Evaluate the influences of some factors as enzyme level, the time of diluting enzyme solution, substrate level and temperature on pH value of RNase from Vietnam Cobra (Naja naja) venom. Results: the changes of pH value for cobra venom RNase can be depended on concentrations of the enzyme and substrate. This variation in the pH value of the enzyme can be explained by existence of this RNase as an enzyme system composed of some interconvertible forms. The interconversion between these enzyme forms is very slow process (counting by hours) in comparison with the rate of reactions
Cobra ; Hydrogen-Ion Concentration ; Ribonucleases

PURPOSE: Breast cancer is a significant health problem worldwide, accounting for a quarter of all cancer diagnoses in women. Current strategies for breast cancer treatment are not fully effective, and there is substantial interest in the identification of novel anticancer agents especially from natural products including toxins. Cytotoxins are polypeptides found in the venom of cobras and have various physiological effects. In the present study, the anticancer potential of cytotoxin-II against the human breast adenocarcinoma cell line (MCF-7) was investigated. METHODS: The cytotoxic effects of cytotoxin-II were determined by morphological analysis and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The mode and mechanism of cell death were investigated via acridine orange/ethidium bromide (AO/EtBr) double staining, flow cytometric analysis of cell death, detection of mitochondrial membrane potential, measurement of intracellular reactive oxygen species (ROS), annexin V/propidium iodide staining, and caspase-9 activity assays. RESULTS: The half maximal inhibitory concentration (IC50) of cytotoxin-II in MCF-7 cells was 4.18+/-1.23 microg/mL, while the value for cisplatin was approximately 28.02+/-1.87 microg/mL. Morphological analysis and AO/EtBr double staining showed typical manifestations of apoptotic cell death (in doses lower than 8 microg/mL). Dose- and time-dependent ROS generation, loss of mitochondrial membrane potential, caspase-9 activation, and cell cycle arrest were observed in their respective tests. CONCLUSION: In conclusion, cytotoxin-II has potent anticancer effects in the MCF-7 cell line, which are induced via the intrinsic pathways of apoptosis. Based on these findings, cytotoxin-II is a suitable choice for breast cancer treatment.
Adenocarcinoma* ; Antineoplastic Agents ; Apoptosis* ; Biological Products ; Breast Neoplasms ; Breast* ; Caspase 9 ; Cell Cycle Checkpoints ; Cell Death ; Cell Line* ; Cisplatin ; Cobra Venoms* ; Cytotoxins ; Diagnosis ; Elapidae ; Female ; Humans ; MCF-7 Cells ; Membrane Potential, Mitochondrial ; Peptides ; Reactive Oxygen Species ; Snakes ; Venoms

Neuro-osteological interactions have an important role in the regulation of bone metabolism and regeneration. Neuropeptides combined with porous biphasic calcium phosphates (BCP) using protein adsorption may contribute to the acceleration of bone formation. In the present study, we investigated the effect of BCP combined with nerve growth factor (NGF) on the growth of osteoblasts in vitro and the combinational therapeutic effect on the repair of calvarial defects in vivo. NGF was separated and purified from Chinese cobra venom using a simplified three-step chromatography method. BCP combined with NGF exerted a potent effect on osteoblast differentiation, as evidenced by enhanced cell proliferation, increased ALP activity and the up-regulated expression of osteogenesis-related genes and proteins. Further, combinational therapy with BCP and NGF improved calvarial regeneration, which was superior to treatment with therapy alone, as observed using imageological and morphological examination and histological and immunohistochemical staining. The results confirmed the effect of neuro-osteological interactions through combinatorial treatment with NGF and BCP to promote osteogenesis and bone formation, which may provide an effective and economical strategy for clinical application.
Acceleration ; Adsorption ; Asian Continental Ancestry Group ; Calcium Phosphates ; Cell Proliferation ; Ceramics* ; Chromatography ; Cobra Venoms ; Elapidae* ; Humans ; In Vitro Techniques ; Metabolism ; Methods ; Nerve Growth Factor* ; Neuropeptides ; Osteoblasts ; Osteogenesis ; Regeneration ; Therapeutic Uses*

Among the enzymes found in snake venom, ribonuclease (RNase) has been known to have the potential effect against cancer and HIV. In a previous report, the author and his colleague have demonstrated that RNase from Vietnamese black cobra (Naja naja) venom differed from all the other identified RNase for its extremely low optimal value of pH. The results in this study showed that it also differed in nonlinear activity dependence on the enzyme concentrations and a sigmoidal curve of saturation with the substrate. This enzyme expressed the maximal activity at the ionic strength of 10 mM of the reaction buffers. Ammonium sulfate entirely suppressed the enzyme activity at the concentration over 70 mM, and sodium chloride reduced the activity by 70% at the level over 100 mM. No magnesium ion was needed for the activation of this RNase.
Snake Venoms ; Snakes ; Animals, Poisonous ; Black Cobra Venom

Bungarus Parvus, a precious animal Chinese medicinal material used in clinical practice, is believed to be first recorded in Ying Pian Xin Can published in 1936. This study was carried out to analyze the names, geographical distribution, morphological characteristics, ecological habits, poisonousness, and medicinal parts by consulting ancient Chinese medical books and local chronicles, Chinese Pharmacopeia, different processing standards of trditional Chinese medicine(TCM) decoction pieces, and modern literatures. The results showed that the earliest medicinal record of Bungarus Parvus was traced to 1894. In 1930, this medicinal material was used in the formulation of Annao Pills. The original animal, Bungarus multicinctus, was recorded by the name of "Bojijia" in 1521. The morphological characteristics, ecological habits, and poisonousness of the original animal are the same in ancient and modern records. The geographical distribution is similar between the ancient records and modern documents such as China Medicinal Animal Fauna. The dried body of young B. multicinctus is used as Bungarus Parvus, which lack detailed references. As a matter of fact, it is still inconclusive whether there are differences between young snakes and adult snakes in terms of active ingredients, pharmacological effects, and clinical applications. This study clarified the medicinal history and present situation of Bungarus Parvus. On the basis of the results, it is suggested that systematic comparison on young and adult B. multicinctus should be carried out to provide references for revising the medicinal parts of B. multicinctus.
Animals ; Bungarus ; Snakes ; China ; Medicine, Chinese Traditional ; Drugs, Chinese Herbal

BACKGROUND AND OBJECTIVES: Hyperacute rejection (HAR) is a major obstacle to successful xenotransplantation of vascularized organs. This study was conducted to observe the effect of hemolysis of perfused human whole blood on pig heart function, and determine the major risk factors for preservation of xenoperfused cardiac function using ex-vivo pig to human xenogeneic cardiac perfusion model. MATERIALS AND METHODS: Harvested pig hearts were perfused with normal human whole blood (group 1), two different types of pre-treated human whole blood (group 2: immunoglobulins were depleted by plasmapheresis, group 3: pre-treated with plasmapheresis, GAS914, cobra venom factor (CVF) and steroid), and normal porcine whole blood as control (group 4) for 3 hours. RESULTS: Duration of heart beat was significantly prolonged in group 2 and group 3. Histological examination showed widespread HAR features but was gradually delayed in groups 2 and 3 compared to group 1. The absolute levels of serum creatine kinase-MB and Troponin I increased gradually, and was lower in group 3. Serum hemoglobin levels were rapidly increased in groups 3 and 4, compared to group 1. Extracellular potassium level increased sharply from the beginning of blood perfusion in groups 1, 2 and 3, compared to group 4. CONCLUSION: Pretreatment of human whole blood, including immunoglobulin depletion, CVF and steroid reduced and delayed the destruction of pig myocardium by HAR. However, the increased extracellular potassium levels in groups 1, 2 and 3 reflected that these treatments could not prohibit myocardial injury by HAR.
Cobra Venoms ; Creatine ; Diphtheria Toxoid ; Extracorporeal Circulation ; Haemophilus Vaccines ; Heart ; Hemoglobins ; Hemolysis ; Humans ; Hyperkalemia ; Immunoglobulins ; Myocardium ; Perfusion ; Plasmapheresis ; Potassium ; Rejection (Psychology) ; Risk Factors ; Transplantation, Heterologous ; Trisaccharides ; Troponin I

PURPOSE: We designed a pig to canine liver xenotransplantation model to study the diverse immunologic and hemodynamic consequences that follow xenotransplantation and hyperacute rejection. METHODS: The animals were divided into two groups: the cobra venom factor and Gadolinium chloride treatment group (CVF+Gd group) (3 cases) and the control group (3 cases). The donor pig's whole liver was harvested, and then the harvested pig's whole liver was transplanted into a dog after the dog underwent left hepatectomy. After reperfusion of the graft, blood samples were taken 20, 40 and 60 minutes after reperfusion, and the liver, lung and kidney tissues were taken 1 hour after reperfusion. RESULTS: In the control group, the grafts showed a patchy hypoperfused liver surface and it felt rubbery solid compared to the CVF+Gd group. The serum total protein, albumin, fibrinogen and platelets decreased abruptly and there were no significant differences between the two groups. The serum PT, PTT and FDP were increased in both groups and the CVF+Gd group showed a more obtuse slope than the control group. We could not find any intravascular pathologic changes on the microscopic findings of the graft. Only scant intravascular fibrin deposition was found. Hepatocellular vacuolization and sinusoidal dilatation were also found. There were patches of necrosis without any zonal distribution, intrasinusoidal neutrophil sequestration and interstitial hemorrhage. These findings were milder in the CVF+Gd group. CONCLUSION: The pig to canine partial auxiliary liver xenotransplantation model is feasible and it is a good model before starting to perform pig to primate liver xenotransplantation. In the CVF+Gd group, pathologic findings like patch hepatocyte necrosis etc. were less severe. As there were no corresponding vascular pathologic findings, these findings are not the direct effect of CVF and gadolinium treatment, and so other factors like Ischemia- reperfusion injury should be considered.
Animals ; Blood Platelets ; Cobra Venoms ; Complement System Proteins ; Dilatation ; Dogs ; Fibrin ; Fibrinogen ; Fluconazole ; Formycins ; Gadolinium ; Hemodynamics ; Hemorrhage ; Hepatectomy ; Hepatocytes ; Humans ; Kidney ; Kupffer Cells ; Liver ; Lung ; Necrosis ; Neutrophils ; Primates ; Rejection (Psychology) ; Reperfusion ; Reperfusion Injury ; Ribonucleotides ; Tissue Donors ; Transplantation, Heterologous ; Transplants

The purpose of this paper is to encapsulate neurotoxin-I (NT-I), a kind of analgesic peptide, into polylactic acid (PLA) nanoparticles (NPs) and to evaluate their transport into the brain after intranasal administration (in) by use of microdialysis sampling technique developed in our laboratory recently. NT-I-NPs (NT-Iradiolabeled with sodium 125I-Iodide) were prepared by a double emulsification solvent evaporation method, and were characterized in terms of surface morphology, particle size distribution, zeta potential and entrapment efficiency. Then, NT-I-NPs were administered intranasally or intravenously to rats and the radioactivities in periaqueductal gray (PAG) were monitored up to 240 min utilizing the microdialysis sampling technique. Nanoparticles prepared were spherical with homogenous size distribution. Their mean particle size and zeta potential measured were (65.3 +/- 10.8) nm and (-28.6 +/- 2.3) mV, respectively. The entrapment efficiency of NT-Iencapsulated into nanoparticles was (35.5 +/- 2.8)%. The brain transport results showed that the time to peak level (Tmax) of NT-I-NPs (in) was (65 +/- 10) min approximately, apparently shorter compared with NT-I-NPs [iv, (95 +/- 10) min] or NT-I [iv, (145 +/- 10) min]. The concentration to peak level (Cmax) and the area under the curves from zero to 4 h (AUC0-4h) of each group followed this order: NT-I-NPs (in) > NT-I-NPs (iv) > NT-I (iv). With nanoparticles as carriers and administered intranasally could be a potential way for centrally active peptides to improve their brain transport. Microdialysis is quite a good technique for the study of drug delivery to the brain.
Administration, Intranasal ; Animals ; Area Under Curve ; Cobra Neurotoxin Proteins ; administration & dosage ; pharmacokinetics ; Drug Delivery Systems ; Lactic Acid ; chemistry ; Male ; Microdialysis ; Nanoparticles ; Particle Size ; Periaqueductal Gray ; metabolism ; Polyesters ; Polymers ; chemistry ; Random Allocation ; Rats ; Rats, Sprague-Dawley

The purpose of the present study is to establish a rapid and effective PCR method for the identification of B. multicinctus. Based on sequence alignment of B. multicinctus and its adulterants, we found that Cyt b gene is a good molecular genetic marker for the authentication of B. multicinctus. On the basis of the sequence data, a pair of highly specialized primers was designed. The templates were extracted by the DNA purification system. Key factors such as annealing temperature, concentration of Taq enzyme and cycle numbers were analyzed and optimized. The modified PCR program consisted of an initial denaturation step at 95 degrees C for 5 min, followed by 30 cycles of 95 degrees C for 30 s and 55 degrees C for 45 s and a final extension at 72 degrees C for 5 min. Thirteen samples of B. multicinctus were identified accurately from their 20 adulterants in 4 hours. The results indicated it is a highly accurate, rapid and applicable method for the authentication of B. multicinctus.
Animals ; Bungarus ; classification ; genetics ; Cytochromes b ; genetics ; DNA Primers ; genetics ; Drug Contamination ; Molecular Sequence Data ; Polymerase Chain Reaction ; methods ; Sequence Alignment ; Sequence Analysis, DNA

OBJECTIVETo develop a convenient and effective method for the identification of Bungarus multicinctus.

METHODBased on the sequence of Cyt b gene fragment of B. multicinctus and its adulterants, a pair of highly specific primer (HJL- and HJH-) were designed for distinguishing B. ulticinctus from other species of snake. To establish specific PCR reaction condition, the primers were employed to amplify the DNA templates extracted from B. multicinctus and 6 other species of snake, under different annealing temperature. Using this method, B. multicinctus was identified from 18 samples bought from many drugstores.

RESULTA 230 bp DNA fragment was amplified from B. multicinctus in PCR with annealed temperature at 67 degrees C, whereas no DNA fragment was amplified from other snake samples under the same reaction condition, B. multicinctus could be clearly distinguished from others by PCR reaction with the highly specific primers. In the present study, 18 sample, bought from different drugstores, were also identified by the highly specific PCR with the primers. The results indicated that 14 samples were B. multicinctus and the other 4 were adulterant, which was consistent with the conclusion of authentication based on morphological.

CONCLUSIONThe primers designed in the present study were highly specific for B. multicinctus.

Animals ; Base Sequence ; Bungarus ; classification ; genetics ; Cytochromes b ; genetics ; DNA ; genetics ; DNA Primers ; Drug Contamination ; Materia Medica ; Molecular Sequence Data ; Polymerase Chain Reaction ; methods ; Sequence Analysis, DNA ; Snakes ; classification ; genetics ; Species Specificity