Reversal of Succinylcholine Induced Apnea with an Organophosphate Scavenging Recombinant Butyrylcholinesterase

Background

Concerns about the safety of paralytics such as succinylcholine to facilitate endotracheal intubation limit their use in prehospital and emergency department settings. The ability to rapidly reverse paralysis and restore respiratory drive would increase the safety margin of an agent, thus permitting the pursuit of alternative intubation strategies. In particular, patients who carry genetic or acquired deficiency of butyrylcholinesterase, the serum enzyme responsible for succinylcholine hydrolysis, are susceptible to succinylcholine-induced apnea, which manifests as paralysis, lasting hours beyond the normally brief half-life of succinylcholine. We hypothesized that intravenous administration of plant-derived recombinant BChE, which also prevents mortality in nerve agent poisoning, would rapidly reverse the effects of succinylcholine.

Methods

Recombinant butyrylcholinesterase was produced in transgenic plants and purified. Further analysis involved murine and guinea pig models of succinylcholine toxicity. Animals were treated with lethal and sublethal doses of succinylcholine followed by administration of butyrylcholinesterase or vehicle. In both animal models vital signs and overall survival at specified intervals post succinylcholine administration were assessed.

Results

Purified plant-derived recombinant human butyrylcholinesterase can hydrolyze succinylcholine in vitro. Challenge of mice with an LD₁₀₀ of succinylcholine followed by BChE administration resulted in complete prevention of respiratory inhibition and concomitant mortality. Furthermore, experiments in symptomatic guinea pigs demonstrated extremely rapid succinylcholine detoxification with complete amelioration of symptoms and no apparent complications.

Conclusions

Recombinant plant-derived butyrylcholinesterase was capable of counteracting and reversing apnea in two complementary models of lethal succinylcholine toxicity, completely preventing mortality. This study of a protein antidote validates the feasibility of protection and treatment of overdose from succinylcholine as well as other biologically active butyrylcholinesterase substrates.     

Arbuscular Mycorrhizal fungi (AMF) protects photosynthetic apparatus of wheat under drought stress

Photosynthesis Research - Drought stress (DS) is amongst one of the abiotic factors affecting plant growth by limiting productivity of crops by inhibiting photosynthesis. Damage due to DS and its...     

Redefining the Ethnographic Object: An Anthropology Museum Turns Fifty

Exhibit A: Objects of Intrigue. Museum of Anthropology, University of British Columbia, Vancouver, March 9, 1999–March 31, 2000.
Objects and Expressions: Celebrating the Collections of the Museum of Anthropology at the University of British Columbia. Vancouver: Museum of Anthropology, University of British Columbia, 1999.     

Characterization of a mercury-reducing Bacillus cereus strain isolated from the Pulicat Lake sediments, south east coast of India

Pulicat Lake sediments are often severely polluted with the toxic heavy metal mercury. Several mercury-resistant strains of Bacillus species were isolated from the sediments and all the isolates exhibited broad spectrum resistance (resistance to both organic and inorganic mercuric compounds). Plasmid curing assay showed that all the isolated Bacillus strains carry chromosomally borne mercury resistance. Polymerase chain reaction and southern hybridization analyses using merA and merB3 gene primers/probes showed that five of the isolated Bacillus strains carry sequences similar to known merA and merB3 genes. Results of multiple sequence alignment revealed 99% similarity with merA and merB3 of TnMERI1 (class II transposons). Other mercury resistant Bacillus species lacking homology to these genes were not able to volatilize mercuric chloride, indicating the presence of other modes of resistance to mercuric compounds.     

Computational identification of key residues regulating fluorescence emission in a red/green cyanobacteriochrome

Cyanobacteriochromes (CBCRs) are linear tetrapyrrole bilin-binding photoreceptors of cyanobacteria that exhibit high spectral diversity, gaining attention in optogenetics and bioimaging applications. Several engineering studies on CBCRs were attempted, especially for designing near-infrared (NIR) fluorescent proteins with longer fluorescence wavelengths. However, despite continuous efforts, a key component regulating fluorescence emission property in CBCRs is still poorly understood. As a model system, we focused on red/green CBCR Slr1393g3, from the unicellular cyanobacterium Synechocystis sp. PCC 6803 to engineer Pr to get far-red light-emitting property. Energy profiling and pairwise structural comparison of Slr1393g3 variants effectively reveal the mutations that are critical to the fluorescence changes. H497 seems to play a key role in stabilizing the chromophore environment, especially the α3 helix, while H495, T499, and Q502 are potential key residues determining fluorescence emission peak wavelength. We also found that mutations of α2 and α4 helical regions are closely related to the chromophore binding stability and likely affect fluorescence properties. Taken together, our computational analysis suggests that the fluorescence of Slr1393g3 is mainly controlled by the stabilization of the chromophore binding pocket. The predicted key residues potentially regulating the fluorescence emission property of a red/green CBCR will be advantageous for designing improved NIR fluorescent protein when combined with in vitro molecular evolution approaches.     

Photosynthetic efficiency in sun and shade plants

Photosynthetica - Photosynthesis is amongst the plant cell functions that are highly sensitive to any type of changes. Sun and shade conditions are prevalent in fields as well as dense forests....     

Role of Ca2+ in induction and secretion of dengue virus-induced cytokines

The present study was undertaken to investigate the role of calcium ions (Ca₂₊) in the induction and secretion of the dengue type 2 virus induced cytotoxic factor and the cytotoxin. This was done by using calcium channel blocking drugs such as verapamil, nifedipine or diltiazem hydrochloride. The production of cytotoxic factor was significantly reduced by treatment of dengue type 2 virus infected mice with verapamil. Similarly, a dosedependent inhibition of the secretion of cytotoxic factor was observed, when spleen cells of the virus-primed mice were treatedin vitro with the 3 calcium channel blockers. The production of cytotoxin by macrophages was abrogated by pretreatment with calcium channel blockers but had little effect on its secretion as shown by treatment of macrophages with verapamil at 1 h after the induction to later periods up to 18 h. The findings thus show that in the induction of both the cytokines Ca₂₊ plays a critical role; on the other hand it is required for the secretion of the cytotoxic factor but not for that of the cytotoxin.     

Critical role of mitochondrial dysfunction and impaired mitophagy in diabetic nephropathy

Mitochondrial dynamics play a critical role in deciding the fate of a cell under normal and diseased condition. Recent surge of studies indicate their regulatory role in meeting energy demands in renal cells making them critical entities in the progression of diabetic nephropathy. Diabetes is remarkably associated with abnormal fuel metabolism, a basis for free radical generation, which if left unchecked may devastate the mitochondria structurally and functionally. Impaired mitochondrial function and their aberrant accumulation have been known to be involved in the manifestation of diabetic nephropathy, indicating perturbed balance of mitochondrial dynamics, and mitochondrial turnover. Mitochondrial dynamics emphasize the critical role of mitochondrial fission proteins such as mitochondrial fission 1, dynamin-related protein 1 and mitochondrial fission factor and fusion proteins including mitofusin-1, mitofusin-2 and optic atrophy 1. Clearance of dysfunctional mitochondria is aided by translocation of autophagy machinery to the impaired mitochondria and subsequent activation of mitophagy regulating proteins PTEN-induced putative kinase 1 and Parkin, for which mitochondrial fission is a prior event. In this review, we discuss recent progression in our understanding of the molecular mechanisms targeting reactive oxygen species mediated alterations in mitochondrial energetics, mitophagy related disorders, impaired glucose transport, tubular atrophy, and renal cell death. The molecular cross talks linking autophagy and renoprotection through an intervention of 5′-AMP-activated protein kinase, mammalian target of rapamycin, and SIRT1 factors are also highlighted here, as in-depth exploration of these pathways may help in deriving therapeutic strategies for managing diabetes provoked end-stage renal disease.     

Purification and characterization of a novel plant-type carbonic anhydrase from Bacillus subtilis

Carbonic anhydrase enzyme, one of the fastest known enzymes, remains largely unexplored in prokaryotes when compared to its mammalian counterparts despite its ubiquity. In this study, the enzyme has been purified from Bacillus subtilis SA3 using sequential Sephadex G-75 chromatography, DEAE cellulose chromatography, and sepharose-4B-L-tyrosinesulphanilamide affinity chromatography and characterized to provide additional insights into its properties. The apparent molecular mass of carbonic anhydrase obtained by SDS-PAGE was found to be approximately 37 kDa. Isoelectric focusing of the purified enzyme revealed an isoelectric point (pI) of around 6.1 when compared with marker. The presence of metal ions such as Zn²⁺, Co²⁺, Cu²⁺, Fe³⁺, Mg²⁺, and anion SO₄⁻ increased enzyme activity while strong inhibition was observed in the presence of Hg²⁺, Cl⁻, HCO₃⁻, and metal chelator EDTA. The optimum pH and temperature for the enzyme were found to be 8.3 and 37°C, respectively. Enzyme kinetics with p-nitrophenyl acetate as substrate at pH 8.3 and 37°C determined the V_max and K_m values of the enzyme to be 714.28 μmol/mg protein/min and 9.09 mM, respectively. The K_i value for acetazolamide was 0.22 mM, compared to 0.099 mM for sulphanilamide. The results from N-terminal amino acid sequencing imply the purified protein is a putative beta-carbonic anhydrase with close similarities to CAs from plants, microorganisms.