The use of extracorporeal membrane oxygenation in pediatric patients with sickle cell disease.

Previous reports have described the use of extracorporeal membrane oxygenation (ECMO) for acute chest syndrome of sickle cell disease (SCD). However, there have been no reports of venoarterial (VA) ECMO for cardiac dysfunction in patients with SCD. We describe a patient with SCD and life-threatening cardiogenic shock who was successfully treated with VA ECMO. Furthermore, SCD patients have unique comorbidities that warrant particular consideration when utilizing ECMO. We discuss these considerations and review the documented experience with ECMO for pediatric SCD patients from the Extracorporeal Life Support Organization (ELSO) registry. From 1990 until 2012, 52% of the 65 pediatric patients with SCD placed on ECMO survived, with 85% of those receiving venovenous (VV) ECMO surviving and 43% of those receiving VA ECMO surviving. However, significant complications, such as bleeding, neurological injury and kidney injury, also occurred with both VV and VA ECMO. Ten percent of SCD patients receiving VA ECMO experienced either a cerebral infarct or hemorrhage; our patient suffered a cerebrovascular accident while on ECMO, though she survived with good neurologic outcome. To our knowledge, this is the first report of a pediatric patient with SCD and cardiogenic shock successfully managed with VA ECMO. In conjunction with the ELSO registry review, this case report suggests that, while VA ECMO can be successfully used in patients with SCD and severe cardiovascular dysfunction, clinicians should also be aware of the potential for serious complications in this high-risk population.

Downregulation of HER2/neu receptor by solamargine enhances anticancer drug-mediated cytotoxicity in breast cancer cells with high-expressing HER2/neu.

Overexpression of HER2/neu is associated with drug resistance and poor outcome in breast cancer. Solamargine (SM), a glycoalkaloid purified from the herb Solanum incanum, exhibits HER2/neu gene modulation of HER2/neu high-expressing human breast cancer cell line ZR-75-1. SM downregulation of HER2/neu gene expression was determined by RT-PCR and Southern hybridization. Additionally, the membrane-bound HER2/neu receptor in highly HER2/neu-expressing breast cancer cells was determined by radioimmunoassay, immunocytochemistry, fluorescent immunocytochemistry, and flow cytometry. SM significantly decreased the number of HER2/neu receptors on the cell membrane. Methotrexate (MTX), 5-florouracil (5-Fu), and cisplatin (CDDP) are commonly used for breast carcinoma treatment in clinics; however, patients with HER2/neu overexpression exhibit resistance to these anticancer drugs. Notably, combination of MTX, 5-Fu, and CDDP with SM individually increased the susceptibility of breast cancer cells to these chemotherapeutic agents. Experimental results indicated that downregulation of HER2/neu by SM might be an effective strategy for enhancing drug susceptibility of breast cancer cells expressing high levels of HER2/neu.

Solamargine induces apoptosis and sensitizes breast cancer cells to cisplatin.

Solamargine (SM), a major steroidal alkaloid glycoside, was purified from Solanum incanum plant. SM exhibited the most cytotoxic effect comparing with that of cisplatin (cDDP), methotrexate (MTX), 5-fluorouracil (5-FU), epirubicin (EPI) and cyclophosphamide (CP) against human breast cancer cells. In this study, SM induces apoptosis of the breast cancer cells and the mechanism was characterized. SM up-regulated the expressions of external death receptors, such as tumor necrosis factor receptor I (TNFR-I), Fas receptor (Fas), TNFR-I-associated death domain (TRADD), and Fas-associated death domain (FADD). SM also enhanced the intrinsic ratio of Bax to Bcl-2 by up-regulating Bax and down-regulating Bcl-2 and Bcl-xL expressions. These effects resulted in the release of mitochondrial cytochrome c and activation of caspase-8, -9 and -3 in the cells, indicating that SM triggered extrinsic and intrinsic apoptotic pathways of breast cancer cells. Similar to function way of SM, cDDP causes cancer cell apoptosis though caspase-8/caspase-3 and Bax/cytochrome c pathways, but the resistance to cDDP is correlated with Bcl-2 and Bcl-xL overexpression. However, the overexpression of Bcl-2 and Bcl-xL can be broken through by SM. The combined treatment of SM and cDDP significantly reduced Bcl-2 and Bcl-xL expressions, and enhanced Bax, cytochrome c, caspase-9 and -3 expressions in breast cancer cells. Thus, the combined use of SM and cDDP may be effective in cDDP-resistant breast cancer.

Identification of functional involvement of tryptophan residues in phospholipase A2 from Naja naja atra (Taiwan cobra) snake venom.

Phospholipase A2 (PLA2) from Naja naja atra snake venom was subjected to Trp modification with 2-nitrophenylsulfenyl chloride (NPS-Cl), and six derivatives were separated by HPLC. The results of amino-acid analysis and sequence determination revealed that Trp-18, Trp-19 and Trp-61 were modified by NPS-Cl. The order of accessibilities of the three Trp residues for NPS-Cl was Trp-18 > Trp-19 > Trp-61. Sulfenylation of Trp-18 caused a 92% drop in enzymatic activity. Modification of Trp-19 and Trp-61 resulted in a decrease in enzymatic activity of PLA2 by 45.5% and 51%, respectively. The enzyme modified on both Trp-18 and Trp-19 or on both Trp-18 and Trp-61 retained little PLA2 activity. It is evident that Trp-18 plays a more crucial function in PLA2 than Trp-19 and Trp-61. Sulfenylation did not significantly affect the secondary structure of the enzyme molecule as revealed by the CD spectra, and Ca2+ binding and antigenicity of sulfenylated PLA2 was unaffected. These observations, together with the fact that Trp-18 is involved in the substrate binding of PLA2, suggest that incorporation of a bulky NPS group on Trp-18 might give rise to a direct distortion of the interaction between substrate and the enzyme molecule. Alternatively, modification of Trp-19 and Trp-61 might indirectly affect the interfacial binding of PLA2 with its substrate.

A novel function of emodin: enhancement of the nucleotide excision repair of UV- and cisplatin-induced DNA damage in human cells.

Nucleotide excision repair (NER) is the main pathway by which mammalian cells remove carcinogenic DNA lesions caused by UV light and many other common mutagens. To explore the effect of emodin on NER, its influence on the repair of UV- and cisplatin-induced DNA damage in human fibroblast cells (WI38) was evaluated. Emodin increased unscheduled DNA synthesis (UDS) of UV-treated cells and reduced cisplatin-induced DNA adducts in WI38 in a concentration-dependent manner, indicating that emodin might promote NER capability in cells. The resultant NER complex is a cooperative assembly of XPF, ERCC1, XPA, RPA, and XPG subunits. The gene regulations of the subunits after emodin treatment were determined by reverse transcription-polymerase chain reaction (RT-PCR) using specific primers. Among the subunits, the expression of ERCC1 in WI38 cells was up-regulated significantly after emodin treatment. All other expressions remained essentially unchanged. In addition, calcium influx in WI38 was increased in proportion to the concentration of emodin. Since UV-induced NER is Ca2+ dependent, elevation of calcium influx may be another mechanism by which emodin facilitates DNA repair. In conclusion, emodin can increase the repair of UV- and cisplatin-induced DNA damage in human cells, and elevated ERCC1 gene expression and Ca2+-mediated DNA repair processes may be involved in the repair mechanism of emodin.

Anticancer activity evaluation of the solanum glycoalkaloid solamargine. Triggering apoptosis in human hepatoma cells.

Solamargine, an herbal and molluscicidal medicine derived from Solanum incanum, is a steroidal alkaloid glycoside. To characterize the anticancer mechanism of solamargine on human hepatoma cells (Hep3B), changes of cell morphology, DNA content, and gene expression of cells after solamargine treatment were studied. The appearance in solamargine-treated cells of chromatin condensation, DNA fragmentation, and a sub-G(1) peak in a DNA histogram suggests that solamargine induces cell death by apoptosis. The maximum number of dead Hep3B cells was detected within 2 hr of incubation with constant concentrations of solamargine, and no further cell death was observed after an extended incubation with solamargine, indicating that the action of solamargine was irreversible. To determine the susceptibility of cell phases to solamargine-mediated apoptosis, Hep3B cells were synchronized at defined cell cycles by cyclosporin A, colchicine, and genistein, followed by solamargine treatment. The IC(50) values of solamargine for control, G(0)/G(1)-, M-, and G(2)/M-synchronized Hep3B cells were 5.0, > 10, 3.7, and 3.1 microg/mL, implying that cells in the G(2)/M phases are relatively susceptible to solamargine-mediated apoptosis. In addition, a parallel up-regulation of tumor necrosis factor receptor (TNFR)-I and -II on Hep3B cells was detected after solamargine treatment, and the solamargine-mediated cytotoxicity could be neutralized with either TNFR-I or -II specific antibody. Therefore, these results reveal that the actions of TNFR-I and -II on Hep3B cells may be independent, and both are involved in the mechanism of solamargine-mediated apoptosis.

High affinity antibody to cobrotoxin prepared from the derivatives of glutaraldehyde-detoxified cobrotoxin.

High affinity antibodies to cobrotoxin were obtained by immunization with derivatives of glutaraldehyde (GA)-modified cobrotoxin. The derivatives completely lost lethality and binding activity to nicotinic acetylcholine receptors (nAChR), but retained the same antigenicity as cobrotoxin toward anti-cobrotoxin antibody. Owing to hyperimmunization with these low toxicity derivatives, a high affinity antibody to cobrotoxin was induced in a short period. We also showed that the derivatives of cobrotoxin may have altered local conformation, and residues which contribute to the intensity of binding between antigen and antibody may consequently be exposed. Hence, the modified derivatives have increased binding affinity to anti-cobrotoxin antibody. In addition, since high affinity antibodies prepared using the derivatives exhibit more potent binding affinity to cobrotoxin than conventional anti-cobrotoxin antibody, the specific neutralizing capacity of the high affinity antibodies is greatly increased. These results lead to the conclusion that the derivatives of GA-modified cobrotoxin have the same antigenicity as the native toxin, and can be used as immunogens for the production of high affinity antibodies to cobrotoxin.

The structural loop II of cobrotoxin is the main binding region for nAChR and epitope in the region is conformation-dependent.

Modification of positively charged residues, Lys and Arg, in cobrotoxin revealed that Lys-27, Lys-47, Arg-28, Arg-30, Arg-33, and Arg-36 of cobrotoxin were essential for the lethality and binding activity to nicotinic acetylcholine receptor (nAChR). The antigenicity of cobrotoxin was drastically diminished when Lys-47, Arg-28, Arg-30, Arg-33, and Arg-36 were modified, while that of the Lys-27-modified derivative was not significantly changed. The CD spectra of cobrotoxin displayed similar patterns after modification of Lys-27, Lys-47, and Arg-28. These findings suggest that Lys-27, Lys-47, and Arg-28 residues may be related to the direct binding to nAChR, and that there is no involvement of Lys-27 in the antigenic determinants of cobrotoxin. Extending the modification to Arg-30, Arg-33, and Arg-36 caused progressive conformational changes of cobrotoxin and resulted in decreased binding activity to antibody and nAChR. This indicates that Arg-30, Arg-33, and Arg-36 may be of structural importance for maintaining the active conformation of cobrotoxin. These results, together with the facts that Tyr-25, Tyr-35, and Trp-29 of cobrotoxin are important in nAChR binding activity, but Trp-29 and Tyr-35 residues are not essential for the antigenicity, suggest that the structural loop II of cobrotoxin is the main binding region for nAChR and the epitope in that region is conformation-dependent.

Determination of soluble nAChR-binding activity of alpha-neurotoxins by an innovative precipitation with DEAE-Sephacel.

A simple and convenient method for determining the binding activity of alpha-neurotoxins toward soluble nicotinic acetylcholine receptor (snAChR) by precipitation with DEAE-Sephacel was established. The determination was carried out by incubation of 125I-neurotoxin with snAChR, followed by precipitation with DEAE-Sephacel. The DEAE-Sephacel particles bind negatively charged snAChR with high affinity and simultaneously precipitate the 125I-neurotoxin bound to the receptors. After centrifugation, the free 125I-neurotoxin in the supernatant was counted, and the amounts of neurotoxins bound to snAChR could be determined. Two alpha-neurotoxins, cobrotoxin and alpha-bungarotoxin, were employed to verify the feasibility of this determination. The different binding characteristics of cobrotoxin and alpha-bungarotoxin to snAChR could be distinguished. This method required only small quantities of DEAE-Sephacel (7 mg), snAChR (0.54 micrograms), and 125I-neurotoxin (90 fmol) for each reaction, and minimized the handling of isotopic materials as compared with the conventional methods. This method is reliable, reproducible, and superior to current methods for the determination of the snAChR-binding activity for alpha-neurotoxins.

Chemical modification of cationic groups of a novel alpha-neurotoxin (Oh-4) from king cobra (Ophiophagus hannah) venom.

The cationic groups of arginine and lysine residues in Oh-4, a novel alpha-neurotoxin from king cobra (Ophiophagus hannah) venom were subjected to modification with p-hydroxyphenylglyoxal (HPG) and trinitrobenzene sulfonate (TNBS), respectively. Monoderivatization of Arg-35, resulted in a drastic loss in neurotoxicity to 25% of the native toxin. The activity was decreased to a greater extent with the derivative extensively modified on Arg-35, -9, and -37. The Arg-35-modified derivative retained about a half of the antigenicity of the native toxin, and extensive modification on Arg-9 and Arg-37 caused a further decrease in the antigenicity of the toxin molecule. Selective trinitrophenylation (TNP-) of Lys-51 caused losses of neurotoxicity and antigenicity by 77 and 83%, respectively. These results indicate that Arg-35 and Lys-51 in Oh-4 have important roles in the neurotoxicity. In contrast to the Arg residues at 9, 35, and 37, Lys-51 plays a more critical role in the antigenicity.

Immunological neutralization of cobrotoxin by its homologous precipitin and non-precipitin antibodies.

Anti-cobrotoxin antibodies can be separated into precipitin and non-precipitin antibodies. The precipitin antibody possesses the same binding affinity to cobrotoxin as non-precipitin antibody, but the neutralizing capability of the latter is superior to that of the former in blocking cobrotoxin binding to nicotinic acetylcholine receptor (nAChR). After preincubation with antibodies, cobrotoxin completely lost its binding activity to nAChR. The dissociation of cobrotoxin-nAChR complex by the antibodies was low, and 60% of the complex formation appeared to be irreversible. These results indicate that the neutralization of cobrotoxin by the antibody may predominantly involve unbound, receptor-free cobrotoxin. The relationships of neutralization capacity and binding affinity as well as bond strength between cobrotoxin and its antibodies are incongruous. Different local conformational changes of a unique Trp in cobrotoxin on binding with the precipitin and non-precipitin antibodies seem to lead to different accessibility for fluorescence quenchers. Characterization of the binding domains by immunoprecipitation with the antibodies correlated with the quenching results. Thus, the binding topography of cobrotoxin may play an important role over the binding affinity and bond strength in neutralization by cobrotoxin antibody.

Analysis of a conformation-independent epitope and a conformational epitope in a protein: a study on cobrotoxin from Taiwan cobra venom.

The antibodies against cobrotoxin were separated into two antibody preparations by successive affinity chromatographies on reduced and S-carboxymethylated (RCM)-cobrotoxin-Sepharose, and cobrotoxin-Sepharose columns. The antibodies (abbreviated as Abcf-i) that bound with the RCM-cobrotoxin-Sepharose were verified to specifically recognize the continuous epitopes of cobrotoxin, which were insensitive to conformational changes. Whilst the antibodies (abbreviated as Abcf-d) that did not bind with the RCM-cobrotoxin-Sepharose column recognized the conformational epitopes in cobrotoxin. The two antibody preparations were employed to screen the antigenic peptides derived from the proteolytic hydrolysate of cobrotoxin and RCM-cobrotoxin. Five antigenic peptides (AP-4, AP-5, AP-10, AP-11, and AP-12) were obtained from the acid protease A-digested hydrolysate of cobrotoxin, and two antigenic peptides (V8-2 and V8-4) were found in the hydrolysate of RCM-cobrotoxin after hydrolysis with Saccharomyces aureus V8 protease. The segments at positions 1-21 and 22-38 encompassed the peptide fractions, AP-4, AP-5, V8-2, and V8-4, that reacted with Abcf-i, indicating that the two segments bore the continuous epitopes of cobrotoxin. Alternatively, AP-10, AP-11, and AP-12 reacted with both Abcf-i and Abcf-d. The structures of the three peptides had a common segment at positions 43-62, suggesting that this region comprised the conformation-independent epitopes as well as conformational epitopes in cobrotoxin. These results reflected that the conformation-independent and conformational epitopes in a protein can be separately identified.

Characterization of epitopes in native and unfolded cobrotoxin: evidence of an immunodominant C-terminal region related to the production of precipitating and non-precipitating antibodies against cobrotoxin.

Rabbits hyperimmunized with cobrotoxin from Taiwan cobra venom produced non-precipitating as well as precipitating antibodies. Both antibody preparations exhibited higher affinity for native cobrotoxin than for reduced and S-carboxymethylated (RCM) cobrotoxin. This indicated that the epitope structures in cobrotoxin are mostly conformation-dependent. In order to identify the conformational epitopes, native cobrotoxin was hydrolyzed with acid protease A, and 12 peptides were obtained on HPLC. Three peptide fragments, AP-10, AP-11, and AP-12, showed pronounced antigenicities toward precipitating as well as non-precipitating antibodies. AP-10, AP-11, and AP-12 contained a common segment in the C-terminal region of cobrotoxin, residues 43 to 62, with intact disulfide linkages. Complete removal of the C-terminal antibodies from antisera and precipitating antibodies on a C-terminal segment-Sepharose affinity column resulted in the loss of their precipitability with cobrotoxin, whilst restoration of precipitability was observed on the addition of the C-terminal antibodies to the C-terminal antibody-depleted antisera and precipitating antibodies. Studies on the antigenic structures of RCM-cobrotoxin revealed that RCM-cobrotoxin contains an immunodominant epitope at positions 22-38. The N-terminal and C-terminal regions of RCM-cobrotoxin encompass other epitopes which exhibit low reactivities toward anti-RCM-cobrotoxin antibodies. However, no precipitated antigen-antibody complexes were observed with the mixture of anti-RCM-cobrotoxin antibodies and RCM-cobrotoxin. These results suggest that the inherently different immunogenicities with different segments might affect the precipitabilities of the resulting antibodies, and that the notable immunogenecity of the C-terminal region is related to the production of precipitating and non-precipitating antibodies against cobrotoxin.

Modulation of epidermal terminal differentiation in patients after long-term topical corticosteroids.

The expression of the various markers for terminal epidermal differentiation in atrophic skin of patients after long-term topical corticosteroids (TCS) was studied by electron microscopy, immunofluorescence using antibody to profilaggrin/filaggrin (PF/FG), immunoperoxidase staining using antibody to involucrin, and oil red O stain for neural lipids of the stratum corneum. Thirty-nine patients were subdivided into two groups: (A) 19 patients suffering from rebound phenomenon after stopping TCS and (B) 20 patients without rebound phenomenon. Biopsy specimens were taken before ending the use of TCS in both groups. In group A, both the morphological markers (including the different epidermal strata, keratohyalin granules, lamellar granules, and cornified cell envelopes) and the molecular markers (including involucrin, PF/FG, and neutral lipids) of terminal epidermal differentiation were significantly suppressed. On the other hand, the differentiational markers in the atrophic skin of patients without rebound phenomena were only slightly altered. These results suggest that potent TCS not only has antiproliferative actions but also inhibits the differentiation of epidermis, resulting in structural defects in the epidermis, especially the stratum corneum.

Permeability barrier abnormality of hairless mouse epidermis after topical corticosteroid: characterization of stratum corneum lipids by ruthenium tetroxide staining and high-performance thin-layer chromatography.

Topical corticosteroids (TCS) are among the most frequently used topical therapeutics. Recently, it has been shown that TCS not only has antiproliferative actions, but also inhibits the differentiation of the epidermis and finally perturbates stratum corneum (s.c.) barrier function. It is well established that epidermal barrier function resides within the intercellular lipids of the SC. However, to date, little is known about the effects of TCS on the structure and composition of s.c. lipids. We therefore used hairless mouse skin to study the sequential changes of the s.c. permeability barrier and their intercellular lipids by ruthenium tetroxide staining and high-performance thin-layer chromatography (HPTLC) during topical use of corticosteroids. The results demonstrated a progressive increase in transepidermal water loss accompanied by a diminution in the SC intercellular lipid lamellae, which showed a normal structure of individual lamella. Analysis of lipid composition by HPTLC after a 6-week application of TCS also showed an obvious decrease in all the main components of s.c. lipids, which are known to constitute the permeability barrier of the skin. In light of these results, our work provides direct morphological evidence that TCS deteriorates the permeability barrier of epidermis when applied to normal skin.

Role of the exon 6 of human PDGF A-chain mRNA in the determination of gene expression by reverse transcription-polymerase chain reaction.

Alternative-spliced PDGF A mRNA is constituted with exon 6-excluded (PDGFA [-6]) and exon 6-included (PDGFA[+6]) transcripts. To define the role of exon 6 in the gene expression of alternative-spliced PDGF A in U937 cell, the exon-junction primers were employed for RT-PCR. The PDGFA[-6] mRNA could be amplified with specific primers at annealing temperatures of 60 degrees C, 65 degrees C, 70 degrees C and 75 degrees C, whereas PDGFA[+6] mRNA could be detected only at 70 degrees C, suggesting that the RNA secondary structure of PDGFA[+6] might affect the RT-PCR reactions. In addition, PDGFA[+6] mRNA was incapable of competing with PDGFA[-6] mRNA for the common primers. A highly complementary and double-stranded RNA structure was formed for PDGF A when the exon 6 was included in the sequence. Since accessibility of a RNA template for in vitro amplification is related to RNA secondary structure, the results derived from RT-PCR and mRNA folding of PDGFA[+6] are essentially consistent. Thus, these results suggest that the exon 6 may affect the determination of gene expression by constricting the RNA secondary structure of PDGF A. The requirement of imperatively high stringency in the hybridization conditions for PDGFA[+6] mRNA may account for the low detection of the transcript in cells by conventional methods.

Roles of the aromatic residues in cobrotoxin in antigenicity and binding activity to nicotinic acetylcholine receptor.

The single Trp-29 in cobrotoxin was modified with 2-hydroxy-5-nitrobenzyl bromide, and Tyr-25 and -35, with tetranitromethane. Although the binding activity of cobrotoxin to the nicotinic acetylcholine receptor (nAChR) was decreased after modification of Trp-29, the antigenicity remained essentially unchanged as measured by a competitive RIA. Tyr-35-nitrated cobrotoxin still retained relatively high antigenicity and potent binding activity to nAChR. However, modification of both Tyr-25 and Tyr-35 led to an altered secondary structure, with greatly reduced binding to antibody and receptor. Hence, Tyr-25 in cobrotoxin is essential for the maintenance of the active conformation for the bindings. Although the non-precipitating antibody against cobrotoxin exhibiting a higher neutralizing capacity than did the precipitating antibody, the binding affinity of the former to cobrotoxin and its modified derivatives was lower than that of the latter.

Immunochemical characterization of alpha-bungarotoxin detoxified with glutaraldehyde.

Modification of alpha-bungarotoxin (alpha-BuTX) with glutaraldehyde (GA), a bifunctional reagent, resulted in the formation of crosslinked derivatives. The intramolecular linked monomeric derivative as well as dimeric and higher molecular weight derivatives arising from intermolecular reactions were separated by gel filtration. The dimer and polymers were completely detoxified and the monomer lost nearly all of its lethal toxicity as well as binding activity to the nicotinic acetylcholine receptor. However, all the modified derivatives retained relatively high antigenic activity towards the antibody against native toxin. Antisera prepared by immunization with the unfractionated modified products of alpha-BuTX contained considerable amounts of a non-precipitating antibody, but much less of a precipitating antibody to alpha-BuTX, while they had the same capacity as that of anti-alpha-BuTX for neutralizing the lethal toxicity of alpha-BuTX. Furthermore, the antisera could be obtained in only half period required for anti-alpha-BuTX sera so that the detoxified GA-alpha-BuTX is an useful immunogen for the production of antibody to alpha-BuTX.

Status of tryptophan residue in cobrotoxin and alpha-bungarotoxin.

Acrylamide quenching studies indicated that the exposure degree of the Trp residue in cobrotoxin was higher than that in alpha-bungarotoxin. The Trp residue of cobrotoxin was in a positively charged environment as revealed by iodide quenching, while the Trp residue of alpha-bungarotoxin was not accessible for iodide. Analysis of hydrophilicity profile and local concentration of positively charged residues of toxin molecule also indicated that Trp in cobrotoxin was in a highly hydrophilic and positively charged environment. Measurement of Trp fluorescence with increasing temperature showed that the stability of environment of Trp in alpha-bungarotoxin was higher than in cobrotoxin. Result of competitive binding for nicotinic acetylcholine receptor (AchR) between cobrotoxin and alpha-bungarotoxin revealed that the molecular interaction of the two toxins with AchR was not the same. These, together with the fact that the cationic groups of the two toxins are involved in the binding with AchR, suggest that the observed different environment surrounded Trp residue and different AchR binding mechanism might fulfill a different requirement of the invariant Trp in the lethality of cobrotoxin and alpha-bungarotoxin.

Role of amino and carboxyl groups of cobrotoxin in the conformational stability and the interaction with acetylcholine receptor.

To study the functional involvements of the common interaction of the Leu-1 alpha-amino group and Asp-58 in cobrotoxin, the lysine epsilon-amino groups of cobrotoxin were initially guanidinated with o-methylisourea. The alpha-amino group of Leu-1 was them modified with TNBS after the guanidination of cobrotoxin. Both modified derivatives displayed no significant changes in the secondary structure and antigenicity of cobrotoxin, whereas the binding affinity for nicotinic acetylcholine receptor (nAChR) was pronouncedly decreased when Leu-1 was modified. Six out of seven free carboxyl groups and the remaining buried Glu-21 carboxyl group of cobrotoxin were modified with glycine methyl ester in the absence and presence of guanidine HCl, respectively. Alternation in the beta-sheet secondary structure of cobrotoxin was observed with the carboxyl-group modified derivatives, which caused a decrease in the binding activity of the toxin molecule to the antibody and nAChR. Moreover, modification of the Glu-21 carboxyl group of cobrotoxin further reduced the nAChR binding activity, while the antigenicity remained unchange. Thus, our results conclude that the Glu-21 residue and the common interaction of the terminal Leu-1 alpha-amino group and the Asp-58 carboxyl group are related to the nAChR-binding activity of cobrotoxin, and the free carboxyl groups in cobrotoxin are conformation-essential.