Fine structure of the rhombencephalic tela of the bullfrog,Rana catesbeiana

Summary The posterior rhombencephalic tela choroidea of the bullfrog was examined by electron microscopy. This membrane, the pia-ependymal roof of the caudal hindbrain, contains a large central region characterized by cuboidal ependymal cells which surround sizable microscopic apertures — the interependymal pores.Ultrastructurally ependymal cells of this area are characterized by infrequent apical microvilli and cilia. They contain irregularly shaped nuclei and few cytoplasmic organelles that are largely apical in position. The most striking feature is an abundance of cytoplasmic filaments forming an extensive cytoskeleton. Laterally these cells are joined by numerous elaborate desmosomes. The majority of the ependymal cells have a basal lamina consisting of single, double, or triple laminae lying parallel to the basal plasma membrane.Several unusual specializations are seen at the margins of the interependymal pores. The ependymal cells have lateral cytoplasmic processes that form the actual border of each pore. These processes originate from the apical surface of the cell and partially enclose an elaborate network of basal lamina associated with the interependymal pores.These findings demonstrate microscopic apertures in the roof of the fourth ventricle in the bullfrog that are associated with an unusual form of supportive ependyma.     

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.     

牛蛙外周血细胞的形态学特点

本研究对雌雄牛蛙(Rana catesbeiana)外周血细胞的组成、形态、大小和数量进行了观察和统计。牛蛙外周血细胞由红细胞、白细胞以及血栓细胞组成,其中红细胞体积最大,平均大小(长径×短径)为(25.68±1.88)μm×(16.49±1.53)μm,扫描电镜下发现红细胞表面光滑;血栓细胞呈卵圆形或纺锤形,其体积最小,平均大小为(8.62±1.04)μm×(7.47±1.11)μm;白细胞由淋巴细胞、单核细胞、浆细胞、嗜中性粒细胞、嗜酸性粒细胞和嗜碱性粒细胞组成,扫描电镜下白细胞表面粗糙不平,有许多不规则的凸起。白细胞中淋巴细胞最多,其中小淋巴细胞约占白细胞的32.66%±4.29%,大淋巴细胞约占6.03%±1.54%;嗜碱性粒细胞最少,只占4.78%±0.83%;浆细胞胞体大小不一,常呈椭圆形,平均大小为(23.51±0.59)μm×(22.86±0.67)μm;此外,牛蛙外周血细胞中单核细胞、淋巴细胞和嗜碱性粒细胞的数量比例以及淋巴细胞和嗜碱性粒细胞的大小均有性别的差异(P0.05)。     

Developmental and diel profiles of plasma corticosteroids in the bullfrog,Rana catesbeiana

Corticosteroids synergize with the thyroid hormone (TH) at late metamorphic stages and might have a role in the hormonal regulation of amphibian metamorphosis. This role could be influenced by diel fluctuations, particularly if the peak of the plasma corticoids changed in relation to the TH peaks. Diel variation in plasma corticosteroids was studied in Rana catesbeiana prometamorphic and climax tadpoles on 18:6, 12:12 and 6:18 light:dark (LD) cycles. Cortisol (hydrocortisone; HC) and aldosterone (ALDO) exhibited different, but LD cycle-specific, circadian fluctuations at prometamorphosis, whereas corticosterone (CORT) was undetectable (less than 1.18 ng/ml). HC, ALDO and CORT rhythms became synchronous at early metamorphic climax on all LD cycles, although the cosinor-derived acrophases, which occurred around the time of the dark:light transition, shifted approximately 6 h earlier from 18L:6D to 6L:18D. On both 18L:6D and 12L:12D, the acrophase of HC changed little from prometamorphosis to climax, whereas that of ALDO underwent a major phase shift. On 6L:18D, both the ALDO and the HC acrophases shifted at climax. These LD cycle-specific phase shifts of the diel rhythms placed the acrophases of the corticoids in different phase relationships to that of the previously determined thyroxine (T(4)) acrophase at climax, and may partially explain the influence of the light regimen on metamorphic timing. The pronounced diel variations in the corticoid concentrations from the troughs to the peaks show that hormone levels are a function of the time of day and the environmental lighting regimen, which need to be taken into account in measuring the level of plasma hormones in amphibians. The 24-h means calculated from the data of all the sampling times showed that only plasma ALDO and CORT, but not HC, rose markedly at climax, although there were significant LD cycle-related differences in the mean levels of both HC and ALDO at prometamorphosis, and in HC at climax. Additional work sampling at mid-light showed that plasma CORT peaked at Stage XXIII, decreased at the end of climax, and remained low in the postmetamorphic froglet at 2.1 ng/ml. In the adult bullfrog, CORT was clearly the predominant corticosteroid at 34.3 ng/ml, whereas HC and ALDO levels were only approximately 1.3 ng/ml.     

Molecular cloning of preprocathepsin E cDNA from the stomach of bullfrog Rana catesbeiana

A cDNA library was constructed from a poly(A)⁺ RNA fraction of the gastric mucosa of bullfrog Rana catesbeiana. We cloned a cDNA encoding preprocathepsin E (Pre-Pro-CE) from the library. The present study is the first demonstration of the Pre-Pro-CE cDNA of lower vertebrate such as amphibian. Amino acid sequence deduced from the cDNA was compared with partial amino acid sequence determined by Edman degradation, suggesting that the cDNA comprises an open reading frame encoding a signal peptide (16 amino acids), a pro-sequence (33 amino acids) and a mature protein region (348 amino acids). Two consensus tri-peptide sequences (FDT and VDT) as active site and positions of seven cysteine residues were conserved in this amphibian CE. Although the bullfrog CE was deduced to contain one potential N-linked glycosylation site, its position (Asn¹³⁹-Leu¹⁴⁰-Thr¹⁴¹) was different from that of mammalian CEs. Molecular phylogenetic analysis showed that the bullfrog Pro-CE belongs to the typical Pro-CE group among various aspartic proteinases.     

Fine structure of the median eminence and the pars nervosa of the bullfrog,Rana catesbeiana

Summary The hypothalamic neurosecretory system of the bullfrog, Rana catesbeiana, was studied with light- and electron microscopy. The median eminence is roughly divided into two portions. The upper portion mostly consists of ependymal cells, glial cells and preoptico-hypophysial nerve tract, whereas in the lower portion, neurosecretory axons, glial cells, processes of glial and ependymal cells, and fine blood vessels of the hypothalamic portal vein are located. A part of the neurosecretory axons of the preoptico-hypophysial tract proceeds to the lower portion of the median eminence. These axons are arranged perpendicularly to the capillaries of the hypothalamic portal vein. The glial cells are densely located in the area of the median eminence where neurosecretory material is abundant. The neurosecretory material in the neurosecretory cells, their axons, the median eminence and the pars nervosa of the bullfrog shows a positive reaction to PAS treatment.The neurohemal area of the median eminence is occupied by many neurosecretory and non-neurosecretory axons, containing neurosecretory granules and/or synaptic vesicles. The axonal portions with the synaptic vesicles which are considered to be the nerve endings abut on the capillaries of the portal system. The size of synaptic vesicles in the axon terminals containing few neurosecretory granules is larger than those in the endings with many neurosecretory granules. Infrequently glial and ependymal processes are interposed between the nerve endings and the capillary wall.In the hilar region of the infundibulum, synapses are frequently observed between the thin fibers with or without neurosecretory granules and dendrites of non-neurosecretory neurons. The probable functions of these synapses are briefly discussed on the basis of our findings. Both in the hilar region of the infundibulum and in the pars nervosa, electron-dense neurosecretory granules of two different sizes were observed. The median eminence contains only one type of granules.The fine structure of the pars nervosa shows similar structures to those of the median eminence. Both in the median eminence and the pars nervosa, the fenestrated endothelium of the capillaries was frequently observed. The thick perivascular connective tissue space containing fibroblasts and collagen fibrils was observed both in the median eminence and the pars nervosa. Vesicles in the cytoplasm of the endothelial cells which appear to take a part in the transendothelial transport were observed.This investigation was supported in part by United States Public Health Service Research Grant, No. A-3678, to Hideshi Kobayashi from the National Institute of Arthritis and Metabolic Diseases and partly by a grant for Fundamental Scientific Research from the Ministry of Education of Japan. The authors wish to express their thanks to Prof. K. Takewaki for his kind encouragement.     

Adrenergic receptors and the regulation of vascular resistance in bullfrog tadpoles (Rana catesbeiana)

Summary Vascular adrenergic sensitivity to exogenous catecholamines was examined in tadpoles of the American bullfrog (Rana catesbeiana), ranging from stage III to XIV. Central arterial blood pressure was measured in decerebrate bullfrog tadpoles to determine a reasonable initial infusion pressure. Solutions of epinephrine and phenylephrine were infused into the vasculature of pithed tadpoles, and the resulting changes in vascular resistance (R _v) were used to construct log dose-response relationships. Epinephrine infusion produced a dose-dependent increase in R _v (EC₅₀=5.3·10^-7 M), which could be reversed by sodium nitroprusside (a smooth muscle relaxant) and blocked by phenoxybenzamine (an -adrenergic antagonist). Larval R _v also increased with infusion of the -agonist phenylephrine (EC₅₀=7.4·10⁸ M). Infusion of 10^-6 M isoproterenol (a -agonist) largely reversed the phenylephrine-induced increase in R _v. These results indicate that the capacity exists for both -mediated vasoconstriction and -mediated vasodilation early in bullfrog ontogeny. Neither initial R _v nor the responses to infused epinephrine or phenylephrine were significantly correlated to development over the range of larval stages used in this study.Abbreviations ECG electrocardiogram - EPI epinephrine - ISO isoproterenol - PHE phenylephrine - POB phenoxybenzamine - R _v vascular resistance - SNP sodium nitroprusside     

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.     

Biomechanics of vibration reception in the bullfrog,Rana catesbeiana

The opercularis system (OPS) of amphibians consists of an opercularis muscle that connects the shoulder girdle skeleton to the operculum, a movable element in the oval window of the otic capsule. The role of the OPS in reception of vibrations was examined in bullfrogs (Rana catesbeiana) tested in various postures that manipulated differential motion between the shoulder girdle (the origin of the opercularis muscle) and skull (including the inner ear). Amplitude and phase relationship of motions of the suprascapular cartilage of the shoulder girdle and the posterior skull were also measured during these tests. 1. Microphonic responses to vertical vibrations from 25-200 Hz were typically highest when frogs were in a normal, sitting posture with the head held off the vibrating platform. Responses from animals in which the head directly contacted the platform were often less (by up to 10 dB at certain frequencies). Responses from all test positions were highest at lower frequencies, especially between 50-100 Hz. 2. Suprascapular accelerations were typically highest in the normal, sitting posture, and at lower frequencies (50-75 Hz) were often greater than that of the vibrating platform by up to 8 dB. The shoulder girdle skeleton of the bullfrog is therefore readily affected by vertical substrate motion. 3. The amplitude of microphonic responses in the different test postures did not correspond well with head acceleration. Rather, response amplitude corresponded best with the absolute difference between shoulder and head motion. For example, in the normal posture, suprascapular motion was much greater than head motion, and responses were relatively high. If only the head was vibrated, head motion was high and shoulder motion low, and responses also were relatively high. If the head and body were vibrated together, their motions were similar, and responses to the same platform accelerations were often reduced. Phase differences between shoulder and head motions were small at the frequencies examined and may be of little functional significance. The importance of differences in shoulder and head motion suggests that the resulting differential motion of the operculum and inner ear fluids can produce waves that stimulate appropriate end organs (such as the saccule). 4. Removal of the opercularis muscle reduced responses up to 18 dB at certain frequencies in some of the test postures. The most significant reductions were observed in those postures with a significant difference between shoulder and head motion (such as the normal posture).(ABSTRACT TRUNCATED AT 400 WORDS)     

Solution structure of the cytotoxic RNase 4 from oocytes of bullfrog Rana catesbeiana

Cytotoxic ribonucleases with antitumor activity are mainly found in the oocytes and early embryos of frogs. Native RC-RNase 4 (RNase 4), consisting of 106 residues linked with four disulfide bridges, is a cytotoxic ribonuclease isolated from oocytes of bullfrog Rana catesbeiana. RNase 4 belongs to the bovine pancreatic ribonuclease (RNase A) superfamily. Recombinant RC-RNase 4 (rRNase 4), which contains an additional Met residue and glutamine instead of pyroglutamate at the N terminus, was found to possess less catalytic and cytotoxic activities than RNase 4. Equilibrium thermal and guanidine-HCl denaturation CD measurements revealed that RNase 4 is more thermally and chemically stable than rRNase 4. However, CD and NMR data showed that there is no gross conformational change between native and recombinant RNase 4. The NMR solution structure of rRNase 4 was determined to comprise three alpha-helices and two sets of antiparallel beta-sheets. Superimposition of each structure with the mean structure yielded an average root mean square deviation (RMSD) of 0.72(+/-0.14)A for the backbone atoms, and 1.42(+/-0.19)A for the heavy atoms in residues 3-105. A comparison of the 3D structure of rRNase 4 with the structurally and functionally related cytotoxic ribonuclease, onconase (ONC), showed that the two H-bonds in the N-terminal pyroglutamate of ONC were not present at the corresponding glutamine residue of rRNase 4. We suggest that the loss of these two H-bonds is one of the key factors responsible for the reductions of the conformational stability, catalytic and cytotoxic activities in rRNase 4. Furthermore, the differences of side-chain conformations of subsite residues among RNase A, ONC and rRNase 4 are related to their distinct catalytic activities and base preferences.     

Cytosolic and mitochondrial phosphoenolpyruvate carboxykinase of the bullfrog,Rana catesbeiana,liver

The mitochondrial and cytosolic phosphoenolpyruvate carboxykinase (GTP: oxaloacetate carboxy-lyase(transphosphorylating), EC 4.1.1.32) occurring in the bullfrog (Rana catesbeiana) liver were studied. The enzymes in the two intracellular compartments of both tadpole and adult frog liver were immunologically identical. Both radioactively-labelled forms of the mitochondrial and cytosolic phosphoenolpyruvate carboxykinase from bullfrog liver were imported at the same rate into intact mitochondria in vitro. The mitochondrial and cytosolic enzyme activities did not respond to the administration of glucagon, glucocorticoid, quinolinate and d-mannoheptulose which are known as enhancers of phosphoenolpyruvate carboxykinase, but were found to increase during natural metamorphosis. The former activity was markedly increased in the tadpoles treated with 3,5,3′-triiodothyronine. It was supposed that in the bullfrog liver the phosphoenolpyruvate carboxykinase localized in the mitochondria is of central importance in phosphoenolpyruvate synthesis from oxaloacetate     

Changes in serum lipoproteins during metamorphosis of the bullfrog,Rana catesbeiana

Serum lipoproteins of the bullfrog, Rana catesbeiana, were studied during metamorphosis. Adult bullfrog has essentially one lipoprotein, designated β-lipoprotein. This β-lipoprotein migrates during electrophoresis to β-globulin region and it has a low hydrated density such that it exhibits floatation in a solvent of density 1.063. On the other hand, tadpole serum has one more lipoprotein, designated as α-lipoprotein, in addition to the β-lipoprotein. The α-lipoprotein migrates to the α-globulin region in zone electrophoresis and corresponds to the so called high density lipoprotein judging from ultracentrifugal behavior. Serum α-lipoprotein disappears and β-lipoprotein content decreases during metamorphosis.     

Metabolism of C26 bile alcohols in the bullfrog, Rana catesbeiana

Metabolism of C26 bile alcohols in the bullfrog, Rana catesbeiana, was studied. [24-14C]-24-Dehydro-26-deoxy-5 beta-ranol (3 alpha,7 alpha,12 alpha-trihydroxy-27-nor-5 beta-cholestan-24-one) was chemically synthesized from [24-14C]cholic acid and incubated with bullfrog liver homogenate fortified with NADPH. 24-Dehydro-26-deoxy-5 beta-ranol was shown to be converted into both 26-deoxy-5 beta-ranol and 24-epi-26-deoxy-5 beta-ranol [(24S)- and (24R)-27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24-tetrols] in addition to 5 beta-ranol [(24R)-27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,26-pentol], which is the major bile alcohol of the bullfrog. [24-3H]-26-Deoxy-5 beta-ranol and [24-3H]-24-epi-26-deoxy-5 beta-ranol were prepared from 24-dehydro-26-deoxy-5 beta-ranol by reduction with sodium [3H] borohydride and administered respectively to two each of four bullfrogs by intraperitoneal injection. After 24 h, labeled 5 beta-ranol was isolated from the bile of the bullfrogs that received [24-3H]-26-deoxy-5 beta-ranol. In contrast little if any radioactivity could be detected in 5 beta-ranol or its 24-epimer after administration of [24-3H]-24-epi-26-deoxy-5 beta-ranol.     

An alkaline thiol proteinase in the liver mitochondria of bullfrog, Rana catesbeiana

The mitoplasts were prepared from bullfrog (Rana catesbeiana) liver mitochondria by treatment with digitonin and were then separated into the matrix and inner membrane fractions. The matrix fraction thus obtained was free of lysosomal contaminations and exhibited a distinct proteinase activity. pH dependency of the matrix proteinase activity measured in the presence and absence of iodoacetamide revealed that the matrix contained at least two kinds of proteinase, a major alkaline thiol proteinase having an optimal pH at 8.5 and a minor neutral proteinase having an optimal pH at 7.5. The major matrix proteinase activity was strongly inhibited by leupeptin, chymostatin, antipain and E64-C, an inhibitor of Ca2+-dependent thiol proteinase, while it was scarcely affected by diethylpyrocarbonate. The activity was also inhibited by DTNB and p-chloromercuribenzoate. Addition of hydrocarbon compounds such as ethylene glycol, glycerol, Triton X-100 and poly (ethylene glycol) to the reaction mixture was found to decrease the matrix proteinase activity. Neither cytochrome c nor glutamate dehydrogenase was hydrolyzed when subjected to the matrix proteinase activity in vitro. On the other hand, cytochrome c oxidase was effectively hydrolyzed, and the enzyme associated with the mitochondrial innermembrane fragments was partially hydrolyzed by the major matrix proteinase activity.     

On the mechanism of respiration in the bullfrog,Rana catesbeiana: A reassessment

The mechanism of respiration in the bullfrog has been analyzed by means of pressure recordings from the buccal cavity, the lungs and the abdominal cavity, by cinematography and cinefluorography, and by electromyography of buccal, laryngeal and abdominal muscles. Gas flow was investigated by putting frogs in atmospheres of changing argon and nitrogen content and monitoring the concentration of the nostril efflux. Three kinds of cyclical phenomena were found. (1) Oscillatory cycles consist of rhythmical raising and lowering of the floor of the mouth, with open nares. They have a definite respiratory function in introducing fresh air into the buccal cavity. (2) Ventilatory cycles involve opening and closing of the glottis and nares and renewal of a portion of the pulmonary gas. More muscles are involved and the pattern of muscular activity is more complex than in the oscillatory cycles. (3) Inflation cycles consist of a series of ventilation cycles, interrupted by an apneic pause. The intensity of the ventilatory cycles increases before this pause and decreases immediately thereafter. This results in a stepwise increase in pulmonary pressure, to a plateau (coincident with the pause) followed by a sudden or stepwise decrease. The respiratory mechanism depends on the activity of a buccal force pump, which determines pulmonary pressure whose level is always slightly less than the peak pressure values of the ventilation cycles. The elevated pulmonary pressure is responsible for the expulsion of pulmonary gas during the second phase of the next ventilation cycle. This pressure is maintained by the elastic fibers (and the smooth masculature) of the lungs.     

性类固醇激素受体ER、AR和PR在牛蛙胃肠胰的定位

本文用免疫组织化学技术检测及光密度定量分析,研究4种性类固醇激素受体在牛蛙(Rana catesbeiana)胃肠胰内的定位及表达的强弱,探讨4种性类固醇激素在牛蛙胃肠胰中的功能。染色结果显示,4种性类固醇激素受体在牛蛙胃肠胰内都有分布,雌雄之间分布差异较小。雌激素受体α(ERα)主要分布在牛蛙胃腺、直肠固有层和胰腺中;雌激素受体β(ERβ)主要分布在食道上皮、直肠固有层和胰腺中;雄激素受体(AR)主要分布在食道上皮、直肠固有层和胰腺中;孕激素受体(PR)主要分布在空肠、食道上皮和胰腺中。光密度检测结果显示,雌激素受体α(ERα)在牛蛙胃和直肠中阳性反应最强,在胰腺中相对较弱。雌激素受体β(ERβ)在牛蛙直肠中阳性反应最强,胰腺和食道次之。雄激素受体(AR)在食道中阳性反应相对较强,其他部位都较弱。孕激素受体(PR)在空肠中的阳性反应较强,其他部位都较弱。雌激素受体α(ERα)在胃中以及雄激素受体(AR)在食道和直肠中的免疫阳性反应均是雌性牛蛙强于雄性牛蛙,雌激素受体β(ERβ)在胰腺中的免疫阳性反应是雄性牛蛙强于雌性牛蛙。4种性类固醇激素受体中,雌激素受体α(ERα)和雌激素受体β(ERβ)在牛蛙胃肠胰中的分布最多,雄激素受体(AR)、孕激素受体(PR)的分布相对较少。性类固醇激素受体主要分布在食道、胃、直肠和胰腺中,其中,分布最多的部位是胃和直肠。4种性类固醇激素受体在牛蛙胃肠胰内的分布表明,性类固醇激素对牛蛙消化功能特别是胃和直肠的功能具有多方面的调节作用。     

Embryonic and larval hemoglobins during the early development of the bullfrog, Rana catesbeiana

The main hemoglobin (Hb) found in Shumway (embryonic) stage 25 bullfrogs is that which we have designated Td-4. The other major tadpole Hbs (Td-1, 2, and 3) predominate during Taylor and Kollros (larval) stages I-XVIII. We propose that Td-4 is an embryonic Hb, whereas Td-1, 2, and 3 are larval (fetal-like) Hbs. Embryonic Hb Td-4 continues to be synthesized during the larval stages. During the larval period, the average peripheral blood Hb profile changes very little with morphological stage or general growth. However, there is great heterogeneity in the embryonic:larval Hb ratio among individual tadpoles of a given stage or weight, apparently due to differential Hb and red cell production by the two active erythropoietic sites, mesonephric kidneys (Td-4), and liver (Td-1, 2, 3).