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Venomous animals immobilize prey using protein toxins that act on ion channels and other targets of biological importance. Broad use of toxins for biomedical research, diagnosis, and therapy has been limited by inadequate target discrimination, for example, among ion channel subtypes. Here, a synthetic toxin is produced by a new strategy to be specific for human Kv1.3 channels, critical regulators of immune T cells. A phage display library of 11,200 de novo proteins is designed using the [alpha]-KTx scaffold of 31 scorpion toxin sequences known or predicted to bind to potassium channels. Mokatoxin-1 (moka1) is isolated by affinity selection on purified target. Moka1 blocks Kv1.3 at nanomolar levels that do not inhibit Kv1.1, Kv1.2, or KCa1.1. As a result, moka1 suppresses CD3/28-induced cytokine secretion by T cells without cross-reactive gastrointestinal hyperactivity. The 3D structure of moka1 rationalizes its specificity and validates the engineering approach, revealing a unique interaction surface supported on an [alpha]-KTx scaffold. This scaffold-based/target-biased strategy overcomes many obstacles to production of selective toxins. mokatoxin | moka1 | phage display | peptide toxin | animal venom doi/10.1073/pnas.0910123106

The channel-forming component of anthrax toxin, [([PA.sub.63]).sub.7], is a heptameric water-soluble protein at neutral pH, but under acidic conditions it spontaneously inserts into lipid bilayers to form a 14-stranded [beta]-barrel ion- conducting channel. This channel plays a vital role in anthrax pathogenesis because it serves as a conduit for the membrane translocation of the two enzymatic components of anthrax toxin, lethal factor and edema factor. Anthrax channels open and close in response to changes in transmembrane voltage, a property shared by several other pore-forming toxins. We have discovered an unexpected phenomenon in cysteine-substituted channels that provides a window into this gating process: their normal voltage-dependent gating can be abolished by reaction with methanethiosulfonate (MTS) reagents or exposure to oxidizing conditions. Remarkably, this perturbation is seen with cysteines substituted at sites all along the ~100 [Angstrom] length of the channel's [beta]-barrel. In contrast, reaction with N-ethylmaleimide, a thiol-reactive compound that does not form a mixed disulfide, does not affect gating at any of the sites tested. These findings, coupled with our biochemical detection of dimers, have led us to conclude that MTS reagents are catalyzing the formation of intersubunit disulfide bonds that lock channels in a conducting state, and that voltage gating requires a conformational change that involves the entire [beta]-barrel.

The facultative methylotroph Methylobacterium extorquens AM1 possesses two pterin-dependent pathways for [C.sub.1] transfer between formaldehyde and formate, the tetrahydrofolate ([H.sub.4]F)-linked pathway and the tetrahydromethanopterin ([H.sub.4]MPT)-linked pathway. Both pathways are required for growth on [C.sub.1] substrates; however, mutants defective for the [H.sub.4]MPT pathway reveal a unique phenotype of being inhibited by methanol during growth on multicarbon compounds such as succinate. It has been previously proposed that this methanol-sensitive phenotype is due to the inability to effectively detoxify formaldehyde produced from methanol. Here we present a comparative physiological characterization of four mutants defective in the [H.sub.4]MPT pathway and place them into three different phenotypic classes that are concordant with the biochemical roles of the respective enzymes. We demonstrate that the analogous [H.sub.4]F pathway present in M. extorquens AM1 cannot fulfill the formaldehyde detoxification function, while a heterologously expressed pathway linked to glutathione and NA[D.sup.+] can successfully substitute for the [H.sub.4]MPT pathway. Additionally, null mutants were generated in genes previously thought to be essential, indicating that the [H.sub.4]MPT pathway is not absolutely required during growth on multicarbon compounds. These results define the role of the [H.sub.4]MPT pathway as the primary formaldehyde oxidation and detoxification pathway in M. extorquens AM1.

Ochratoxin A is an important nephrotoxic and nephrocarcinogenic mycotoxin, produced by Aspergillus ochraceus as a polyketide-derived secondary metabolite. A portion of a putative polyketide synthase gene (pks) involved in the biosynthesis of this mycotoxin was cloned by using a suppression subtractive hybridization PCR-based approach. The predicted amino acid sequence of the 1.4 kb clone shared 28-35% identity to acyl transferase regions from fungal polyketide synthases found in the databases. Based on reverse transcription PCR studies, the pks gene is expressed only under ochratoxin A permissive conditions and only during the early stages of the mycotoxin synthesis. A mutant in which the pks gene has been interrupted cannot synthesize ochratoxin A. This report is the first of the cloning and characterization of a gene involved in ochratoxin A biosynthesis.

Although N[H.sub.2]-terminal mutant huntingtin (htt) fragments cause neurological disorders in Huntington's disease (HD), it is unclear how toxic htt fragments are generated and contribute to the disease process. Here, we report that complex N[H.sub.2]-terminal mutant htt fragments smaller than the first 508 amino acids were generated in htt-transfected cells and HD knockin mouse brains. These fragments constituted neuronal nuclear inclusions and appeared before neurological symptoms. The accumulation and aggregation of these htt fragments were associated with an age-dependent decrease in proteasome activity and were promoted by inhibition of proteasome activity. These results suggest that decreased proteasome activity contributes to late onset htt toxicity and that restoring the ability to remove N[H.sub.2]-terminal fragments will provide a more effective therapy for HD than inhibiting their production.

Undimerized [beta]-tubulin is toxic in the yeast S. cerevisiae. It can arise if levels of [beta]-tubulin and [alpha]-tubulin are unbalanced or if the tubulin heterodimer dissociates. We are using the toxicity of [beta]-tubulin to understand early steps in microtubule morphogenesis. We find that deletion of PLP1 suppresses toxic [beta]-tubulin formed by disparate levels of [alpha]- and [beta]-tubulin. That suppression occurs either when [alpha]-tubulin is modestly underexpressed relative to [beta]-tubulin or when [beta]-tubulin is inducibly and strongly overexpressed. Plp1p does not affect tubulin expression. Instead, a significant proportion of the undimerized [beta]-tubulin in plp1[DELTA] cells is less toxic than that in wild-type cells. It is also less able to combine with [alpha]-tubulin to form a heterodimer. As a result, plp1[DELTA] cells have lower levels of heterodimer. Importantly, plp1[DELTA] cells that also lack Pac10, a component of the GimC/PFD complex, are even less affected by free [beta]-tubulin. Our results suggest that Plp1p defines a novel early step in [beta]-tubulin folding.

Shiga toxins (Stxs) produced by enterohaemorrhagic Escherichia coil or Shigella dysenteriae damage human endothelial cells predominantly in cooperation with pro-inflammatory cytokines, such as TNF-[alpha]. However, in this study, in vitro IFN-[gamma] pre-treatment resulted in human lung microvascular endothelial cells becoming over 10 000-fold less sensitive to Stxs. In contrast, in their basal condition, they were extremely sensitive to Stxs. Interestingly, TNF-[alpha] addition to IFN-[gamma] reverted the Stx-resistant phenotype, which corresponded with its well-established enhancing effect on Stx toxicity. Toxin binding to the cell was barely affected by IFN-[gamma]. Also, the toxin uptake in the Stx-resistant phenotype was more than 100-fold greater than that of normal cells, when compared at Stx concentrations resulting in equivalent degrees of cell damage. Protein synthesis was inhibited by nearly 90 % in the Stx-resistant phenotype after 24 h toxin exposure. This indicated that the intracellular toxin was active as an N-glycosidase, while cells were still over 60 % viable, suggesting a possible unknown cytotoxic function of Stx. In conclusion, this study shows a unique effect of IFN-[gamma] in the suppression of the toxicity of Stxs in a human microvascular endothelial cell model and the involvement of a novel mechanism in this suppression.

D-Glucose uptake in germinating wild-type Aspergillus nidulans conidia is an energy-requiring process mediated by at least two transport systems of differing affinities for glucose: a low-affinity system ([K.sub.m]~1.4 mM) and a high-affinity system ([K.sub.m]~16 [mu]M). The low-affinity system is inducible by glucose; the high-affinity system is subject to glucose repression effected by the carbon catabolite repressor CreA and is absent in sorA3 mutant conidia, which exhibit resistance to L-sorbose toxicity. An intermediate-affinity system ([K.sub.m]~400 [mu]M) is present in sorA3 conidia germinating in derepressing conditions, creA derepressed mutants show enhanced sensitivity to L-sorbose. The high-affinity uptake system appears to be responsible for the uptake of this toxic sugar.

Vibrio cholerae of both biotypes produce a soluble [Zn.sup.2+]-dependent metalloprotease: haemagglutinin/protease (Hap), encoded by hapA. Hap has been shown to have mucinolytic and cytotoxic activity. These activities are likely to play an important role in the pathogenesis of cholera and the reactogenicity of attenuated vaccine strains. Production of Hap requires transcriptional activation by the HapR regulator and is repressed by glucose. The present study shows that mucin purified from two sources, bile salts, and growth at 37[degrees]C enhanced Hap protease production. Analysis of hapA and hapR promoter fusions with the lacZ gene showed both promoters to be activated in a cell-density-dependent pattern. Glucose repressed and mucin induced the hapA promoter by a HapR-independent mechanism. Bile had no effect on either hapR or hapA promoter activity. Expression of hapA was required for vibrios to translocate through a mucin-containing gel. These results suggest Hap to play an important role in cholera pathogenesis by promoting mucin gel penetration, detachment and spreading of infection along the gastrointestinal tract.

Bacillus sphaericus, a bacterium of biotechnological interest due to its ability to produce mosquitocidal toxins, is unable to use sugars as carbon source. However, ptsHI genes encoding HPr and El proteins belonging to a PTS were cloned, sequenced and characterized. Both HPr and El proteins were fully functional for phosphoenolpyruvate-dependent transphosphorylation in complementation assays using extracts from Staphylococcus aureus mutants for one of these proteins. HPr ([His.sub.6]) was purified from wild-type and a Ser46/GIn mutant of B. sphaericus, and used for in vitro phosphorylation experiments using extracts from either B. sphaericus or Bacillus subtilis as kinase source. The results showed that both phosphorylated forms, P-Ser46-HPr and P-His15-HPr, could be obtained. The findings also proved indirectly the existence of an HPr kinase activity in B. sphaericus. The genetic structure of these ptsHI genes has some unusual features, as they are co-transcribed with genes encoding metabolic enzymes related to N-acetylglucosamine (GlcNAc) catabolism (nagA, nagB and an undetermined orr2). In fact, this bacterium was able to utilize this amino sugar as carbon and energy source, but a ptsH null mutant had lost this characteristic. Investigation of GlcNAc uptake and streptozotocin inhibition in both a wild-type and a ptsH null mutant strain led to the proposal that GlcNAc is transported and phosphorylated by an [Ell.sup.Nag] element of the PTS, as yet uncharacterized. In addition, GlcNAc-6phosphate deacetylase and GlcN-6-phosphate deaminase activities were determined; both were induced in the presence of GlcNAc. These results, together with the authors' recent findings of the presence of a phosphofructokinase activity, are strongly indicative of a glycolytic pathway in B. sphaericus. They also open new possibilities for genetic improvements in industrial applications.

Evolution of resistance by pests is the main threat to long-term insect control by transgenic crops that produce Bacillus thuringiensis (Bt) toxins. Because inheritance of resistance to the Bt toxins in transgenic crops is typically recessive, DNA-based screening for resistance alleles in heterozygotes is potentially much more efficient than detection of resistant homozygotes with bioassays. Such screening, however, requires knowledge of the resistance alleles in field populations of pests that are associated with survival on Bt crops. Here we report that field populations of pink bollworm (Pectinophora gossypiella), a major cotton pest, harbored three mutant alleles of a cadherin-encoding gene linked with resistance to Bt toxin Cry1Ac and survival on transgenic Bt cotton. Each of the three resistance alleles has a deletion expected to eliminate at least eight amino acids upstream of the putative toxin-binding region of the cadherin protein. Larvae with two resistance alleles in any combination were resistant, whereas those with one or none were susceptible to Cry1Ac. Together with previous evidence, the results reported here identify the cadherin gene as a leading target for DNA-based screening of resistance to Bt crops in lepidopteran pests.

Methanol intoxication produces toxic injury to the retina and optic nerve, resulting in blindness. The toxic metabolite in methanol intoxication is formic acid, a mitochondrial toxin known to inhibit the essential mitochondrial enzyme, cytochrome oxidase. Photobiomodulation by red to near-IR radiation has been demonstrated to enhance mitochondrial activity and promote cell survival in vitro by stimulation of cytochrome oxidase activity. The present studies were undertaken to test the hypothesis that exposure to monochromatic red radiation from light-emitting diode (LED) arrays would protect the retina against the toxic actions of methanol-derived formic acid in a rodent model of methanol toxicity. Using the electroretinogram as a sensitive indicator of retinal function, we demonstrated that three brief (2 min, 24 s) 670-nm LED treatments (4 J/[cm.sup.2]), delivered at 5, 25, and 50 h of methanol intoxication, attenuated the retinotoxic effects of methanol-derived formate. Our studies document a significant recovery of rod- and cone-mediated function in LED-treated, methanol-intoxicated rats. We further show that LED treatment protected the retina from the histopathologic changes induced by methanol-derived formate. These findings provide a link between the actions of monochromatic red to near-IR light on mitochondrial oxidative metabolism in vitro and retinoprotection in vivo. They also suggest that photobiomodulation may enhance recovery from retinal injury and other ocular diseases in which mitochondrial dysfunction is postulated to play a role.

Excess lipid accumulation in non-adipose tissues is associated with insulin resistance, pancreatic [beta]-cell apoptosis and heart failure. Here, we demonstrate in cultured cells that the relative toxicity of two common dietary long chain fatty acids is related to channeling of these lipids to distinct cellular metabolic fates. Oleic acid supplementation leads to triglyceride accumulation and is well tolerated, whereas excess palmitic acid is poorly incorporated into triglyceride and causes apoptosis. Unsaturated fatty acids rescue palmitate-induced apoptosis by channeling palmitate into triglyceride pools and away from pathways leading to apoptosis. Moreover, in the setting of impaired triglyceride synthesis, oleate induces lipotoxicity. Our findings support a model of cellular lipid metabolism in which unsaturated fatty acids serve a protective function against lipotoxicity though promotion of triglyceride accumulation.

Stress granules (SGs) are formed in the cytoplasm in response to various toxic agents, and are believed to play a critical role in the regulation of mRNA metabolism during stress. In SGs, mRNAs are stored in an abortive translation initiation complex that can be routed to either translation initiation or degradation. Here, we show that G3BP, a phosphorylation-dependent endoribonuclease that interacts with RasGAP, is recruited to SGs in cells exposed to arsenite. G3BP may thus determine the fate of mRNAs during cellular stress. Remarkably, SG assembly can be either dominantly induced by G3BP overexpression, or on the contrary, inhibited by expressing a central domain of G3BP. This region binds RasGAP and contains serine 149, whose dephosphorylation is induced by arsenite treatment. Critically, a phosphomimetic mutant (S149E) fails to oligomerize and to assemble SGs, whereas a nonphosphorylatable G3BP mutant (S149A) does both. These results suggest that G3BP is an effector of SG assembly, and that Ras signaling contributes to this process by regulating G3BP dephosphorylation.

The Foxl1 gene, which encodes a winged helix transcriptional regulator, is expressed in the mesenchymal layer of developing and mature gastrointestinal tract. Foxl1-deficient mice exhibit various defects not only in the epithelial layer of the gastrointestinal tract but also in gut-associated lymphoid tissues. In the small intestine of Foxl1-deficient mice, the formation of Peyer's patches is affected, particularly in the caudal region. This alteration is shown to be due to the delayed formation of Peyer's patches organizing centers as revealed by the expressions of VCAM1 and IL-7 receptor [alpha]-chain at 17.5 days postcoitus. Peyer's patch defects are concordant with the significantly decreased expression of Lymphotoxin [beta]-receptor in the caudal region of fetal intestine. Foxl1 is suggested to regulate the responsiveness of fetal intestinal mesenchymal cells to inductive signals mediated by Lymphotoxins during Peyer's patch organogenesis. In addition, constitutive outgrowth of colonic patches due to defects in radioresistant stromal components of colonic patches are seen in Foxl1-deficient mice. Because of the functional similarities of hypertrophic colonic patches to those seen in hapten-induced experimental colitis, this hypertrophy is suggested to involve Lymphotoxin [beta]-receptor signaling. Together, the data suggest that Foxl1 might be involved in cellular responses of gut-associated lymphoid tissues dependent upon the Lymphotoxins/Lymphotoxin [beta]-receptor axis. Keywords: Foxl1; Peyer's patch; Colonic patch; Gut mesenchyme; Lymphotoxins; Mouse

Toxigenic Vibrio cholerae cause cholera, a severe diarrheal disease responsible for significant morbidity and mortality worldwide. Two determinants, cholera enterotoxin (CT) and toxin coregulated pilus (TCP) are critical factors responsible for this organism's virulence. The genes for these virulence determinants belong to a network of genes (the ToxR regulon) whose expression is modulated by transcriptional regulators encoded by the toxRS, tcpPH, and toxT genes. To define the ToxR regulon more fully, mutants defective in these regulatory genes were transcriptionally profiled by using V. cholerae genomic microarrays. This study identified 13 genes that were transcriptionally repressed by the toxT mutation (all involved in CT and TCP biogenesis), and 27 and 60 genes that were transcriptionally repressed by the tcpPH and toxRS mutations, respectively. During the course of this analysis, we validated the use of a genomic DNA-based reference sample as a means to standardize and normalize data obtained in different microarray experiments. This method allowed the accurate transcriptional profiling of V. cholerae cells present in stools from cholera patients and the comparison of these profiles to those of wild-type and mutant strains of V. cholerae grown under optimal conditions for CT and TCP expression. Our results suggest that vibrios present in cholera stools carry transcripts for these two virulence determinants, albeit at relatively low levels compared with optimal in vitro conditions. The transcriptional profile of vibrios present in cholera stools also suggests that the bacteria experienced conditions of anaerobiosis, iron limitation, and nutrient deprivation within the human gastrointestinal tract.

Bacillus thuringiensis (Bt) crystal proteins are pore-forming toxins used as insecticides around the world. Previously, the extent to which these proteins might also target the invertebrate phylum Nematoda has been mostly ignored. We have expressed seven different crystal toxin proteins from two largely unstudied Bt crystal protein subfamilies. By assaying their toxicity on diverse free-living nematode species, we demonstrate that four of these crystal proteins are active against multiple nematode species and that each nematode species tested is susceptible to at least one toxin. We also demonstrate that a rat intestinal nematode is susceptible to some of the nematicidal crystal proteins, indicating these may hold promise in controlling vertebrate-parasitic nematodes. Toxicity in nematodes correlates with damage to the intestine, consistent with the mechanism of crystal toxin action in insects. Structure-function analyses indicate that one novel nematicidal crystal protein can be engineered to a small 43-kDa active core. These data demonstrate that at least two Bt crystal protein subfamilies contain nematicidal toxins.

Little is known about the mechanisms converting psychosocial stress into cellular dysfunction. Various genes, up-regulated in atherosclerosis but also by psychosocial stress, are controlled by the transcription factor nuclear factor [kappa]B (NF-[kappa]B). Therefore, NF-[kappa]B is a good candidate to convert psychosocial stress into cellular activation. Volunteers were subjected to a brief laboratory stress test and NF-[kappa]B activity was determined in peripheral blood mononuclear cells (PBMC), as a window into the body and because PBMC play a role in diseases such as atherosclerosis. In 17 of 19 volunteers, NF-[kappa]B was rapidly induced during stress exposure, in parallel with elevated levels of catecholamines and cortisol, and returned to basal levels within 60 min. To model this response, mice transgenic for a strictly NF-[kappa]B-Controlled [beta]-globin transgene were stressed by immobilization. Immobilization resulted in increased [beta]-globin expression, which could be reduced in the presence of the [alpha]1-adrenergic inhibitor prazosin. To define the role of adrenergic stimulation in the up-regulation of NF-[kappa]B, THP-1 cells were induced with physiological amounts of catecholamines for 10 min. Only noradrenaline resulted in a dose- and time-dependent induction of NF-[kappa]B and NF-[kappa]B-dependent gene expression, which depended on pertussis-toxin-sensitive G protein-mediated phosphophatidylinositol 3-kinase, Ras/Raf, and mitogen-activated protein kinase activation. Induction was reduced by [[alpha].sub.1]- and [beta]-adrenergic inhibitors. Thus, noradrenaline-dependent adrenergic stimulation results in activation of NF-[kappa]B in vitro and in vivo. Activation of NF-[kappa]B represents a downstream effector for the neuroendocrine response to stressful psychosocial events and links changes in the activity of the neuroendocrine axis to the cellular response.

The protective antigen (PA) of the anthrax toxin binds to a cell surface receptor and thereby allows lethal factor (LF) to be taken up and exert its toxic effect in the cytoplasm. Here, we report that clustering of the anthrax toxin receptor (ATR) with heptameric PA or with an antibody sandwich causes its association to specialized cholesterol and glycosphingolipid-rich microdomains of the plasma membrane (lipid rafts). We find that although endocytosis of ATR is slow, clustering it into rafts either via PA heptamerization or using an antibody sandwich is necessary and sufficient to trigger efficient internalization and allow delivery of LF to the cytoplasm. Importantly, altering raft integrity using drugs prevented LF delivery and cleavage of cytosolic MAPK kinases, suggesting that lipid rafts could be therapeutic targets for drugs against anthrax. Moreover, we show that internalization of PA is dynamin and Eps15 dependent, indicating that the clathrin-dependent pathway is the major route of anthrax toxin entry into the cell. The present work illustrates that although the physiological role of the ATR is unknown, its trafficking properties, i.e., slow endocytosis as a monomer and rapid clathrin-mediated uptake on clustering, make it an ideal anthrax toxin receptor.

The sulfur nutrition of five benthic animals living in or on reductive, tidal-flat sediments has been studied by using the sulfur isotopic signatures of their soft tissues. The mean [delta][sup.34]S values (+6.1 [per thousand] to +13.8 [per thousand], relative to the Canyon Diablo troilite reference) of these animals' tissues are lower than those of many common marine animals, which have values close to those of seawater sulfate-sulfur (+21 [per thousand]). This indicates that these animals use a [sup.34]S-depleted sulfur source, which may be derived from bacterial sulfide in the sediments (less than -20[per thousand]). The animals that inhabit such sediments are adapted to an anoxic environment, where toxic hydrogen sulfide prevails. Due to the sulfide-rich habitat, benthic animals are expected to assimilate hydrogen sulfide directly during detoxification, or to prefer a diet which has a low [delta][sup.34]S signature. Variations in [delta][sup.34]S values of the sampled molluscs were fairly small, whereas migratory shrimp and fish tended to have larger variations, implying that migratory animals feed on diverse prey, while the molluscs feed on a common food, or their assimilative capacity for sulfide-sulfur does not vary among individuals. A gastropod inhabiting the surface of the reductive sediment showed a distinctively low [delta][sup.34]S value (+5.8 [per thousand] to + 6.4 [per thousand]), indicating that the animal fed on detritus from reeds and halophytes with low [delta][sup.34]S values (-19.1 [per thousand] to -2.1[per thousand]). This study suggests that some benthic animals incorporate sulfide-sulfur for their nutrition and that sulfur isotope composition can be a useful indicator in the analysis of the food habitat of animals.

Sponges are sessile filter feeders that are exposed to toxic compounds/xenobiotics and a huge number of diverse pro- and eukaryotic organisms occurring in the aqueous milieu. To cope with these threats, sponges have developed a variety of chemical and immunological defense systems. Among them are potent secondary metabolites or toxins. The demosponge Suberites domuncula contains the bioactive compound okadaic acid; concentrations of [approximately equal to] 50 ng/g ([approximately equal to] 60 nM) of wet tissue have been quantified by LC/MS. In the present study, we sought to determine whether okadaic acid is beneficial for this sponge. Antibodies were raised against okadaic acid and used to localize this compound in situ. It could be demonstrated that the polyclonal antibodies/ antiserum stained the sponge cells (endopinacocytes) which form the epithelium of the lacunae and the water channels. They also reacted with bacteria present in sponge tissue. Okadaic acid was found to augment the immune response of S. domuncula against the bacterial endotoxin lipopolysaccharide (LPS). If primmorphs, a special form of 3-D aggregates, are incubated with LPS at a concentration of 3 [micro]g/ml, a reduction in the incorporation rate of [[sup.3]H] phenylalanine is measured. In the presence of okadaic acid, this effect is measured already at a concentration of 1 [micro]g/ml, suggesting that okadaic acid sensitizes this (immune) response. Okadaic acid is a known inhibitor of protein phosphatases which, as shown here, cause an increased phosphorylation of p38, a central kinase of the MAP kinase pathway; this may explain the sensitive response of the sponge cells to LPS. At higher concentrations, okadaic acid induces apoptosis, as was demonstrated here by a differential expression of the Bcl-2 homolog and the caspase gene. It is concluded that okadaic acid has two functions in the sponge; first, at low concentrations (< 100 nM) a stimulation of the defense system against bacteria, and second, at concentrations above 500 nM, a differential effect on the pro-/anti-apoptotic genes.

Sublethal effects on larval crabs upon exposure to toxic dinoflagellates were examined in the laboratory in early 1999. Specifically, oxygen consumption rates and geotaxis responses were determined for stage 1 larvae of the crabs Cancer oregonensis (Dana) and C. magister Dana that were exposed to non-toxic (Alexandrium tamarense, strain 115) or toxic (A. fundyense, strain 1719) dinoflagellates or to freshly hatched nauplii of the brine shrimp Artemia sp. In C. oregonensis, larvae exposed to the toxic dinoflagellate showed reduced rates of oxygen consumption compared to those exposed to non-toxic dinoflagellates or brine shrimp nauplii. Larvae exposed to a filtrate of the non-toxic dinoflagellate showed no change in oxygen consumption, but a reduced rate when exposed to filtrate from the toxic alga at densities > 5x[10.sup.2] cells [ml.sup.-1]. In C. magister, larvae exposed to the non-toxic A. tamarense or the toxic A. fundyense had reduced oxygen consumption rates. Larvae exposed to filtrates of non-toxic and toxic dinoflagellates had no change in oxygen consumption. In geotaxis tests, C. oregonensis larvae exposed for 1 day to the toxic A. fundyense reduced their level of locomotion compared to those exposed to non-toxic A. tamarense or to brine shrimp nauplii. C. magister larvae showed no change in activity after a 1-day exposure to the toxic A. fundyense. After a 4-day exposure to A. fundyense, C. magister larvae had much reduced locomotion. Reduced locomotory activity in larvae exposed to toxic algae is consistent with the changes in oxygen consumption rates. Responding to exposure to toxic algae by reducing locomotion may affect vertical migration in these negatively buoyant crab larvae, resulting in sinking below a toxic alga bloom, at least temporarily.

Research has been conducted on Vibrio cholerae O1 El Tor strains. Results demonstrate that the copies of CTX prophage integrate in the chromosome of some Vibrio cholerae O1 El Tor strains.

Results demonstrate that the mu-conotoxin SmIIIAfrom Conus stercusmuscarum inhibits irreversibly the tetrodotoxin-resistant sodium channels in frog neurons, indicating to its unique ligand property for voltage-gated sodium channels. The peptide differs from other mu-conotoxins by lacking hydroxyproline.

Research has been conducted on peptides from tarantula venom. The isolation and characterization of these peptides have been carried out and the results demonstrate that they belong to the inhibitory cystine knot family of peptide toxins which interact with voltage-gated ion channels.

Research has been conducted on phormidolide, a cyanobacterium Phormidium sp. isolate. The study of structure elucidation of this macrolide including absolute stereochemistry is presented.

Research has been conducted on Bacillus thuringiensis Cry1 toxins. The pore formation and binding abilities of Cry1A and Cry1Fa B. thuringiensis toxins have been investigated via the use of brush border membrane vesicles prepared from Heliothis virescens strains, and the results demonstrate that the reduction in toxin binding is responsible for increased resistance to Cry1 toxins from H. virescens strain.

Research has been conducted on Vibrio vulnificus. The development of the molecular detection method for estimation of the public health risk associated with the presence of V. vulnificus is described, and the results demonstrate that special attention should be paid to the presence of pathogenic viable but nonculturable cells in environmental samples.

Research has been conducted on microcystin-LR from a naive human semisynthetic phage display library. The isolation of the recombinant phage-antibody clones against this microcystin, the expression of some phage antibodies as soluble single-chain antibody fragments and the characterization of these antibodies via the use of competition enzyme-linked immunosorbent assay are described.

Research has been conducted on murien hydrolases found in various bacteria. The study of these hydrolases has been carried out via the analysis of Escherichia coli mutants constructed with delitions in multiple murein hydrolases, and the results demonstrate that these hydrolase mutants are sensitive to high-molecular-weight antibiotics which are usually not effective against E. coli.

Research has been conducted on sigma factor Clostridium difficile TxeR for toxin gene expression. The role of environmental conditions in tox gene expression and the possibility that these conditions control TxeR synthesis and regulate tox genes indirectly have been tested, and the results are reported.

Research has been conducted on fish pathogen Aeromonas salmonicida subsp. salmonicida. The hypothesis that type III secretion system is present in this bacterium has been tested, and the results demonstrate that knockout mutagenesis of the ycsV homologue in A.salmonicida subsp. salmonicida prevents AexT toxin secretion.

Research has been conducted on Bacillus thuringiensis strains. Bacillus thuringiensis acrystalliferrous mutant strain which produces brown soluble pigment identified as beta-exotoxin I has been investigated, and the results demonstrate that the genes involed in beta-exotoxin I production have been identified via the use of the random mutant library and the mini-transposon delivery system.

Research has been conducted on the polypeptide toxin and its analogue. The binding of these compounds has been investigated via the use of thermodynamic mutant cyclic analyses and the results are discussed.

Research has been conducted on the exotoxin A regulation in Pseudomonas aeruginosa. The role of pvdS in this regulation during chronic lung infections associated with the genetic disease cystic fibrosis has been investigated and the results are reported.

Research describes isolation and cloning of two toxins, pseudechetoxin and pseudecin from Pseudechis australis and P. porphyriacus, respectively. Results show that the toxins exhibit high homology and block the cyclic nucleotide-gated ion channels with different affinities.

Hematopoietic stem cells (HSCs) can be identified by a 'side population' (SP) phenotype. Previous studies have implicated the ATP binding cassette transporter genes Mdr1a/1b and/or Bcrp1 in the SP phenotype. To define the relative role of these transporters, we generated Bcrp1 null mice and evaluated HSCs both functionally and phenotypically. Loss of Bcrp1 gene expression, but not Mdr1a/1b, led to a significant reduction in the number of SP cells in the bone marrow and in skeletal muscle. In the bone marrow, there was a nearly absolute loss of lineage negative, c-Kit-positive, Sca-1-positive SP cells, and the residual SP cells were depleted of repopulating cells in a transplant assay, demonstrating that Bcrp1 expression is necessary for the SP phenotype in HSCs. Furthermore, Bcrp1 null hematopoietic cells were significantly more sensitive to mitoxantrone in drug-treated transplanted mice. These results show that Bcrp1 gene expression alone defines the SP stem cell phenotype, and suggest that the physiological function of Bcrp1 expression in HSCs is to provide protection from cytotoxic substrates.

Research has been conducted on the marine natural alkaloid haliclamine A. The synthesis of this alkaloid has been carried out via the use of thiophene chemistry and Zincke procedure in constructing primary amines of the bis-pyridinium macrocycle and the details are reported.

Huntington's disease and several other neurological diseases are caused by expanded polyglutamine [poly(Gln)] tracts in different proteins. Mechanisms for expanded (>36 Gln residues) poly(Gln) toxicity include the formation of aggregates that recruit and sequester essential cellular proteins [Preisinger, E., Jordan, B. M., Kazantsev, A. & Housman, D. (1999) Phil. Trans. R. Soc. London B 354, 1029-1034; Chen, S., Berthelier, V., Yang, W. & Wetzel, R. (2001)J. Mol. Biol. 311, 173-182] and functional alterations, such as improper interactions with other proteins [Cummings, C. J. & Zoghbi, H. Y. (2000) Hum. Mol. Genet. 9, 909-916]. Expansion above the 'pathologic threshold' ([approximately equal to] 36 Gln) has been proposed to induce a conformational transition in poly(Gln) tracts, which has been suggested as a target for therapeutic intervention. Here we show that structural analyses of soluble huntingtin exon 1 fusion proteins with 16 to 46 glutamine residues reveal extended structures with random coil characteristics and no evidence for a global conformational change above 36 glutamines. An antibody (MW1) Fab fragment, which recognizes full-length huntingtin in mouse brain sections, binds specifically to exon 1 constructs containing normal and expanded poly(Gln) tracts, with affinity and stoichiometry that increase with poly(Gln) length. These data support a 'linear lattice' model for poly(Gln), in which expanded poly(Gln) tracts have an increased number of ligand-binding sites as compared with normal poly(Gln). The linear lattice model provides a rationale for pathogenicity of expanded poly(Gln) tracts and a structural framework for drug design.