UNASSIGNED: Bungarus multicinctus is one of the top ten venomous snakes in China. Its venom is mainly neurotoxin-based. Novel antivenom drugs need to be further researched and developed.
UNASSIGNED: This study aimed to explore the molecular mechanism of Cynanchum paniculatum in treating Bungarus multicinctus bites based on network pharmacology. Material and methods. The potential active ingredients of Cynanchum paniculatum were screened and their SDF structures were obtained using the PubChem database and imported into the SwissTargetPrediction database, and targets were obtained for the antitoxin effects of Cynanchum paniculatum in the treatment of Bungarus multicinctus bites. The Cynanchum paniculatum-active compound-potential target network and protein-protein interaction network were constructed by using Cytoscape software, and then biological function analysis and KEGG pathway enrichment analysis were performed using the DAVID.
UNASSIGNED: Seven potential active components (cynapanoside C, cynatratoside B, tomentolide A, sitosterol, sarcostin, tomentogenin, and paeonol) and 286 drug targets were obtained, including 30 key targets for the treatment of bungarotoxin toxicity. The active components mainly acted on PIK3CA, MAPK1, MAP2K1, JAK2, FYN, ACHE, CHRNA7, CHRNA4, and CHRNB2, and they antagonized the inhibitory effect of bungarotoxin on the nervous system through cholinergic synapses and the neurotrophin signaling pathway.
UNASSIGNED: Cynanchum paniculatum exerts a therapeutic effect on Bungarus multicinctus bites through multiple active components, multiple targets, and multiple pathways. The findings provide a theoretical basis for the extraction of active components of Cynanchum paniculatum and for related antivenom experiments.

Bungarus multicinctus is the most venomous snake distributed in China and neighboring countries of Myanmar, Laos, north Vietnam and Thailand. The high mortality rate of B. multicinctus envenomation is attributed to the lethal components of α-, β-, γ- and κ- bungarotoxins contained in the venom. Although anti-B. multicinctus sera were produced in Shanghai, Taiwan and Vietnam, the most widely clinic used product was term as B. multicinctus antivenin and manufactured by Shanghai Serum Bio-technology Co. Ltd. In the present investigation, high purity α-, β- and γ-bungarotoxins were separately isolated from B. multicinctus crude venom. Rabbit anti- α-, β- and γ-bungarotoxin antisera were prepared by common methods, respectively. LD50 values of α-, β- and γ-bungarotoxins were systematically determined via three administration pathways (intraperitoneal, intramuscular and intravenous injections) in Kunming mice. LD50 values of β-bungarotoxin were closely related with injection routines but those of both α- and γ-bungarotoxins were not dependent on the injection routines. Commercial B. multicinctus antivenin showed strong immunoreaction with high molecular weight fractions of the B. multicinctus but weakly recognized low molecular weight fractions like α- and γ-bungarotoxins. Although B. multicinctus antivenin showed immunoreaction with high molecular weight fractions of Bungarus fasciatus, Naja atra, Ophiophagus hannah venoms but the antivenin only demonstrated animal protection efficacy against O. hannah venom. These results indicated that the high molecular weight fractions of the O. hannah played an important role in venom lethality but those of B. fasciatus and N. atra did not have such a role.

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell surface mobility(1-7). To study receptor cell surface localization and endocytosis, the technique described here combines the use of fluorescent α-bungarotoxin with cells expressing constructs containing an α-bungarotoxin (Bgt) binding site (BBS). The BBS (WRYYESSLEPYPD) is based on the α subunit of the muscle nicotinic acetylcholine receptor, which binds Bgt with high affinity(8,9). Incorporation of the BBS site allows surface localization and measurements of receptor insertion or removal with application of exogenous fluorescent Bgt, as previously described in the tracking of GABAA and metabotropic GABAB receptors(2,10). In addition to the BBS site, we inserted a pH-sensitive GFP (pHGFP(11)) between amino acids 4 and 5 of the mature GABAAR subunit by standard molecular biology and PCR cloning strategies (see Figure 1)(12). The BBS is 3\' of the pH-sensitive GFP reporter, separated by a 13-amino acid alanine/proline linker. For trafficking studies described in this publication that are based on fixed samples, the pHGFP serves as a reporter of total tagged GABAAR subunit protein levels, allowing normalization of the Bgt labeled receptor population to total receptor population. This minimizes cell to cell Bgt staining signal variability resulting from higher or lower baseline expression of the tagged GABAAR subunits. Furthermore the pHGFP tag enables easy identification of construct expressing cells for live or fixed imaging experiments.

Alcohol abuse and alcoholism are serious and costly problem in USA. Thus, the development of anti-alcoholism agents could be very significant. The understanding of the neurochemical basis underlying the addictive properties of drugs of abuse is imperative for the development of new pharmacological means to reverse the addictive state, prevent relapse or to reduce the intake of addictive compounds. The nicotinic acetylcholine receptors (nAChRs) are important therapeutic targets for various diseases. Recent studies have revealed that the alpha3beta2, alpha3beta3, and alpha6 subunits of nAChR protein family might be pharmacological targets for developing new drugs in the treatment of alcoholism. We have performed computational homology modeling of the alpha3beta2, alpha3beta3, and alpha6 subunits of human nACHRs based upon the recently determined crystal structure of the extracellular domain (ECD) of the mouse nAChR alpha1 subunit complexed with alpha-bungarotoxin at 1.94 A resolution. For comparison, we also built the ECD models of alpha4beta2, and alpha7 subunits of human nACHRs which are neurochemical targets for cessation of smoking. The three-dimensional (3D) models of the ECD of the monomer, and pentamer of these human nAChR were constructed. The docking of the agonist in the ligand-binding pocket of the human nAChR dimers was also performed. Since the nAChR ligand-binding site is a useful target for mutagenesis studies and the rational design of drugs against various diseases, these models provide useful information for future investigation.

Homomeric alpha7 nicotinic acetylcholine receptors are a well-established, pharmacologically distinct subtype. The more recently identified alpha9 subunit can also form functional homopentamers as well as alpha9alpha10 heteropentamers. Current fluorescent probes for alpha7 nicotinic ACh receptors are derived from alpha-bungarotoxin (alpha-BgTx). However, alpha-BgTx also binds to alpha9* and alpha1* receptors which are coexpressed with alpha7 in multiple tissues. We used an analog of alpha-conotoxin ArIB to develop a highly selective fluorescent probe for alpha7 receptors. This fluorescent alpha-conotoxin, Cy3-ArIB[V11L;V16A], blocked ACh-evoked alpha7 currents in Xenopus laevis oocytes with an IC(50) value of 2.0 nM. Observed rates of blockade were minute-scale with recovery from blockade even slower. Unlike FITC-conjugated alpha-BgTx, Cy3-ArIB[V11L;V16A] did not block alpha9alpha10 or alpha1beta1deltaepsilon receptors. In competition binding assays, Cy3-ArIB[V11L;V16A] potently displaced [(125)I]-alpha-BgTx binding to mouse hippocampal membranes with a K(i) value of 21 nM. Application of Cy3-ArIB[V11L;V16A] resulted in specific punctate labeling of KXalpha7R1 cells but not KXalpha3beta2R4, KXalpha3beta4R2, or KXalpha4beta2R2 cells. This labeling could be abolished by pre-treatment with alpha-cobratoxin. Thus, Cy3-ArIB[V11L;V16A] is a novel and selective fluorescent probe for alpha7 receptors.

Polybia-MPII (INWLKLGKMVIDAL-NH2), a mastoparan isolated from the crude venom of the swarming wasp Polybia paulista, was injected into the left hind limb of Swiss white mice. Between 3 h and 21 days later the mice were killed and the soleus muscles from both hind limbs were removed. Sections of the muscles were made for transmission electron microscopy and immunocytochemistry. Transmission electron microscopy showed that both the volume fraction occupied by synaptic vesicles and synaptic vesicle density was greatly reduced after exposure to Polybia-MPII, although there was no significant structural damage to the plasma membrane of the terminal boutons and mitochondria were indistinguishable from those in normal, control boutons. Immunocytochemistry revealed that in control muscles 99% of motor end plates identified by the positive labelling of acetylcholine receptors by TRITC-alpha-bungarotoxin co-labelled with anti-synaptophysin antibody, but this figure fell by 30% in muscles exposed to the toxin. These changes were transient. They were maximal at 6 h and fully reversed by 3 days. At no time was axonal labelling with anti-neurofilament antibodies affected by exposure to Polybia-MPII. We conclude that mastoparan Polybia-MPII is a minor neurotoxin and suggest that its neurotoxic activity is unlikely to be of clinical significance.

BACKGROUND: The alpha7 nicotinic acetylcholine receptors (nAChRs) play an important role in the pathophysiology of neuropsychiatric diseases such as schizophrenia and Alzheimer\'s disease. However, there are currently no suitable positron emission tomography (PET) radioligands for imaging alpha7 nAChRs in the intact human brain. Here we report the novel PET radioligand [11C]CHIBA-1001 for in vivo imaging of alpha7 nAChRs in the non-human primate brain.
RESULTS: A receptor binding assay showed that CHIBA-1001 was a highly selective ligand at alpha7 nAChRs. Using conscious monkeys, we found that the distribution of radioactivity in the monkey brain after intravenous administration of [11C]CHIBA-1001 was consistent with the regional distribution of alpha7 nAChRs in the monkey brain. The distribution of radioactivity in the brain regions after intravenous administration of [11C]CHIBA-1001 was blocked by pretreatment with the selective alpha7 nAChR agonist SSR180711 (5.0 mg/kg). However, the distribution of [11C]CHIBA-1001 was not altered by pretreatment with the selective alpha4beta2 nAChR agonist A85380 (1.0 mg/kg). Interestingly, the binding of [11C]CHIBA-1001 in the frontal cortex of the monkey brain was significantly decreased by subchronic administration of the N-methyl-D-aspartate (NMDA) receptor antagonist phencyclidine (0.3 mg/kg, twice a day for 13 days); which is a non-human primate model of schizophrenia.
CONCLUSIONS: The present findings suggest that [11C]CHIBA-1001 could be a novel useful PET ligand for in vivo study of the receptor occupancy and pathophysiology of alpha7 nAChRs in the intact brain of patients with neuropsychiatric diseases such as schizophrenia and Alzheimer\'s disease.

Transverse cryosections, 6-8 mum thick, were cut from unfixed biventer cervicis muscles of chicks and quadriceps muscles of humans, mounted on glass slides and incubated for 1h in either isotonic phosphate buffered saline, pH 7.3 (PBS), or crude venom of venom of Pseudechis colletti at concentrations between 2.1 and 210 microgml(-1) in PBS. They were then exposed to a fluoresceine-conjugated alpha-bungarotoxin to label ACh receptor sites. Exposure to the crude venom of P. colletti prevented the labelling of acetylcholine (ACh) receptors in chick muscle in a dose-dependant manner; at a concentration of 2.1 microgml(-1) labelling fell by 20% and at a concentration of 21 microgml(-1) by more than 90%. In contrast, exposure to the venom at concentrations as high as 210 microgml(-1) had no effect on receptor labelling in human skeletal muscle. The results suggest that ACh receptors in human skeletal muscle are relatively resistant to the postsynaptically active neurotoxins in the venom of P. colletti. The data explain the apparent anomaly that the venom blocks neuromuscular transmission in isolated nerve-muscle preparations of the chick whilst human subjects of envenoming bites by P. colletti exhibit no overt signs of neurotoxicity.

BACKGROUND: Immunotherapy for treatment of snake bites has been based on mammalian IgG. Recently, polyvalent ovine Fab has become available. However, papain, used in the Fab fragmentation process, is a human allergen. Avian eggs are a source of antibodies and a truncated version of IgY, IgY(DeltaFc), is found in ducks. In this study, we induced duck antibodies by using detoxified cobra and krait venoms and then purified IgY(DeltaFc) antibodies from the hyperimmune duck egg yolk.
METHODS: Ducks were used for immunization and their eggs were collected for antibody production. ICR strain female mice were used in the in vivo neutralization test. Monovalent antivenoms to Formosan cobra venom and Formosan multi-banded krait venom were raised and purified from hyper-immune duck egg yolk individually. The LD(50) of venoms were determined by subcutaneous injection of different venom doses into the mice. The survival/death ratios were recorded after 24 hours.
RESULTS: The antibody purified from egg yolk showed high titer response to its immunogen (cobra or krait venom) by an ELISA. Overall, the antibodies from duck eggs efficiently protected mice from envenomations.
CONCLUSIONS: The antivenoms purified from the egg yolk of ducks immunized with cobra venom and krait venom neutralized the lethal effects of these venoms with good efficacy in a mouse model. The antivenoms were effective in neutralizing lethality in mice injected at 4xLD(50) of venoms.
CONCLUSIONS: These results indicate that antibodies derived from ducks can serve as a new source for the generation of antivenoms.

We studied the role of the cytoplasmic regions adjacent to the M3 and M4 transmembrane segments of alpha7 nicotinic receptors in the expression of functional channels. For this purpose, a total of 50 amino acids were mutated throughout the mentioned regions. Mutants close to M3, from Arg294 to Leu321, showed slight modifications in the levels of alpha-bungarotoxin binding sites and acetylcholine-evoked currents. Exceptions were mutants located at two clusters (His296 to Pro300 and Ile312 to Trp316), which exhibited low expression levels. In addition, some mutants showed altered functional responses. Many mutants close to M4 showed increased receptor expression, especially the ones located at the hydrophobic face of a putative amphipathic helix. This effect seems to be the consequence of a combination of increased receptor biosynthesis, higher transport efficiency and delayed degradation, such that we postulate that elements in the amphipathic domain strongly influence receptor stability. Finally, some mutants in this region showed altered functional responses: elimination of positively charged residues (Arg424 and Arg426) increased currents, whereas the opposite was observed upon suppression of negatively charged ones (Glu430 and Glu432). These results suggest that the cytoplasmic regions close to M3 and M4 play important structural and functional roles.