A novel protein toxin from the deadly box jellyfish (Sea Wasp, Habu-kurage) Chiropsalmus quadrigatus

The deadly box jellyfish (Sea Wasp, Habu-kurage in Japanese) Chiropsalmus quadrigatus Haeckel (Cubozoa) is distributed widely in the tropical Pacific region. In Japan, three fatal cases due to stings from this species have been reported officially. We successfully isolated C. quadrigatus toxin-A (CqTX-A, 44 kDa), a major proteinaceous toxin, for the first time, from the nematocysts of C. quadrigatus. CqTX-A showed lethal toxicity to crayfish when administered via intraperitoneal injection (LD50 = 80 microg/kg) and hemolytic activity toward 0.8% sheep red blood cells (ED50 = 160 ng/ml). Furthermore, we sequenced the cDNA encoding CqTX-A. The deduced amino acid sequence of CqTX-A (462 amino acids) showed 25.2% and 21.6% sequence similarity to Carybdea rastoni toxins (CrTXs) and Carybdea alata toxin-A (CrTX-A), respectively, which are Cubozoan jellyfish toxins.     

Effect of pH on the conformation and stability of the plant toxin ricin

Intrinsic protein fluorescence of native plant toxin and its isolated subunits were studied. The effect of pH was studied on: conformation of ricin and its A- and R-chains; affinity to galactose of ricin and its binding B-subunit. At two pH 5.0 and 7.0, the structural stability of toxin and subunits was estimated according to denaturational action of guanidine chloride. It was demonstrated that position of maximum and the spectrum shape of fluorescence of native toxin and catalytical A-subunit insignificantly depends on pH in the range of 3-8, whereas sufficient changes of the separameters for the ricin B-chain reveal structural transition at pH 4-5. The affinity of galactose of ricin and its isolated B-chain depends on pH, the maximal binding is observed at pH 7. The structural stability of ricin and isolated chains significantly differs at pH 7.5 and 5.0, thus the structure stability of ricin and A-chain increases, and that of B-chain decreases at pH 5.0.     

The influence of anti-(ricin toxin A chain) monoclonal antibodies on the pharmacokinetics of ricin toxin A chain and recombinant ricin A chain in mice

Summary Two monoclonal antibodies against ricin toxin A chain (RTA) have been examined for their effects on the blood survival and biodistribution of RTA and recombinant ricin A chain in mice. When admixed with the toxins at 1:1 molar ratios prior to intravenous injection, the antibodies prolonged blood survival and whole-body retention of both species of RTA, and this was due essentially to reduced renal clearance of the toxins. Immune complexes were identified by gel filtration chromatography and immune precipitation with anti-IgG antiserum in mixtures prior to injection and in the serum of mice injected with the mixtures. An irrelevant monoclonal antibody showed no complex formation, and no effect on biodistribution. These studies have shown that immune complexes formed between monoclonal antibodies and protein antigens of molecular mass up to at least 30 kDa survive in the circulation, rather than being cleared by the reticuloendothelial system. Such antibodies could be used to modulate the biodistribution of toxic molecules such as ribosome-inhibiting proteins like RTA. This might be exploited therapeutically, for example in the construction of bispecific antibodies against ribosomal inhibiting proteins and tumour-associated antigens.     

A novel biosensor selective for organoarsenicals

Organoarsenicals used as herbicides and growth promoters for farm animals are degraded to inorganic arsenic. Available bacterial whole-cell biosensors detect only inorganic arsenic. We report a biosensor selective for the trivalent organoarsenicals methylarsenite and phenylarsenite over inorganic arsenite. This sensor may be useful for detecting degradation of arsenic-containing herbicides and growth promoters.     

A hand-held surface plasmon resonance biosensor for the detection of ricin and other biological agents

There is an ongoing need for field-deployable biosensor devices. We have constructed a fully self-contained, hand-held biosensor, based on the surface plasmon resonance technique. The dimensions of the sensor unit are 15 x 8 cm, the weight is 600 g and it is powered by a 9 V battery. We have characterised the responsiveness of the sensor using calibrated sucrose solutions and were able to measure changes as small as 3.3 x 10(-6) refractive index units. To demonstrate functionality of the sensor, we have prepared surfaces with an antibody fragment specific for the biological toxin ricin. We were able to detect ricin at 200 ng/mL in 10 min, which is approximately 2500 times less than the minimum lethal dose. We were also able to verify positive binding within a second 10 min window. This sensor demonstrates important steps required for the development of fully integrated, hand-held sensor devices and will form the basis of a multi-analyte system, to be developed in the near future. It also represents the first completely hand-held SPR device, not requiring external power or a computer connection to operate.     

Biologically active A-chain of the plant toxin ricin expressed from a synthetic gene in Escherichia coli

To assess the biological activity and pharmacokinetic properties of nonglycosylated ricin A-chain (RA), we have obtained the polypeptide following expression of a synthetic 842-bp RA gene in Escherichia coli. Expression of the gene was carried out using the phage T5 PN25 promoter fused to the E. coli lac operator. The RA polypeptide was synthesized in a completely soluble form and was purified in one step by immunoabsorption. It was shown to be as cytotoxic for a human cell line as both native RA and chemically deglycosylated native RA. Reconstituted whole ricin and an immunotoxin containing the recombinant RA were also biologically active. Immunotoxins made with recombinant and deglycosylated RA had similar clearance rates in vivo showing, after a short period of rapid elimination, stabilities far higher than that of an immunotoxin made with native RA. Our results show that the complete elimination of sugar side chains from the RA is not sufficient to entirely eradicate the rapid initial in vivo clearance of RA-based biologicals.     

A novel nanolayer biosensor principle

A method for eliminating the mass transport limitation on biosensor surfaces is introduced. The measurement of macromolecular binding kinetics on plane surfaces is the key objective of many evanescent wave (e.g. total internal reflection fluorescence (TIRF)), and surface plasmon resonance (SPR) based biosensor systems, allowing the determination of binding constants within minutes or hours. However, these methods are limited in not being rigorously applicable to large macromolecules like proteins or DNA, since the on-rates are transport limited due to a Nernst diffusion layer of 5-10 microm thickness. Thus, for the binding of fibrinogen (340 kDa) to a surface current SPR biosensors will show a mass transport coefficient of ca. 2 x 10(-6) m/s. In a novel approach with an immiscible fluid vesicle (e.g. air bubble), it has been possible to generate nanoscopic fluid films of ca. 200 nm thickness on the sensor surface of an interfacial TIRF rheometer system. The thickness of the liquid film can be can be easily probed and measured by evanescent wave technology. This nanofilm technique increases the mass transport coefficient for fibrinogen to ca. 1 x 10(-4) m/s eliminating the mass transport limitation, making the binding rates reaction-rate limited. From the resulting exponential kinetic functions, lasting only 20-30s, the kinetic constants for the binding reaction can easily be extracted and the binding constants calculated. As a possible mechanism for the air bubble effect it is suggested that the aqueous fluid flow in the rheometer cell is separated by the air bubble below the level of the Nernst boundary layer into two independent laminar fluid flows of differing velocity: (i) a slow to stationary nanostream ca. 200 nm thick strongly adhering to the surface; and (ii) the bulk fluid streaming over it at a much higher rate in the wake of the air bubble. Surprising properties of the nanofluidic film are: (i) its long persistence for at least 30-60s after the air bubble has passed (2.5s); and (ii) the absence of solute depletion. It is suggested that a new liquid-liquid interface (i.e. a "vortex sheet") between the two fluid flows plays a decisive role, lending metastability to the nanofluidic film and replenishing its protein concentration via the vortices-thus upholding exponential binding kinetics. Finally, the system relaxes via turbulent reattachment of the two fluid flows to the original velocity profile. It is concluded that this technique opens a fundamentally novel approach to the construction of macromolecular biosensors.     

A novel fluorescent protein-based biosensor for gram-negative bacteria

Site-directed mutagenesis of enhanced green fluorescent protein (EGFP) based on rational computational design was performed to create a fluorescence-based biosensor for endotoxin and gram-negative bacteria. EGFP mutants (EGFP(i)) bearing one (G10) or two (G12) strands of endotoxin binding motifs were constructed and expressed in an Escherichia coli host. The EGFP(i) proteins were purified and tested for their efficacy as a novel fluorescent biosensor. After efficient removal of lipopolysaccharide from the E. coli lysates, the binding affinities of the EGFP(i) G10 and G12 to lipid A were established. The K(D) values of 7.16 x 10(-7) M for G10 and 8.15 x 10(-8) M for G12 were achieved. With high affinity being maintained over a wide range of pH and ionic strength, the binding of lipid A/lipopolysaccharide to the EGFP(i) biosensors could be measured as a concentration-dependent fluorescence quenching of the EGFP mutants. The EGFP(i) specifically tagged gram-negative bacteria like E. coli and Pseudomonas aeruginosa, as well as other gram-negative bacteria in contaminated water sampled from the environment. This dual function of the EGFP(i) in detecting both free endotoxin and live gram-negative bacteria forms the basis of the development of a novel fluorescent biosensor.     

Mannose receptor-mediated uptake of ricin toxin and ricin A chain by macrophages. Multiple intracellular pathways for a chain translocation

The role of the high mannose carbohydrate chains in the mechanism of action of ricin toxin was investigated. Ricin is taken up by two routes in macrophages, by binding to cell surface mannose receptors, or by binding of the ricin galactose receptor to cell surface glycoproteins. Removal of carbohydrate from ricin by periodate oxidation led to a large loss in toxicity via both routes of uptake by an effect on the B chain not due to a loss of galactose binding affinity. These data suggest that the carbohydrate chains of ricin B chain may be required for full toxicity. The pathway of uptake of ricin by the macrophage mannose receptor was found to differ in several respects from uptake via the galactose-specific pathway. Analysis of intoxication of macrophages by ricin in the presence of ammonium chloride suggested that mannose receptor bound ligand passes through acidic vesicles prior to translocation, unlike galactose bound ligand. Intoxication by ricin via galactose-specific uptake was potentiated by swainsonine but not by castanospermine, suggesting that ricin may be attacked by an endogenous mannosidase within the cell, and that ricin passes through either a lysosomal or a Golgi compartment prior to translocation.     

Ribonuclease activity associated with the 60S ribosome-inactivating proteins ricin A, phytolaccin and Shiga toxin

All purified preparations of the ribosome-inactivating proteins ricin A, phytolaccin and Shiga toxin were shown to exhibit ribonuclease activity with 5S or 5.8S rRNA substrates. These toxin species generated reproducible patterns of RNA fragments distinct for each toxin species while multiple preparations of a single toxin species yielded similar RNA fragment patterns. The heat inactivation profile of Shiga toxin was identical for its RNase and protein synthesis inhibitory activities. These data are the first to indicate that the ribosome-inactivating catalytic toxins, in addition to alpha-sarcin, exhibit RNase activity. These results suggest RNase activity may be responsible for ribosome-inactivation catalyzed by ricin, phytolaccin and Shiga toxin proteins.     

Disruption of the Golgi apparatus by brefeldin A inhibits the cytotoxicity of ricin, modeccin, and Pseudomonas toxin

We have studied the cytotoxicity of ricin in cells treated with brefeldin A (BFA), which dramatically disrupts the structure of the Golgi apparatus causing Golgi content and membrane to redistribute to the ER. BFA inhibits the cytotoxicity of ricin in Chinese hamster ovary, normal rat kidney, and Vero cells and abolishes the enhancement of ricin cytotoxicity by NH4Cl, nigericin, swainsonine, and tunicamycin or by a mutation in endosomal acidification. BFA protects cells from the cytotoxicities of modeccin and Pseudomonas toxin, but has no effect on the intoxication by diphtheria toxin. Pretreatment of BFA does not protect cells from ricin treatment in the absence of BFA. Our results suggest that ricin, modeccin, and Pseudomonas toxin share a common pathway of intracellular transport from endosomes to the Golgi region where they are released into the cytosol. In contrast, the lack of protection of Vero cells from diphtheria toxin by BFA indicates that diphtheria toxin is released from acidified endosomes without involving the Golgi region.     

A novel multi-walled carbon nanotube-based biosensor for glucose detection

The bioelectrochemical characteristics of a novel multi-walled carbon nanotube (MWNT)-based biosensor for glucose detection are studied and compared with those of glassy carbon (GC)-based biosensor. The MWNT-based biosensor exhibits a strong glucose response at applied potentials of 0.65 and 0.45 V versus Ag/AgCl, respectively, while GC-based biosensor shows a weak glucose response at 0.65 V and no response at 0.45 V. Besides, the MWNT-based biosensor shows a high stability of 86.7% of the initial activity to glucose after four-month storage, much higher than 37.2%, the corresponding value for a GC-based biosensor. The detection mechanism of the MWNT-based biosensor is also discussed in detail.     

A novel liposome-based electrochemical biosensor for the detection of haemolytic microorganisms

Summary A biosensor strategy for rapid amperometric detection of organisms was investigated in which a redox mediator was entrapped within liposomes. The selective release of mediator by haemolytic bacteria, followed by signal generation, confers differentiation between haemolytic and non-haemolytic species. The potential of this approach is illustrated by results for various strains of Listeria monocytogenes, Listeria welshimeri and Escherichia coli.     

Somatic cell mutants resistant to ricin, diphtheria toxin, and to immunotoxins

The human B-cell line Namalwa expresses the common acute lymphoblastic leukemia antigen (CALLA). Frame-shift mutants in Namalwa cell cultures were generated with ICR-191, and mutants were then selected for resistance to ricin or resistance to a conjugate of ricin with the anti-CALLA antibody J5 in the presence of lactose. Three mutants were found that were resistant to ricin and were in addition shown to be resistant to diphtheria toxin, to a J5-ricin conjugate, and to a conjugate between ricin B-chain and gelonin. The mutants, however, were sensitive to a J5-gelonin conjugate. These mutants expressed high levels of CALLA and/or receptors for ricin, and their cell-free translation systems appeared to be as sensitive to the inhibitory action of ricin A-chain and of gelonin as the translation system of wild-type Namalwa cells. The behavior of these mutants was consistent with the hypothesis that these cells possess an alteration of their surface that impedes the passage of ricin and diphtheria toxin across the plasma membrane. A fourth mutant was found to bind reduced quantities of ricin and was resistant to ricin but was sensitive to J5-ricin. The properties of this cell line provide evidence that the binding of antibody-ricin conjugates to cells via the ricin moiety may be prevented without impeding the cytotoxicity of the conjugates.     

Transport of protein toxins into cells: pathways used by ricin,cholera toxin and Shiga toxin

Ricin, cholera, and Shiga toxin belong to a family of protein toxins that enter the cytosol to exert their action. Since all three toxins are routed from the cell surface through the Golgi apparatus and to the endoplasmic reticulum (ER) before translocation to the cytosol, the toxins are used to study different endocytic pathways as well as the retrograde transport to the Golgi and the ER. The toxins can also be used as vectors to carry other proteins into the cells. Studies with protein toxins reveal that there are more pathways along the plasma membrane to ER route than originally believed.     

A novel biosensor for mercuric ions based on motor proteins

We explored the potential of contractile proteins, actin and myosin, as biosensors of solutions containing mercuric ions. We demonstrate that the reaction of HgCl2 with myosin rapidly inhibits actin-activated myosin ATPase activity. Mercuric ions inhibit the in vitro analog of contraction, namely the ATP-initiated superprecipitation of the reconstituted actomyosin complex. Hg reduces both the rate and extent of this reaction. Direct observation of the propulsive movement of actin filaments (10 nm in diameter and 1 microm long) in a motility assay driven by a proteolytic fragment of myosin (heavy meromyosin or HMM) is also inhibited by mercuric ions. Thus, we have demonstrated the biochemical, biophysical and nanotechnological basis of what may prove to be a useful nano-device.