-1, a lipid raft protein, from mouse brain extract prepared with 2% SDS. These results indicate that this simple protocol has potential applications for excellent immunoaffinity separation of the less-soluble proteins in diverse cells and tissues.

The antimicrobial melittin-related peptides (MRPs) isolated from skin extracts of the Japanese frogs, Rana sakuraii and Rana tagoi, show amino acid sequence similarity with melittin from the venom of honeybees but the evolutionary relationship between the amphibian and insect peptides is unknown. cDNA clones encoding the MRP precursor (preproMRP) were obtained from R. sakuraii and R. tagoi skin total RNA. The nucleotide and deduced amino acid sequences of the clones indicated that the preproMRPs are organized like typical amphibian antimicrobial peptide precursors, with a highly conserved signal peptide, a more variable intervening sequence, and a hypervariable mature peptide region. This organization is markedly different from that of prepromelittin, in which the melittin sequence is flanked by multiple Xaa-Pro and Xaa-Ala dipeptides. The data indicate, therefore, that the genes encoding frog skin MRPs are not orthologous to the genes encoding melittins from bee venom. In adult R. sakuraii specimens, preproMRP gene transcripts were detected in total RNA from skeletal muscle as well as skin but not from heart, stomach, small intestine, or liver. In R. tagoi, preproMRP mRNA was not detected in skin prior to the onset of metamorphosis, but its level increased markedly during metamorphosis reaching a maximum at the stages of metamorphic climax.

The anal fin in Japanese medaka, Oryzias latipes, is a typical sexual secondary character. In the present study, we focused on this organ and examined the effects of low doses of a natural estrogen, 17beta-estradiol (E(2)), and an environmental xenoestrogen, bisphenol A (BPA), in vivo by monitoring estrogen receptor (ER) alpha gene expression. Groups of adult male and female medaka were immersed in 10(-9) M E(2) or 10(-10) to 10(-8) M BPA and the levels of ERalpha gene transcripts in the anal fins were measured by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). One day of treatment with each concentration of BPA examined and 10(-9) M E(2) increased the levels of ERalpha mRNA in female anal fins by 3-fold as compared with controls. In the male specimens, neither 10(-9) M E(2) nor 10(-10) M BPA showed remarkable effects on the anal fins as compared with the results in females, but 10(-9) and 10(-8) M BPA increased the levels of ERalpha mRNA by 2.3- and 3.3-fold with 1 day of exposure, respectively. The present results showed that medaka anal fins may be a sensitive bio-indicator for screening of environmental estrogenic chemicals.

We investigated effects of different concentrations (10(-7) - 10(-5) M) of bisphenol A (BPA), which is known as an estrogenic and anti-thyroid hormonal endocrine disrupter, on the expression of thyroid hormone receptor (TR) alpha and beta and retinoid X receptor (RXR) gamma mRNA in tails of stage 52-54 Xenopus tadpoles in organ culture in the presence or absence of different concentrations of triiodo-thyronine (T(3)). In the absence of T(3), BPA at any concentration examined did not show remarkable effects on tail length but blocked 10(-7) M T(3)-induced tail resorption in a concentration-dependent manner. Semi-quantitative analyses of TRalpha and TRbeta mRNAs by RT-PCR in the tail specimens indicated that BPA shows an apparent antagonistic effect towards the receptors and reduced their mRNA levels relative to controls. When administered together with 10(-7) M T(3), the antagonistic effects of BPA were detected more clearly and dose-dependently. While BPA prevented the autoinduction of both TRalpha and TRbeta genes by T(3), the effect was less marked on TRalpha than on TRbeta. BPA also moderately suppressed RXRgamma gene expression. Gene expression of RXRgamma, a partner for heterodimer formation of TRs, was supressed by T(3) alone and also by BPA alone, but no additive effects were observed so far as studied. The present study indicates that a relatively low concentration of BPA, 10(-7) M, as compared with those examined previously (10(-5) to 10(-4) M) by us and other investigators, acts as an antagonist of T(3) through suppression of TRalpha and TRbeta gene expression in Xenopus tail in culture.

The 24 amino-acid residue antimicrobial peptide, brevinin-1 is synthesized in the skins of a wide range of species of Eurasian and North American frogs belonging to the genus Rana . All previously characterized brevinin-1 peptides contain the cyclic heptapeptide domain Cys 18 -(Xaa) 4 -Lys-Cys 24 at the COOH-terminus of the molecule. Four structurally related peptides were isolated from an extract of the skin of the Ryukyu brown frog Rana okinavana. The amino acid sequences of the peptides [Phe-(Xaa) 4 -Ile-(Xaa) 2 -Leu-Ala-Lys-Gly-Leu-Pro-Ser-Leu-Ile-Xaa-Leu-Xaa-Lys-Lys·NH 2 ] identified them as members of the brevinin-1 family that lacked the COOH-terminal cyclic domain but contained a C-terminally α-amidated residue. It is suggested, as one possibility, that the Cys 18 in the brevinin-1 consensus sequence has been deleted and the Cys 24 residue has mutated to a glycine that acts as substrate for peptidyl-glycine α-amidating monooxygenase. The peptides potently inhibited the growth of Escherichia coli and Staphylococcus aureus confirming that a cyclic domain is not necessary for antimicrobial activity. A fifth peptide (SFLNFFKGAA 10 KNLLAAGLDK 20 LKCKISGTQC 30 ), that also displayed broad-spectrum antimicrobial activity, was isolated from the skin extract and showed structural similarity with members of the ranatuerin-2 family previously isolated from the skin of North American ranid frogs.

An extract of the skin of Schlegel’s green tree frog, Rhacophorus schlegelii (Anura: Rhacophoridae), contained a protein that inhibited the growth of the Gram-negative bacterium Escherichia coli but was inactive against the Gram-positive bacterium Staphylococcus aureus. The protein was purified to near homogeneity by reverse-phase HPLC and amino acid sequence analysis of the products of an endoproteinase Glu-C digest identified the protein as histone H2B. The complete primary structure of the 125 amino acid residue Rhacophorus histone H2B was determined by nucleotide sequence analysis of a cloned cDNA encoding the protein. Mass spectrometry demonstrated that the protein isolated from the skin was not post-translationally modified. Histone fragments with antimicrobial activity were not identified in the Rhacophorus skin extract nor were cationic, α-helical antimicrobial peptides of the kind isolated from the skins of several other frog families. The data provide further evidence that histones play a role in the defense against microorganisms.

Two peptides with antimicrobial and cytolytic properties were purified from an extract of the skin of Tago's brown frog Rana tagoi. The primary structure of one peptide (FLPILGKLLS(10)GIL.NH(2)) identifies it as a member of the temporin family, whereas the second peptide (AIGSILGALA(10)KGLPTLISWI(20)KNR.NH(2)) displays 78% sequence identity to melittin from the venom of the honeybee Apis florea. Compared with melittin, the melittin-related peptide (MRP) was equipotent in inhibiting the growth of the Gram-positive bacterium Staphylococcus aureus, 5-fold less potent against the Gram-negative bacterium Escherichia coli and against the fungal pathogen, Candida albicans. MRP was 13-fold less hemolytic than melittin against human erythrocytes and 4- and 5-fold less cytolytic against mouse EL4 T-lymphoma-derived cells and L929 fibroblasts, respectively. However, at non-cytotoxic concentrations (or=8 microM), MRP did not protect HeLa cells from cell death produced by human rhinovirus type 2 infection.

During a large-scale in vitro translation analysis of a human full-length cDNA bank, we found many clones producing in vitro translation products showing ladder bands on a fluorogram with the equidistance of about 9 kDa at the position larger than the molecular mass expected from the open reading frame. We have analyzed a clone showing a typical pattern of the ladder bands. This clone encoded a 188-amino acid polypeptide containing a putative transmembrane domain. A green fluorescent protein-tagged polypeptide expressed in COS7 cells was localized in the endoplasmic reticulum and the Golgi apparatus. The ladder bands were observed in a rabbit reticulocyte lysate system, but not in a wheat germ extract system. Addition of the glutathione S-transferase-fused ubiquitin into the lysate caused upward shifts of the ladder bands. Addition of microsomal membranes prevented the formation of the ladder bands. Time course experiments demonstrated that the in vitro translation products increased in the presence of microsomal membranes, but were gradually degraded in their absence. These results suggest that the ladder formation resulted from the ubiquitination of misfolded polypeptide that failed to translocate to its proper position, and that an exclusion mechanism of misfolded membrane protein works in the rabbit reticulocyte lysate system.

During large-scale in vitro translation analysis of a human full-length cDNA bank, we found a clone producing a remarkably smaller translation product than that expected from the open reading frame. The cDNA encodes a polyubiquitin, UbC, composed of nine tandem repeats of the ubiquitin unit. The bank contained twelve UbC cDNAs including four full-length ones. Sequencing analysis of these clones showed that UbC cDNAs can be classified into two types, UbC1 and UbC2, in each of which there are six polymorphic nucleotide variations. The present UbC cDNA was in vitro translated in a rabbit reticulocyte or wheat germ extract to produce a free ubiquitin labeled with [35S]methionine. The labeled ubiquitin could be used as a substrate for thiol ester formation with ubiquitin-activating enzyme E1 or ubiquitin-conjugating enzyme E2.

The neurohypophysis of Xenopus and that of Ranidae and Bufonidae contain hydrin 1 (vasotocinyl-Gly-Lys-Arg) and hydrin 2 (vasotocinyl-Gly), respectively. In order to test the aldosterone-releasing activity of arginine vasotocin (AVT) and hydrin 1, purification of these peptides from an acid-extract of the neurointermediate lobe of Xenopus laevis was performed using an ODS-silica cartridge and reverse-phase and ion-exchange HPLC columns. As a result, an additional AVT-related peptide was newly found. Amino-acid analysis revealed that this peptide is vasotocinyl Gly-Lys (AVT-GK). The aldosterone-releasing activity of AVT-GK was equivalent to that of hydrin 1 (AVT-GKR) and lower than that of AVT. Like AVT and AVT-GKR, AVT-GK were effective in stimulating water flux from the isolated urinary bladder of the toad. Since AVT-GK is regarded as an intermediate between hydrin 1 and hydrin 2 in terms of its C-terminal form, it was designated hydrin 1′.

The joining peptide (JP) and the N- terminal peptide of proopiomelanocortin (NPP) were isolated from an acid-acetone extract of the distal lobe of the pituitary of the bullfrog, Rana catesbeiana, and purified by gel filtration and reverse-phase high performance liquid chromatography. The amino acid sequence of the bullfrog JP resembled the sequences of the JPs of Rana ridibunda (86% similarity) and Xenopus laevis (54% similarity), as deduced from the nucleotide sequences of their cDNAs. The amino acid sequence of bullfrog NPP showed 100%, 85%, and 50% similarity with those of Rana ridibunda, Xenopus laevis, and human NPPs, respectively. Administration of bullfrog NPP (0.05–5 μg/ml) to perifused Rana ridibunda interrenal slices induced a dose-dependent stimulation of corticosterone and aldosterone release. The present results indicate that the primary structure of NPP has been highly conserved during evolution. These data also reveal that NPP, which has no sequence homology with ACTH, exhibits a substantial corticotropic activity.

Two major components which stimulate aldosterone release from Xenopus adrenocortical tissue were isolated from an acid-acetone extract of the neurointermediate lobes of the bullfrog (Rana catesbeiana) using C18 Sep-Pak cartridges, Sephadex G-50, and reverse-phase HPLC columns. One of the components was identified as arginine vasotocin (AVT) from its HPLC profile and amino acid sequence analysis. The other was an AVT-like decapeptide with an extra glycine residue at the C-terminus of nonamidated AVT, which was recently termed hydrin 2. The yields of these two peptides were almost the same. They also showed equipotent activity in stimulating water flux from the isolated urinary bladder of the toad (Bufo japonicus).