Arachnid toxinology in Australia: from clinical toxicology to potential applications.

The unique geographic isolation of Australia has resulted in the evolution of a distinctive range of Australian arachnid fauna. Through the pioneering work of a number of Australian arachnologists, toxinologists, and clinicians, the taxonomy and distribution of new species, the effective clinical treatment of envenomation, and the isolation and characterisation of the many distinctive neurotoxins, has been achieved. In particular, work has focussed on several Australian arachnids, including red-back and funnel-web spiders, paralysis ticks, and buthid scorpions that contain neurotoxins capable of causing death or serious systemic envenomation. In the case of spiders, species-specific antivenoms have been developed to treat envenomed patients that show considerable cross-reactivity. Both in vitro and clinical case studies have shown they are particularly efficacious in the treatment of envenomation by spiders even from unrelated families. Despite their notorious reputation, the high selectivity and potency of a unique range of toxins from the venom of Australian arachnids will make them invaluable molecular tools for studies of neurotransmitter release and vesicle exocytosis as well as ion channel structure and function. The venoms of funnel-web spiders, and more recently Australian scorpions, have also provided a previously untapped rich source of insect-selective neurotoxins for the future development of biopesticides and the characterisation of previously unvalidated insecticide targets. This review provides a historical viewpoint of the work of many toxinologists to isolate and characterise just some of the toxins produced by such a unique group of arachnids and examines the potential applications of these novel peptides.     

Paralytic shellfish toxin profiles and toxin variability of the genus Alexandrium (Dinophyceae) isolated from the Southeast China Sea.

Paralytic shellfish toxin (PST) profiles of 16 Alexandrium isolates from the Southeast China Sea were analyzed by high-pressure liquid chromatography. Toxin content and composition of three A. tamarense isolates, ATDH01, ATGX02 and ATMJ02, were also investigated at different growth phases and under various culture conditions. Our results showed that six strains of A. affine were non-toxic, while 10 strains of A. tamarense and A. catenella were toxic. These toxic isolates grown in the same culture conditions consistently produced an unusually high proportion of the N-sulfocarbamoyl toxin C1/2 (around 60-80% of total toxins) and medium amounts of gonyautoxin GTX5 (around 15-30% of total) with only trace quantities (<5% of total) of other saxitoxin derivatives (i.e. GTX1, GTX3, GTX4 and neoSTX). The toxin composition of three A. tamarense isolates did not vary with the growth phases, although higher toxin contents (Qt, fmolcell(-1)) were found in the exponential phase. Variations in temperature, salinity and nutrient levels affected toxin content of three A. tamarense isolates but they did not have pronounced effects on the toxin composition (mole %). These results indicate that toxin composition remained relatively constant under various culture conditions, suggesting that toxin composition could be used as a stable biomarker for the Alexandrium species in this region. However, comparison of toxin profiles between isolates from different localities require special caution since isolates even from the same region can have distinct toxin profiles.     

Shiga toxin: expression, distribution, and its role in the environment

In this review, we highlight recent work that has increased our understanding of the production and distribution of Shiga toxin in the environment. Specifically, we review studies that offer an expanded view of environmental reservoirs for Shiga toxin producing microbes in terrestrial and aquatic ecosystems. We then relate the abundance of Shiga toxin in the environment to work that demonstrates that the genetic mechanisms underlying the production of Shiga toxin genes are modified and embellished beyond the classical microbial gene regulatory paradigms in a manner that apparently "fine tunes" the trigger to modulate the amount of toxin produced. Last, we highlight several recent studies examining microbe/protist interactions that postulate an answer to the outstanding question of why microbes might harbor and express Shiga toxin genes in the environment.     

Cardiovascular, haematological and neurological effects of the venom of the Papua New Guinean small-eyed snake (Micropechis ikaheka) and their neutralisation with CSL polyvalent and black snake antivenoms.

Cardiovascular and haematological effects of venom of the small-eyed Snake (Micropechis ikaheka) were examined in ventilated anaesthetised piglets. Neurotoxic effects were examined in chick biventer cervicis nerve-muscle preparations. Immunoreactivity of venom was tested against the monovalent antivenom components in a CSL Ltd Venom Detection Kit. Neutralisation was tested in vivo and in vitro with CSL Ltd polyvalent snake and Black Snake (Pseudechis australis) antivenoms. Venom in 0.1% bovine serum albumin in saline was infused into piglets in doses 1-2000 microg/kg. Pulmonary hypertension (P= 0.0007) and depression of cardiac output (P= 0.002) were observed up to 3 h after 150-160 microg/kg. The concentration of plasma free-haemoglobin increased more than 50-fold, indicating haemolysis. Neither coagulopathy nor thrombocytopenia occurred. Creatine phosphokinase and serum potassium levels did not increase suggesting absence of acute rhabdomyolysis. The venom caused post-synaptic neurotoxicty. Immunoreactivity of venom with Black Snake antivenom was observed at very high venom concentrations. Cardiovascular effects were absent and haemolysis was less after venom was pre-incubated at 37 degrees C for 30 min with polyvalent antivenom. Neutralisation by Black Snake antivenom was less effective. The neurotoxicity was neutralised by polyvalent or Black Snake antivenoms. Human envenomation may be treated with CSL Ltd polyvalent snake antivenom.     

Effects of Lonomia obliqua (lepidoptera, saturniidae) toxin on clotting, inflammatory and antibody responsiveness in genetically selected lines of mice.

Lines of mice genetically selected for high (H) or low (L) antibody response and for maximal (AIRMAX) or minimal (AIRMIN) acute inflammatory reaction, in which the opposite extreme potentialities have been clearly defined, offer an appropriate model for investigating the environmental and genetic factors acting on innate and adaptative immunobiological functions. This model has been successfully employed to study the resistance or susceptibility against pathogens and/or toxins. It had been demonstrated that the skin contact with Lonomia obliqua caterpillar bristles induces local inflammation and may elicit severe hemorrhagic disorders. In the present study, blood coagulation time, and the acute inflammatory reaction were scored 24 h after injection of the Lonomia bristles crude extract in a subcutaneous dorsal air pouch. The acute inflammation was determined by the leukocyte concentration in the local exudates. The highest interline differences were observed between the AIRMAX (10(6) cells/ml) and AIRMIN (2 x 10(5) cells/ml) and this distinct expression involves the number of monocytes, eosinophils and mainly neutrophils. Regarding coagulation, the highest interline difference was observed between the HIII and LIII mice, and the F1)[LIII x HIII] hybrids showed the overdominance of the fast clotting character. The adaptative immune response was evaluated by comparing the anti-Lonomia bristle extract IgG titer among the lines: the antibody titers were higher in the H lines than in the L ones and equivalent in the AIRMAX and AIRMIN mice, in accordance to the phenotype profiles generated by the distinct selective processes. The genetically selected mice lines-AIRMAX, AIRMIN, HI, HIII, HG, LIII and LG-showed an almost continuous distributions for inflammation, coagulation time and IgG antibody titers, being the interline variances always higher than the intraline ones for the individually measured phenotypes. Altogether, these results suggest the independent polygenic regulation of these traits, being indicative of the genetic control to Lonomia toxin innate and adaptative sensitivity in humans.     

Isolation and chemical characterization of a toxin isolated from the venom of the sea snake, Hydrophis torquatus aagardi

Sea snakes (family: Hydrophiidae) are serpents found in the coastal areas of the Indian and Pacific Oceans. There are two subfamilies in Hydrophiidae: Hydrophiinae and Laticaudinae. A toxin, aagardi toxin, was isolated from the venom of the Hydrophiinae snake, Hydrophis torquatus aagardi and its chemical properties such as molecular weight, isoelectric point, importance of disulfide bonds, lack of enzymatic activity and amino acid sequence were determined. The amino acid sequence indicated a close relationship to the primary structure of other Hydrophiinae toxins and a significant difference from Laticaudinae toxins, confirming that primary toxin structure is closely related to sea snake phylogenecity.     

Mojave toxin in venom of Crotalus helleri (Southern Pacific Rattlesnake): molecular and geographic characterization.

Mojave toxin (MT) was detected in five of 25 Crotalus helleri (Southern Pacific rattlesnake) sampled using anti-MT antibodies and nucleotide sequence analysis. All of the venoms that were positive for MT were collected from Mt San Jacinto in Riverside Co., California. Since this population is geographically isolated from C. scutulatus scutulatus (Mojave rattlesnake), it is unlikely that this finding is due to recent hybridization. MT concentration differences between C. helleri and C. s. scutulatus reflected the presence of 'isoforms' of the toxin in the venom. Whereas C. s. scutulatus generally has several isoforms of the toxin (detected by Western blotting), only one 'isoform' that focused at pI 5.1 was detected in C. helleri. Both acidic and basic subunits of MT sequences were obtained from C. helleri DNA with primers specific for MT, but only from snakes that had MT in their venom. The sequence identity of the C. helleri acidic subunit to the C. s. scutulatus subunit was 84.9%, whereas the sequence identity of the C. helleri basic subunit was 97% to the C. s. scutulatus basic subunit. Using casein, fibrin, and hide powder azure as substrates, assays for proteolytic activity suggested that C. helleri possesses several different types of metalloproteinases in their venom. However, proteolytic activity was not detected, or present in reduced amounts, in specimens having MT. Clinical neurotoxicity following envenomation by certain populations of C. helleri may be due to MT.     

Mipartoxin-I, a novel three-finger toxin, is the major neurotoxic component in the venom of the redtail coral snake Micrurus mipartitus (Elapidae)

The major venom component of Micrurus mipartitus, a coral snake distributed from Nicaragua to northern South America, was characterized biochemically and functionally. This protein, named mipartoxin-I, is a novel member of the three-finger toxin superfamily, presenting the characteristic cysteine signature and amino acid sequence length of the short-chain, type-I, α-neurotoxins. Nevertheless, it varies considerably from related toxins, with a sequence identity not higher than 70% in a multiple alignment of 67 proteins within this family. Its observed molecular mass (7030.0) matches the value predicted by its amino acid sequence, indicating lack of post-translational modifications. Mipartoxin-I showed a potent lethal effect in mice (intraperitoneal median lethal dose: 0.06 μg/g body weight), and caused a clear neuromuscular blockade on both avian and mouse nerve-muscle preparations, presenting a post-synaptic action through the cholinergic nicotinic receptor. Since mipartoxin-I is the most abundant (28%) protein in M. mipartitus venom, it should play a major role in its toxicity, and therefore represents an important target for developing a therapeutic antivenom, which is very scarce or even unavailable in the regions where this snake inhabits. The structural information here provided might help in the preparation of a synthetic or recombinant immunogen to overcome the limited venom availability.     

Tx2-6 toxin of the Phoneutria nigriventer spider potentiates rat erectile function.

The venom of the spider Phoneutria nigriventer contains several toxins that have bioactivity in mammals and insects. Accidents involving humans are characterized by various symptoms including penile erection. Here we investigated the action of Tx2-6, a toxin purified from the P. nigriventer spider venom that causes priapism in rats and mice. Erectile function was evaluated through changes in intracavernosal pressure/mean arterial pressure ratio (ICP/MAP) during electrical stimulation of the major pelvic ganglion (MPG) of normotensive and deoxycorticosterone-acetate (DOCA)-salt hypertensive rats. Nitric oxide (NO) release was detected in cavernosum slices with fluorescent dye (DAF-FM) and confocal microscopy. The effect of Tx2-6 was also characterized after intracavernosal injection of a non-selective nitric oxide synthase (NOS) inhibitor, L-NAME. Subcutaneous or intravenous injection of Tx2-6 potentiated the elevation of ICP/MAP induced by ganglionic stimulation. L-NAME inhibited penile erection and treatment with Tx2-6 was unable to reverse this inhibition. Tx2-6 treatment induced a significant increase of NO release in cavernosum tissue. Attenuated erectile function of DOCA-salt hypertensive rats was fully restored after toxin injection. Tx2-6 enhanced erectile function in normotensive and DOCA-salt hypertensive rats, via the NO pathway. Our studies suggest that Tx2-6 could be important for development of new pharmacological agents for treatment of erectile dysfunction.     

Australian scorpion stings: a prospective study of definite stings.

There is little information on scorpion stings in Australia. The aim of this study is to describe the circumstances and clinical effects of stings by Australian scorpions. Cases of scorpion stings were collected prospectively from calls and presentations to Australian poison information centres and emergency departments from February 2000 to April 2002. Only definite scorpion stings where the scorpion was immediately collected and expertly identified were included. There were 95 patients, 33 males and 62 females, with a mean age of 32 (SD 19.5; range 1-71) and 23 children (age<15 years). Three families of scorpions caused all stings: Buthidae (79), Bothruiridae (11, all Cercophonius spp.) and Urodacidae (five, all Urodacus spp.). The majority of stings (76%) were by one genus of scorpion Lychas spp. Seventy one percent of stings occurred between 6pm and 8am and 82 (86%) occurred indoors. Sixty percent of stings occurred on distal limbs. The median duration of effects was 6 h (interquartile range (IQR): 1-24 h). Immediate localised pain occurred in all cases and was severe in 76 cases (80%). Other local effects included red mark/redness (66%), tenderness (35%), numbness (12%) and paraesthesia (11%). Minor systemic effects (nausea, headache and malaise) occurred in 11% of cases. There were no deaths or major systemic envenoming. Less severe effects were observed for the larger Urodacus species, compared to Lychas spp. Scorpion stings in Australia do not appear to cause severe or life-threatening effects, even in children. This differs from other parts of the world, where severe envenoming is reported. The major clinical effect is severe pain, consistent with other scorpion stings. Most stings occurred indoors and at night.     

Comparison of paralytic shellfish poisoning toxin between carnivorous crabs (Telmessus acutidens and Charybdis japonica) and their prey mussel (Mytilus galloprovincialis) in an inshore food chain.

Paralytic shellfish poisoning toxin in two shore crab species, Telmessus acutidens and Charybdis japonica, were compared with the toxin in the prey mussel Mytilus galloprovincialis and causative dinoflagellates Alexandrium tamarense, all having been collected at Onahama, Fukushima Prefecture, in the northern part of Japan. When the toxicities were detected in mussels by mouse bioassays, 73.7% of the sampled T. acutidens were toxic in the hepatopancreas. T. acutidens has been found to become toxic for three years, therefore, it can be concluded that the crab commonly and repeatedly accumulate the toxins via the food chain at Onahama. C. japonica was also expected to be a possible vector species, because small quantities of the toxins were detected in eight specimens of the crab by HPLC analysis. By the comparison of the toxin profiles in the dinoflagellates, mussels and the crab T. acutidens, reductive conversions of GTX1 and GTX4 were observed when the toxins passed through the three species in the food chain. But increases of STX and neoSTX by further reductive process were not observed in the crab. The absence of the STX group toxins in the crab suggests that the crab eliminates the toxin before such reductive process occur.     

Cardiovascular effects and lethality of venom from nematocysts of the box-jellyfish Chiropsalmus quadrigatus (Habu-kurage) in anaesthetized rats.

Haemodynamic effects of saline-extracted venom from nematocysts isolated from Chiropsalmus quadrigatus (Habu-kurage) were studied in anaesthetized rats. Intravenous administration of venom (0.2-5 microg protein/kg) produced immediately dose-dependent hypertension and bradycardia. Femoral blood flow transiently increased but calculated femoral vascular conductance decreased. Changes caused by 1 microg/kg of venom were reproducible, and were not affected by prazosin, atropine or BQ123 (ET(A) receptor antagonist) but were significantly attenuated by nicardipine. At doses over 2 microg/kg, hypotension and a decrease in pulse pressure were observed subsequent to transient hypertension. In 5 of 8 rats received 5 microg/kg venom and 6 of 6 rats at 10 microg/kg, death due to irreversible cardiac arrest occurred within 30 min after intravenous injection. However, during nicardipine infusion, venom (10 microg/kg) exerted only modest effects and the rats survived. Heating venom (50 degrees C for 10 min) before injection practically abolished the haemodynamic effects of 10 microg/kg venom, indicating its thermolability. Data show that C. quadrigatus venom has both vasoconstrictor and cardiodepressive effects in rats, and suggest that a calcium channel blocker can protect against the cardiovascular and lethal effects of the venom.     

Isolation and pharmacological effects of leptoxin, a novel proteic toxin from Leptodactylus pentadactylus skin secretion

The in vivo and in vitro pharmacological effects of leptoxin, one of the most lethal protein toxins known at present date (LD₅₀ 0.5 ± 0.03 μg/kg i.v., mice) isolated from Leptodactylus pentadactylus skin secretion, were studied. In rats, leptoxin (1.0 μg/kg, i.v.) induced cardiorespiratory collapse with abundant tracheal secretion followed by sudden death. The cardiovascular shock, pulmonary edema and mortality were not prevented by pretreating the animals with effective doses of pharmacological blockers, i.e., atropine with or without bilateral vagotomy, phentolamine, propranolol, hexamethonium, captopril, dexamethasone, indomethacin, L-NAME, promethazine, Ginkgolide BN-52021 or tezosentan. Pulmonary macroscopic examination revealed increased tracheobronchial secretion, hemorrhagic areas and edema. Microscopic examination showed intense vascular congestion, alveolar and septal interstitial hemorrhage and alveolar edema, without infiltrated inflammatory cells. Leptoxin increased pulmonary index (0.67 ± 0.09 vs. 1.55 ± 0.24; p < 0.05) and the Evans blue concentration in the bronchoalveolar fluid (1.24 ± 0.17 vs. 4.17 ± 1.47 μg/μL; p < 0.01) and in the lung parenchyma (40.73 ± 3.27 vs. 65.33 ± 4.51 μg/μL; p < 0.03). Leptoxin increased the pulmonary perfusion pressure from 13.7 ± 5.3 to 54.0 ± 6.3 mmHg. It also induced a vasoconstrictor effect in the perfused mesenteric vascular bed that could be explained by a hyperreactivity to phenylephrine. Thus, the results suggest that leptoxin-induced death occurs by acute pulmonary edema due to increased microvascular pulmonary pressure evoked by direct vasoconstriction. Despite its strong toxicity, the role of leptoxin in L. pentadactylus skin remains unknown.     

Neocuproine,a selective Cu(I) chelator,and the relaxation of rat vascular smooth muscle by S-nitrosothiols

1. A study has been made of the effect of neocuproine, a specific Cu(I) chelator, on vasodilator responses of rat isolated perfused tail artery to two nitrosothiols: S-nitroso-N-acetyl-D,L-penicillamine (SNAP) and S-nitroso-glutathione (GSNO).
2. Bolus injections (10 μl) of SNAP or GSNO (10⁻⁷–10⁻³ M) were delivered into the lumen of perfused vessels pre-contracted with sufficient phenylephrine (1–7 μM) to develop pressures of 100–120 mmHg. Two kinds of experiment were made: SNAP and GSNO were either (a) pre-mixed with neocuproine (10⁻⁴ M) and then injected into arteries; or (b) vessels were continuously perfused with neocuproine (10⁻⁵ M) and then injected with either pure SNAP or GSNO.
3. In each case, neocuproine significantly attenuated vasodilator responses to both nitrosothiols, although the nature of the inhibitory effect differed in the two types of experiment. We conclude that the ability of exogenous nitrosothiols to relax vascular smooth muscle in our ex vivo model is dependent upon a Cu(I) catalyzed process. Evidence is presented which suggests that a similar Cu(I)-dependent mechanism is responsible for the release of NO from endogenous nitrosothiols and that this process may assist in maintaining vasodilator tone in vivo.

     

An in vivo examination of the stability of venom from the Australian box jellyfish Chironex fleckeri.

We have previously characterised the pharmacological activity of a number of jellyfish venoms with a particular emphasis on the profound cardiovascular effects. It has been suggested that jellyfish venoms are difficult to work with and are sensitive to pH, temperature and chemical changes. The current study aimed to examine the working parameters of the venom of the Australian box jellyfish Chironex fleckeri to enable fractionation and isolation of the toxins with cardiovascular activity. C. fleckeri venom was made up fresh each day and subjected to a number of different environments (i.e. a pH range of 5-9 and a temperature range of 4-30 degrees C). In addition, the effect of freeze drying and reconstituting the venom was investigated. Venom (50 microg/kg, i.v.) produced a transient hypertensive response followed by cardiovascular collapse in anaesthetised rats. This biphasic response was not significantly effected by preparation of the venom at a pH of 5, 7 or 9. Similarly, venom (50 microg/kg, i.v.) did not display a loss of activity when exposed to temperatures of 4, 20 or 30 degrees C for 1.5h. However, the cardiovascular activity was abolished by boiling the venom. Freeze drying, and then reconstituting, the venom did not significantly affect its cardiovascular activity. However, repeated freeze drying and reconstituting of extracted venom resulted in a significantly loss of activity. This study provides a more detailed knowledge of the parameters in which C. fleckeri venom can be used and, while supporting some previous studies, contradicts some of the perceived problems of working with the venom.     

Toxicity and toxin identification in Colomesus asellus, an Amazonian (Brazil) freshwater puffer fish.

Toxicity and toxin identification in Colomesus asellus, an Amazonian (Brazil) freshwater puffer fish. By using four different techniques--mouse bioassay, ELISA, HPLC and mass spectrometry-we evaluated the toxicity in the extracts of C. asellus, a freshwater puffer fish from the rivers of the Amazon, and identified for the first time the components responsible for its toxicity. The T20G10 monoclonal antibody raised against TTX, and employed in an indirect competitive enzyme immunoassay, showed very low affinity for the C. asellus extracts, indicating that TTX and its analogs are not the main toxic components of the extracts. This antibody was efficient in detecting presence of TTX in a total extract of Sphoeroides spengleri, which is one of the most toxic puffer fish found in the Atlantic coast. Extracts of C. asellus were toxic when administered intraperitonially into mice with an average toxicity of 38.6+/-12 mouse unit (MU)/g, while HPLC analysis indicated a lower toxin content (7.6+/-0 5MU/g). The HPLC profile showed no traces of TTX, but only the presence of PSPs (STX, GTX 2 and GTX 3). These toxins were also confirmed by electrospray ionization mass spectrometry.     

PnTx4-3, a new insect toxin from Phoneutria nigriventer venom elicits the glutamate uptake inhibition exhibited by PhTx4 toxic fraction.

Several pools of neurotoxic peptides obtained from fractionated Phoneutria nigriventer venom induce different toxicological effects. One of them, PhTx4, is highly toxic towards insects and displays only a slight toxicity when injected in mice. Also, this fraction contains a class of peptides that are able to inhibit glutamate uptake in preparations of mammalian central nervous systems (CNS). In this work a new toxin called PnTx4-3 was isolated from the PhTx4 fraction by reverse phase and anion exchange steps using high performance liquid chromatography (HPLC). Edman sequencing of PnTx4-3 revealed that it was a polypeptide of 48 amino acid residues, containing 10 cysteines cross-linked by five disulfide bridges. The molecular mass measured by ES-Q-TOF mass spectrometry was 5199.49+/-0.64 Da, which is very close to the calculated mass from amino acid sequence (5199.99 Da). This toxin induces immediate excitatory effects when injected intrathoracically in house flies and cockroaches. Intracerebroventricular injections of 30 microg of PnTx4-3 in mice resulted in no apparent signs of intoxication. In order to make an orthologous comparison, pharmacological characterisation were carried out in rat brain synaptosomes by using [3H]-L-glutamate, showed that the whole PhTx4 fraction as well as the pure toxins PnTx4-3, Tx4(6-1) and Tx4(5-5) obtained of this fraction, were able to inhibit the glutamate uptake in the micromolar concentration range. PnTx4-3 inhibits the glutamate uptake in a dose dependent manner, with an IC50 of approximately 1 microM. PnTx4-3 is highly homologous to the Tx4(6-1) and Tx4(5-5) toxins previously described from the same fraction.     

Isoform-specific effects of a novel BmK 11(2) peptide toxin on Na channels.

BmK 11(2) is a 7216Da polypeptide toxin purified from the venom of the scorpion Buthus martensii Karsch. Nanomolar concentrations of the toxin prolong amphibian nerve action potentials without attenuation of the amplitude. The pharmacological action of the toxin and its sequence similarity to other alpha-scorpion toxins suggest that BmK 11(2) selectively alters voltage-gated Na channels. In order to test whether BmK 11(2) preferentially modulates the gating or kinetics of certain channel isoforms, we applied BmK 11(2) to muscle, heart and neuronal Na channels. 100nM BmK 11(2) increased the peak current amplitude of skeletal muscle (micro1) and neuronal (N1E-115) Na currents by 40 and 20%, respectively, and reduced the cardiac Na (hH1) current by 15%. The toxin slowed current decay of all isoforms, most prominently in N1E-115 (tau(BmK)/tau(Control)=12), micro1 (11), and less so for hH1 (1.3). BmK 11(2) shifted the voltage dependence of activation of micro1 and N1E-115 currents. BmK 11(2) had no effect on steady-state inactivation, use-dependent availability, and the kinetics of entry into slowly recovering inactivated states.     

Induction of apoptosis/necrosis in various human cell lineages by Haemophilus ducreyi cytolethal distending toxin.

We investigated the impact of highly purified Haemophilus ducreyi cytolethal distending toxin (HdCDT) on the apoptosis and necrosis of various human cells; including myeloid cells, epithelial cells, keratinocytes, and primary fibroblasts. The levels of apoptosis and necrosis induced in these cells were compared to those induced by HdCDT in human T cells and in the Jurkat T cell line. Levels of caspase-3 activity were measured, and membrane changes like phosphatidylserine (PS) translocation was evaluated after double-staining with the fluorescein isothiocyanate (FITC)-labeled annexin V and propidium iodide (PI) using flow cytometry. HdCDT induced various degrees of apoptosis and necrosis in dose- and time-dependent manners in cells of various lineages. Early and late apoptosis (annexin V-stained cells) were induced in more than 90% of T cells and monocytes after treatment with 100 ng/ml HdCDT for 24 and 48 h, respectively. The corresponding numbers for epithelial cells, keratinocytes, and fibroblasts were 26-32% after treatment with 100 ng/ml HdCDT for 48 h. HdCDT appears to eliminate effectively by inducing apoptosis those cells that are involved in immune responses. Epithelial cells, keratinocytes and fibroblasts, which are important for the healing of chancroid ulcers, are eliminated by apoptosis or necrosis after contact with HdCDT, albeit slower and to a lesser extent than T cells.     

The loss of PSP toxin production in a formerly toxic Alexandrium lusitanicum clone.

Toxin production has always been considered a constitutive characteristic of dinoflagellates in the genus Alexandrium. Here we demonstrate that saxitoxin production can be lost by an Alexandrium species during routine culture maintenance. This is the first report of any marine saxitoxin-producing alga ever to have completely lost the ability to produce toxins. A clonal toxic isolate of Alexandrium lusitanicum from Portugal has been maintained in culture since 1962. In 1992, a subculture was established and sent to a different laboratory. Recent comparisons of the parental strain and the subculture revealed that the former had lost its toxicity, whereas the latter still produces saxitoxins. This loss of toxicity was confirmed by three independent toxin detection methods: mouse bioassay, mouse neuroblastoma assay and HPLC. Sequence analyses of different rRNA domains demonstrated that the toxic and non-toxic cultures are genetically identical for those markers. Morphological analysis showed that both cultures have the same plate tabulation and are A. lusitanicum. These results strongly argue that the loss of toxicity is not a result of a culturing artifact or mistake, such as mislabeling or contamination. The clonal cultures also show a significant difference in growth. Possible explanations for the change include genetic mutations or the effects of prolonged treatment of the non-toxic culture with antibiotics.