Abstract
Background
The hemolytic activity of skin secretions obtained by stimulating the frog Kaloula pulchra hainana with diethyl ether was tested using human, cattle, rabbit, and chicken erythrocytes. The skin secretions had a significant concentration-dependent hemolytic effect on erythrocytes. The hemolytic activity of the skin secretions was studied in the presence of osmotic protectants (polyethylene glycols and carbohydrates), cations (Mg2+, Ca2+, Ba2+, Cu2+, and K+), or antioxidants (ascorbic acid, reduced glutathione, and cysteine).
Results
Depending on their molecular mass, osmotic protectants effectively inhibited hemolysis. The inhibition of skin hemolysis was observed after treatment with polyethylene glycols (1000, 3400, and 6000 Da). Among divalent cations, only 1 mM Cu2+ markedly inhibited hemolytic activity. Antioxidant compounds slightly reduced the hemolytic activity.
Conclusions
The results suggested that skin secretions of K. pulchra hainana induce a pore-forming mechanism to form pores with a diameter of 1.36-2.0 nm rather than causing oxidative damage to the erythrocyte membrane.
Amphibian; Skin secretions; Hemolysis; Pore-forming
Background
The skin of amphibians has numerous biochemical and physiological functions to assure
amphibian survival. Several bioactive components with specialized functions and
molecular structures have been isolated and purified from amphibian skin [ 1Clarke BT. The natural history of amphibian skin secretions, their
normal functioning and potential medical applications. Biol Rev Camb Philos Soc.
1997;19(3):365–379. doi: 10.1017/S0006323197005045.
https://doi.org/10.1017/S000632319700504...
, 2Duellman WE, Trueb L, editor. Biology of Amphibians. New York:
McGraw-Hill; 1986. pp. 197–228. ].
Some of these components and their analogs have been used for treating diseases such
as microbial infections and burns [ 1Clarke BT. The natural history of amphibian skin secretions, their
normal functioning and potential medical applications. Biol Rev Camb Philos Soc.
1997;19(3):365–379. doi: 10.1017/S0006323197005045.
https://doi.org/10.1017/S000632319700504...
, 3Lu CX, Nan KJ, Lei Y. Agents from amphibians with anticancer
properties. Anticancer Drugs. 2008;19(10):931–939. doi:
10.1097/CAD.0b013e3283139100.
https://doi.org/10.1097/CAD.0b013e328313...
]. Thus, increasing numbers of studies have
focused on amphibian skin secretions to identify biologically active proteins and
peptides [ 1Clarke BT. The natural history of amphibian skin secretions, their
normal functioning and potential medical applications. Biol Rev Camb Philos Soc.
1997;19(3):365–379. doi: 10.1017/S0006323197005045.
https://doi.org/10.1017/S000632319700504...
, 4Conlon JM, Iwamuro S, King JD. Dermal cytolytic peptides and the
system of innate immunity in anurans. Ann N Y Acad Sci. 2009;19:75–82. doi:
10.1111/j.1749-6632.2008.03618.x.
https://doi.org/10.1111/j.1749-6632.2008...
].
Cnidarian venom is an abundant source of numerous bioactive molecules such as
pore-forming proteins, small cytotoxic peptides, 5-hydroxytryptamine, and histamine.
Of the cytolytic, hemolytic, and neurotoxic effects of Cnidarian venom, hemolytic
activity is the most commonly investigated [ 5Alvarez C, Mancheño JM, Martinez D, Tejuca M, Pazos F, Lanio ME.
Sticholysins, two pore-forming toxins produced by the Caribbean Sea anemone
Stichodactyla helianthus: their interaction with membranes.
Toxicon. 2009;19(8):1135–1147. doi:
10.1016/j.toxicon.2009.02.022.
https://doi.org/10.1016/j.toxicon.2009.0...
- 7Lassen S, Wiebring A, Helmholz H, Ruhnau C, Prange A. Isolation of a
Nav channel blocking polypeptide from Cyanea capillata
medusae-a neurotoxin contained in fishing tentacle isorhizas. Toxicon.
2012;19(6):610–616. doi: 10.1016/j.toxicon.2012.02.004.
https://doi.org/10.1016/j.toxicon.2012.0...
]. Hemolysis induced by Cnidarian venom
toxins has been particularly investigated to identify targets and the attachment of
proteins to cell membranes [ 8Edwards LP, Whitter E, Hessinger DA. Apparent membrane
pore-formation by Portuguese Man-of-war (Physalia physalis)
venom in intact cultured cells. Toxicon.2002;19(9):1299–1305. doi:
10.1016/S0041-0101(02)00138-1.
https://doi.org/10.1016/S0041-0101(02)00...
, 9Frazão B, Vasconcelos V, Antunes A. Sea anemone (cnidaria, anthozoa,
actiniaria) toxins: an overview. Mar Drugs.
2012;19(8):1812–1851. ].
Kaloula pulchra hainana (Anura) is an endemic amphibian found in
the low elevation regions of Hainan Island of China. These frogs always live near
pools, which is a harsh environment with numerous pathogens. When these frogs are
stimulated, their belly bulges and white secretion is released from their skin. In a
previous study, we purified and characterized a 23-kDa trypsin inhibitor from the
skin secretions of K. pulchra hainana , designated K.
pulchra hainana trypsin inhibitor (KPHTI) [ 10Zhang Y, Wang M, Wei S. Isolation and characterization of a trypsin
inhibitor from the skin secretions of Kaloula pulchra hainana.
Toxicon. 2010;19(4):502–507. doi:
10.1016/j.toxicon.2010.05.006.
https://doi.org/10.1016/j.toxicon.2010.0...
].
Skin secretions of the frog K. pulchra hainana also exhibit hemolytic activity. This study aimed to establish basic information on erythrocyte hemolysis and cell membrane peroxidation induced by these skin secretions. To determine the toxicological properties of the skin secretions, we investigated the effects of different factors on erythrocyte hemolysis, including osmotic protectants, cations, antioxidants, and chelating agents.
Methods
Materials
N-acetyl-D-galactosamine (NAGA), N-methylmannopyranose, D-glucose, D-trehalose, D-lactose, reduced glutathione (GSH), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (USA). Polyethylene glycols (PEGs) of different molecular masses (300, 400, 1000, 3400, and 6000 Da) were obtained from Fluka (USA). The protein concentrations of skin secretions were determined using a protein assay kit (Bio-Rad, USA) with BSA as a standard [ 11Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;19:265–275. ]. All other reagents used were of the highest purity available.
Preparation of skin secretions
Adult specimens of K. pulchra hainana of both genders (n = 10; weight range: 80–120 g) were collected in Danzhou City, Hainan Province in southern China. The skin of the frogs was briefly stimulated with diethyl ether and then washed with 50 mM Tris–HCl buffer (pH 7.8) containing 0.1 M NaCl and 5 mM ethylene diamine tetraacetic acid (EDTA). The secretions were centrifuged at 10,000 g for 20 minutes at 4°C to remove insoluble materials. The supernatant was collected, lyophilized, and stored at −80°C until use. Before an experiment, the lyophilized skin secretions were dissolved in phosphate-buffered saline (PBS) (137 mM NaCl, 1.5 mM KH2PO4, 2.7 mM KCl, 8.1 mM Na2HPO4) and then dialyzed against PBS.
Hemolysis determinations
The hemolytic activity of skin secretions was determined using human, cattle,
rabbit, and chicken erythrocytes, as reported by Liu et al . [
12Liu SB, He YY, Zhang Y, Lee WH, Qian JQ, Lai R, Jin Y. A novel
non-lens betagamma-crystallin and trefoil factor complex from amphibian skin and
its functional implications.PLoS One. 2008;19(3):e1770. doi:
10.1371/journal.pone.0001770.
https://doi.org/10.1371/journal.pone.000...
]. Erythrocytes from these species
were washed with PBS until the supernatant was clear and then resuspended in
PBS. Erythrocyte suspensions (5 × 106 cells/mL) were
incubated with different concentrations of skin secretions (0.28, 0.56, 1.4,
2.8, 4.2, and 5.6 μg/mL) at 37°C for 30 minutes
and then centrifuged at 1000 g for 5 minutes at 4°C to
precipitate intact erythrocytes and debris. The supernatants were assayed for
absorbance at 540 nm to determine the percentage of hemoglobin released
from the lysed erythrocytes. We defined 100% lysis as the absorbance of a
supernatant obtained using 1% Triton X-100 instead of test samples. The
hemolytic activity of the skin secretions was expressed as the percentage of
absorbance compared with that observed after 100% lysis induced by Triton X-100.
The supernatant of an untreated erythrocyte suspension in PBS was used as a
spectrophotometric blank.
Determination of membrane pore diameters
The diameters of membrane pores induced by skin secretions were determined as
described previously [ 13Scherrer R, Gerhardt P. Molecular sieving by the Bacillus
megaterium cell wall and protoplast. J Bacteriol.
1971;19(3):718–735. ]. In brief,
40 mOsm PEGs of various molecular masses (300, 400, 1000, 3400, and
6000 Da) were added to PBS to counteract the osmotic pressure of
hemoglobin [ 14Freedman JC, Hoffman JF. Ionic and osmotic equilibria of human red
blood cells treated with nystatin. J Gen Physiol. 1979;19(2):157–185. doi:
10.1085/jgp.74.2.157.
https://doi.org/10.1085/jgp.74.2.157...
]. Following this, by
changing the concentration of NaCl, the total osmotic pressure of the
extracellular fluid was adjusted to 295 mOsm. After human erythrocytes
were suspended in a PEG solution (5 × 106 cells/mL), the
hemolytic activity of skin secretions was determined as described above. The
hydrodynamic diameters of PEG 300, 400, 1000, 3400, and 6000 were 1.16, 1.36,
2.0, 3.8, and 5.8 nm, respectively [ 13Scherrer R, Gerhardt P. Molecular sieving by the Bacillus
megaterium cell wall and protoplast. J Bacteriol.
1971;19(3):718–735. , 15Sabirov RZ, Krasilnikov OV, Ternovsky VI, Merzliak PG. Relation
between ionic channel conductance and conductivity of media containing different
nonelectrolytes. A novel method of pore size determination. Gen Physiol Biophys.
1993;19(2):95–111. ].
Osmotic protectants
The following osmotic protectants were used: 5 mM NAGA, 10 mM N-methylmannopyranose, 25 mM D-glucose, 25 mM D-trehalose, and 25 mM D-lactose. Each of these was added to erythrocytes suspended in PBS. After skin secretions (5.6 μg/mL) were added and incubated at 37°C for 30 minutes, the hemolytic activity was determined as described above. For osmotic protectants with large molecular masses, erythrocytes were preincubated with PEG 6000 for 10 minutes, following which erythrocytes were removed and resuspended in PBS. Following this, skin secretions (0.7, 1.4, or 5.6 μg/mL) were added and hemolytic activity was determined.
Cations and EDTA
Different cations (KCl, BaCl2, MgCl2, CuSO4, or CaCl2) were individually mixed with erythrocyte suspensions, following which skin secretions (1.4 or 5.6 μg/mL) were added and incubated at 37°C for 30 minutes and hemolytic activity was determined. The final concentration of CaCl2, BaCl2, MgCl2, and KCl was 10 mM, whereas that of CuSO4 was 1 mM. The effect of EDTA (0.1 mM, 0.2 mM, or 1 mM) was also determined.
Antioxidants
Ascorbic acid, GSH, and cysteine were used to assess possible erythrocyte cell membrane oxidative damage induced by skin secretions. An antioxidant (2 mM) was added to an erythrocyte suspension, following which skin secretions were added (5.6 μg/mL) and hemolytic activity was determined.
Statistical analysis
Results are given as means ± standard error (SE) for ten experiments. Results for different experimental conditions were compared by Student’s t-tests. A p-value < 0.05 was considered significant [ 16Zar JH. Biostatistical analysis. New Jersey: Prentice-Hall; 1984. pp. 717–732. ].
Results
Hemolytic activity of K. Pulchra hainana skin secretions
The frog skin secretions induced the hemolysis of erythrocytes from different species in a dose-dependent manner (Figure 1 ). The sensitivities of these erythrocytes to the secretions were different; the most sensitive erythrocytes were human erythrocytes. For human erythrocytes, the percent hemolysis was 19.8 ± 3% with 0.28 μg/mL of skin secretions, while total hemolysis was 90.3 ± 4% with 5.6 μg/mL of skin secretions. Among the four species examined, chicken erythrocytes were the least susceptible to these skin secretions; the hemolytic activity was approximately 60.5 ± 2% with the highest concentration of skin secretions (5.6 μg/mL).
Hemolytic activity of skin secretions from Kaloula pulchra hainana tested with erythrocytes from different species: human (□), cattle (Δ), rabbit (○), and chicken (▼ ).
The hemolytic activity of the skin secretions was tested in the presence of PEGs with different hydrodynamic diameters: 300, 400, 1000, 3400, and 6000 Da. These were used to estimate pore diameters in erythrocyte cell membranes. Hemolysis induced by the skin secretions was not affected by treatment with PEG 300, it was partially inhibited by PEG 400, and markedly inhibited by treatment with PEGs 1000, 3400, and 6000 (p < 0.05, compared with no PEGs; Figure 2 ).
Osmotic protection against hemolytic activity using a series of polyethylenglycols (PEGs; 300, 400, 1000, 3400, and 6000 Da). Aliquots of skin secretions from Kaloula pulchra hainana (0.7, 1.4, or 5.6 μg/mL) were added to erythrocyte suspensions containing PEGs at a final concentration of 25 mM (n = 10). **p < 0.01.
Effects of osmotic protectants on hemolytic activity
Erythrocyte suspensions were first pretreated with 25 mM PEG 6000 for 5 minutes, following which erythrocytes were removed and resuspended in PBS. Following this, skin secretions (0.7, 1.4, or 5.6 μg/mL) were added to test their hemolytic activities. The erythrocytes were hemolyzed equally compared with controls, which supported that the larger PEG molecules did not bind to the membrane to reduce the interaction between the cell membrane and skin secretions. However, because PEG 6000 is an osmotic protectant, it maintained the medium as more hypertonic and blocked membrane pores induced by the skin secretions.
Compared with the controls, the osmotic protectants with small molecular masses, including NAGA, D-glucose, methylmannopyranose, trehalose, and lactose, did not significantly inhibit the hemolytic activity of the skin secretions (Table 1 ).
Effect of different agents on the hemolytic activity of skin secretions of Kaloula pulchra hainana
Effects of cations and EDTA on hemolytic activity
The effects of different cations on the hemolytic activity of the skin secretions were assessed using the divalent cations Ca2+, Mg2+, Ba2+, and Cu2+ and the monovalent cation K+. Except for Cu2+, these cations at concentrations of < 10 mM did not affect the hemolytic activity compared with the control (data not shown). Cu2+ at a concentration of 1 mM significantly inhibited the hemolytic activity at skin secretion concentrations of 1.4 and 5.6 μg/mL, whereas 10 mM Mg2+, Ca2+, and K+ had slight inhibitory effects (Figure 3 ). EDTA at any of the concentrations used did not inhibit hemolysis (Table 1 ).
Effects of cations (10 mM Ca 2+ , 10 mM Ba 2+ , 10 mM Mg 2+ , 1 mM Cu 2+ , and 10 mM K + ) on the hemolytic activity of skin secretions of Kaloula pulchra hainana . Each cation was incubated with erythrocyte suspensions, following which 1.4 or 5.6 μg/mL of skin secretions was added (n = 10), *p < 0.05.
Effects of antioxidants on hemolytic activity
The antioxidants cysteine, GSH, and ascorbic acid (2 mM) reduced the hemolysis induced by skin secretions, with results ranging from 14 to 20% (Table 1 ). However, these results were not significantly different from those of controls.
Discussion
Amphibian skin is a convenient source of substantial amounts of granular gland
secretions from which numerous components with specialized functions have been
isolated and characterized [ 1Clarke BT. The natural history of amphibian skin secretions, their
normal functioning and potential medical applications. Biol Rev Camb Philos Soc.
1997;19(3):365–379. doi: 10.1017/S0006323197005045.
https://doi.org/10.1017/S000632319700504...
, 2Duellman WE, Trueb L, editor. Biology of Amphibians. New York:
McGraw-Hill; 1986. pp. 197–228. , 17You D, Hong J, Rong M, Yu H, Liang S, Ma Y, Yang H, Wu J, Lin D, Lai
R. The first gene-encoded amphibian neurotoxin. J Biol Chem.
2009;19(33):22079–22086. doi: 10.1074/jbc.M109.013276.
https://doi.org/10.1074/jbc.M109.013276...
,
18Zhang YX, Chen CW, Wang M, Wei SS, Guan H, Chi TT, Qi XZ, Hu WT.
Purification and characterization of albumin from frog skin of
Duttaphrynus melanostictus. Protein J.2011;19(7):464–470.
doi: 10.1007/s10930-011-9349-6.
https://doi.org/10.1007/s10930-011-9349-...
]. In a previous study, we found that
the skin secretions from K. pulchra hainana exhibited diverse
biological activities, including protease inhibitory, cytotoxic, and hemolytic
activities [ 10Zhang Y, Wang M, Wei S. Isolation and characterization of a trypsin
inhibitor from the skin secretions of Kaloula pulchra hainana.
Toxicon. 2010;19(4):502–507. doi:
10.1016/j.toxicon.2010.05.006.
https://doi.org/10.1016/j.toxicon.2010.0...
].
Hemolysis can be induced by several protein toxins from animals, plants, and
microbes, particularly marine animals [ 19Parker MW, Feil SC. Pore-forming protein toxins: from structure to
function. Prog Biophys Mol Biol. 2005;19(1):91–142. doi:
10.1016/j.pbiomolbio.2004.01.009.
https://doi.org/10.1016/j.pbiomolbio.200...
].
Some of these venoms affect biological membranes by inducing the formation of pores
or channels in natural and model bilayer lipid membranes [ 20García-Sáez AJ, Buschhorn SB, Keller H, Anderluh G, Simons K,
Schwille P. Oligomerization and pore formation by equinatoxin II inhibit
endocytosis and lead to plasma membrane reorganization. J Biol Chem.
2011;19(43):37768–37777. doi: 10.1074/jbc.M111.281592.
https://doi.org/10.1074/jbc.M111.281592...
- 22Savva CG, Fernandes da Costa SP, Bokori-Brown M, Naylor CE, Cole AR,
Moss DS, Titball RW, Basak AK. Molecular architecture and functional analysis of
NetB, a pore-forming toxin from Clostridium perfringens. J Biol
Chem. 2013;19(5):3512–3522. doi: 10.1074/jbc.M112.430223.
https://doi.org/10.1074/jbc.M112.430223...
]. Thus,
hemolytic activity induced by protein toxins has been used as a sensitive
toxicological tool to investigate the targeting and attachment of proteins to cell
membranes [ 15Sabirov RZ, Krasilnikov OV, Ternovsky VI, Merzliak PG. Relation
between ionic channel conductance and conductivity of media containing different
nonelectrolytes. A novel method of pore size determination. Gen Physiol Biophys.
1993;19(2):95–111. ].
We used a series of PEGs (300, 400, 1000, 3400, and 6000 Da) with different
hydrodynamic diameters to determine their effects on erythrocyte hemolysis induced
by the skin secretions of K. pulchra hainana [ 15Sabirov RZ, Krasilnikov OV, Ternovsky VI, Merzliak PG. Relation
between ionic channel conductance and conductivity of media containing different
nonelectrolytes. A novel method of pore size determination. Gen Physiol Biophys.
1993;19(2):95–111. , 23Tejuca M, Dalla SM, Potrich C, Alvarez C, Menestrina G. Sizing the
radius of the pore formed in erythrocytes and lipid vesicles by the toxin
sticholysin I from the sea anemoneStichodactyla helianthus. J
Membr Biol. 2001;19(2):125–135. doi: 10.1007/s00232-001-0060-y.
https://doi.org/10.1007/s00232-001-0060-...
]. The small polymers PEG 300 did not affect hemolysis, while PEG 400 only partially
inhibited hemolysis. However, hemolysis was significantly inhibited by treatment
with larger PEGs of 1000, 3400, and 6000 Da (Figure 2 ). We deduced that these skin secretions had
induced the formation of transmembrane pores in erythrocyte membranes. In contrast,
small osmolytes, including glucose, trehalose, lactose, methylmanopyranose, and
galactosamine, did not affect hemolysis (Table 1 ). Thus, we hypothesize that the hydrophilic pores in
erythrocyte cell membranes induced by these skin secretions caused a colloid osmotic
burst that resulted in erythrocyte lysis. The diameters of these pores were
approximately 1.36-2.0 nm based on the hydrodynamic diameters of PEG 400 and
1000 of 1.36 nm and 2.0 nm, respectively [ 24Rinaldi AC, Mangoni ML, Rufo A, Luzi C, Barra D, Zhaos H, Kinnunen
PK, Bozzi A, Di Giulio A, Simmaco M. Temporin-L: antimicrobial, haemolytic and
cytotoxic activities, and effects on membrane permeabilization in lipid
vesicles. Biochem J. 2002;19(Pt 1):91–100. ].
In addition to pore-forming mechanisms, lipid peroxidation of erythrocyte membranes
plays an important role in the hemolysis induced by hemolytic protein toxins,
resulting in cell membrane disorder [ 19Parker MW, Feil SC. Pore-forming protein toxins: from structure to
function. Prog Biophys Mol Biol. 2005;19(1):91–142. doi:
10.1016/j.pbiomolbio.2004.01.009.
https://doi.org/10.1016/j.pbiomolbio.200...
,
20García-Sáez AJ, Buschhorn SB, Keller H, Anderluh G, Simons K,
Schwille P. Oligomerization and pore formation by equinatoxin II inhibit
endocytosis and lead to plasma membrane reorganization. J Biol Chem.
2011;19(43):37768–37777. doi: 10.1074/jbc.M111.281592.
https://doi.org/10.1074/jbc.M111.281592...
]. The antioxidant compounds GSH,
cysteine, and ascorbic acid only minimally reduced the hemolytic activity of these
skin secretions. From these results, we deduced that these skin secretions induced
erythrocyte lysis by inducing pore formation in bilayer lipid membranes rather than
causing oxidative damage.
βγ-CAT, a protein purified from the skin secretions of the frog
Bombina maxima , has potent hemolytic activity and induces the
formation of membrane pores with diameters of approximately 2.0 nm [ 12Liu SB, He YY, Zhang Y, Lee WH, Qian JQ, Lai R, Jin Y. A novel
non-lens betagamma-crystallin and trefoil factor complex from amphibian skin and
its functional implications.PLoS One. 2008;19(3):e1770. doi:
10.1371/journal.pone.0001770.
https://doi.org/10.1371/journal.pone.000...
]. Antimicrobial peptides from amphibian
skin also exhibit hemolytic activity. In that report, the antimicrobial activity of
the skin secretions was not detected for some gram-positive bacteria and
gram-negative bacteria, suggesting that hemolysis was not caused by antimicrobial
peptides. Preincubating these skin secretions with trypsin or collagenase resulted
in a significant loss of hemolytic activity (unpublished observations). Proteins in
K. pulchra hainana skin secretions were analyzed by sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Two major bands were
identified with molecular weights of approximately 28–60 kDa and
17–25 kDa (unpublished observations). However, we have no data
regarding which proteins contributed to the hemolytic activity of these skin
secretions.
Cations, particularly Ca2+, affect the hemolytic activities of sea anemone
toxins, although different results are obtained depending on the specimens and toxin
structures [ 25Yu H, Li C, Li R, Xing R, Liu S, Li P. Factors influencing
haemolytic activity of venom from the jellyfish Rhopilema esculentum
Kishinouye. Food Chem Toxicol.2007;19(7):1173–1178. doi:
10.1016/j.fct.2006.12.025.
https://doi.org/10.1016/j.fct.2006.12.02...
- 27Marino A, Morabito R, Pizzata T, La Spada G. Effect of various
factors on Pelagia noctiluca (Cnidaria, Scyphozoa) crude
venom-induced haemolysis. Comp Biochem Physiol A Mol Integr Physiol.
2008;19(1):144–149. doi: 10.1016/j.cbpa.2008.06.013.
https://doi.org/10.1016/j.cbpa.2008.06.0...
]. In our study, the hemolytic activity of skin secretions
was slightly affected by 10 mM Ca2+, whereas it was significantly
inhibited by 1 mM Cu2+. The chelating agent EDTA at any tested
concentration did not produce any effects, suggesting that cations were not
necessary for the hemolytic activity of these skin secretions.
Conclusion
In this study, we analyzed the effects of osmotic protectants, cations, and antioxidants on erythrocyte hemolysis induced by the skin secretions from the frog K. pulchra hainana . We observed that osmotic protectants of high molecular mass inhibited this hemolytic activity. Cu2+ also significantly inhibited the hemolytic activity. We deduced that these skin secretions induced erythrocytes lysis by a pore-forming mechanism in the bilayer lipid membrane. This hypothesis needs to be explored in detail in future investigations.
We are grateful to Miss Kelly Zheng, Li XC and Dr. Liao CH for reviewing the manuscript and giving some valuable suggestions.
- Clarke BT. The natural history of amphibian skin secretions, their normal functioning and potential medical applications. Biol Rev Camb Philos Soc. 1997;19(3):365–379. doi: 10.1017/S0006323197005045.
» https://doi.org/10.1017/S0006323197005045 - Duellman WE, Trueb L, editor. Biology of Amphibians. New York: McGraw-Hill; 1986. pp. 197–228.
- Lu CX, Nan KJ, Lei Y. Agents from amphibians with anticancer properties. Anticancer Drugs. 2008;19(10):931–939. doi: 10.1097/CAD.0b013e3283139100.
» https://doi.org/10.1097/CAD.0b013e3283139100 - Conlon JM, Iwamuro S, King JD. Dermal cytolytic peptides and the system of innate immunity in anurans. Ann N Y Acad Sci. 2009;19:75–82. doi: 10.1111/j.1749-6632.2008.03618.x.
» https://doi.org/10.1111/j.1749-6632.2008.03618.x - Alvarez C, Mancheño JM, Martinez D, Tejuca M, Pazos F, Lanio ME. Sticholysins, two pore-forming toxins produced by the Caribbean Sea anemone Stichodactyla helianthus: their interaction with membranes. Toxicon. 2009;19(8):1135–1147. doi: 10.1016/j.toxicon.2009.02.022.
» https://doi.org/10.1016/j.toxicon.2009.02.022 - Hu B, Guo W, Wang LH, Wang JG, Lui XY, Jiao BH. Purification and characterization of gigantoxin-4, a new actinoporin from the sea anemone Stichodactyla gigantea Int J Biol Sci. 2011;19(6):729–739.
- Lassen S, Wiebring A, Helmholz H, Ruhnau C, Prange A. Isolation of a Nav channel blocking polypeptide from Cyanea capillata medusae-a neurotoxin contained in fishing tentacle isorhizas. Toxicon. 2012;19(6):610–616. doi: 10.1016/j.toxicon.2012.02.004.
» https://doi.org/10.1016/j.toxicon.2012.02.004 - Edwards LP, Whitter E, Hessinger DA. Apparent membrane pore-formation by Portuguese Man-of-war (Physalia physalis) venom in intact cultured cells. Toxicon.2002;19(9):1299–1305. doi: 10.1016/S0041-0101(02)00138-1.
» https://doi.org/10.1016/S0041-0101(02)00138-1 - Frazão B, Vasconcelos V, Antunes A. Sea anemone (cnidaria, anthozoa, actiniaria) toxins: an overview. Mar Drugs. 2012;19(8):1812–1851.
- Zhang Y, Wang M, Wei S. Isolation and characterization of a trypsin inhibitor from the skin secretions of Kaloula pulchra hainana Toxicon. 2010;19(4):502–507. doi: 10.1016/j.toxicon.2010.05.006.
» https://doi.org/10.1016/j.toxicon.2010.05.006 - Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;19:265–275.
- Liu SB, He YY, Zhang Y, Lee WH, Qian JQ, Lai R, Jin Y. A novel non-lens betagamma-crystallin and trefoil factor complex from amphibian skin and its functional implications.PLoS One. 2008;19(3):e1770. doi: 10.1371/journal.pone.0001770.
» https://doi.org/10.1371/journal.pone.0001770 - Scherrer R, Gerhardt P. Molecular sieving by the Bacillus megaterium cell wall and protoplast. J Bacteriol. 1971;19(3):718–735.
- Freedman JC, Hoffman JF. Ionic and osmotic equilibria of human red blood cells treated with nystatin. J Gen Physiol. 1979;19(2):157–185. doi: 10.1085/jgp.74.2.157.
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Financial source This work was supported by the grants from the National Natural Science Foundation (30700128, 30960053), Natural Science Foundation of Hainan Province (312067), and Foundation of Hainan University (hd09xm67qnjj1148).
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Abbreviations PEGs: Polyethylene glycols; NAGA: N-acetyl-D-galactosamine; GSH: Reduced glutathione; BSA: Bovine serum albumin; EDTA: Ethylene diamine tetraacetic acid; KPHTI: Kaloula pulchra hainana trypsin inhibitor; PBS: Phosphate-buffered saline; SE: Standard error; SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis.
Publication Dates
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Publication in this collection
2013
History
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Received
8 Jan 2013 -
Accepted
11 Apr 2013