Tunable Surface and Matrix Chemistries in Optically Printed (0-3) Piezoelectric Nanocomposites.

In this work, the impacts of varying surface modification, matrix parameters, and fabrication conditions on the performance of optically printed (0-3) piezoelectric polymer nanocomposites are examined. For example, we find that a 75% reduction in nanoparticle edge-length boosted the piezoelectric coefficient (d) by over 100%. By optimizing the composition and fabrication conditions, 10% by mass loading barium titanate nanocomposites are able to yield d values of ∼80 pC/N compared to <5 pC/N when parameters are not optimized.

Production of vaccines against leading biowarfare toxins can utilize DNA scientific technology.

There are a significant number of different natural toxins that are potential biological warfare agents against which a vaccine is needed. DNA science has been a key to the development of potential vaccines against the top threat toxin and should contribute such effects for other toxin's vaccines. Several different DNA technological scientific techniques have been used to accomplish the general goals of (1) cloning of the toxin or large toxin fragments, (2) altering the specific gene sequence to obtain high level expression of vaccine candidate production in alternate species (3) placement of the vaccine gene in very different presentation types of species.

Vaccination of mice with DNA encoding a large fragment of botulinum neurotoxin serotype A.

The potential utility of using DNA vaccination to protect mice from the microbial neurotoxin, botulinum toxin type A, was evaluated. A synthetically derived gene encoding a carboxyl-terminal 50 kDa fragment of the toxin was placed in two sites in the DNA inoculation vehicle pCMVint-BL (Vical), one predicted to lead to MHC I processing (pJT-1 construct) and the other to direct MHC II processing (pJT-2 construct). Mice were then inoculated at 3 week intervals with these two constructs and with the vehicle alone and evaluated for protection from botulinum toxin by i.

Antibodies and T cells against synthetic peptides of the C-terminal domain (Hc) of botulinum neurotoxin type A and their cross-reaction with Hc.

Seventeen peptides containing T cell and/or antibody (Ab) epitopes previously localized on Hc of botulinum neurotoxin type A were used in SJL and BALB/c mice as immunogens either individually or as an equimolar mixture of groups that contained epitopes of T cells, Abs or both, to determine their abilities to generate T cells and/or Abs that recognize intact Hc. In SJL, peptide 897-915 which included both T cell and Ab epitopes, elicited Abs that cross-reacted very strongly with Hc. In BALB/c, peptides 869-887, 883-901, 981-999 and 1275-1296 which contained Ab epitopes generated Abs that cross-reacted strongly with Hc.

Immune recognition of botulinum neurotoxin type A: regions recognized by T cells and antibodies against the protective H(C) fragment (residues 855-1296) of the toxin.

Botulism toxicity is caused by botulinum neurotoxins (BoNTs), a group of protein neurotoxins produced by Clostridium botulinum. Recent studies have shown that immunization with a C-terminal fragment [H(C), residues 855-1296] of BoNT type A (BoNT/A) affords excellent protection against BoNT/A toxicity. The present work was carried out in order to map the molecular and cellular immunological recognition of H(C).

Localization of the regions on the C-terminal domain of the heavy chain of botulinum A recognized by T lymphocytes and by antibodies after immunization of mice with pentavalent toxoid.

We have mapped the regions recognized by T and/or B cells (Abs) on the C-terminal domain (Hc) of the heavy chain of botulinum neurotoxin serotype A (BoNT/A) after immunization of two inbred mouse strains with pentavalent toxoid (BoNTs A, B, C, D and E). Using a set of synthetic overlapping peptides, encompassing the entire Hc domain (residues 855-1296), we demonstrated that T cells of Balb/c (H-2d) mice, primed with one injection of toxoid, recognized two major regions within residues 897-915 and 939-957. After multiple inoculations with toxoid, T cells of Balb/c expanded their recognition ability and responded very well to challenge with peptide 1261-1279 and moderately to stimulation with peptide 1149-1167.

Mapping of the antibody-binding regions on botulinum neurotoxin H-chain domain 855-1296 with antitoxin antibodies from three host species.

Botulism due to food poisoning is caused mainly by protein toxins, botulinum neurotoxins (BoNTs), produced by Clostridium botluinum in seven known immunological serotypes. These are the most potent toxins and poisons known. BoNT effects blockade of neuromuscular transmission by preventing neurotransmitter release.

Expression of a large, nontoxic fragment of botulinum neurotoxin serotype A and its use as an immunogen.

Using the polymerase chain reaction, a large fragment of botulinum toxin was placed in two expression systems, one designed to produce a fusion protein product and another designed to produce only the toxin fragment. Expression of the fragment in the latter system was inconsistent. Expression of the fusion protein was easily measurable by ELISA.

Protective vaccination with a recombinant fragment of Clostridium botulinum neurotoxin serotype A expressed from a synthetic gene in Escherichia coli.

A completely synthetic gene encoding fragment C, a approximately 50-kDa fragment, of botulinum neurotoxin serotype A was constructed from oligonucleotides. The gene was expressed in Escherichia coli, and full-sized product was produced as judged by Western blot (immunoblot) analysis. Crude extracts of E.

Antibodies having markedly different effects on enzymatic activity and induction of acetylcholine release by two presynaptically-acting phospholipase A2 neurotoxins.

The enzymatic and acetylcholine-releasing activities of two presynaptically-acting phospholipase A2 neurotoxins (pseudexin B and scutoxin) were studied in a synaptosomal fraction. Scutoxin (100 nM) induced greater [14C]acetylcholine release than did pseudexin B (100 nM). Both toxins caused fatty acid production in the synaptosomal fraction, although pseudexin B was more active than scutoxin.

Monoclonal antibodies to VRV-PL-VIIIa, a basic multitoxic phospholipase A2 from Vipera russelli venom.

VRV-PL-VIIIa, the most basic phospholipase A2 (PLA2) from the venom of Vipera russelli, induces multiple toxic effects, including neurotoxicity, myotoxicity, edema and hemorrhage. Rabbit polyclonal anti-serum was raised against VRV-PL-VIIIa. The antiserum cross-reacted in enzyme-linked immunosorbant assay (ELISA) with two other PLA2 from the same venom, VRV-PL-V and VRV-PL-VI, and with ammodytoxin A, caudoxin and crotoxin.

Primary sequence determination of the most basic myonecrotic phospholipase A2 from the venom of Vipera russelli.

The most basic phospholipase A2 (PLA2), VRV-PL-VIIIa, was purified from (Sri Lankan) Vipera russelli venom. It is a major component of the venom, contributing over 40% to the whole venom PLA2 activity. The purity of VRV-PL-VIIIa was ascertained by electrophoresis and by reverse phase high-pressure liquid-chromatography (RP-HPLC).

Effects of steroids on association of T-2 toxin with mammalian cells.

The effects of steroids on the association of T-2 toxin with cultured cells were evaluated. Preincubating cells with certain steroids led to a time- and concentration-related increase in total T-2-cell association. At maximally effective concentrations, the increase in association was 300-500%.

Specific antibodies against the Zn(2+)-binding domain of clostridial neurotoxins restore exocytosis in chromaffin cells treated with tetanus or botulinum A neurotoxin.

Although tetanus and botulinum A neurotoxins are ineffective in cultured chromaffin cells, they will inhibit carbachol-induced release of noradrenaline provided they gain access to the cytosol either through artificial pores generated in the plasma membrane or by binding to incorporated exogenous gangliosides. The block of exocytosis persists for weeks followed by a slow recovery of cell function. When specific anti-botulinum A toxin antibodies are introduced into cells through pores after manifestation of the block by botulinum A neurotoxin, restoration of exocytotic function is accelerated and fully reestablished within 4 days.

Effects of myonecrotic snake venom phospholipase A2 toxins on cultured muscle cells.

We have attempted to establish a cell culture model suitable for molecular mechanism of action studies of necrotic phospholipases A2 (PLA2). Three myonecrotic PLA2 were purified, one basic PLA2 from Naja nigricollis venom and two basic PLA2 (VRV-PL-V and VRV-PL-VIIIa) from Vipera russelli venom. The effects of these PLA2 on several established muscle cell lines were evaluated.

Effect of emetine on T-2 toxin-induced inhibition of protein synthesis in mammalian cells.

Chinese hamster ovary cells were used to examine the effect of emetine upon the toxicity of T-2 toxin and several related trichothecene inhibitors of polypeptide synthesis. Emetine inhibited protein synthesis and T-2 toxin-cell association in a concentration-dependent manner. The dose-response curves for these two effects were nearly identical.

Effects of emetine on the specific association of T-2 toxin with mammalian cells.

The effects of emetine on the association of T-2 toxin with Chinese hamster ovary cells were examined. T-2 toxin-cell association at both 4 degrees C and 37 degrees C was reduced by up to 90% after preincubation of cells with emetine. Emetine-induced reduction in T-2 toxin-cell association was time-, temperature-, and concentration-dependent.

Binding of myotoxin a to cultured muscle cells.

The binding of radiolabeled myotoxin a to various cultured cell lines was evaluated. One rat skeletal muscle-derived cell line, L8, bound substantially more myotoxin a than did all all other cell lines examined. Several biophysical parameters of myotoxin a-L8 binding were determined.

Identification of the site at which phospholipase A2 neurotoxins localize to produce their neuromuscular blocking effects.

Experiments were conducted on mouse hemidiaphragm preparations using five phospholipase A2 neurotoxins of differing chain structures and antigenicities [notexin (one chain); crotoxin (two chains not covalently bound), beta-bungarotoxin (two chains covalently bound); taipoxin (three chains), and textilotoxin (five chains; one copy each of three chains and two copies of a fourth chain)]. Three clostridial neurotoxins (botulinum neurotoxin types A and B, and tetanus toxin) were used in comparison experiments. Phospholipase A2 neurotoxins produced concentration-dependent blockade of neuromuscular transmission.

Epitope mapping of snake venom phospholipases A2 with pseudexin monoclonal antibodies.

Fifteen different monoclonal antibodies, developed against a pseudexin A, B, and C mixture, were screened for linear epitope recognition. Peptides (9-mers) spanning pseudexin B were synthesized on alanine-derivatized polyethylene pins and subsequently probed with antibody. Four antibodies recognized linear epitopes of pseudexin A, pseudexin B, and also nonidentical sequences found in other phospholipases A2 (PLA2S) as determined by enzyme-linked immunosorbent assays.

Preparation and characterization of monoclonal antibodies against pseudexin.

Fifteen hybridoma cell lines secreting monoclonal antibodies against pseudexin were developed. The cell lines were grown as ascites tumors and the resulting antibodies were purified by Protein A affinity-chromatography. Several of the antibodies exhibited extensive ELISA cross-reactions with different phospholipase A2 toxins from various snake venoms, while other of the antibodies reacted only with the pseudexins.

Cross-neutralizations of phospholipase A2 neurotoxins from snake venoms.

Rabbit antisera were produced against several purified phospholipase A2 neurotoxins from snake venoms. The neutralizing and cross-neutralizing capacities of these antisera were evaluated using mice. In all except one case, homologous antisera neutralized the lethal effects of the neurotoxins.

Epitope mapping studies of snake venom phospholipase A2 using monoclonal antibodies.

Fifteen different monoclonal antibodies developed against pseudexin, a snake venom phospholipase A2 with presynaptic neurotoxicity, were screened for linear epitope recognition. Peptides (9-mers) spanning pseudexin were synthesized by using alanine-derivatized polyethylene pins and subsequently probed with antibody. Four antibodies bound to toxin peptides and were detected with an enzyme-linked immunosorbent assay.