Previously, we have isolated authentic bombesin and another bombesin like peptide named bombesin like immunoreactivity (BLI)-K2 from the skin of Korean fire-bellied toad, Bombina orientalis. In the present study, we have newly purified three heterogeneous forms of BLI named BLI-K3, BLI-K4, and BLI-K5 from side fractions obtained in previous isolation of bombesin like peptide. The BLIs were separated into five peaks on a column of C18 preparative HPLC. Among them, three minor peaks containing BLI-K3, K4, and K5 were purified by means of sequential chromatography on the columns of SP cation exchange HPLC and C18 reverse phase HPLC. The purified BLI-K3 and K4 showed high binding affinity to an anti-bombesin serum (LBE 2G-2) with binding potency of 72 and 95%, respectively, relative to that of bombesin. However, they did not possess any distinctive biological activity of bombesin like peptide. On the contrary, the biological activity of BLI-K5 was similar to that of bombesin but its binding affinity to an anti-bombesin serum was low. The results indicate that three heterogeneous forms of BLI were coexpressed with bombesin and BLI-K2 in the skin of B. orientalis. All forms of the purified BLI in the present study were immunologically active but only BLI-K5 possessed the distinctive biological activity of bombesin like peptide.
Anura* ; Bombesin* ; Chromatography ; Chromatography, High Pressure Liquid ; Population Characteristics ; Skin*

The furcocercus cercariae of Neodiplostomum seoulense (Digenea: Neodiplostomidae) penetrate the skins of tadpoles and shed their tails. The speculated mechanism of this tail loss was physical efforts required to produce a vigorous zigzag motion during skin penetration; no other mechanism has been proposed. We examined the relationship between the host serum and cercarial tail loss. Cercariae of N. seoulense were collected from experimentally infected Segmentina hemisphaerula, and lots of 300 cercariae were cultured in medium 199 contained several types of sera. Cercarial tail degradation was induced in all media, but all the cercariae cultured except those cultured in media containing fetal bovine serum (FBS) died within 48 hr. After 72 hr cultivation in media containing FBS, cercarial tail degradation was induced in 67.0%; in continuous cultivation 13.3% of larvae survived for 7 days. Tail degradation did not occur in the absence of serum and when serum was heat inactivated at 56 degrees C for 30 min. The addition of 20 mM ethylenediaminetetraacetic acid (EDTA) blocked cercarial tail degradation completely. Moreover, the addition of 20 mM MgCl2 restored tail degradation blocked by EDTA. These results suggest that the alternative complement pathway is related with the N. seoulense cercarial tail degradation induced by serum.
Trematoda/*physiology ; Tail/*physiology ; Larva/parasitology ; Complement System Proteins/immunology/*physiology ; Anura/parasitology ; Animals

Electron microscopy on the skin of young frogs, Rana temporaria, has been carried out with particular reference to cellular attachment sites. For the first time now several technical developments allow a more detailed visualization of the fine structure within the cellular attachment sites as well as making it possible to show the ultrastructural morphology of the junctional complexes, and to demonstrate that the desmosomes are regularly distributed around each skin cell, especially in the S. granulosum. The relations of these findings to those of previous investigations concerning the functional organization of the junctional complexes and to the findings in skin cancer from a cellular adhesion view point have been briefly discussed.
Animal ; Anura ; Cell Membrane ; Epithelial Cells* ; Microscopy, Electron ; Skin/cytology* ; Skin Neoplasms/etiology

The purpose of the present study was to examine the kinetic process of hemoglobin (Hb) carrying and releasing oxygen. Under the standard conditions (pH 7.4, Po(2) 20 mmHg, 20 °C) the blood samples of chicken, rabbit, frog and carp were equilibrated in oxygen content analyzer with calibrated gas mixture A (0.5% CO(2) and 99.5% N(2)). Then the blood samples were exposed to gas mixture B (21% O(2), 0.5% CO(2) and 78.5% N(2)). After equilibration, the blood samples were exposed to gas mixture A again. During the whole process, Po(2) of blood samples was detected in real-time. The time spent in blood Po(2) changing from 0 to 21 kPa was recorded carefully. The results indicated that the kinetic curve of Hb carrying oxygen presented a shape of "S". It was similar to the Hb oxygen dissociation curve (Hb ODC). Based on the curve, T(50), a new kinetic parameter, was established. T(50) is the time of 50% O(2) saturation of Hb. It can reflect the efficiency of Hb carrying oxygen. Through comparing of T(50), the efficiency of Hb carrying oxygen among 4 species of animals was: frog < carp < rabbit < chicken. In the phase I of Hb carrying and releasing oxygen kinetic curve, the slope in carp was much larger than that in rabbit; the time [(1 411±6) s] of Hb releasing oxygen in chicken was longer than that in other 3 animals. These differences reflected the variety of efficiency of Hb carrying and releasing oxygen. In addition, the kinetic features of Hb carrying oxygen were likely to become an important index to evaluate the function of Hb carrying oxygen, especially in evaluating the ability of artificial blood substitute. On the basis of the analysis of the kinetic curve of Hb carrying oxygen and Hb ODC, another new important efficacy parameter E(50) was proposed. E(50) reflects the relationship between the time of 50% O(2) saturation of Hb and environmental Po(2). E(50) can be used as a synthetic index to assess the efficiency of Hb carrying oxygen.
Animals ; Anura ; Carps ; Chickens ; Hemoglobins ; metabolism ; Hydrogen-Ion Concentration ; Kinetics ; Oxygen ; metabolism ; Rabbits

OBJECTIVE: To quantitatively investigate the effects of Ringer's solution with different concentrations of alcohol (1%～80%) on biphasic compound action potentials (AP) from frog sciatic nerve trunk, and their recoveries from alcohol effects. METHODS: Individual segments of frog sciatic nerve trunk with a length of 6 to 8 cm were prepared. Ringer's solution with different concentrations of alcohol (0%, 1%, 2%, 4%, 8%, 16%, 32%, 48%, 64% and 80%) was applied onto the segment of the trunk between the stimulus and ground electrodes via an agent reservoir which was newly armed in a nerve trunk shielded chamber for 5 minutes. The nerve trunk was respectively electro-stimulated to generate the biphasic compound AP which was recorded using the experimental system of BL-420F. This was followed by 5 times washout plus 5 min administration with Ringer's solution before recovery recording of AP. RESULTS: Compared to normal Ringer's solution, Ringer's solution with alcohol at ≤4% did not have dramatic impacts on the AP amplitude and conduction velocity, while Ringer's solution with alcohol at ≥8% there was significant decrease in these two parameters. Ringer's solution with alcohol at the conentrations of 16%, 32% and ≥48% could prevent a small proportion (30%), a large proportion (90%) and all (100%) of sciatic nerve trunks, respectively, from generating AP. Washout with normal Ringer's solution after alcohol application at the concentration of ≤32%, AP could totally recover to normal status. While alcohol at the concentration of 48%, 64% and 80%, the probabilities to regenerate APs were 90%, 40% and 0%, and the AP amplitudes were decreased to 60%, 36% and 0%, respectively. After washout, AP conduction velocity showed no difference with alcohol at the concentration of ≤8% when compared with that before washout, while it could not be recovered to normal under alcohol at ≥16%. CONCLUSION Ringer's solution with different concentrations of alcohol exerts different effects on biphasic compound AP amplitude and conduction velocity. Hopefully, our findings could be helpful for the alcoholic usage and its recovery from alcoholic damage.
Action Potentials ; Animals ; Anura ; Ethanol ; pharmacology ; Ringer's Solution ; pharmacology ; Sciatic Nerve ; drug effects

This study was carried out to observe the ultrastructure of tongue mast cells in frog, Bombina orientalis. In the light microscopy, mast cells were round, oval, spindle-like and irregular shape and occasionally had one or more tail-like long cytoplasmic processes. Mast cells had many red granules stained with toluidine blue in the cytoplasm. In the electron microscopy, mast cells had membrane-bounded granules and poorly developed organelles. The granules were composed of two parts, low and dense electron density. In high magnification the dense part in the middle region of granule had honey comb-like crystalline structure.
Anura* ; Crystallins ; Cytoplasm ; Honey ; Mast Cells* ; Microscopy ; Microscopy, Electron ; Organelles ; Tolonium Chloride ; Tongue*

Full evidence and obvious reasons made it possible to arrive at the conclusion that the nature of transmission upon cerebrospinal neurons is overwhelmingly mechanical, not only in the periphery- between various receptors and afferent nerve terminals, and between surrounding tissues and free nerve endings- but also in the cerebral cortex. When viewed from the standpoint of the everchanging patterns of natural mechanical stimuli, the neurons in the conscious cerebral cortex and the pain endings in an acute inflammatory locus have the same situation very much in common. It is quite likely that natural mechanical stimuli dominate over cerebrospinal nervous phenomena and physiologists have been watching the missing mechanism at work in every experiment upon afferent nerve terminals and cerebral cortex that they have done. The terms "psychic tension" and "central excitatory state" comparable to muscular tonus are of interest because they involve the use of mathematical techniques in psychology and neurophysiology. They are capable of becoming weak or strong, and they serve as an inner stimulus to give impetus to behavior. Unfortunately, however, it is an elusive inner stimulus, and it defies a lucid definition. But natural mechanical stimuli embody the psychic tension and the central excitatory state ultimately. It seems now that we just found a place where constant complaints against neurophysiology and physiological psychology are ventilated. We may conclude that natural mechanical stimuli are the leading direct stimuli to cerebrospinal neurons in the human body, and the plastic and developmental nervous phenomena and mental phenomena can be explained objectively by a familliar datum of mechanical energy and that we can reasonably expect the day of regarding material world and spiritual world in the monistic conception of matter-energy system.
Animals ; Anura ; Cerebral Cortex/*physiology ; Human ; In Vitro ; Motor Neurons/physiology ; Nerve Endings/physiology ; Receptors, Sensory/*physiology ; Spinal Cord/*physiology

OBJECTIVETo study the bioaffinity between 8 bufadienolides(Bu) and tumor cells and analyze the correlation between the bioaffinity and the anti-tumor activities of Bu.

METHODMix and cultivate the chloroform extract of Chansu and MGC-803. Measure the content of 8 Bu in supernatant and cells using HPLC and calculate their affinity rate.

RESULTThe coefficient correlation between the decrease of Bu in cell supernatant after affinity and its MGC-803 restrictive activities, and between the cotent percentage of the free Bu in free cells with its MGC-803 restrictive activities, and between the difference between the decrease and the percentage and its MGC-803 restrictive activities is r = 0.82 (P < 0.05), r = -0.04 and r = 0.83 (P < 0.05) respectively.

CONCLUSIONEight Bu have different levels of affinity with MGC-803 which correlate with their anti-tumor activities.

Amphibian Venoms ; chemistry ; Animals ; Antineoplastic Agents ; isolation & purification ; pharmacology ; Anura ; Bufanolides ; isolation & purification ; pharmacology ; Cell Line, Tumor ; Chromatography, High Pressure Liquid ; methods ; Neoplasms ; drug therapy

In order to test the equilibrium solubility of puerarin in different solvents and solubilizer,cilia toxicity and irritation of these excipient, the balance method, toad in the ciliary body toxicity and rat nasal mucosa irritation were used respectively. Results showed that puerarin solubility was 56.44 g x L(-1) in combined solvent of 30% PEG200 and 10% Kolliphor HS 15. With normal saline solution as negative control and sodium deoxycholate as positive control, the effects of 30% PEG200, 30% PEG 400, 10% Kolliphor HS 15 and combination of 30% of PEG200 and 10% Kolliphor HS 15 on toad palate cilium were observed and cilia movement duration was recorded. The results indicated that there was no significant difference in cilia movement duration among 30% PEG200, 10% Kolliphor HS 15 and normal saline group. The rats long-term nasal mucous membrane irritation of 30% PEG 400, 10% Kolliphor HS 15, which had no cilia toxicity, was studied, with normal saline solution as negative control. There were no significant difference revealed on rat nasal mucosa epithelial thickness among 30% PEG 400, 10% Kolliphor HS 15 and normal saline. Above researches showed 30% PEG 400, 10% Kolliphor HS 15 was ideal for solubility of puerarin nasal drops and showed a lower cilia toxicity and irritation, and can be used as the solvent and solubilizer of puerarin nasal drops.
Administration, Intranasal ; methods ; Animals ; Anura ; Cilia ; chemistry ; Female ; Isoflavones ; chemistry ; Male ; Nasal Mucosa ; Polyethylene Glycols ; chemistry ; Rats ; Rats, Sprague-Dawley ; Solubility ; Solvents ; chemistry

OBJECTIVETo explore a new experimental method for screening of allergens in post-market traditional Chinese medicine injections by confirming allergens in Huachansu injection.

METHODFirst of all, the serum of patients allergic to Huachansu injection were collected, at the same time, the dubious allergen was conjugated to bovine serum albumin (BSA) by EDC coupling procedure to form complete antigen (BNP-BSA), which makes it possible to reproduce the allergic reaction of Huachansu injection in vitro. The histamine liberation ratio, the level of TNF-alpha and Histamine released from RBL-2H3 mast cell were detected; the above data were compared with those obtained in vivo.

RESULTThe difference of the histamine liberation ratio, the levels of TNF-alpha and histamine of the resibufogenin-BSA group, group of patients allergic to Huachansu injection were not significant compared with those of normal control group. However, there were significant difference in those data among the cinobufagin-BSA group, the blank control and normal control group (P<0.05).

CONCLUSIONThe allergen in the serum collected from patients allergic to Huachansu injection is resibufogenin.

Allergens ; adverse effects ; immunology ; Amphibian Venoms ; adverse effects ; immunology ; Animals ; Anura ; Bufanolides ; adverse effects ; immunology ; Drug Hypersensitivity ; immunology ; Histamine Release ; Humans ; Mast Cells ; immunology ; Medicine, Chinese Traditional