Final Protein Concentration Research Articles

Structural Basis for Universal Corrinoid Recognition by the Cobalamin Transport Protein Haptocorrin

Cobalamin (Cbl; vitamin B12) is an essential micronutrient synthesized only by bacteria. Mammals have developed a sophisticated uptake system to capture the vitamin from the diet. Cbl transport is mediated by three transport proteins: transcobalamin, intrinsic factor, and haptocorrin (HC). All three proteins have a similar overall structure but a different selectivity for corrinoids. Here, we present the crystal structures of human HC in complex with cyanocobalamin and cobinamide at 2.35 and 3.0 Å resolution, respectively. The structures reveal that many of the interactions with the corrin ring are conserved among the human Cbl transporters. However, the non-conserved residues Asn-120, Arg-357, and Asn-373 form distinct interactions allowing for stabilization of corrinoids other than Cbl. A central binding motif forms interactions with the e- and f-side chains of the corrin ring and is conserved in corrinoid-binding proteins of other species. In addition, the α- and β-domains of HC form several unique interdomain contacts and have a higher shape complementarity than those of intrinsic factor and transcobalamin. The stabilization of ligands by all of these interactions is reflected in higher melting temperatures of the protein-ligand complexes. Our structural analysis offers fundamental insights into the unique binding behavior of HC and completes the picture of Cbl interaction with its three transport proteins.

TBid Undergoes Multiple Conformational Changes at the Membrane Required for Bax Activation

Bid is a Bcl-2 family protein that promotes apoptosis by activating Bax and eliciting mitochondrial outer membrane permeabilization (MOMP). Full-length Bid is cleaved in response to apoptotic stimuli into two fragments, p7 and tBid (p15), that are held together by strong hydrophobic interactions until the complex binds to membranes. The detailed mechanism(s) of fragment separation including tBid binding to membranes and release of the p7 fragment to the cytoplasm remain unclear. Using liposomes or isolated mitochondria with fluorescently labeled proteins at physiological concentrations as in vitro models, we report that the two components of the complex quickly separate upon interaction with a membrane. Once tBid binds to the membrane, it undergoes slow structural rearrangements that result in an equilibrium between two major tBid conformations on the membrane. The conformational change of tBid is a prerequisite for interaction with Bax and is, therefore, a novel step that can be modulated to promote or inhibit MOMP. Using automated high-throughput image analysis in cells, we show that down-regulation of Mtch2 causes a significant delay between tBid and Bax relocalization in cells. We propose that by promoting insertion of tBid via a conformational change at the mitochondrial outer membrane, Mtch2 accelerates tBid-mediated Bax activation and MOMP. Thus the interaction of Mtch2 and tBid is a potential target for therapeutic control of Bid initiated cell death.

Convergent Replication of Mouse Synthetic Prion Strains

Prion diseases are neurodegenerative disorders characterized by the aberrant folding of endogenous proteins into self-propagating pathogenic conformers. Prion disease can be initiated in animal models by inoculation with amyloid fibrils formed from bacterially derived recombinant prion protein. The synthetic prions that accumulated in infected organisms are structurally distinct from the amyloid preparations used to initiate their formation and change conformationally on repeated passage. To investigate the nature of synthetic prion transformation, we infected mice with a conformationally diverse set of amyloids and serially passaged the resulting prion strains. At each passage, we monitored changes in the biochemical and biological properties of the adapting strain. The physicochemical properties of each synthetic prion strain gradually changed on serial propagation until attaining a common adapted state with shared physicochemical characteristics. These results indicate that synthetic prions can assume multiple intermediate conformations before converging into one conformation optimized for in vivo propagation.

Differential expression of two isolates of beak and feather disease virus capsid protein in Escherichia coli

Expression of recombinant beak and feather disease virus (BFDV) capsid-associated protein (Cap) has relied on inefficient techniques that typically produce low yields or use specialized expression systems, which greatly increase the cost and expertise required for mass production. An Escherichia coli system was used to express recombinant BFDV Cap derived from two isolates of BFDV, from a Long-billed Corella (Cacatua tenuirostris) and an Orange-bellied parrot (OBP; Neophema chrysogaster). Purification by affinity and size exclusion chromatography was optimized through an iterative process involving screening and modification of buffer constituents and pH. A buffer containing glycerol, β-mercaptoethanol, Triton X-100, and a high concentration of NaCl at pH 8 was used to increase solubility of the protein. The final concentration of the corella-isolated BFDV protein was fifteen- to twenty-fold greater than that produced in previous publications using E. coli expression systems. Immunoassays were used to confirm the specific antigenicity of recombinant Cap, verifying its validity for use in continued experimentation as a potential vaccine, a reagent in diagnostic assays, and as a concentrated sample for biological discoveries.

Opticin Exerts Its Anti-angiogenic Activity by Regulating Extracellular Matrix Adhesiveness

Opticin is an extracellular matrix glycoprotein that we identified associated with the collagen network of the vitreous humor of the eye. Recently, we discovered that opticin possesses anti-angiogenic activity using a murine oxygen-induced retinopathy model: here, we investigate the underlying mechanism. Using an ex vivo chick chorioallantoic membrane assay, we show that opticin inhibits angiogenesis when stimulated by a range of growth factors. We show that it suppresses capillary morphogenesis, inhibits endothelial invasion, and promotes capillary network regression in three-dimensional matrices of collagen and Matrigel(TM). We then show that opticin binds to collagen and thereby competitively inhibits endothelial cell interactions with collagen via α(1)β(1) and α(2)β(1) integrins, thereby preventing the strong adhesion that is required for proangiogenic signaling via these integrins.

Specific Recognition of Biologically Active Amyloid-β Oligomers by a New Surface Plasmon Resonance-based Immunoassay and an in Vivo Assay in Caenorhabditis elegans

Soluble oligomers of the amyloid-β (Aβ) peptide play a key role in the pathogenesis of Alzheimer's disease, but their elusive nature makes their detection challenging. Here we describe a novel immunoassay based on surface plasmon resonance (SPR) that specifically recognizes biologically active Aβ oligomers. As a capturing agent, we immobilized on the sensor chip the monoclonal antibody 4G8, which targets a central hydrophobic region of Aβ. This SPR assay allows specific recognition of oligomeric intermediates that rapidly appear and disappear during the incubation of synthetic Aβ(1-42), discriminating them from monomers and higher order aggregates. The species recognized by SPR generate ionic currents in artificial lipid bilayers and inhibit the physiological pharyngeal contractions in Caenorhabditis elegans, a new method for testing the toxic potential of Aβ oligomers. With these assays we found that the formation of biologically relevant Aβ oligomers is inhibited by epigallocatechin gallate and increased by the A2V mutation, previously reported to induce early onset dementia. The SPR-based immunoassay provides new opportunities for detection of toxic Aβ oligomers in biological samples and could be adapted to study misfolding proteins in other neurodegenerative disorders.

Efficient lysozyme refolding at a high final concentration and a low dilution factor

Of the various protein refolding methods, direct dilution is one of the simplest and easiest for scaling up the refolding process. However, it requires a large amount of refolding buffer, often utilizes a number of chemicals, and results in a low final protein concentration. In this report, we demonstrate that reduced dithiothreitol (DTTred), a carryover from denaturation, is a crucial and adverse factor in lysozyme refolding. Accordingly, we proposed a method of using high concentration of oxidized glutathione (GSSG) in the refolding buffer to eliminate excess DTTred and aid in the refolding of lysozyme. The efficiency of this method is 84%, which resulted in a high final refolded protein concentration of 1.5g/l and required only a low dilution factor (4×). Furthermore, compared with the traditional 50× direct dilution (resulting in a similar yield of 74%), the low dilution factor required much less GSSG and other constituents.

Soluble Monomeric IgG1 Fc

Antibody fragments are emerging as promising biopharmaceuticals because of their relatively small size and other unique properties. However, compared with full-size antibodies, these antibody fragments lack the ability to bind the neonatal Fc receptor (FcRn) and have reduced half-lives. Fc engineered to bind antigens but preserve interactions with FcRn and Fc fused with monomeric proteins currently are being developed as candidate therapeutics with prolonged half-lives; in these and other cases, Fc is a dimer of two CH2-CH3 chains. To further reduce the size of Fc but preserve FcRn binding, we generated three human soluble monomeric IgG1 Fcs (mFcs) by using a combination of structure-based rational protein design combined with multiple screening strategies. These mFcs were highly soluble and retained binding to human FcRn comparable with that of Fc. These results provide direct experimental evidence that efficient binding to human FcRn does not require human Fc dimerization. The newly identified mFcs are promising for the development of mFc fusion proteins and for novel types of mFc-based therapeutic antibodies of small size and long half-lives.

Abstract 106: Corticosteroid Treatment Induces Structural and Functional Changes in High-Density Lipoproteins

Rationale: We have previously shown that high-density lipoproteins (HDL) have potent anti-inflammatory properties. HDL levels are decreased in most inflammatory diseases. Corticosteroid therapy modifies lipoproteins profiles in subjects with chronic obstructive pulmonary disease (COPD) but results from these studies are inconsistent. Here, we hypothesize that in COPD subjects, prednisone treatment induces beneficial structural and functional changes in HDL thus improving HDL anti-inflammatory properties. Objectives: To elucidate the effect of prednisone on (i) HDL size, composition and subpopulation distribution (ii) the lipid and lipoprotein profiles of subjects with COPD and (iii) HDL anti-inflammatory properties. Methods: We recruited COPD subjects (n=11) treated with prednisone and people treated with antibiotics (n=6) for lower respiratory tract infections (control). Blood samples were collected on days 1, 5 of prednisone treatment and 6 weeks post-treatment. The treatment started with a high-dose (>50 mg) and was reduced over a 5-7 day treatment period. We used gel-filtration chromatography to analyse plasma lipoprotein profiles. ApoA-I, apoA-II, apoB levels were determined immunoturbidometrically; total cholesterol, HDL-C, triglyceride and phospholipid concentrations were determined colorimetrically; and size distribution and surface charge of HDL were determined by 2-D gel electrophoresis. HDL was isolated from plasma and incubated for 16h on human endothelial cells (final protein concentration 1 mg/ml). Cells were further stimulated for 5h with TNF-α (0.2 ng/ml). Adhesion molecule expression (ICAM-1 and VCAM-1) was assessed by flow cytometry. Results: Plasma from prednisone treated subjects showed increased apoA-I (36%), apo-B (51%), total cholesterol (13%) and phospholipid (37%) levels and decreased plasma triglycerides (41%) relative to 6 weeks post-treatment. HDL-C levels were higher in subjects that received high-dose prednisone (0.94±0.21 mmol/L) compared to 6 weeks post-treatment (0.65±0.06 mmol/L) and antibiotic-treated subjects (0.43±0.17 mmol/L). HDL diameter in prednisone treated subjects increased from 7.9 nm to 10 nm. HDL from prednisone-treated subjects suppressed ICAM-1 (70%) and VCAM-1 (65%) protein expression more effectively than the HDL from 6-weeks post-treatment (25% and 12% respectively) or the HDL from the antibiotics-treated subjects (36% and 24% respectively) (p<0.05 for all). Conclusions: In this study, we have shown the evidence that high-dose, short-term prednisone treatment increases HDL levels and improves their anti-inflammatory properties.

The fibrillogenic L178H variant of apolipoprotein A-I forms helical fibrils

A number of amyloidogenic variants of apoA-I have been discovered but most have not been analyzed. Previously, we showed that the G26R mutation of apoA-I leads to increased β-strand structure, increased N-terminal protease susceptibility, and increased fibril formation after several days of incubation. In vivo, this and other variants mutated in the N-terminal domain (residues 26 to ∼90) lead to renal and hepatic accumulation. In contrast, several mutations identified within residues 170 to 178 lead to cardiac, laryngeal, and cutaneous protein deposition. Here, we describe the structural changes in the fibrillogenic variant L178H. Like G26R, the initial structure of the protein exhibits altered tertiary conformation relative to wild-type protein along with decreased stability and an altered lipid binding profile. However, in contrast to G26R, L178H undergoes an increase in helical structure upon incubation at 37°C with a half time (t1/2) of about 12 days. Upon prolonged incubation, the L178H mutant forms fibrils of a diameter of 10 nm that ranges in length from 30 to 120 nm. These results show that apoA-I, known for its dynamic properties, has the ability to form multiple fibrillar conformations, which may play a role in the tissue-specific deposition of the individual variants.

Urocortins Improve Dystrophic Skeletal Muscle Structure and Function through Both PKA- and Epac-Dependent Pathways

In Duchenne muscular dystrophy, the absence of dystrophin causes progressive muscle wasting and premature death. Excessive calcium influx is thought to initiate the pathogenic cascade, resulting in muscle cell death. Urocortins (Ucns) have protected muscle in several experimental paradigms. Herein, we demonstrate that daily s.c. injections of either Ucn 1 or Ucn 2 to 3-week-old dystrophic mdx(5Cv) mice for 2 weeks increased skeletal muscle mass and normalized plasma creatine kinase activity. Histological examination showed that Ucns remarkably reduced necrosis in the diaphragm and slow- and fast-twitch muscles. Ucns improved muscle resistance to mechanical stress provoked by repetitive tetanizations. Ucn 2 treatment resulted in faster kinetics of contraction and relaxation and a rightward shift of the force-frequency curve, suggesting improved calcium homeostasis. Ucn 2 decreased calcium influx into freshly isolated dystrophic muscles. Pharmacological manipulation demonstrated that the mechanism involved the corticotropin-releasing factor type 2 receptor, cAMP elevation, and activation of both protein kinase A and the cAMP-binding protein Epac. Moreover, both STIM1, the calcium sensor that initiates the assembly of store-operated channels, and the calcium-independent phospholipase A(2) that activates these channels were reduced in dystrophic muscle by Ucn 2. Altogether, our results demonstrate the high potency of Ucns for improving dystrophic muscle structure and function, suggesting that these peptides may be considered for treatment of Duchenne muscular dystrophy.

Cutting Off Functional Loops from Homodimeric Enzyme Superoxide Dismutase 1 (SOD1) Leaves Monomeric β-Barrels

Demetallation of the homodimeric enzyme Cu/Zn-superoxide dismutase (SOD1) is known to unleash pronounced dynamic motions in the long active-site loops that comprise almost a third of the folded structure. The resulting apo species, which shows increased propensity to aggregate, stands out as the prime disease precursor in amyotrophic lateral sclerosis (ALS). Even so, the detailed structural properties of the apoSOD1 framework have remained elusive and controversial. In this study, we examine the structural interplay between the central apoSOD1 barrel and the active-site loops by simply cutting them off; loops IV and VII were substituted with short Gly-Ala-Gly linkers. The results show that loop removal breaks the dimer interface and leads to soluble, monomeric β-barrels with high structural integrity. NMR-detected nuclear Overhauser effects are found between all of the constituent β-strands, confirming ordered interactions across the whole barrel. Moreover, the breathing motions of the SOD1 barrel are overall insensitive to loop removal and yield hydrogen/deuterium protection factors typical for cooperatively folded proteins (i.e. the active-site loops act as a "bolt-on" domain with little dynamic influence on its structural foundation). The sole exceptions are the relatively low protection factors in β-strand 5 and the turn around Gly-93, a hot spot for ALS-provoking mutations, which decrease even further upon loop removal. Taken together, these data suggest that the cytotoxic function of apoSOD1 does not emerge from its folded ground state but from a high energy intermediate or even from the denatured ensemble.

A Study of the Spatial Protein Organization of the Postsynaptic Density Isolated from Porcine Cerebral Cortex and Cerebellum

Postsynaptic density (PSD) is a protein supramolecule lying underneath the postsynaptic membrane of excitatory synapses and has been implicated to play important roles in synaptic structure and function in mammalian central nervous system. Here, PSDs were isolated from two distinct regions of porcine brain, cerebral cortex and cerebellum. SDS-PAGE and Western blotting analyses indicated that cerebral and cerebellar PSDs consisted of a similar set of proteins with noticeable differences in the abundance of various proteins between these samples. Subsequently, protein localization in these PSDs was analyzed by using the Nano-Depth-Tagging method. This method involved the use of three synthetic reagents, as agarose beads whose surface was covalently linked with a fluorescent, photoactivable, and cleavable chemical crosslinker by spacers of varied lengths. After its application was verified by using a synthetic complex consisting of four layers of different proteins, the Nano-Depth-Tagging method was used here to yield information concerning the depth distribution of various proteins in the PSD. The results indicated that in both cerebral and cerebellar PSDs, glutamate receptors, actin, and actin binding proteins resided in the peripheral regions within ∼ 10 nm deep from the surface and that scaffold proteins, tubulin subunits, microtubule-binding proteins, and membrane cytoskeleton proteins found in mammalian erythrocytes resided in the interiors deeper than 10 nm from the surface in the PSD. Finally, by using the immunoabsorption method, binding partner proteins of two proteins residing in the interiors, PSD-95 and α-tubulin, and those of two proteins residing in the peripheral regions, elongation factor-1α and calcium, calmodulin-dependent protein kinase II α subunit, of cerebral and cerebellar PSDs were identified. Overall, the results indicate a striking similarity in protein organization between the PSDs isolated from porcine cerebral cortex and cerebellum. A model of the molecular structure of the PSD has also been proposed here.

Linking assimilate supply and seed developmental processes that determine soybean seed composition

The concentrations of protein and oil in soybean ( Glycine max L.) seeds are important factors determining the quality of soybean meal and its competitiveness in international markets. Studies aimed at understanding genetic factors and/or environmental conditions that distinguish high levels of protein and oil accumulation from those that result in deficient levels typically analyze mature seeds. This approach inevitably confounds maternal, zygotic, and environmental effects on seed component accumulation. We have attempted to resolve these effects by manipulating assimilate supply per seed and quantifying the impact directly on the rate and duration of protein, oil, and residual (predominately carbohydrate) accumulation. Genotypes varying in final seed protein concentration were grown in the field and exposed to treatments to increase (depodding) or decrease (shading) assimilate supply per seed during seed filling. Depodding increased the rate of protein, oil, and residual accumulation. Protein accumulation was most responsive, resulting in a dramatic increase in final protein concentration, particularly in the low-protein genotypes. Shading decreased oil accumulation with less impact on protein and residual content, leading to a reduction in final oil concentration. Rates of protein accumulation explained most of the variation in final contents rather than duration. For each seed component, a single hyperbolic function described the relationship between assimilate supply per seed and the rate of component accumulation for all genotypes and treatments. These results underscore the importance of evaluating genetic and environmental effects directly on seed component accumulation, and confirm the predominant influence of assimilate supply per seed on soybean seed composition. Theoretical modeling showed that further increases in seed protein concentration cannot rely on more assimilates supply and that future increases will depend on increases in resource use efficiency and/or changes in partitioning.

Human cytochrome P450 and UDP-glucuronosyltransferase reaction phenotyping of MPC-3100, an oral HSP90 inhibitor.

e13019 Background: MPC-3100 is an oral synthetic inhibitor of HSP90, in a phase I safety study in cancer patients. Major cytochrome P450 (CYP450) and UDP-glucuronosyltransferase (UGT) isozymes were identified to define the inclusion and exclusion criteria necessary to ensure patient safety. Methods: CYP450 isozymes responsible for the metabolism of MPC-3100 were assessed by two methods. The first entailed the use of recombinant human Supersomes (1A2, 2C8, 2C9, 2C19, 2D6 and 3A4) suspended in phosphate buffer (pH 7.4) with 40 pmoles of CYP450. An NADPH regenerating system was used with a 5 minute pre-incubation prior to adding 10 µM MPC-3100. The second used human donor liver microsomes suspended in phosphate buffer (pH 7.4) with 0.5 mg/mL of protein. The microsomes were pre-incubated with CYP450 isozyme selective chemical inhibitors prior to addition of 10 µM MPC-3100 and NADPH. UGT isozymes responsible for glucuronidation of MPC-3100 and a major oxidative metabolite were identified using recombinant UGT Supersomes suspended in Tris-HCl buffer (pH 7.5) at a final protein concentration of 1 mg/mL. The UGT Supersomes were pre-incubated with alamethicin and the reaction was initiated with UDPGA. The disappearance of MPC-3100 and formation of metabolites was monitored by HPLC-ESI-MS/MS. Results: MPC-3100 was metabolized by CYP450 1A2, 2C19, 2D6 and 3A4, but not by 2C8 or 2C9. With the use of selective chemical inhibitors in pooled human liver microsomes, the percent of MPC-3100 metabolized at 40 minutes was inhibited greatest by azamulin (31.4%), ketoconazole (20.9%) and quinidine (5.5%). MPC-3100 was glucuronidated by UGT 1A3, 1A8 and 1A4, but not by 1A1, 1A6, 1A7, 1A9, 1A10, 2B4, 2B5, 2B15, and 2B17. The major oxidative metabolite of MPC-3100 was a substrate for UGT 1A3, 1A7, 1A9, 1A10, 2B4, 2B17 with 1A1, 1A8, and 2B7 predominating. Conclusions: CYP450 3A4 is the primary isozyme involved in the metabolism of MPC-3100. UGT 1A4 is the major isozyme responsible for direct glucuronidation of MPC-3100; whereas multiple UGTs (1A1, 1A8, and 2B7) are capable of glucuronidating the major oxidative metabolite. Therefore current clinical studies preclude co-administration of potent CYP3A4 inhibitors with MPC-3100.

Recombinant soluble glypican 3 protein inhibits the growth of hepatocellular carcinoma in vitro

showing that hepatocellularcarcinoma (HCC) cells infected with lentivirus for soluble glypican 3 (sGPC3) expressionhad a lower proliferation rate. The authors indicated that the growth inhibition might be dueto the sGPC3 protein secreted by infected HCC cells. Here we provide direct evidenceshowing that the sGPC3 protein does in fact inhibit the growth of HCC cells

Recovery of Small Infectious PrPres Aggregates from Prion-infected Cultured Cells

Prion diseases are characterized by deposits of abnormal conformers of the PrP protein. Although large aggregates of proteinase K-resistant PrP (PrP(res)) are infectious, the precise relationships between aggregation state and infectivity remain to be established. In this study, we have fractionated detergent lysates from prion-infected cultured cells by differential ultracentrifugation and ultrafiltration and have characterized a previously unnoticed PrP species. This abnormal form is resistant to proteinase K digestion but, in contrast to typical aggregated PrP(res), remains in the soluble fraction at intermediate centrifugal forces and is not retained by filters of 300-kDa cutoff. Cell-based assay and inoculation to animals demonstrate that these entities are infectious. The finding that cell-derived small infectious PrP(res) aggregates can be recovered in the absence of strong in vitro denaturating treatments now gives a biological basis for investigating the role of small PrP aggregates in the pathogenicity and/or the multiplication cycle of prions.

Morphology and structure of lipoproteins revealed by an optimized negative-staining protocol of electron microscopy

Plasma lipoprotein levels are predictors of risk for coronary artery disease. Lipoprotein structure-function relationships provide important clues that help identify the role of lipoproteins in cardiovascular disease. The compositional and conformational heterogeneity of lipoproteins are major barriers to the identification of their structures, as discovered using traditional approaches. Although electron microscopy (EM) is an alternative approach, conventional negative staining (NS) produces rouleau artifacts. In a previous study of apolipoprotein (apo)E4-containing reconstituted HDL (rHDL) particles, we optimized the NS method in a way that eliminated rouleaux. Here we report that phosphotungstic acid at high buffer salt concentrations plays a key role in rouleau formation. We also validate our protocol for analyzing the major plasma lipoprotein classes HDL, LDL, IDL, and VLDL, as well as homogeneously prepared apoA-I-containing rHDL. High-contrast EM images revealed morphology and detailed structures of lipoproteins, especially apoA-I-containing rHDL, that are amenable to three-dimensional reconstruction by single-particle analysis and electron tomography.

Haemonchus contortus: in vivo anthelmintic activity of Eugenia dysenterica DC. and Caryocar brasiliense Cambess leaves in sheep

Internal parasites represent the largest threat to productivity and economic gain for Brazilian sheep producers. The incidence of resistant populations to available anthelmintics have increased. In this work, „santa inês“ breed sheep were fed with powder extract of E. dysenterica and C. brasiliense leaves, which were dried at 37°C, grounded and mixed together. These leaves were added to animal feed (dose of 1,2g/kg). The sheep weight on the 1th, 7th and 14th day was measured; as well the fecal egg counts per gram (epg) and blood tests were undertaken. To provide nutritional information, the powder extract was analyzed. A reduction of 81% in epg was observed on the 14th day. The blood test showed considerable decrease on eosinophils levels in treated animals. Bromatology analysis of powder leaves added to animal feed in comparison with pattern animal feed, showed no significant variation on the level of fatty acids. The levels of protein and minerals decreased 7% and 17%, respectively; however, the final protein concentration was 22%, whereas 12% represents the normal concentration used by most producers. An increase of 37% of fiber was observed, but no feed consumption decrease was noticed. Despite the variations of chemical composition, after 14 days, no significant weight loss was observed (0,6%) in treated animals. In conclusion, the powder extract used here, could represent an alternative natural source for anthelmintic compounds.

Allosteric Block of KCa2 Channels by Apamin

Activation of small conductance calcium-activated potassium (K(Ca)2) channels can regulate neuronal firing and synaptic plasticity. They are characterized by their high sensitivity to the bee venom toxin apamin, but the mechanism of block is not understood. For example, apamin binds to both K(Ca)2.2 and K(Ca)2.3 with the same high affinity (K(D) approximately 5 pM for both subtypes) but requires significantly higher concentrations to block functional current (IC(50) values of approximately 100 pM and approximately 5 nM, respectively). This suggests that steps beyond binding are needed for channel block to occur. We have combined patch clamp and binding experiments on cell lines with molecular modeling and mutagenesis to gain more insight into the mechanism of action of the toxin. An outer pore histidine residue common to both subtypes was found to be critical for both binding and block by the toxin but not for block by tetraethylammonium (TEA) ions. These data indicated that apamin blocks K(Ca)2 channels by binding to a site distinct from that used by TEA, supported by a finding that the onset of block by apamin was not affected by the presence of TEA. Structural modeling of ligand-channel interaction indicated that TEA binds deep within the channel pore, which contrasted with apamin being modeled to interact with the channel outer pore by utilizing the outer pore histidine residue. This multidisciplinary approach suggested that apamin does not behave as a classical pore blocker but blocks using an allosteric mechanism that is consistent with observed differences between binding affinity and potency of block.