CNTN6 mutations are risk factors for abnormal auditory sensory perception in autism spectrum disorders.

Contactin genes CNTN5 and CNTN6 code for neuronal cell adhesion molecules that promote neurite outgrowth in sensory-motor neuronal pathways. Mutations of CNTN5 and CNTN6 have previously been reported in individuals with autism spectrum disorders (ASDs), but very little is known on their prevalence and clinical impact. In this study, we identified CNTN5 and CNTN6 deleterious variants in individuals with ASD. Among the carriers, a girl with ASD and attention-deficit/hyperactivity disorder was carrying five copies of CNTN5. For CNTN6, both deletions (6/1534 ASD vs 1/8936 controls; P=0.00006) and private coding sequence variants (18/501 ASD vs 535/33480 controls; P=0.0005) were enriched in individuals with ASD. Among the rare CNTN6 variants, two deletions were transmitted by fathers diagnosed with ASD, one stop mutation CNTN6W923X was transmitted by a mother to her two sons with ASD and one variant CNTN6P770L was found de novo in a boy with ASD. Clinical investigations of the patients carrying CNTN5 or CNTN6 variants showed that they were hypersensitive to sounds (a condition called hyperacusis) and displayed changes in wave latency within the auditory pathway. These results reinforce the hypothesis of abnormal neuronal connectivity in the pathophysiology of ASD and shed new light on the genes that increase risk for abnormal sensory perception in ASD.

Patterns of placental development evaluated by X chromosome inactivation profiling provide a basis to evaluate the origin of epigenetic variation.

BACKGROUND: Inactivation of the maternally or paternally derived X chromosome (XCI) initially occurs in a random manner in early development; however as tissues form, a 'patchiness' will occur in terms of which X is inactivated if cells positioned near each other are clonally descended from a common precursor. Determining the relationship between skewed XCI in different tissues and in different samples from the same tissue provides a molecular assessment of the developmental history of a particular tissue that can then be used to understand how genetic and epigenetic variation arises in development. METHODS: XCI skewing was evaluated in and compared between amnion, chorion, trophoblast and mesenchyme using multiple sampling sites from 14 term placentae. XCI was also evaluated in chorionic villus samples obtained at multiple sites and depths from four additional term placentae. The pattern of variation was then compared with methylation variation associated with the H19/IGF2 imprinting control region (ICR); promoter regions of KISS1, PTPN6, CASP8 and APC; and LINE-1 elements. RESULTS: Mean placental level of skewing for amnion and chorion are correlated, consistent with a common developmental origin of at least a component of these membranes from inner cell mass derivatives subsequent to XCI, while trophoblast appears to be derived independently, consistent with its origin from the trophectoderm. Villus samples taken from different depths spanning the fetal to maternal side of the placenta were highly clonally related. Comparing patterns of clonal growth identified through XCI to the distribution of epigenetic variation in other genomic regions suggests that some variation arises early in development (e.g. LINE-1 methylation), whereas other variation arises predominantly after villus tree formation (e.g. methylation at H19/IGF2 ICR). CONCLUSIONS: The patterns of XCI skewing are consistent with a model whereby each biopsied site of chorionic villi represents one or a few individual villus trees, each of which is clonally derived from only one or a few precursor cells. Sampling of placentae to evaluate changes associated with clinical pathology should be done with consideration of the tree-to-tree differences. A limitation of this study is the small number of placentas used and therefore placental-specific differences in variation could not be assessed.

The utility of quantitative methylation assays at imprinted genes for the diagnosis of fetal and placental disorders.

An imbalance of imprinted gene expression within 11p15.5 is observed in Beckwith-Wiedemann syndrome (BWS), as well as in a variety of placental abnormalities including complete hydatidiform mole (CHM), placental mesenchymal dysplasia (PMD) and triploidy. To facilitate the diagnosis of epigenetic errors and chromosomal imbalance of 11p15.5, we validated a pyrosequencing assay to measure methylation at KvDMR1 using blood samples from 13 BWS cases, 8 of which showed reduced methylation as compared to control blood. An imbalance between maternal and paternal genomes as is found in triploidy, CHM or PMD was also associated with altered KvDMR1 methylation. A reciprocal pattern of methylation was obtained in the triploid cases by assaying the proximal 11p15.5 ICR associated with H19. To distinguish chromosome 11 specific alterations from whole genome imbalance, other imprinted differentially methylated regions (DMRs) can be utilized. Thus, pyrosequencing assays for DMRs associated with SGCE, SNRPN, and MEST were also compared for their utility in diagnosing parental imbalance in placental samples. While each of these assays could successfully distinguish parental origin of triploidy, SGCE showed the clearest separation between groups. The combined use of a chromosome 11p15.5 assay (e.g. KvDMR1 or H19-ICR) and non-chromosome 11 assay (e.g. SGCE) provides a potentially valuable diagnostic tool in the rapid screening of methylation errors in placental disorders. These results also show the maintenance of imprinting status at these loci in the human placenta, even in the presence of abnormal pathology.

Predictive impact of rare genomic copy number variations in siblings of individuals with autism spectrum disorders.

Identification of genetic biomarkers associated with autism spectrum disorders (ASDs) could improve recurrence prediction for families with a child with ASD. Here, we describe clinical microarray findings for 253 longitudinally phenotyped ASD families from the Baby Siblings Research Consortium (BSRC), encompassing 288 infant siblings. By age 3, 103 siblings (35.8%) were diagnosed with ASD and 54 (18.8%) were developing atypically. Thirteen siblings have copy number variants (CNVs) involving ASD-relevant genes: 6 with ASD, 5 atypically developing, and 2 typically developing. Within these families, an ASD-related CNV in a sibling has a positive predictive value (PPV) for ASD or atypical development of 0.83; the Simons Simplex Collection of ASD families shows similar PPVs. Polygenic risk analyses suggest that common genetic variants may also contribute to ASD. CNV findings would have been pre-symptomatically predictive of ASD or atypical development in 11 (7%) of the 157 BSRC siblings who were eventually diagnosed clinically.

A review of stonefish venoms and toxins.

Venoms from stonefish (genus Synanceja) have marked effects on the cardiovascular and neuromuscular systems and on vascular permeability; the venoms also exhibit haemolytic and hyaluronidase activity. Recently, a toxic protein, stonustoxin (SNTX), was purified from the venom of S. horrida: the primary lethal action of SNTX has been attributed to its potent endothelium-dependent vasorelaxant activity causing a rapid, marked and irreversible hypotension; the other actions of SNTX resemble those of the stonefish crude venoms.

Plasminogen activators t-PA, u-PA and its inhibitor (PAI) in normal males and females.

We determined the plasma levels of tissue plasminogen activator (t-PA), plasminogen activator inhibitor (PAI) activity and their antigen levels including urokinase plasminogen activator (u-PA) in 33 male and 27 female normal subjects. Males had mean t-PA activity of 0.50 iu/ml which was significantly lower (p less than 0.01) than the females 0.64 iu/ml. Males had higher (p less than 0.001) mean PAI activity (15.5 AU/ml) as compared to females 10.3 AU/ml. The respective mean levels of t-PA and PAI antigen were significantly higher (p less than 0.01) in males (8.1 ng/ml and 17.6 ng/ml) than in females (6.2 ng/ml and 12.1 ng/ml). The mean u-PA level in males was 1.54 ng/ml which was significantly higher (p less than 0.01) than in females with 1.02 ng/ml. In post-venous occlusion studies, females had a greater mean response of 8.6 fold in t-PA activity as compared to males with a mean of 4.5 fold increase. The mean t-PA antigen response in males was 2.0 fold increase as compared to 2.6 fold increase in the females. No significant responses were seen in both sexes in either PAI activity or antigen levels when compared with the resting state. In zymography studies, free t-PA, its inhibitor complexes and u-PA were demonstrated in the euglobulin fractions of stored plasma. This study demonstrates that significant differences in t-PA, u-PA and PAI exist between male and female subjects which should be taken into account when determining their levels in clinical conditions.

Cytologic features of Kimura's disease in fine-needle aspirates. A study of eight cases.

Kimura's disease is a chronic inflammatory disorder of unknown etiology, presenting usually as painless subcutaneous swellings in the head and neck region or in the salivary glands. The cytologic features of fine-needle aspirates of eight cases of Kimura's disease were studied with reference to the histologic appearance of the subsequent surgical specimens. In the cytologic smears, the prominent feature was the presence of significant numbers of eosinophils in a background of lymphoid cells. Fragments of collagenous tissue and Warthin-Finkeldey polykaryocytes occasionally were seen. In the cell block, vascular proliferation and fibrosis were useful features, providing further support to the diagnosis. The constellation of these features is characteristic of Kimura's disease and should suggest this diagnosis in the appropriate clinical setting. For initial diagnosis, excisional biopsy is important for the exclusion of malignant lymphoma, histiocytosis X, angiolymphoid hyperplasia with eosinophilia and other reactive lymphadenopathies. Nonetheless, fine-needle aspiration cytology may be valuable in the diagnosis of recurrent lesions of Kimura's disease and may spare the patient from repeated biopsies.

Inhibition of sodium-dependent uptake processes in purified rat brain synaptosomes by Lophozozymus pictor toxin and palytoxin.

To get an insight into the mechanism of neurotoxicity exhibited by Lophozozymus pictor toxin (LPTX) and the toxin isolated from P.caribaeorum (C-PTX) studies were carried out on the effect of these toxins on the uptake of selected substrates (neurotransmitters, amino acids and glucose) in isolated nerve endings. The toxins were found to inhibit the uptake of gamma-aminobutyric acid (GABA), noradrenaline, choline, L-leucine and 2-deoxy-D-glucose in rat brain synaptosomes. LPTX- or C-PTX-induced inhibition of synaptosomal uptake was reduced in the absence of Na+ in the assay medium. Synaptosomes exposed to LPTX and C-PTX release K+ in a dose-dependent manner. Ouabain, a selective inhibitor of the plasma membrane Na+, K(+)-ATPase could inhibit LPTX- and C-PTX-induced K+ efflux from synaptosomes and alleviate the toxin-induced inhibition of synaptosomal GABA uptake. It appears that the induction of ionic flux is the primary cause of toxicity by these toxins leading to the inhibition of Na(+)-dependent uptake processes in synaptosomes. The antagonistic action of ouabain suggests the involvement of the membrane sodium pump in the development of cytotoxicity.

Solitary necrotic nodule of the liver: parasitic origin?

AIMS: To report further cases of solitary necrotic nodule of the liver and to study its nature. METHODS: Seven nodules were retrieved from 4000 necropsy and surgical liver specimens coming to light over the past five years. All of them satisfied the diagnostic criteria of solitary necrotic nodule: a solid lesion with a central necrotic core and a hyalinised fibrotic capsule containing elastic fibres. Their clinicopathological features were reviewed. RESULTS: The nodules were incidental findings at surgery or necropsy in four men and three women whose ages ranged from 48 to 79 years (mean 63.7 years). Four were found in the right lobe and three in the left. Six were subcapsular and only one deep in the parenchyma, with sizes ranging from 0.3-2.5 cm. Each of them was solitary, well demarcated, and round to oval with a firm, whitish rim and a core of yellowish white cheese-like to solid material. In addition to the basic architecture, there were a number of common and undescribed histological features: presence of varying numbers of small mural vessels with intimal fibrosis and obliteration, presence of cholesterol clefts and foamy cells among necrotic material, and sparsity of inflammatory cells. In the two cases where ghosts of degenerated cells and partially preserved liver reticulin pattern were noted, worms were identified, one being Clonorchis sinensis. CONCLUSIONS: The entity is believed to be a "burnt-out phase" of a variety of benign lesions. Parasitic infestation is another possible cause, and presence of ghosts of degenerate cells, partially preserved liver reticulin pattern, cholesterol clefts and foamy cells among necrotic material are auxiliary features pointing to such an aetiology. The variation in morphological fine details reflects both the lesion's diverse pathogenesis and the fact that it can be of varying duration.

Effects of thymosin alpha1 on the development of amoeboid microglial cells in the corpus callosum of neonatal BALB/c and athymic mice.

The present study investigated the effects of intraperitoneal injections of thymosin alpha1 on the supraventricular amoeboid microglial cells (SAMC) in the newborn athymic and normal BALB/c mice. The microglial cells labelled by the lectin GSA I-B4 and the antibody Mac-1 showed a 27% reduction in number in the athymic mice receiving thymosin alpha1 injections compared with those receiving vehicle injections, and a 37% reduction in BALB/c mice receiving thymosin alpha1 injections compared with those receiving vehicle injections. Some of the SAMC in both BALB/c and athymic mice receiving thymosin alpha1 injections became ramified, while the remainder still exhibited their normal amoeboid appearance with few filopodial processes. Ultrastructurally, the lectin reaction product was confined to the plasma membrane and some cytoplasmic vacuoles of labelled SAMC. In both BALB/c and athymic mice, some labelled microglial cells became slender or elongated after thymosin alpha1 injections. Also their cytoplasm was reduced and contained fewer organelles. Radioimmunoassay of the plasma of thymosin alpha1 and vehicle-injected mice showed that there was a significant increase in the cortisol level in BALB/c (P < 0.01) and athymic (P < 0.001) mice 5 days after thymosin alpha1 injections, compared with that of the control mice. The results point to a strong correlation between the reduction of SAMC and the increased level of plasma cortisol. Supporting this is the fact that cortisol is known to suppress the production of monocytes considered to be the precursors of amoeboid microglia.

Synaptosomal binding of 125I-labelled daboiatoxin, a new PLA2 neurotoxin from the venom of Daboia russelli siamensis.

Daboiatoxin (DbTx), the PLA2 neurotoxin from Daboia russelli siamensis venom, was shown to bind specifically and saturably to rat cerebrocortical synaptosomes and synaptic membrane fragments. Two families of binding sites were detected by equilibrium binding analysis in the presence and absence of Ca2+. Scatchard analysis of biphasic plateaus revealed Kdl 5 nM and Bmax1, 6 pmoles/mg protein, and Kd2 80 nM and Bmax2 20 pmoles/mg protein, respectively, for the high- and low-affinity binding sites. The binding of 125I-DbTx to synaptosomes did not show marked dependence on Ca2+, Mg2+, Co2+ and Sr2+. Native DbTx was the only strong competitor to 125I-DbTx synaptosomal binding (IC50 12.5 nM, KI 5.5 nM). Two other crotalid PLA2 neurotoxins, crotoxin CB and mojave toxin basic subunit, and nontoxic C. Atrox PLA2 enzyme, were relatively weaker inhibitors, while two viperid PLA2 neurotoxins, ammodytoxin A and VRV PL V, were very weak inhibitors. Crotoxin CA was a poor inhibitor even at microM concentrations, whereas no inhibitory effect at all was observed with crotoxin CACB, ammodytoxin C, VRV PL VIIIa, taipoxin, beta-bungarotoxin, or with PLA2 enzymes from N. naja venom, E. schistosa venom, bee venom and porcine pancreas. All other pharmacologically active ligands examined (epinephrine, norepinephrine, histamine, choline, dopamine, serotonin, GABA, naloxone, WB-4101, atropine, hexamethonium and alpha-bun-garotoxin) also failed to interfere with 125I-DbTx binding. As those competitors that showed partial inhibition were effective only at microM concentration range compared to the Kd (5 nM) of 125I-DbTx synaptosomal binding, DbTx could well recognize a different neuronal binding site. Rabbit anti-DbTx polyclonal antisera completely blocked the specific binding. When a range of Ca2+ and K+ channels modulators were examined, Ca2+ channel blockers (omega-conotoxins GVIA and MVIIC, taicatoxin, calciseptine and nitrendiprene) did not affect the binding even at high concentrations, while charybdotoxin was the only K+ channel effector that could partially displace 125I-DbTx synaptosomal binding amongst the K+ channel blockers tested (apamin, dendrotoxin-I, iberiotoxin, MCD-peptide, 4-aminopyridine and tetraethylammonium), suggesting that neither K+ nor Ca2+ channels are associated with DbTx binding sites.

Role of free thiol groups in the biological activities of stonustoxin, a lethal factor from stonefish (Synanceja horrida) venom.

Stonustoxin (SNTX) is a two-subunit protein purified from the venom of a stonefish, Synanceia horrida. It has potent lethal activity and is also a membrane pore-forming cytolysin. The role of thiol groups in the biological activities of SNTX was investigated. Both the hemolytic and lethal activities of SNTX were potentiated by the reducing agent, dithiothreitol (DTT). The hemolytic activity of SNTX was sensitive to the modification of thiol groups by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). The loss of haemolytic activity correlated with the number of thiol groups that were titrated with DTNB. Thiol modification of SNTX with DTNB also inhibited its lethality. These inhibitory effects of thiol modification could be reversed by reduction with DTT. It was also found that the haemolytic activity of SNTX could not be inhibited by cholesterol. These observations indicate that free thiol groups play an important role in the haemolytic activity and lethality of SNTX but unlike other thiol-activated cytolysins, SNTX was not inhibited by cholesterol. Thus, SNTX may represent a new class of cytolytic toxin.

Production and characterization of monoclonal antibodies against stonustoxin from Synanceja horrida.

Stonustoxin (SNTX), a lethal factor purified from the venom of stonefish Synanceja horrida, is a protein (148,000 mol. wt) existing as a dimer comprising two subunits (alpha and beta) of mol. wts 71,000 and 79,000, respectively. Its LD50 (i.v.) is 17 ng/g in mice and it causes haemolysis of rat and rabbit erythrocytes in vitro. Eight monoclonal antibodies (Mabs) against SNTX have been developed using the Balb/C mouse. These Mabs have been purified by Protein G affinity membrane disc chromatography. They were all classified as IgG1 with half of them having kappa and the rest lambda light chains. They had affinity constants ranging from 3.75 x 10(-9) to 9.74 x 10(-9) M. Six were able to protect mice from a challenge of a lethal dose of SNTX. However, not all protective Mabs were able to neutralize the haemolytic effect in vitro. Only four Mabs (31A, 32B, 38A and 46A) could inhibit rat and rabbit erythrocyte haemolysis, while one Mab (43D) offered partial inhibition and another Mab (8A) did not inhibit haemolysis at all. The non-protective Mabs (43B and 44G) were also incapable of neutralizing haemolysis. Five epitopes were recognized by the eight Mabs. Four Mabs (31A, 32B, 38A and 46A) were found to have similar epitope specificity while the rest were directed at different epitopes on the SNTX molecule. Thus these results suggest that the domain on the SNTX molecule responsible for lethality is probably distinct from the domain important for in vitro haemolytic activity.

A major lethal factor of the venom of Burmese Russell's viper (Daboia russelli siamensis): isolation, N-terminal sequencing and biological activities of daboiatoxin.

A major lethal factor, daboiatoxin (DbTx), showing strong PLA2 activity (specific activity 91.7 nmoles/min/mg), was purified to homogeneity from the venom of Burmese Russell's viper (Daboia r. siamensis) by a combination of gel filtration on Sephadex G-75 and ion-exchange chromatography on CM-Sephadex C-25, followed by purification on high-performance gel filtration Shim-pack Diol-150 column. DbTx is a single-chain PLA2 toxin with approximate mol. wt 15,000 as determined by HPLC gel filtration and SDS-PAGE. It constitutes 12% of total venom protein and is the main lethal component of Burmese Russell's viper venom with an LD50 i.p. (0.05 mg/kg) 12-fold greater than that of the whole venom (LD50 i.p. 0.6 mg/kg). DbTx produces neurotoxic symptoms in mice and exhibits potent oedema-inducing activity (minimum oedema dose 0.05 microgram), indirect haemolytic activity and a strong myonecrotic activity, but no haemorrhagic activity. DbTx is cytotoxic to HeLa cells causing cytolysis of the cells 24 hr post-exposure to toxin (50 micrograms/ml). The first 20 N-terminal amino acid sequence (NFFQF AEMIV KMTGK EAVHS) shows a significant resemblance to those of the PLA2s from the venoms of Bulgarian viper (V. a. ammodytes) and Taiwan Russell's viper (V. r. formosensis).

Effects of stonustoxin (lethal factor from Synanceja horrida venom) on platelet aggregation.

Stonustoxin (SNTX) is a 148,000 mol. wt lethal factor isolated from Synanceja horrida venom. It induces species-restricted red cell haemolysis which correlates with its effects on platelet aggregation in whole blood. SNTX induced a concentration-dependent and irreversible platelet aggregation in rabbit or rat but not in human or mouse whole blood. The degree of haemolysis and platelet aggregation induced by SNTX in rabbit or rat whole blood were concentration dependent. At concentrations of SNTX where only slight or no haemolysis was observed, no platelet aggregation occurred. Although SNTX itself could not induce platelet aggregation in rabbit or rat platelet-rich plasma (PRP), it had biphasic effects on collagen- or ADP-induced platelet aggregation in PRP. It inhibited collagen- or ADP-induced platelet aggregation at high concentrations (0.08-0.8 micrograms/ml) but the response was potentiated by lower stonustoxin concentrations (0.008-0.016 micrograms/ml). The inhibition of collagen- or ADP-induced platelet aggregation might be due to the lytic activity of SNTX on the platelets.

Isolation of a novel fluorescent toxin from the coral reef crab, Lophozozymus pictor.

A novel toxin has been isolated from an aqueous extract of the coral reef crab Lophozozymus pictor by a combination of ion exchange and two-dimensional thin layer chromatography. The isolated toxin exhibited bioactivities similar to palytoxin (toxicity response of mice, cytotoxicity towards HeLa cells and release of potassium ions from rat erythrocytes). Unlike palytoxin it gave an intense blue fluorescence under ultraviolet light. A fluorescence scan showed that the toxin had a maximum excitation wavelength at 285 nm and a maximum emission wavelength at 355 nm. The toxin was distinguishable from palytoxin when analysed by high performance capillary electrophoresis and reverse phase high performance liquid chromatography.

Stonustoxin: effects on neuromuscular function in vitro and in vivo.

Stonustoxin (8-50 micrograms/ml) produced a rapid and concentration-dependent rise in tension (contracture) of the electrically stimulated mouse hemidiaphragm followed by a gradual waning of tension from the peak to the baseline; the nerve-evoked and the directly (muscle)-evoked twitches of the hemidiaphragm were also progressively and irreversibly blocked in a time- and concentration-dependent manner. Stonustoxin (22 and 44 micrograms/ml) produced a similar rapid rise in tension of the chick biventer cervicis muscle as well as irreversible and concentration-dependent blockade of nerve-evoked twitches and contractures produced by acetylcholine (200 microM), carbachol (8 microM) and KCl (40 mM). The muscle contracture produced by stonustoxin was blocked by dantrolene sodium (6 microM) but not by tubocurarine (15 microM). Moreover, stonustoxin (40 micrograms/ml) did not inhibit nerve conduction in the toad sciatic nerve and stonustoxin (60 micrograms/ml) did not exhibit any anticholinesterase activity. The inhibition of neuromuscular function by stonustoxin in the mouse hemidiaphragm and chick biventer cervicis muscle can therefore be attributed to some irreversible myotoxic action(s) of the toxin, whereas the stonustoxin-induced muscle contractures could have been mediated via depolarization of muscle fibres.

Stonustoxin: a highly potent endothelium-dependent vasorelaxant in the rat.

Stonefish venom has been documented to cause marked hypotension and respiratory difficulties in envenomed animals. Stonustoxin, a lethal protein recently isolated from the venom of the stonefish Synanceja horrida produced hypotension and, at concentrations above 20 micrograms/kg, death in anaesthetized rats, with no observable effects on nerve-evoked twitches of the tibialis and diaphragm muscles. Stonustoxin (20-160 ng/ml) induced endothelium-dependent relaxations of rat thoracic aortae precontracted with noradrenaline. Higher concentrations induced relaxations followed by contractions. Methylene blue, haemoglobin and the specific NO-synthase inhibitor L-NG-nitro arginine methyl ester inhibited stonustoxin-induced relaxations, while the cyclooxygenase inhibitor indomethacin was without effect. The results of the present study show that stonustoxin causes marked vasorelaxation of the rat isolated aorta, which appears to be due to the release of endothelium-derived relaxing factor (probably nitric oxide or nitric oxide-yielding substances) from the vascular endothelium, and this may be responsible for the in vivo hypotensive and lethal actions of stonustoxin and of stonefish venom.

A genomic region encoding stonefish (Synanceja horrida) stonustoxin beta-subunit contains an intron.

The polymerase chain reaction (PCR) has been used to amplify a 1899 base pair fragment from stonefish genomic DNA. A comparison of the translated nucleotide sequence of this product with the separately determined N-terminal amino acid sequence of the beta-subunit reveals the presence of a 416 bp intron at Gly 18. The nucleotide sequence following this intron encodes 476 amino acids whose sequence showed no homology to other known toxins. This region, however, contained amino acid sequences identical to internal peptide sequences determined separately from the toxin's beta-subunit.

Lophozozymus pictor toxin: a fluorescent structural isomer of palytoxin.

Purified Lophozozymus pictor toxin (LPTX) shares many properties similar to palytoxin (PTX). LPTX and palytoxin isolated from Palythoa caribaeorum (C-PTX) have similar mol. wts of approx. 2680 on ionspray mass spectrometry (MS). In addition, antibodies against PTX could recognize and bind LPTX. Mixed mode high-performance liquid chromatography (HPLC) of LPTX, C-PTX and H-PTX (isolated from Palythoa tuberculosa) showed a major PTX component common to all three with the characteristic PTX-like UV spectrum at a retention time (Rt) of 17 min. However, LPTX exhibits fluorescence but PTX of equivalent toxicity does not. LPTX showed a unique peak at Rt of approx. 22 min on mixed mode HPLC. In addition, LPTX and C-PTX showed different ion fragmentation patterns on MS/MS. These results suggest that LPTX and the palytoxins are structural isomers, containing at least one difference which gives rise to fluorescence in LPTX.