The alpha-subunit of glycoprotein hormones exists in the prolactin secretory granules of the bullforg (Rana catesbeiana) pituitary gland

Summary Our recent finding that the number of immunoreactive -subunit cells was invariably greater than the total number of immunoreactive gonadotropin (GTH) and thyrotropin (TSH) cells in the bullfrog (Rana catesbeiana) pituitary gland raises the possibility that the -subunit also exists in pituitary cells other than GTH and TSH cells. The present study demonstrates that there are a considerable number of immunoreactive prolactin (PRL) cells that are also stained with antibody against the -subunit when adjacent sections are immunocytochemically examined. Neither immunoreactive growth hormone nor adrenocorticotropin cells are stained with the antibody against the -subunit. The specificity of the antibody against the -subunit and of that against PRL was demonstrated by preabsorption test, non-competitive binding test, and immunoblot analysis. Double-immunolabeling with gold particles of different sizes for the -subunit and PRL revealed that most of the immunolabeled PRL-secretory granules are also labeled with the -subunit antibody. The gold particles indicating the presence of the -subunit were mostly found in the peripheral zone of the secretory granules.     

Molecular cloning and expression of prohormone convertases PC1 and PC2 in the pituitary gland of the bullfrog, Rana catesbeiana

We cloned cDNAs encoding PC1 and PC2 from a cDNA library constructed for the anterior pituitary gland of the bullfrog (Rana catesbeiana) and sequenced them. The bullfrog PC1 cDNA consisted of 2972 base pairs (bp) with an open reading frame of 2208 bp and encoded a protein of 736 amino acids, including a putative signal peptide of 26 amino acids. The protein showed a high homology to R. ridibunda PC1 (95.1%) and mammalian PC1 (72.6%). The bullfrog PC2 cDNA consisted of 2242 bp with an open reading frame of 1914 bp and encoded a protein of 638 amino acids, including a putative signal peptide of 23 amino acids. This protein showed a high homology to R. ridibunda PC2 (95.5%) and mammalian PC2 (84.8%). The catalytic triad of serine proteinases of the subtilisin family was found at Asp-168, His-209, and Ser-383 in the PC1 protein and at Asp-167, His-208, and Ser-384 in the PC2 protein. In situ hybridization staining revealed that PC2 mRNA was detected in corticotrope cells of the tadpoles, but not in those of the adults. In the adult, only PC1 mRNA was detected in the pars distalis but both PC1 and PC2 mRNAs were detected in the pars intermedia. The data also showed that PC1 mRNA was expressed in gonadotrope cells.     

Presence of sauvagine-like epitopes in the interrenal gland of the bullfrog Rana catesbeiana

Immunocytochemistry was used to investigate the presence of corticotropin-releasing factor-like peptides in the interrenal (adrenal) glands of the bullfrog Rana catesbeiana by using specific antisera raised against synthetic nonconjugated rat/human corticotropin-releasing factor, urotensin I, and sauvagine. From these three antisera, covering a broad range of corticotropin-releasing factor-like immunoreactivities, only the sauvagine antiserum gave positive immunoreactivity. Sauvagine immunoreactivity was found in cortical cells grouped into cords in the renal zone of the interrenal gland. The central and subcapsular cords were less stained. Tyrosine hydroxylase-positive chromaffin cells were not sauvagine-immunoreactive. The immunoreactivity was abolished, in all cases, by previous immunoabsorption of the sauvagine antiserum with synthetic sauvagine (0.1 7M), but it was not eliminated by sucker (Catostomus commersoni) urotensin I, sole (Hippoglossoides elassodon) urotensin I, sucker corticotropin-releasing factor, rat/human corticotropin-releasing factor, or ovine corticotropin-releasing factor (0.1-10 7M). In a sauvagine radioimmunoassay, interrenal extracts displaced ¹²⁵I-sauvagine from antiserum only partially, and not in parallel with the sauvagine standard curve. The results suggest that the sauvagine immunoreactivity in the R. catesbeiana interrenal gland may represent a novel sauvagine-like peptide.     

Immunocytochemical localization of secretory phospholipase A(2)-like protein in the pituitary gland and surrounding tissue of the bullfrog, Rana catesbeiana.

Previously, we obtained a protein that has considerable amino acid sequence homology with secretory phospholipase A(2) (PLA(2)) from a bullfrog pituitary fraction obtained during the purification of thyrotropin (TSH). Subsequently, partial amino acid sequence (N-terminal 45 amino acid residues) analysis revealed this protein to be identical to the N-terminal amino acid sequence of otoconin-22, the major protein of aragonitic otoconia in the Xenopus saccule. In this study we developed an antibody against the N-terminal peptide of the bullfrog protein and applied it for immunocytochemical study of the pituitary and its surrounding tissue. Western blotting analysis showed that this antibody recognizes a 20.4-kD protein that has a molecular mass close to that of otoconin-22. Immunohistochemical reaction with the antibody was not found in any anterior pituitary cells but was intense in the monolayer epithelial cells of the endolymphatic sac surrounding the pituitary gland, which is a major storage site of calcium carbonate in amphibians. An electron microscopic study revealed that the cuboidal cells in the endolymphatic sac contained large, polymorphic secretory granules in their apical cytoplasm. Immunogold particles indicating the presence of a PLA(2)-like protein were observed predominately in these secretory granules. These findings support the view that this PLA(2)-like protein obtained during purification of TSH was derived from the endolymphatic sac adhering to the pituitary and that this protein is a bullfrog otoconin. (J Histochem Cytochem 49:631-637, 2001)     

Glomerular permeability in the bullfrog Rana catesbeiana

Filtration studies suggest similar size pores in the glomerular filters of mammals and amphibians. However, the glomerular wall in the bullfrog exhibits several structural features not found in mammals. The subendothelial space of the basement membrane is often greatly enlarged and infiltrated by cellular elements. The lamina densa of the basement membrane shows extensive variation in thickness and packing of its filaments. On the other hand, the epithelial slits in the bullfrog are closed by a slit diaphragm which appears similar in size and structure to the slit diaphragm in mammals. Horse spleen ferritin, a protein with a hydrodynamic radius of 61 A, was used as an ultrastructural tracer to determine whether the highly variable structure of the basement membrane renders this layer more permeable than its mammalian counterpart. Within 10 min after intravenous injection, ferritin was found throughout the basement membrane and often in clusters within the subepithelial layer adjacent to the slit diaphragm. Virtually no ferritin was found within the urinary space, podocytes, or cells of the proximal tubule. Ferritin distribution was the same in both superficial glomeruli and more deeply lying glomeruli regardless of the method of fixation. These results indicate that in the bullfrog the slit diaphragm is a principal filtration barrier to ferritin and thus to smaller plasma proteins.     

Synergistic cytotoxicity of Rana catesbeiana ribonuclease and IFN-gamma on hepatoma cells

RC-RNase purified from Rana catesbeiana (bullfrog) oocytes is a pyrimidine-guanine sequence-specific ribonuclease. RC-RNase is derived from the RNase superfamily genes exerting distinct ribonucleolytic activity and possesses cytotoxicity to tumor cells, but rarely to primary cells. In this study, we utilized RC-RNase to function with antiproliferative cytokines. The combination with TNF-alpha or TNF-beta would not aggravate cell death. However, the combination with IFN-gamma could induce synergistic cytotoxicity verified by XTT assays toward three hepatoma cell lines bearing different differentiation stages. The distinct cytotoxicity from RC-RNase or RC-RNase/IFN-gamma on different hepatoma cells was correlated with the differentiation extent but not the proliferation rate of the cells. Despite the synergistic cytotoxicity and severe mitochondrial disruptions in the RC-RNase/IFN-gamma-treated cells, we scarcely detected any significant feature of apoptosis or necrosis by FACS analysis on annexin-V/propidium iodide staining. The mechanisms of cell death triggered by RC-RNase or RC-RNase/IFN-gamma require further investigation.     

Involution of gonadotrophic cells of the pituitary gland in the male frog, Rana esculenta, after section of the pituitary stem]

One and two months after section of the hypophyseal stalk in the male frog. Rana esculenta, and involution of numerous gonadotrophic cells, contrasting with the normal aspect of the eosinophilic cells can be observed.     

Dye-coupling among frog (Rana catesbeiana) taste disk cells

• 1.1. Dye-coupling among taste disk cells in the bullfrog fungiform papillae was examined histologically by injecting a fluorescent dye (Lucifer yellow) into the cell, and the effects of the dye-coupling on depolarizing responses induced by taste stimuli were studied electrophysiologically.
• 2.2. With dye injection into a taste cell, dye-coupling was found between taste cells (23%) or between taste cell and supporting cell (28%). With dye injection into a supporting cell, dye-coupling was found between supporting cells (34%) or between supporting cell and taste cell (27%).
• 3.3. Depolarizing responses recorded from either a taste cell or a supporting cell to stimulation with 0.5 M NaCl or 10 mM quinine-HCl were the same in amplitude whether the dye-coupling to another cell was present or not. On the other hand, depolarizing responses recorded from a taste cell for 0.5 mM acetic acid became significantly larger when dye-coupled to a supporting cell.
• 4.4. It is concluded that gustatory transduction for acid stimuli is influenced by supporting cells coupled to taste cells.

     

Tympanic sound radiation in the bullfrog Rana catesbeiana

Members of the Rana catesbeiana clade display sexually dimorphic eardrums. In this species assemblage the eardrum of males can be 50% larger than in females of the same body size. There has been, however, no apparent functional explanation for this dimorphism. Measurements of the acoustical coupling (transfer function) of internally generated sound to the enlarged eardrum of male bullfrogs (R. catesbeiana) show distinct energy peaks coincident with those observed in the spectral envelopes of the release and mating calls. Moreover, when the tympanic membranes are artificially damped the spectrum of the release call is drastically altered and the total amount of power radiated decreases substantially. These observations point to a previously unsuspected role for the ears in the sound broadcasting process of the bullfrog and possibly other anurans with similarly modified tympanic membranes. Accepted: 19 July 1997     

Determination of hemoglobin expression patterns in erythroid cells of Rana catesbeiana tadpoles

1. Rana catesbeiana (bullfrog) tadpoles are heterogeneous in the relative amounts of four major tadpole hemoglobins (Hbs), as well as in the relative amounts of two tadpole red blood cell types in the peripheral blood. 2. Previous work has shown that this heterogeneity is present at all stages of larval development and growth. 3. Although some tadpoles lack one of the Hbs in their peripheral blood (i.e. the electrophoretically slowest form, Td-4), the missing Hb can be found in the erythropoietic organ from which it emanates (the kidneys), indicating that the heterogeneity results from quantitative differences in gene expression. 4. We wished to know whether this in vivo regulation is subject to external environmental perturbation and report that tadpoles of known Hb phenotypes regenerate precisely the pre-anemia Hb profile during early as well as late stages of recovery from phenylhydrazine-induced anemia. 5. These and other results indicate that the in vivo mechanism for regulating the pattern of Hb expression has become firmly determined in the erythropoietic system by the earliest larval stage of development.     

Ultrastructure of the cortical cell of the interrenal gland of the American bullfrog (Rana catesbeiana)

Summary The cortical cell of the interrenal gland of the American bullfrog, Rana catesbeiana, was examined in the electron microscope. These cells occur in small groups and cords and are quite irregular in shape. The cortical cell is reminiscent of adrenal cortical cells from other vertebrates. Liposomes are variable in size and density. Smooth endoplasmic reticulum is scant in amount and predominantly of the fine tubular type. Mitochondria have vesicular cristae, a dense matrix, and occasionally have blebs, vacuoles, and myelin-like whorls at their surfaces. Intimate morphological relationships are found among the Golgi apparatus, lysosomes, and liposomes, and among Golgi vacuoles, mitochondria, and liposomes. In addition microfibrils are a prominent feature of the cortical cell. The biochemical events of steroidogenesis in amphibia and other vertebrates are discussed in relationship to the organellar interrelations found in the bullfrog interrenal cortical cells. Based on the available chemical and morphological information a scheme is proposed of movement of the steroidal intermediates through the cell that tentatively identifies the localizations of the various enzyme systems involved in corticosteroidogenesis from acetate to corticosterone and aldosterone.Supported in part by N.I.H. Grant RR 06138. Health Sciences Advancement Award.     

Pharmacokinetics of kanamycin in the bullfrog, Rana catesbeiana

1. Kanamycin disposition was studied in bullfrogs (Rana catesbeiana) following single doses IP. Both plasma t1/2 and Vd of the drug increased with increasing time after drug indicating redistribution and tight binding of kanamycin to deep tissue compartments. 2. Kanamycin was eliminated unchanged with a t1/2 plasma = 27 hr; perilymph = 89 hr; endolymph = 183 hr; aqueous humor = 54 hr; and CSF = 58 hr. 3. Kanamycin was absorbed by frogs from environmental water. 4. Environmental conditions must be carefully specified and monitored, as well as the physiological state of the animals when studying the effects of drugs on Amphibia.     

Ultrastructure of the synaptic ribbons in photoreceptor cells of Rana catesbeiana revealed by freeze-etching and freeze-substitution

Summary The three-dimensional structure of synaptic ribbons in photoreceptor cells of the frog retina was studied with freeze-etching and freeze-substitution methods, combined with a rapid-freezing technique. Although the synaptic ribbon consisted of two electron-dense plaques bisected by an electron-lucent layer in conventional thin sections, such lamellar nature was not so evident in freeze-etched replicas. The cytoplasmic surfaces of the synaptic ribbon presented an extremely regular arrangements of small particles 4–6 nm in diameter. Fine filaments 8–10 nm in diameter and 30–50 nm in length connected synaptic vesicles and the ribbon surface. These connections were mediated by large particles on both ends of the filaments. Approximately 3–5 filaments attached to one synaptic vesicle. Synaptic ribbons were anchored to a characteristic meshwork underlying the presynaptic membrane via another group of similar fine filaments. The meshwork seemed to be an etched replicated image of the presynaptic archiform density observed in thin sections.     

Arachnoid mater of the bullfrog,Rana catesbeiana

Summary In the bullfrog, the meninges surrounding the central nervous system include an arachnoid mater that contains layers of cells with abundant intermediate filaments (IFs) having unique organizational characteristics. This membrane contains an inner lamina of cells that resemble fibroblasts and an outer lamina of flattened cells that are almost filled with IFs. The IFs of the outer arachnoid are arranged in compact, arching bundles that lie parallel to the outer surface of the central nervous system. Thus, sections cut tangentially to the membrane reveal bending of filament bundles, whereas transverse sections do not. In some cells bordering the subdural space, bundles of filaments are organized into highly-ordered spiral arrays. Attachments to the numerous desmosomes and, apparently, to the nuclear envelope suggest anchoring of cytoplasmic structures by the IF system. Microtubules occur primarily near the plasma membrane and the nucleus. Numerous caveolae also are associated with the plasma membrane.The unusual abundance, organization, and cytoplasmic relations of IFs in the bullfrog arachnoid suggest that this membrane may serve as an important model for study of fundamental cytoskeletal relations and function.     

Caspase activation in response to cytotoxic Rana catesbeiana ribonuclease in MCF-7 cells

The thylakoid (Delta)pH-dependent pathway transports folded proteins. Identified components include Hcf106 and Tha4. Orthologs of these proteins plus a membrane protein called TatC are essential for the homologous bacterial Tat system. Here we report identification of a chloroplast TatC (cpTatC). cpTatC is an integral thylakoid membrane protein as determined by in vitro chloroplast import and immunoblotting. Antibody to cpTatC specifically inhibited the thylakoid (Delta)pH-dependent pathway in vitro. cpTatC is present in about the same quantity as estimated translocation sites, whereas Hcf106 and Tha4 are present in 5-8-fold excess. These results are relevant to mechanistic models for this system.     

牛蛙(Rana catesbeiana)染色体研究

对牛蛙（Rana catesbeiana,Shaw）的染色体组型进行了研究。观察骨髓的C-中期细胞,结果证明牛蛙的体细胞染色体数目2n=26,其中有5对大型染色体和8对小型染色体,可以分成A、B、C3个组,雌性和雄性个体间没有发现异型性染色体。上述结果与前人的研究结果基本一致。但是作者发现牛蛙骨髓细胞的第7、8、10、12号染色体上均有次缢痕。牛蛙的核型为2n：26=22m＋4sm。     

Localization of brush border cytoskeletal proteins in gastric oxynticopeptic cells from the bullfrog Rana catesbeiana

The contribution of brush border cytoskeletal proteins (actin, villin, fimbrin, and brush border myosin-1) to organization of the cytoskeletal network underlying apical plications of oxynticopeptic cells was examined by immunohistochemical techniques in frozen sections of gastric mucosa from the bullfrog, Rana catesbeiana. Apical localization of F-actin with phalloidin in oxynticopeptic cells inhibited with cimetidine revealed small, punctate domains within the apical cytoplasm that were consistent with the presence of short microvilli revealed by electron microscopy. Localization of F-actin in cells stimulated with forskolin was limited to a wide continuous band of cytoplasm corresponding to the location of numerous long surface folds. Inhibition of protein synthesis with cycloheximide did not prevent acid secretion or formation of actin filaments within surface folds in stimulated oxynticopeptic cells, suggesting that the formation of filaments does not require actin synthesis. Staining of gastric mucosae with fluorescent DNase-1 demonstrated that oxynticopeptic cells possess an unusually large pool of non-filamentous actin. Taken together, these results suggest that actin-filament formation in stimulated cells occurs by polymerization of an existing pool of non-filamentous actin. Localization of antibodies specific for villin and fimbrin revealed that these proteins were present within intestinal absorptive cells and gastric surface and neck cells but were not present within inhibited or stimulated oxynticopeptic cells. Brush border myosin-1, present in intestinal absorptive cells, was not present in gastric epithelium. Thus, we propose that actin-containing projections in oxynticopeptic cells are not organized like intestinal microvilli and that filament formation occurs after stimulation by modulating intracellular pools of filamentous and non-filamentous actin.