Stereospecific action of pyrethroid insecticides on the gamma-aminobutyric acid receptor-ionophore complex.

The potent alpha-cyano-3-phenoxybenzyl pyrethroids, including cypermethrin, deltamethrin, and fenvalerate, act stereospecifically to inhibit binding to rat brain synaptic membranes of sulfur-35-labeled t-butylbicyclophosphorothionate, a new radioligand for the picrotoxinin binding site. Scatchard analysis indicates that picrotoxinin inhibition of t-butylbicyclophosphorothionate binding is competitive whereas cypermethrin inhibition possibly involves a closely associated site in the gamma-aminobutyric acid receptor-ionophore complex. Studies with 37 pyrethroids reveal an absolute correlation, that is, no false positives or negatives, between mouse intracerebral toxicity and in vitro inhibition: all toxic cyano compounds but none of their nontoxic stereoisomers are inhibitors; cis isomers are more potent than trans isomers as both toxicants and inhibitors; and noncyano pyrethroids are much less potent or are inactive.

The structure of the fusion glycoprotein of Newcastle disease virus suggests a novel paradigm for the molecular mechanism of membrane fusion.

BACKGROUND: Membrane fusion within the Paramyxoviridae family of viruses is mediated by a surface glycoprotein termed the "F", or fusion, protein. Membrane fusion is assumed to involve a series of structural transitions of F from a metastable (prefusion) state to a highly stable (postfusion) state. No detail is available at the atomic level regarding the metastable form of these proteins or regarding the transitions accompanying fusion. RESULTS: The three-dimensional structure of the fusion protein of Newcastle disease virus (NDV-F) has been determined. The trimeric NDV-F molecule is organized into head, neck, and stalk regions. The head is comprised of a highly twisted beta domain and an additional immunoglobulin-like beta domain. The neck is formed by the C-terminal extension of the heptad repeat region HR-A, capped by a four-helical bundle. The C terminus of HR-A is encased by a further helix HR-C and a 4-stranded beta sheet. The stalk is formed by the remaining visible portion of HR-A and by polypeptide immediately N-terminal to the C-terminal heptad repeat region HR-B. An axial channel extends through the head and neck and is fenestrated by three large radial channels located approximately at the head-neck interface. CONCLUSION: We propose that prior to fusion activation, the hydrophobic fusion peptides in NDV-F are sequestered within the radial channels within the head, with the central HR-A coiled coil being only partly formed. Fusion activation then involves, inter alia, the assembly of a complete HR-A coiled coil, with the fusion peptides and transmembrane anchors being brought into close proximity. The structure of NDV-F is fundamentally different than that of influenza virus hemagglutinin, in that the central coiled coil is in the opposite orientation with respect to the viral membrane.

Sub-micellar phospholipid accelerates amyloid formation by apolipoprotein C-II.

Lipid-free human apolipoprotein C-II (apoC-II) forms amyloid fibrils with characteristic beta-structure. This conformation is distinct from the alpha-helical fold of lipid-bound apoC-II. We have investigated the effect of the short-chain phospholipid, dihexanoylphosphatidylcholine (DHPC) on amyloid formation by apoC-II. The alpha-helical content of apoC-II increases in the presence of micellar DHPC (16 mM) and amyloid formation is inhibited. However, at sub-micellar DHPC concentrations (below 8 mM) amyloid formation is accelerated 6 fold. These results suggest that individual phospholipid molecules in vivo may exert significant effects on amyloid folding pathways.

Orientation of antigen binding sites in dimeric and trimeric single chain Fv antibody fragments.

Electron microscopy of dimeric and trimeric single chain antibody Fv fragments (scFvs) complexed with anti-idiotype Fab fragments was used to reveal the orientation of antigen binding sites. This is the first structural analysis that discloses the multivalent binding orientation of scFv trimers (triabodies). Three different scFv molecules were used for the imaging analysis; NC10 scFv-5 and scFv-0, with five- and zero-residue linkers respectively between the VH and VL domains, were complexed with 3-2G12 anti-idiotype Fab fragments and 11-1G10 scFv-0 was complexed with NC41 anti-idiotype Fab fragments. The scFv-5 molecules formed bivalent dimers (diabodies) and the zero-linker scFv-0 molecules formed trivalent trimers (triabodies). The images of the NC10 diabody-Fab complex appear as boomerangs, not as a linear molecule, with a variable angle between the two Fab arms and the triabody-Fab complexes appear as tripods.

Structure-activity correlations for interactions of bicyclophosphorus esters and some polychlorocycloalkane and pyrethroid insecticides with the brain-specific t-butylbicyclophosphorothionate receptor.

[35S]t-Butylbicyclophosphorothionate or [35S]TBPS is an improved radioligand for the picrotoxinin binding site in rat brain synaptic membranes. The toxic isomers of the hexachlorocyclohexanes, polychlorobornanes, and chlorinated cyclodienes displace [35S]TBPS with a stereospecificity and potency generally correlated with their mammalian toxicity. In a few cases this correlation is improved by correction for metabolic activation or detoxification on using a coupled brain receptor/liver microsomal oxidase system. The alpha-cyano-3-phenoxybenzyl pyrethroids, although less potent, inhibit [35S]TBPS binding in a stereospecific manner correlated with their toxicity. Scatchard analyses indicate that these three classes of polychlorocycloalkane insecticides act at the TBPS binding site within the gamma-aminobutyric acid (GABA) receptor-ionophore complex whereas the alpha-cyano pyrethroids interact with a closely associated site. These insecticides and TBPS analogs may serve as useful probes further to elucidate the topography of the TBPS binding site and its relationship to the chloride channel.

Interactions of lindane, toxaphene and cyclodienes with brain-specific t-butylbicyclophosphorothionate receptor.

Three major classes of chlorinated hydrocarbon insecticides, i.e., the lindane/hexachlorocyclohexane, toxaphene and aldrin/dieldrin types, are potent, competitive, and stereospecific inhibitors of t-butylbicyclophosphorothionate (TBPS) binding to brain-specific sites, thereby indicating an action at the gamma-aminobutyric acid (GABA)-regulated chloride channel. The most inhibitory and toxic of four isomers of hexachlorocyclohexane is lindane and of greater than 188 components of toxaphene is 2,2,5-endo, 6-exo,8,9,9,10-octachlorobornane. 12-Ketoendrin (IC50 = 36 nM) is twice as active as the most potent previously known inhibitor of TBPS binding and it is also the most inhibitory and toxic of 22 cyclodienes examined. Within each of these three series of polychlorocycloalkanes the mammalian toxicity is closely related to the potency for inhibition of TBPS binding. A modified receptor assay incorporating liver microsomes and reduced nicotinamide-adenine dinucleotide phosphate compensates in part for oxidative detoxification and bioactivation. Specific TBPS binding is reduced in a dose-dependent manner in dieldrin-poisoned rats. DDT, mirex and kepone are not inhibitors of TBPS binding, even at 10 microM.

Similar properties of [35S]t-butylbicyclophosphorothionate receptor and coupled components of the GABA receptor-ionophore complex in brains of human, cow, rat, chicken and fish.

No significant differences are evident in the specific binding characteristics of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) to EDTA/water-dialyzed P2 membranes of human, cow, rat, chicken and fish brain. This species similarity includes dissociation constants of 61-77 nM at 37 degrees C, maximum receptor densities of 3-7 pmol/mg protein, and sensitivity to inhibition or displacement by gamma-aminobutyric acid (GABA), two cage convulsants (picrotoxinin and t-butylbicycloorthobenzoate) and the insecticide [1R,cis, alpha S]-cypermethrin, indicating a constancy during vertebrate evolution of the [35S]TBPS binding site and its coupling with other components of the GABA receptor-ionophore complex. As a possible exception, chicken and fish brain membranes appear to be less sensitive than the others to the insecticide alpha-endosulfan. Human and rat preparations are also essentially identical relative to the inhibition of radioligand binding by two GABA mimetics (muscimol and 3-amino-propanesulfonic acid), six other cage convulsants (including examples of three classes of polychlorocycloalkane insecticides), a potent anthelmintic agent (Ivermectin), dimethylbutylbarbiturate, the convulsant benzodiazepine Ro 5-3663, and ethanol. The findings to date with [35S]TBPS and the GABA receptor-ionophore complex in rat brain membranes are therefore generally applicable to human preparations. Cow brain is an appropriate source for large scale preparations in receptor purification studies since it is essentially identical to human and rat preparations in all parameters examined. Species differences in sensitivity to the toxic effects of the convulsants and polychlorocycloalkane insecticides considered are apparently not attributable to receptor site specificity.

Approach to decreased hearing.

This article is designed to review proper procedure by an emergency or primary care physician when evaluating a patient with decreased hearing. It will cover the anatomy and physiology of hearing, as well as the evaluation and initial management of conductive and sensorineural hearing loss.

Sporulation in Hansenula wingei is induced by nitrogen starvation in maltose-containing media.

The sexually agglutinative yeast Hansenula wingei lives in association with bark beetles that inhabit coniferous trees. This yeast was induced to sporulate by malt extract, which contains a high percentage of maltose (50%) and a low percentage of nitrogen (0.5%). A solution of 1.5% maltose without any growth factors also induced ascosporogenesis in H. wingei. Thus, only a carbon source is required for sporulation as in Saccharomyces. However, potassium acetate did not induce sporulation in H. wingei as it does in S. cerevisiae. Instead, disaccharides (such as maltose, sucrose, or cellobiose) promote sporulation better than either monosaccharides (such as dextrose, fructose, or mannose) or respiratory substrates (such as ethanol or glycerol). The specificity of disaccharides in promoting sporulation in H. wingei may be considered an adaptation since these disaccharides are present in the natural environment of this yeast. In addition, the specificity of disaccharides may be related to the induction of the disaccharidase because cells precultured on dextrose sporulate well on maltose, but cells precultured on maltose sporulate poorly on maltose. When (NH(4))(2)SO(4) was added at a low concentration (3 mM) to synthetic sporulation medium (1.5% maltose solution), sporulation was abolished, whereas other salts and nitrogen sources inhibited to a lesser extent and vitamins and trace elements had no effect. Oxygen was required for sporulation, as expected for an obligate aerobe. Maximal sporulation was achieved in 2% malt extract broth at high cell density (10(9) cells per ml), pH 5, and 25 degrees C. By using these optimal physiological conditions and hybrid strains selected from an extensive genetic breeding program, about 30% asci (10% tetrads) were obtained routinely. Thus, the genetics of cell recognition in this yeast can now be studied.

Fate of inhaled hexachlorocyclopentadiene in albino rats and comparison to the oral and IV routes of administration.

Hexachlorocyclopentadiene (C56) is extremely toxic when inhaled by rats, but only moderately toxic when administered orally. The difference in toxicity as a function of route of exposure was studied by determining the absorption, distribution, and ultimate fate of C56 following acute oral, inhalation, and iv exposure. For rats treated with a single oral dose (5 micrograms/kg) of 14C-C56, radiocarbon was primarily excreted in the 0-72 hr feces (68.2%). Animals treated with equivalent inhaled and iv doses eliminated 23.1 and 31.4% of the dose in the 0-72 hr feces, respectively. Urinary radiocarbon was equivalent to 24.4, 23.1, and 31.4% of the dose for oral, inhalation and iv treatments, respectively. Biliary excretion following all exposure routes was between 9 and 18% of the dose, indicating that the large amount of radiocarbon in the feces of orally treated rats was due to poor absorption of C56 from the gastrointestinal tract. For all exposure routes, the lungs appeared to be a major site of C56 toxicity upon post-mortem examination. This study suggests that the direct damage to lung tissue by inhaled C56 and low bioavailability of orally administered C56 contribute substantially to the difference in toxicity between oral and inhalation exposure to C56. A recently developed inhalation exposure system which allows quantitative administration of toxicant vapors to rats is also described in this report.

Benzodiazepine anticonvulsant action: gamma-aminobutyric acid-dependent modulation of the chloride ionophore.

Brain-specific benzodiazepine receptors are allosterically coupled to chloride ionophore-associated binding sites for sulfur-35-labeled t-butylbicyclophosphorothionate. The specific binding of t-butylbicyclophosphorothionate to fresh unwashed rat cortical membranes is inhibited by nanomolar concentrations of five benzodiazepine agonists but not by the antagonist Ro 15-1788. Their inhibitory potencies in this assay are closely related to their antimetrazol activities. Studies with solubilized receptor-complex preparations establish an absolute requirement for gamma-aminobutyric acid (3 to 10 microM), strongly suggesting that the antagonism of metrazol-induced seizures by the benzodiazepines involves an action on the chloride ionophore mediated through the low affinity gamma-aminobutyric acid receptor.

Modulation of the chloride ionophore by benzodiazepine receptor ligands: influence of gamma-aminobutyric acid and ligand efficacy.

t-Butylbicyclophosphorothionate (TBPS) produces dose-dependent enhancement of [3H]propyl beta-carboline-3-carboxylate ([3H]PCC, 40 pM) binding to the benzodiazepine1 (BZ1) receptor subtype in hippocampus. Furthermore, TBPS enhancement of [3H]PCC binding was antagonized by micromolar concentrations of gamma-aminobutyric acid (GABA) in a way reversible by bicuculline. BZ receptor ligands that are "GABA positive" (i.e., enhance GABA neurotransmission) allosterically inhibited [35S]TBPS binding, whereas "GABA-negative" ligands (i.e., inhibit GABA neurotransmission) produced the opposite effect. The efficacy of the ligands as modulators of [35S]TBPS binding was consistent with their reported in vivo pharmacology. The effects of positive and negative ligands on [35S]TBPS binding were modulated by micromolar concentrations of GABA. Examination of the kinetics of [35S]TBPS binding suggested the presence of slowly and rapidly dissociating components. The GABA-positive clonazepam stabilized the rapidly dissociating component of [35S]TBPS binding, whereas methyl beta-carboline-3-carboxylate had a similar effect on the slowly dissociating component. It is speculated that the slowly dissociating component of [35S]TBPS binding is associated with a closed chloride channel, whereas the opposite is proposed for the rapidly dissociating component. The differential effects of GABA-positive versus GABA-negative ligands on [35S]TBPS binding and the modulatory effect of GABA provide further evidence to suggest that [35S]TBPS labels a site near the chloride ionophore linked to the GABA-BZ receptor complex.

Chemotaxis of Bdellovibrio bacteriovorus toward pure compounds.

Positive chemotaxis by Bdellovibrio bacteriovorus strain UKi2 was measured for 139 compounds. Twenty-one compounds were attractants; sensitive attraction was elicited by acetate, propionate, thioacetate, malonate, cis-oxalacetate, D-glucose-6-phosphate, acetyl coenzyme A, ammonium ion, barium ion, manganous ion, and potassium ion. Several of the attractants for B. bacteriovorus strain UKi2 also were attractants to strains 6-5-S and 114; however, strains 109D and 109J were not attracted by the compounds tested. Of 33 compounds tested, 8 were repellents for B. bacteriovorus strain UKi2: n-caproate, alanine, isoleucine, leucine, phenylalanine, tyrosine, cobaltous chloride, and hydronium ion. None of the organic repellents for strain UKi2 elicited repulson from strains 114 or 109D. However, all three strains of Bdellovibrio show aerotaxis. Several compounds were tested for their effects on viability and predacious growth of B. bacteriovorus strain UKi2. No simple correlation was found between attraction or repulsion and benefit or harm to bdellovibrios. The data are consistent with the view that in nature, the greatest survival value of chemotaxis for bdellovibros may be in aerotaxis, attraction to certain inorganic ions and acetate, and repulsion by hydronium ion.

ScFv multimers of the anti-neuraminidase antibody NC10: shortening of the linker in single-chain Fv fragment assembled in V(L) to V(H) orientation drives the formation of dimers, trimers, tetramers and higher molecular mass multimers.

Synthetic genes encoding single-chain variable fragments (scFvs) of NC10 anti-neuraminidase antibody were constructed by joining the V(L) and V(H) domains with linkers of fifteen, five, four, three, two, one and zero residues. These V(L)-V(H) constructs were expressed in Escherichia coli and the resulting proteins were characterized and compared with the previously characterized NC10 scFv proteins assembled in V(H)-V(L) orientation. Size-exclusion chromatography and electron microscope images of complexes formed between various NC10 scFvs and anti-idiotype Fab' were used to analyse the oligomeric status of these scFvs. The result showed that as the linker length between V(L) and V(H) was reduced, different patterns of oligomerization were observed compared with those with V(H)-V(L) isomers. As was the case for V(H)-V(L) orientation, the scFv-15 V(L)-V(H) protein existed mainly as a monomer whereas dimer (diabody) was a predominant conformation for the scFv-5, scFv-4 and scFv-3 V(L)-V(H) proteins. In contrast to the V(H)-V(L) isomer, direct ligation of V(L) to V(H) led to the formation of predominantly a tetramer (tetrabody) rather than to an expected trimer (triabody). Furthermore, the transition between dimers and higher order oligomers was not as distinct as for V(H)-V(L). Thus reducing the linker length in V(L)-V(H) from three to two residues did not precisely dictate a transition between dimers and tetramers. Instead, two-residue as well as one-residue linked scFvs formed a mixture of dimers, trimers and tetramers.

Human apolipoprotein C-II forms twisted amyloid ribbons and closed loops.

Human apolipoprotein C-II (apoC-II) self-associates in solution to form aggregates with the characteristics of amyloid including red-green birefringence in the presence of Congo Red under cross-polarized light, increased fluorescence in the presence of thioflavin T, and a fibrous structure when examined by electron microscopy. ApoC-II was expressed and purified from Escherichia coli and rapidly exchanged from 5 M guanidine hydrochloride into 100 mM sodium phosphate, pH 7.4, to a final concentration of 0.3 mg/mL. This apoC-II was initially soluble, eluting as low molecular weight species in gel filtration experiments using Sephadex G-50. Circular dichroism (CD) spectroscopy indicated predominantly unordered structure. Upon incubation for 24 h, apoC-II self-associated into high molecular weight aggregates as indicated by elution in the void volume of a Sephadex G-50 column, by rapid sedimentation in an analytical ultracentrifuge, and by increased light scattering. CD spectroscopy indicated an increase in beta-sheet content, while fluorescence emission spectroscopy of the single tryptophan revealed a blue shift and an increase in maximum intensity, suggesting repositioning of the tryptophan into a less polar environment. Electron microscopy of apoC-II aggregates revealed a novel looped-ribbon morphology (width 12 nm) and several isolated closed loops. Like all of the conserved plasma apolipoproteins, apoC-II contains amphipathic helical regions that account for the increase in alpha-helix content on lipid binding. The increase in beta-structure accompanying apoC-II fibril formation points to an alternative folding pathway and an in vitro system to explore the general tendency of apolipoproteins to form amyloid in vivo.

scFv multimers of the anti-neuraminidase antibody NC10: length of the linker between VH and VL domains dictates precisely the transition between diabodies and triabodies.

Single-chain Fv antibody fragments (scFvs) incorporate a polypeptide linker to tether the VH and VL domains together. An scFv molecule with a linker 5-12 residues long cannot fold into a functional Fv domain and instead associates with a second scFv molecule to form a bivalent dimer (diabody). Direct ligation of VH and VL domains further restricts association and forces three scFv molecules to associate into a trivalent trimer (triabody). We have defined the effect of linker length on scFv association by constructing a series of scFvs from anti-neuraminidase antibody NC10 in which the linker varied from one to four glycine residues. NC10 scFv molecules containing linkers of three and four residues showed a strong preference for dimer formation (diabodies), whereas a linker length of one or two glycine residues prevented the formation of diabodies and directed scFv association into trimers (triabodies). The data suggest a relatively strict transition from dimer (diabody) to trimer (triabody) upon reduction of the linker length from three to two glycine residues. Modelling studies are consistent with three residues as the minimum linker length compatible with diabody formation. Electron microscope images of complexes formed between the NC10 scFv multimers and an anti-idiotype Fab' showed that the dimer was bivalent for antigen binding and the trimer was trivalent.

t-[3H]butylbicycloorthobenzoate: new radioligand probe for the gamma-aminobutyric acid-regulated chloride ionophore.

t-[3H]Butylbicycloorthobenzoate [( 3H]TBOB; 22 Ci/mmol) was prepared by reductive dechlorination of its 4-chlorophenyl analog with tritium gas. This new radioligand binds reversibly to fresh washed rat brain P2 membranes in 500 mM NaCl plus 50 mM sodium-potassium phosphate buffer (pH 7.4) at 25 degrees C, with 80-90% specific relative to total binding, a KD of 61 +/- 15 nM, and a Bmax of 1.6 +/- 0.5 pmol/mg of protein. [3H]TBOB association with its binding site(s) is monophasic, but its dissociation is biphasic. The binding characteristics of [3H]TBOB are essentially identical to those of t-[35S]butylbicyclophosphorothionate [( 35S]TBPS) with respect to pH dependence, stimulation by anions, regional distribution in the brain, and pharmacological profile. Saturation analyses and dissociation studies further indicate that TBOB and TBPS have a common binding site. However, binding of the two radioligands differs in respect to temperature effects. In contrast to [35S]TBPS, which exhibits negligible binding at 0 degrees C, [3H]TBOB binds to rat brain membranes at 0, 25, and 37 degrees C with similar KD values. [3H]TBOB with its long radioactive half-life and temperature-independent KD is a valuable supplement to [35S]TBPS in further biochemical and pharmacological characterization of the gamma-aminobutyric acid receptor-ionophore complex.

Characterization of high affinity [3H]pirenzepine and (-)-[3H] quinuclidinyl benzilate binding to muscarinic cholinergic receptors in rabbit peripheral lung.

We have characterized the binding of the selective muscarinic antagonist [3H]pirenzepine ([3H])PZ) and the classical muscarinic antagonist (-)-[3H]quinuclidinyl benzilate ((-)-[3H]QNB) to muscarinic cholinergic sites in rabbit peripheral lung membranes. For both radioligands, high affinity binding with pharmacologic specificity was demonstrated. The high affinity Kd for [3H]PZ binding determined from saturation isotherms was 4.5 nM and the Kd for (-)-[3H]QNB binding was 6.2 pM. Comparison of the total binding capacity values determined by saturation experiments with [3H] PZ and (-)-[3H]QNB demonstrates that approximately 78% of the total muscarinic binding sites in rabbit peripheral lung bind [3H]PZ with high affinity. There was no significant effect of the guanine nucleotide, guanyl-5'-yl imidodiphosphate, on the inhibition of (-)-[3H]QNB binding by the muscarinic agonist carbachol in peripheral lung membranes. If the pulmonary muscarinic receptor with high affinity for PZ proves to have an important role in bronchoconstriction, its characterization could result in the development of more selective bronchodilators.

Cloning, expression, and crystallization of the fusion protein of Newcastle disease virus.

We have recently reported the X-ray crystal structure of a fragment of the fusion protein (F) of Newcastle disease virus (NDV). This work describes the methodology involved in the production and crystallization of that protein in recombinant form. The full-length cDNA of NDV-F was cloned and the ectodomain expressed in both CHO-K1 and Lec-3.2.8.1 cells. The recombinant protein, secreted as a single-chain polypeptide F0', was purified using a c-myc antibody affinity column followed by gel filtration chromatography. Electron microscopic imaging showed the F0' product to consist of unaggregated club-shaped particles. Trypsin treatment of F0' could be used to produce disulfide-linked F2 and F1' chains. However, imaging revealed extensive rosette-like aggregation of the trypsin-treated material, indicative of a conformational change. Only the non-trypsin-treated product was thus suitable for crystallization and two crystal forms were obtained, diffracting to ca. 3.5 and 4.0 A, respectively. Both crystal forms were used in the structure determination.