Growth potential of Rana tigerina skin lipids in cell cultures

Summary This study involves the investigation of the lipid composition of the skin of Rana tigerina which has a significant healing capacity. The results indicated that the lipid extract enhanced keratinocyte and fibroblast cell proliferation progressively and were found to be much more efficient in comparison to agents known to cause cell proliferation and to be anti-inflammatory such as hydrocortisone. Cell proliferation was dose dependent and suppression occurred only at very high doses. [³H]thymidine incorporation studies produced the same results. Because proliferation, migration, and differentiation of the basal cells is essential for initiation and progression of wound healing, any agent enhancing their proliferation would hasten the healing process. This paper therefore aims at elucidating the effect of composition of the total lipid extract confirming the efficacy of frog skin in wound healing and thereby providing an understanding of the natural mechanism of healing.     

A lectin from the thigh muscle of Rana tigerina

Lectin activity has been detected in the thigh muscle extracts of Rana tigerina, which was found to agglutinate both trypsinized and untrypsinized rabbit erythrocytes. The lectin has been purified to homogeneity by MEPBS (0.01 M phosphate-buffered saline (pH 7.2) with 4 mM beta-mercaptoethanol) buffer extraction of the tissue and affinity chromatography on acid-treated Sepharose 6B. The molecular weight (Mr) of the purified lectin was determined by SDS-polyacrylamide gel electrophoresis and gel filtration on Sephadex G-75, which gave values of 15,500 +/- 1000 and 32,000 +/- 1000, respectively, suggesting that the lectin is a dimer. Amino acid composition data of the lectin has revealed that it contains a high proportion of glycine and alanine, and low amounts of sulphur-containing amino acids. Hapten-inhibition study of this lectin has shown that it is galactose-specific. Hemagglutination activity of the lectin can also be inhibited by beta-galactoside containing oligosaccharides.     

Tigerinins: novel antimicrobial peptides from the Indian frog Rana tigerina

Four broad-spectrum, 11 and 12 residue, novel antimicrobial peptides have been isolated from the adrenaline-stimulated skin secretions of the Indian frog Rana tigerina. Sequences of these peptides have been determined by automated Edman degradation, by mass spectral analysis and confirmed by chemical synthesis. These peptides, which we have named as tigerinins, are characterized by an intramolecular disulfide bridge between two cysteine residues forming a nonapeptide ring. This feature is not found in other amphibian peptides. Conformational analysis indicate that the peptides tend to form beta-turn structures. The peptides are cationic and exert their activity by permeabilizing bacterial membranes. Tigerinins represent the smallest, nonhelical, cationic antimicrobial peptides from amphibians.     

Role of temperature in regulation of ovarian cycle in bull frog Rana tigerina

Effect of temperature on the ovarian cycle was studied in R. tigerina by exposing them to (1) constant low (22 degrees C) temperature during preparatory (active vitellogenic growth) phase (March-May) when the mean ambient temperature ranged from 26 degrees-28 degrees C and (2) to constant high (30 degrees +/- 1 degrees C) temperature during postbreeding regression phase (August-November) when the mean ambient temperature ranged from 22 degrees-26 degrees C. The ovaries of initial controls (biopsy samples taken prior to the commencement of the experiment) in March contained only first growth phase (FGP) oocytes with a maximum size range of 361-480 microns in diameter. In the frogs exposed to constant low temperature for 2 months, only 7% of FGP oocytes were recruited to second growth phase (SGP) with a mean largest diameter of 631 microns compared to 31% large SGP oocytes with a mean diameter of 1114 microns in the frogs collected from natural fields. The number of atretic follicles (AF) was lower and fat body weights were significantly higher in low temperature exposed frogs. The exposure of the frogs to constant high temperature during postbreeding months caused an increase in the mean diameter and number of large FGP oocytes, numerical increase in AF and decrease in fat body weights over corresponding controls maintained at room temperature. The pituitary gonadotrophs of these frogs showed stimulatory changes such as increase in cell size and appearance of secretory granules in the cytoplasm. The results suggest that in R. tigerina high temperature stimulates oocyte growth while low temperature retards it.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chromatin organization and basic nuclear proteins in the male germ cells of Rana tigerina

The process of chromatin condensation during spermiogenesis in Rana tigerina is similar to the heterochromatization in somatic cells, where 30 nm fibers are coalesced together into a dense mass in spermatozoa without changing their initial size and nucleosomal organization. This conclusion was supported by the finding that the full set of core histones (H2A, H2B, H3, H4) are still present in sperm chromatin, but histone H1 is replaced by its variant, H1V. Rabbit anti-sera were raised against histone H3, H1, H1V, and H5 (H1 variant in chick erythrocyte). Anti-histone H1 antiserum cross-reacted with histone H1V, which implied the presence of a common epitope. Anti-histone H1V and H5 also showed cross-reaction with each other but not with histone H1, which implied the presence of a common epitope not shared by histone H1. Immunocytochemical studies, using the above antibodies as probes, showed that histones H3 is present in all steps of spermatogenic and spermiogenic cells, and somatic cells including red blood cells, Sertoli cells, and Leydig cells, while histone H1 is present in all of the cells mentioned except in spermatozoa where it is replaced by histone H1V. Histone H1V appears in the early spermatids starting from spermatid 1 (St1), and it persists throughout the course of spermatid differentiation into spermatozoa. Histone H1V is also found in chromosomes of metaphase spermatocyte and red blood cells. Thus histone H1V may cause the final and complete condensation of chromatin in Rana spermatozoa, a process which is similar to the heterochromatization occurring in somatic cells such as metaphase chromosome and chick erythrocyte nucleus.     

Virulence properties of motile aeromonads isolated from farmed frogs Rana tigerina and R. rugulosa

Virulence factors were compared in Aeromonas species isolated from clinically normal and septicaemic farmed frogs from Thailand. Haemolysin activities against frog erythrocytes were significantly different within the collection of aeromonads. Groups of high haemolytic activity (unspeciated Aeromonas, Au), moderate haemolytic activity (A. hydrophila), and low haemolytic activity (A. veronii biovar sobria, A. veronii biovar veronii, A. caviae, A. schubertii) were noted. DNA colony hybridisation studies revealed that Au isolates possessed a haemolysin gene (ASH1) which was not present in any of the other Thai aeromonads or type strains tested. Elastinolytic activity was demonstrated in 90% of the Au isolates, 60% of the A. hydrophila isolates and in none of the other motile aeromonads. The cytotoxic activity of the Aeromonas isolates varied according to the source of cells used in the assays. Cells from rainbow trout were extremely sensitive to Au toxins but less so to toxins produced by other species. In contrast mammalian cells showed very little sensitivity to Au toxins but were more sensitive to toxins produced by A. hydrophila. Selection of suitable assay substrates is therefore important.     

Mediation of cholino-piperine like receptors by extracts of Piper nigrum induces melanin dispersion in Rana tigerina tadpole melanophores

Aim: The present study was carried out to determine the effects of lyophilized dried fruit extracts of Piper nigrum and pure piperine on the tadpole melanophores of frog Rana tigerina which offer excellent in vitro opportunities for studying the effects of pharmacological and pharmaceutical agents. The nature of specific cellular receptors present on the neuro-melanophore junction and their involvement in pigmentary responses has been explored.

Material: Effects of lyophilized extracts of P. nigrum and pure piperine were studied on the isolated tail melanophores of tadpoles of the frog R. tigerina as per the modified method.

Results: The extract of P. nigrum and its active ingredient piperine caused significant melanin dispersal responses leading to darkening of the tail melanophores, which were completely antagonized by atropine and hyoscine. These per se melanin dispersal effects were also found to be markedly potentiated by neostigmine an anticholinesterase agent.

Conclusion: It appears that the melanin dispersal effects of the extracts of P. nigrum and pure piperine leading to skin darkening are mediated by cholinergic muscarinic or piperine-like receptors having similar properties.     

Studies on type I collagen in skin fibroblasts cultured from twins with lethal osteogenesis imperfecta

Studies on type I procollagen produced by skin fibroblasts cultured from twins with lethal type II of osteogenesis imperfecta (OI) showed that biosynthesis of collagen (measured by L-[5-(3)H]proline incorporation into proteins susceptible to the action of bacterial collagenase) was slightly increased as compared to the control healthy infant. SDS/PAGE showed that the fibroblasts synthesized and secreted only normal type I procollagen. Electrophoretic analysis of collagen chains and CNBr peptides showed the same pattern of electrophoretic migration as in the controls. The lack of posttranslational overmodification of the collagen molecule suggested a molecular defect near the amino terminus of the collagen helix. Digestion of OI type I collagen with trypsin at 30 degrees C for 5 min generated a shorter than normal alpha2 chain which melted at 36 degrees C. Direct sequencing of an asymmetric PCR product revealed a heterozygous single nucleotide change C-->G causing a substitution of histidine by aspartic acid in the alpha2 chain at position 92. Pericellular processing of type I procollagen by the twin's fibroblasts yielded a later appearance of the intermediate pC-alpha1(I) form as compared with control cells.     

泽蛙的骨骼系统研究

研究我国产泽蛙（Rana limnocharis）的骨骼系统,包括对各部分形态和结构的详细描述。蝶筛骨结构特别,与已报道的粗皮蛙（R．rugosa）很不相同;第8椎体双凹型,属参差型椎体;左上喙骨在中央区部分重叠在右上喙骨之外边缘上,显示为不完全的固胸型,肩胸骨基部分叉;两性成体的掌、指以及踱、趾各关节下瘤的皮下均有一游离的小骨块,但1龄幼体在该位处则缺如,目前尚未见到有关此小骨块的资料。     

Studies on the effect of the pyrethroid insecticide Decamethrin on ionic transport through the in vitro skin of Rana esculenta

The effect of the insecticide Decamethrin on ionic transport through the isolated skin of Rana esculenta was studied; the skins were bathed with 1-2 mEq Na2SO4 or choline-Cl solutions (exterior), and with Ringer normal (interior). Under open circuit (OC) conditions, mucosal Decamethrin (10(-6) M), did not provoke changes in Na+ fluxes. At 10(-5) M there was a slight inhibition of the JoNa+ after 30 min. The Cl- fluxes did not change. With longer treatments (60-90 min, OC, 10(-5) M) the JnNa+ was inhibited; at 10(-4) M it was augmented. The JnH+ was not affected. Serosal Decamethrin did not modify Na+ and H+ fluxes. In short circuit conditions, Decamethrin (10(-5) M) in the mucosal face inhibited the JnNa+; the JnH+ did not change in these conditions. The abscence of interaction of mucosal Decamethrin with Amiloride was shown.     

Studies on the activation energy and deuterium isotope effect of human skin collagenase on homologous collagen substrates

The activation energy (EA) and solvent-deuterium kinetic isotope effect (kH/kD) of human skin fibroblast collagenase were studied on the homologous human type I, II, and III collagens in both native and denatured states. Values for EA on human type I and II collagens in solution were 47,000 and 61,000 cal, respectively. The Arrhenius plot for type III collagen, unlike that for the other types, was characterized by a break in EA at approximately 26 degrees C. At temperatures below this point, EA was 42,500 cal; at higher temperatures, EA fell to 29,500 cal. This latter value, intermediate between type I collagen monomers and denatured random gelatin alpha chains, appears to result from a further opening in the already loosened helix of the type III collagen molecule in the region of the 3/4:1/4 collagenase cleavage site. The EA of trypsin on native human type III collagen was also measured and found to be 70,000 cal. This high value calls into question the role of serine proteases in the physiologic degradation of this substrate; a much higher energy expenditure was required for trypsin to cleave type III collagen than for the fibroblast collagenase. Reaction velocity on human collagen types I-III in solution was slowed 15-35% (kH/kD = 1.2-1.5) by the substitution of deuterium for hydrogen in the solvent buffer. This value was far lower than that observed following the aggregation of solution monomers into insoluble fibrils (kH/kD = 9). Denaturation of triple helical monomers into random gelatin alpha chains eliminated any slowing by deuterium, and kH/kD was 1.0 in all cases. Since the same peptide bond hydrolysis accompanies the cleavage of all these forms of the collagen substrate, it would appear that the role of water at the rate-limiting step of collagen degradation may not reside in the hydrolysis of a peptide bond per se, but rather may reflect the difficulty in transporting water molecules to the site of such catalysis, especially following fibril aggregation.     

黑斑蛙的减数分裂研究

本文研究了黑斑蛙的减数分裂,发现其性染色体所形成的性二价体主要呈末端与末端联接,浓缩期占79.6％,中期Ⅰ占75％,这进一步证明黑斑蛙确实存在XY型性别决定机制,这种XY型性染色体虽形态相同,但已发生了质的分化,可能是同型异质。黑斑蛙的性染色体并不形成性泡,少数二价体有中间交叉。