Bullfrog Eggs Research Articles

Histological changes in the bullfrog (Lithobates catesbeianus) myocardium induced by severe hypoxia during embryonic development

Amphibians are organisms that suffer high damage from ecological imbalance, since they necessarily have aquatic reproduction and larval phase, being, due to the fragility of this development stage, more susceptible to harmful changes in the environment. The occurrence of environmental hypoxia during development can play an important role in the involvement of serious abnormalities of the central nervous system and heart defects. Bullfrog eggs (Lithobates catesbeianus) (n = 24) were kept in aquariums, with a volume of 5 L, and were divided into two experimental groups: Control Group and Hypoxia Group. The treated groups were kept in experimental conditions during the initial two days of development, being then relocated to aquariums with dechlorinated water and constant aeration. After 12 days the tadpoles had their measurements of mass and length taken and then they were anesthetized by hypothermia, euthanized, and fixed for histological preparation. Animals exposed to hypoxia showed a reduction in body mass and length, as well as a decrease in the mean thickness of the trabeculae of the cardiac tissue. The results show that the acute exposure of bullfrog tadpoles to severe hypoxia has deleterious effects on their development.

EXPOSURE EFFECT TO GLYPHOSATE BASED HERBICIDE - ROUNDUP ORIGINAL ® - AND NUTRITIONAL THERAPY WITH FOLIC ACID AND SELENIUM ON BULLFROG (LITHOBATES CATESBEIANUS, SHAW - 1802) CARDIAC HYTOGENESIS

Amphibians are susceptible to toxicity, due to easy cutaneous absorption of chemicals present in environment. Using of glyphosate-based herbicides is very common and becomes susceptible to be carrying by surface waters to breeding sites. Glyphosate has oxidizing properties and inhibits the action of key enzymes for regulation of cell cycle. Folic acid and selenium have antioxidant properties, which may help to reduce oxidative stress generated by contact with pesticides. Bullfrog eggs (n = 48) were kept in aquariums with constant aeration and controlled temperature (25 ± 2 ° C) with light and dark cycle of 12/12 hours. Subjects were divided in: Control Group (CG, n = 12), Group Exposed to Roundup Original ® (GR, n = 12); group Treat with Folic Acid Exposed to Roundup Original ® (GAFR, n = 12) and Group Treated With Selenium Exposed to Roundup Original ® (GSR n = 12). Larvae were maintained under experimental conditions during the first three days of development, and then relocated to aquariums with dechlorinated water. After 12 days, tadpoles had their mass and length measured, then were anesthetized by hypothermia and fixed in order to histological preparation. Animals exposed to herbicide exhibited decreased body mass, length and reduction in cardiomyocyte diameter, while folic acid and selenium treated animals showed no changes in body measurements neither in trabeculae of heart tissue. Results show that glyphosate has detrimental effects to development of bullfrog tadpoles and periconceptional supplementation with antioxidants can mitigate damages.

Sialic Acid-Binding Lectin from Bullfrog Eggs Exhibits an Anti-Tumor Effect Against Breast Cancer Cells Including Triple-Negative Phenotype Cells.

Sialic acid-binding lectin from Rana catesbeiana eggs (cSBL) is a multifunctional protein that has lectin and ribonuclease activity. In this study, the anti-tumor activities of cSBL were assessed using a panel of breast cancer cell lines. cSBL suppressed the cell growth of all cancer cell lines tested here at a concentration that is less toxic, or not toxic at all, to normal cells. The growth suppressive effect was attributed to the cancer-selective induction of apoptosis. We assessed the expressions of several key molecules associated with the breast cancer phenotype after cSBL treatment by western blotting. cSBL decreased the expression level of estrogen receptor (ER) α, while it increased the phosphorylation level of p38 mitogen-activated protein kinase (MAPK). cSBL also suppressed the expression of the progesterone receptor (PgR) and human epidermal growth factor receptor type 2 (HER2). Furthermore, it was revealed that cSBL decreases the expression of the epidermal growth factor receptor (EGFR/HER1) in triple-negative breast cancer cells. These results indicate that cSBL induces apoptosis with decreasing ErbB family proteins and may have great potential for breast cancer chemotherapy, particularly in triple-negative phenotype cells.

Sialic acid-binding lectin from bullfrog eggs inhibits human malignant mesothelioma cell growth in vitro and in vivo.

Malignant mesothelioma is an aggressive cancer that results from exposure to asbestos. The therapeutic options for this type of cancer are limited; therefore, the development of novel therapeutic agents is urgently required. Sialic acid-binding lectin isolated from Rana catesbeiana oocytes (cSBL) is a novel therapeutic candidate for cancer, which exhibits antitumor activity mediated through RNA degradation. In the present study, we evaluated the effect of cSBL in vitro and in vivo. Xenograft-competent H2452 and MSTO human mesothelioma cell lines were treated with cSBL, and the pathway by which cSBL induces apoptosis was analyzed. In vivo studies were performed using nude mice inoculated with one of the two cell lines, and the effects of cSBL and pemetrexed were monitored simultaneously. Furthermore, the pharmacological interactions between the three agents (pemetrexed, cisplatin and cSBL) were statistically assessed. It was demonstrated that cSBL treatments caused morphological and biochemical apoptotic changes in both cell lines. Caspase cascade analysis revealed that an intrinsic pathway mediated cSBL-induced apoptosis. The administration of cSBL significantly inhibited tumor growth in two xenograft models, without any adverse effects. Furthermore, the combination index and dose reduction index values indicated that the cSBL + pemetrexed combination showed the highest synergism, and thus potential for reducing dosage of each drug, compared with the other combinations, including the existing pemetrexed + cisplatin regimen. cSBL exerted prominent antitumor effects on malignant mesothelioma cells in vitro and in vivo, and showed favorable effects when combined with pemetrexed. These results suggest that cSBL has potential as a novel drug for the treatment of malignant mesothelioma.

RNase activity of sialic acid-binding lectin from bullfrog eggs drives antitumor effect via the activation of p38MAPK to caspase-3/7 signaling pathway in human breast cancer cells.

Sialic acid-binding lectin obtained from bullfrog eggs (SBL) induces cell death in cancer cells but not in normal cells. This antitumor effect is mediated through its ribo-nuclease (RNase) activity. However, the underlying molecular mechanisms remain unclear. We found that the p38 mitogen-activated protein kinase (MAPK) signaling pathway was activated when SBL induced cell death in three human breast cancer cell lines: SK-BR-3, MCF-7, and MDA-MB231. The suppression of p38 MAPK phosphorylation by a p38 MAPK inhibitor as well as short interference RNA knockdown of p38 MAPK expression significantly decreased cell death and increased the cell viability of SBL-treated MDA-MB231 cells. H103A, an SBL mutant lacking in RNase activity, showed decreased SBL-induced cell death compared with native SBL. However, the loss of RNase activity of SBL had no effect on its internalization into cells. The H103A mutant also displayed decreased phosphorylation of p38 MAPK. Moreover, SBL promoted caspase-3/7 activation followed by a cleavage of poly (ADP-ribose)-polymerase, whereas the SBL mutant, H103A, lost this ability. The SBL-induced caspase-3/7 activation was suppressed by the p38 MAPK inhibitor, SB203580, as well as pan-caspase inhibitor, zVAD-fmk. In the presence of zVAD-fmk, the SBL-induced cell death was decreased. In addition, the cell viability of SBL-treated MDA-MB231 cells recovered by zVAD-fmk treatment. Taken together, our results suggest that the RNase activity of SBL leads to breast cancer cell death through the activation of p38 MAPK followed by the activation of caspase-3/7.

Could High Salinity Be Used to Control Bullfrogs in Small Ponds?

Abstract We examined survival of bullfrog (Rana catesbeiana) eggs and tadpoles at 3 ppt and 6 ppt salinity in the laboratory to determine if low-level salinity could be used to eradicate bullfrogs from small ponds that contain native fishes. Bullfrog eggs and tadpoles 9 months old) appeared unaffected by 14 days of 6 ppt salinity. Salinity of 3 ppt did not impact survival of bullfrog tadpole eggs or tadpoles at any of the life stages we tested. Adding salt to ponds in the early spring to increase salinity to 6 ppt may be a cost effective way to eradicate bullfrogs from small ponds without harming native fishes.

Invasion of American bullfrogs along the Yellowstone River

The American bullfrog (Lithobates catesbeianus) is a globally distributed invasive species that was introduced to the Yellowstone River floodplain of Montana. Knowledge about floodplain habitat features that allow for bullfrog persistence and spread will help identify effective control strategies. We used field surveys in 2010, 2012 and 2013 to describe bullfrog spread in the Yellowstone River floodplain and the habitat features that are associated with bullfrog occupancy and colonization. Bullfrogs in our study area expanded from ~ 60 km in 2010 to 106 km in 2013, and are spreading to up- and downstream habitats. The number of breeding sites (i.e., presence of bullfrog eggs or larvae) increased from 12 sites in 2010 to 45 sites in 2013. We found that bullfrogs were associated with deeper waters, emergent vegetation and public-access sites, which are habitat features that characterize permanent waters and describe human-mediated introductions. Control strategies that reduce the hydroperiod of breeding sites may help to limit bullfrog persistence and spread, while an increase in public outreach and education may help prevent further bullfrog introductions at public-access sites.

Involvement of ER stress in apoptosis induced by sialic acid-binding lectin (leczyme) from bullfrog eggs

Sialic-acid binding lectin (SBL) isolated from bullfrog (Rana catesbeiana) oocytes is a multifunctional protein which has lectin activity, ribonuclease activity and cancer-selective antitumor activity. It has been reported that SBL induces apoptosis accompanied by rigid mitochondrial perturbation, which indicates mediation of the intrinsic pathway. However, the mechanism of the antitumor effect of SBL has not been fully elucidated. We report, here, that ER stress is evoked in SBL-treated cells. We show that caspase-4, an initiator caspase of ER stress-mediated apoptosis was activated, and inhibition of caspase-4 resulted in significant attenuation of apoptosis induced by SBL. We analyzed the precise mechanism of activation of the caspase cascade induced by SBL, and found that caspase-9 and -4 are activated upstream of activation of caspase-8. Further study revealed that SBL induces the mitochondrial and ER stress-mediated pathways independently. It is noteworthy that SBL can induce cancer-selective apoptosis by multiple apoptotic signaling pathways, and it can serve as a candidate molecule for anticancer drugs in a novel field.

New small-scale cross-axis coil planet centrifuge: Partition efficiency and application to purification of bullfrog ribonuclease

The new small-scale cross-axis coil planet centrifuge (X-axis CPC) previously designed and fabricated in our laboratory has a distinctive feature such that four separation columns of similar weight are mounted symmetrically around the rotary frame to achieve stable balancing of the centrifuge under a high revolution speed. In this column layout, neighboring columns must be rotated in the opposite direction if viewed from the center of the centrifuge to avoid twisting the interconnecting flow tubes. The effect of rotational direction of the columns on the partition efficiency was evaluated with separation of a set of test samples such as cytochrome c, myoglobin, and lysozyme using an aqueous–aqueous polymer phase system composed of 12.5% (w/w) polyethylene glycol (PEG) 1000 and 12.5% (w/w) dibasic potassium phosphate under 1000 rpm of column revolution. A series of experiments was performed using a set of two diagonally located columns (connected in series) each consisting of five coiled layers of 1 mm I.D. with a total capacity of 27.0 mL. Both right- and left-handed coils were tested each under the optimized conditions for choice of mobile phase and direction of the column rotation so that the satisfactory volume of the mobile phase was retained in the column by the aid of Archimedean screw effect. The results of these studies showed that one particular combination of handedness of the coil and direction of the rotation yielded the best peak resolution for each mobile phase. In order to demonstrate the capability of the apparatus, the purification of ribonuclease (RNase) from the extract of bullfrog egg, sialic acid binding lectin (cSBL), was carried out using both organic–aqueous and aqueous–aqueous polymer phase systems. When using the 16.0% (w/w) PEG 1000–6.3% (w/w) dibasic potassium phosphate–6.3% (w/w) monobasic potassium phosphate system, cSBL was successfully separated from other proteins present in the extract while commercial RNase A was eluted at near the solvent front by the lower phase mobile. The cSBL retained its native RNase activity. The overall results demonstrated that the present new small-scale X-axis CPC is useful for the purification of bioactive compounds without loss of their native activities.

Purification of a 63 kDa beta-D-galactoside binding lectin from cuttlefish, Todarodes pacificus.

A beta-D-galactoside binding lectin (TPL) was newly purified from the body walls of the cuttlefish, Todarodes pacificus with lactosyl-agarose affinity column chromatography and reversed-phase HPLC. Many of the biological properties of the lectin were similar to those of the representative animal beta-D-galactoside binding lectin, "galectin". However, with a molecular mass of 63 kDa under reducing and non-reducing conditions on SDS-PAGE, TPL is larger than any previously reported galectins. The native molecular mass of TPL was estimated to be about 60 kDa by gel filtration, suggesting that it exists as a monomer and that a single polypeptide containing at least two sugar binding sites. The antisera raised against TPL did not crossreact with bull frog egg galectin-1, suggesting that TPL is immunologically distinct from the lower vertebrate galectin.

Crystal structure of bullfrog egg lectin

Crystal structure of bullfrog egg lectin

3-Trifluoromethyl-4-nitrophenol (TFM) toxicity and hepatic microsomal UDP-glucuronyltransferase activity in larval and adult bullfrogs

Effects of TFM (3-trifluoromethyl-4-nitrophenol) were observed in bullfrog ( Rana catesbeiana) larvae and adults in vivo by aqueous and intraperitoneal (i.p.) exposures. UDP-glucuronyltransferase (UDPGT) kinetics were examined in vitro from larval and adult hepatic microsomes. The effect of TFM on bullfrog egg development was also examined. Bullfrog larvae and adults showed similar sensitivities to TFM by i.p. exposure ( LD 50 s = 11.62 and 15.35 mg/kg , respectively), however, larvae were more sensitive than adults in aqueous exposures ( LC 50 s = 0.95 and 12.99 mg/l , respectively). Bullfrog egg development was arrested at the blastula stage at ≥ 1.0 mg/l TFM. No microscopic lesions were observed in tissues from TFM-exposed larvae. TFM-UDPGT kinetics in bullfrog larvae and adults differed significantly ( P < 0.05). V max for TFM-UDPGT in bullfrog larvae and adults was calculated to be 1.05 and 2.45 nmol/min · mg, respectively; the apparent K m for bullfrog larvae and adults was 38 and 64 μM, respectively. However, the enzyme V max K m ratio between the two life stages was not significantly different. Based on data from this and other studies, greater sensitivity to aqueous TFM in gill-respiring larvae (compared with adults) does not primarily appear to be a function of differences in UDPGT-mediated metabolism, but rather may reflect differences in other processes such as uptake.

Primary Structure of a Ribonuclease from Bullfrog (Rana catesbeiana) Liver1

A pyrimidine base-specific ribonuclease was purified from bullfrog (Rana catesbeiana) liver by means of CM-cellulose column chromatography and affinity chromatography on heparin-Sepharose CL-6B, which gave single band on SDS-slab electrophoresis. The primary structure of the bullfrog liver RNase was determined. It consisted of 111 amino acid residues, including 8 half-cystine residues. From the sequence, it was concluded that three disulfide bridges in RNase A were conserved in the bullfrog RNase, that a disulfide bridge in RNase A [Cys65-Cys126 (RNase A numbering)] was deleted, and that a new disulfide bridge was created in the C-terminal part of the enzyme. In this frog RNase, the amino acid residues thought to be essential for catalysis in bovine pancreatic RNase A were conserved except for Asp121 (RNase A numbering). The sequence homology of the bullfrog liver RNase with bovine pancreatic RNase A was 30.6%. The sequence of bullfrog liver RNase was very similar to those of lectins obtained from bullfrog egg by Titani et al. [Biochemistry (1988) 26, 2189-2194] and R. japonica egg by Kamiya et al. [Seikagaku (in Japanese) (1989) 60, 733; and personal communication from Kamiya, Y., Oyama, F., Oyama, R., Sakakibara, F., Nitta, K., Kawauchi, H., and Titani, K.]. The sequence homology between the bullfrog liver RNase and the two lectins was 70.2 and 64.8%, respectively.

18] Purification of a multifunction protein bearing carbamyl-phosphate synthase, aspartate transcarbamylase, and dihydroorotase enzyme activities from mutant hamster cells

This chapter describes the purification of a multifunctional protein bearing carbamyl-phosphate synthase, aspartate transcarbamylase, and dihydroorotase enzyme activities from mutant hamster cells. Carbamyl-P synthase, aspartate transcarbamylase, and dihydroorotase, the first three enzymes of the pathway for the de novo synthesis of pyrimidine nucleotides, are partially purified in low yield from Drosophila melanogaster, bullfrog eggs, rat liver, hematopoietic mouse spleen, mouse Ehrlich ascites cells, and human lymphocytes and are extensively purified from mouse ascites hepatoma cells. In all cases, all three activities are associated with a single multi-enzyme complex of high molecular weight. The three enzymes are linked covalently as a multifunctional protein, providing a simple explanation for the coordinate control of their synthesis in a series of mutant cell lines that overaccumulate the complex to varying degrees. The entire procedure can be completed in less than 24 hours with a yield of about 40 mg of pure protein from about 15 ml of packed cells.

Cleavage in the Indian bull-frog eggs

Cleavage in the Indian bull-frog eggs