Lung endothelial cells are sensitive to epsilon toxin from Clostridium perfringens.

The pore-forming protein epsilon toxin (Etx) from Clostridium perfringens produces acute perivascular edema affecting several organs, especially the brain and lungs. Despite the toxin evident effect on microvasculature and endothelial cells, the underlying molecular and cellular mechanisms remain obscure. Moreover, no Etx-sensitive endothelial cell model has been identified to date. Here, we characterize the mouse lung endothelial cell line 1G11 as an Etx-sensitive cell line and compare it with the well-characterized Etx-sensitive Madin-Darby canine kidney epithelial cell line. Several experimental approaches, including morphological and cytotoxic assays, clearly demonstrate that the 1G11 cell line is highly sensitive to Etx and show the specific binding, oligomerization, and pore-forming activity of the toxin in these cells. Recently, the myelin and lymphocyte (MAL) protein has been postulated as a putative receptor for Etx. Here, we show the presence of Mal mRNA in the 1G11 cell line and the presence of the MAL protein in the endothelium of some mouse lung vessels, supporting the hypothesis that this protein is a key element in the Etx intoxication pathway. The existence of an Etx-sensitive cell line of endothelial origin would help shed light on the cellular and molecular mechanisms underlying Etx-induced edema and its consequences.

Epsilon toxin from Clostridium perfringens induces cytotoxicity in FRT thyroid epithelial cells.

Epsilon toxin (Etx) is produced by Clostridium perfringens and induces enterotoxemia in ruminants. Etx crosses the blood-brain barrier, binds to myelin structures, and kills oligodendrocytes, inducing central nervous system demyelination. In addition, Etx has a cytotoxic effect on distal and collecting kidney tubules. There are few cell lines sensitive to Etx. At present, the most sensitive in vitro model for Etx is the Madin-Darby canine kidney (MDCK) cell line, where Etx oligomerizes and forms a pore with consequent ion efflux and cell death. Although the Etx receptor has not yet been fully clarified, it is known that caveolin 1 and 2 potentiate Etx cytotoxicity and oligomerization, and more recently, the myelin and lymphocyte (MAL) protein has been implicated in Etx binding and activity. Here, we studied the effect of Etx on Fischer rat thyroid cells (FRT) and observed similar effects as those seen in MDCK cells. Etx incubated with FRT cells showed binding to the plasma membrane, and western blotting assays revealed oligomeric complex formation. Moreover, cytotoxic assays on FRT cells after Etx incubation indicated cell death at a similar level as in MDCK cells. In addition, a luminescent ATP detection assay revealed ATP depletion in FRT cells after Etx exposure. Previous studies have reported that FRT cells do not express caveolins and do not form caveolae but express MAL protein in glycolipid-enriched membrane microdomains. Our results indicate that caveolins are not directly implicated in Etx cytotoxicity, supporting the notion that the MAL protein is involved in Etx action. In addition, a cell line of thyroid origin is described for the first time as a good model to study Etx action.

Cellular Redox Systems Impact the Aggregation of Cu,Zn Superoxide Dismutase Linked to Familial Amyotrophic Lateral Sclerosis.

Protein misfolding is implicated in neurodegenerative diseases such as ALS, where mutations of superoxide dismutase 1 (SOD1) account for about 20% of the inherited mutations. Human SOD1 (hSOD1) contains four cysteines, including Cys(57) and Cys(146), which have been linked to protein stability and folding via forming a disulfide bond, and Cys(6) and Cys(111) as free thiols. But the roles of the cellular oxidation-reduction (redox) environment in SOD1 folding and aggregation are not well understood. Here we explore the effects of cellular redox systems on the aggregation of hSOD1 proteins. We found that the known hSOD1 mutations G93A and A4V increased the capability of the thioredoxin and glutaredoxin systems to reduce hSOD1 compared with wild-type hSOD1. Treatment with inhibitors of these redox systems resulted in an increase of hSOD1 aggregates in the cytoplasm of cells transfected with mutants but not in cells transfected with wild-type hSOD1 or those containing a secondary C111G mutation. This aggregation may be coupled to changes in the redox state of the G93A and A4V mutants upon mild oxidative stress. These results strongly suggest that the thioredoxin and glutaredoxin systems are the key regulators for hSOD1 aggregation and may play critical roles in the pathogenesis of ALS.

Dysfunction of spatacsin leads to axonal pathology in SPG11-linked hereditary spastic paraplegia.

Hereditary spastic paraplegias are a group of inherited motor neuron diseases characterized by progressive paraparesis and spasticity. Mutations in the spastic paraplegia gene SPG11, encoding spatacsin, cause an autosomal-recessive disease trait; however, the precise knowledge about the role of spatacsin in neurons is very limited. We for the first time analyzed the expression and function of spatacsin in human forebrain neurons derived from human pluripotent stem cells including lines from two SPG11 patients and two controls. SPG11 patients'-derived neurons exhibited downregulation of specific axonal-related genes, decreased neurite complexity and accumulation of membranous bodies within axonal processes. Altogether, these data point towards axonal pathologies in human neurons with SPG11 mutations. To further corroborate spatacsin function, we investigated human pluripotent stem cell-derived neurons and mouse cortical neurons. In these cells, spatacsin was located in axons and dendrites. It colocalized with cytoskeletal and synaptic vesicle (SV) markers and was present in synaptosomes. Knockdown of spatacsin in mouse cortical neurons evidenced that the loss of function of spatacsin leads to axonal instability by downregulation of acetylated tubulin. Finally, time-lapse assays performed in SPG11 patients'-derived neurons and spatacsin-silenced mouse neurons highlighted a reduction in the anterograde vesicle trafficking indicative of impaired axonal transport. By employing SPG11 patient-derived forebrain neurons and mouse cortical neurons, this study provides the first evidence that SPG11 is implicated in axonal maintenance and cargo trafficking. Understanding the cellular functions of spatacsin will allow deciphering mechanisms of motor cortex dysfunction in autosomal-recessive hereditary spastic paraplegia.

Altered thiol chemistry in human amyotrophic lateral sclerosis-linked mutants of superoxide dismutase 1.

Neurodegenerative diseases share a common characteristic, the presence of intracellular or extracellular deposits of protein aggregates in nervous tissues. Amyotrophic Lateral Sclerosis (ALS) is a severe and fatal neurodegenerative disorder, which affects preferentially motoneurons. Changes in the redox state of superoxide dismutase 1 (SOD1) are associated with the onset and development of familial forms of ALS. In human SOD1 (hSOD1), a conserved disulfide bond and two free cysteine residues can engage in anomalous thiol/disulfide exchange resulting in non-native disulfides, a hallmark of ALS that is related to protein misfolding and aggregation. Because of the many competing reaction pathways, traditional bulk techniques fall short at quantifying individual thiol/disulfide exchange reactions. Here, we adapt recently developed single-bond chemistry techniques to study individual disulfide isomerization reactions in hSOD1. Mechanical unfolding of hSOD1 leads to the formation of a polypeptide loop held by the disulfide. This loop behaves as a molecular jump rope that brings reactive Cys-111 close to the disulfide. Using force-clamp spectroscopy, we monitor nucleophilic attack of Cys-111 at either sulfur of the disulfide and determine the selectivity of the reaction. Disease-causing mutations G93A and A4V show greatly altered reactivity patterns, which may contribute to the progression of familial ALS.

Anatomical and histological study of human deep fasciae development.

PURPOSE: To characterize the connective tissue found between the subcutaneous adipose tissue and the underlying muscle tissue in different regions and at different stages of human fetal development. We aim to identify its structural similarities to adult deep fascia, and to establish its role in myofascial development. METHODS: Samples from the arm, forearm, low back and thigh regions (from sites topographically homologous to the adult deep fascia) of five fetus body donors were obtained to perform gross anatomy dissection and histologic sections. Sections were stained with hematoxylin-eosin and Masson trichrome stain to observe their overall structure. Antiserum to protein S100 was used to analyze the presence and distribution of nerve fibers, and immunohistochemistry processing with Tcf4 marker was used to ensure fibroblast activity. RESULTS: Gross anatomy and histological sections of fetal samples showed the presence of connective tissue topographically and morphologically equivalent to adult deep fasciae. Developing blood vessels and nerves were found evenly distributed within the connective tissue during early development and in the portion adjacent to the muscle at later stages. The presence of Tcf4+ fibroblasts was confirmed in all analyzed mesenchymal connective tissue. CONCLUSIONS: Deep fascia is present from week 21 of human development in the lower back and upper and lower limbs. Blood vessels and nerves develop parallel to it and occasionally cross it from the deep to superficial plane. The presence of Tcf4+ fibroblasts in the deep fascia suggests a crucial role for this structure in muscle morphogenesis.

Combined intermittent hypobaric hypoxia and muscle electro-stimulation: a method to increase circulating progenitor cell concentration?

BACKGROUND: Our goal was to test whether short-term intermittent hypobaric hypoxia (IHH) at a level well tolerated by healthy humans could, in combination with muscle electro-stimulation (ME), mobilize circulating progenitor cells (CPC) and increase their concentration in peripheral circulation. METHODS: Nine healthy male subjects were subjected, as the active group (HME), to a protocol involving IHH plus ME. IHH exposure consisted of four, three-hour sessions at a barometric pressure of 540 hPa (equivalent to an altitude of 5000 m). These sessions took place on four consecutive days. ME was applied in two separate 20-minute periods during each IHH session. Blood samples were obtained from an antecubital vein on three consecutive days immediately before the experiment, and then 24 h, 48 h, 4 days, 7 days and 14 days after the last day of hypoxic exposure. Four months later a control study was carried out involving seven of the original subjects (CG), who underwent the same protocol of blood samples but without receiving any special stimulus. RESULTS: In comparison with the CG the HME group showed only a non-significant increase in the number of CPC CD34+ cells on the fourth day after the combined IHH and ME treatment. CONCLUSION: CPC levels oscillated across the study period and provide no firm evidence to support an increased CPC count after IHH plus ME, although it is not possible to know if this slight increase observed is physiologically relevant. Further studies are required to understand CPC dynamics and the physiology and physiopathology of the hypoxic stimulus.

Multidisciplinary approach to the persistent double distal tendon of the biceps brachii.

PURPOSE: The aim of this study is to correlate the ultrasound (US) appearance of the persistent double or bifid distal tendon of the biceps brachii muscle with anatomical and histological data. This will provide a new model to study the pathological distal biceps brachii tendon (DBBT). METHODS: The DBBT of 20 cadaveric elbows were examined with linear array broadband US transducers (frequency band 14-6 MHz) using an anterior approach. Trypan blue dye was injected underneath the paratenon under US guidance in 16 specimens. After they were dissected, five of them were processed to obtain histological slices stained with hematoxylin-eosin and antiserum to protein S100. RESULTS: At US, the DBBT is a tendon in which the fascicles are organized in two different hyperechoic components separated by a hyperechoic septum related to the endotenon. The endotenon is lax, flexible, and makes folding and gliding of the two portions feasible. The DBBT is surrounded by a hyperechoic paratenon adjacent to the tendon surface, which is only differentiable by US when dye is interposed between such structures. CONCLUSIONS: The connective septum of endotenon located between the two main components of the DBBT is responsible for the US image of two separate tendons and functionally enables it to work as two separate entities, thus allowing respective folding and gliding. The paratenon surrounding the lacertus fibrosus and the DBBT plays an important stabilization role, enabling them to change shape and arrangement during joint motion. It is also an important conduit for nerves and blood vessels.

Human shoulder anatomy: new ultrasound, anatomical, and microscopic perspectives.

This study aimed to describe the shoulder anatomy, together with the anatomical relationships in adults and early stages of development. The shoulder muscles were studied from ultrasound, anatomical, and microscopic perspectives in a sample of 34 human shoulders. Thickness measurements were taken of the tendons and fasciae of the subscapularis, long head tendon of the biceps brachii, supraspinatus, infraspinatus, and teres minor. Ultrasound and dissection techniques are strongly correlated. However, the measurements obtained from the dissection technique were superior to those obtained from the ultrasound in all cases, except for the thickness of the long head tendon of the biceps brachii, the teres minor tendon, and the fascia thickness of the infraspinatus. In addition, the study of shoulder anatomy revealed no differences between females and males. Relevant findings from dissection included a clear overlap between the infraspinatus and supraspinatus, which shared tendon fibers, and a similar connection between the transverse ligament of the long head tendon of the biceps brachii and the subscapularis, which created a more interconnected shoulder function. The study of the anatomical measurements shows an underestimation of the shoulder measurements in the ultrasound compared with the dissection technique, but a high correlation between the measurements made by the two techniques. We present reference values for the tendon and fascia thicknesses of the rotator cuff, with no differences observed by gender. The relationships between shoulder structures described in the anatomical study imply as well that, in the event of an injury, adjacent tissues may be affected. This extended information may facilitate future optimal clinical explorations.

What Is New about the Semimembranosus Distal Tendon? Ultrasound, Anatomical, and Histological Study with Clinical and Therapeutic Application.

The semimembranosus muscle inserts into several tendons that are associated with some pathologies. Although ultrasound is useful for studying, diagnosing, and managing these pathologies, the correct interpretation of any images requires a clear knowledge of the related anatomical structures and the inter-related functions. We studied 38 cryopreserved non-paired knees from adult anatomical specimens and 4 non-paired knees from 29 to 38-week-old fetuses. The semimembranosus muscle and its tendons were located, observed, and injected under ultrasound guidance. The macroscopic anatomy was studied using dissection and anatomical cuts and the tendons were analyzed histologically. Measurements of muscle were taken 10 cm from the medial epicondyle and just before the tendon divided. The ultrasound facilitated the identification of the different divisions of the tendon of semimembranosus muscle and the rotation of the muscle and tendon from medial to posterior. An anatomical study confirmed this rotation and revealed an average width, thickness, and diameter of 38.29 mm, 14.36 mm, and 112.64 mm, respectively. Important relationships were observed between the divisions of the main tendons and the medial collateral ligament, the posterior side of the knee and popliteus muscle. This information can help to explain knee pathologies and facilitate rehabilitation after surgery.

The soleus muscle: MRI, anatomic and histologic findings in cadavers with clinical correlation of strain injury distribution.

OBJECTIVE: The purpose of this study was to describe the normal anatomy of the soleus muscle using magnetic resonance (MR) imaging, anatomic dissection and histologic correlation in cadavers. The second objective of this study was to analyse the morphometry of the soleus muscle in normal volunteers. The final objective was to undertake a retrospective review of soleal strain injuries confirmed with MR imaging, with correlation made between the cadaveric anatomic findings and the MR imaging features. MATERIALS AND METHODS: Eleven fresh cadaveric legs were studied using a high resolution 3.0 T (T) MR imaging scanner to obtain images in the axial, coronal and sagittal planes. After imaging, six specimens were dissected and evaluated by histological analysis, with the remaining five specimens then frozen and cut into axial sections. The corresponding levels on the MR examination were then compared with the levels of anatomic sectioning. MR imaging was also used to examine the soleus muscle in both legs of 20 healthy volunteers. Finally, 55 clinical cases of soleus muscle strains diagnosed between October 2006 and January 2011 that had also previously undergone MR imaging were re-evaluated. The location of strain injury was reviewed and correlated with the anatomic information that had been revealed in the anatomic component of our study. RESULTS: Dissection of the soleus muscle revealed two proximal intramuscular aponeuroses (medial and lateral) that are formed as a direct continuation of the surrounding epimysium. From an anatomic, functional and pathologic perspective, these aponeuroses are considered in this study as intramuscular tendons, however they have been not previously described as such. These tendons penetrate deep into the muscle belly, from which the proximal muscle fibres of the soleus arise. Inferiorly, these muscle fibres insert onto a long distal central tendon that becomes confluent with the overlying distal tendon of gastrocnemius to form the Achilles tendon. Significant differences between the length of the central tendon on the right side (31.35 cm) and the left side (30.36 cm) were observed (p = .002), as well as the length of insertion of this tendon onto the Achilles tendon on the right side (7.19 cm) compared with the left (7.94 cm) (p = .02). The retrospective analysis identified five sites within the soleus where strains were distributed: musculotendinous junction sites (proximal medial strains accounting for 25.5% of all injuries, proximal lateral strains accounting for 12.7% and distal central tendon strains accounting for 18.2%) and myofascial sites (anterior strains accounting for 21.8% of all injuries and posterior strains accounting for 21.8%). Strains of the proximal medial musculotendinous junction were the most common of soleal muscle injuries, comprising 56.4% of all cases. CONCLUSION: Current information on the detailed anatomy of the soleus muscle in the anatomic and radiological literature is lacking. Knowledge of this anatomy accounts for the distribution of sports-induced injuries within the soleus muscle-tendon unit and therefore assists in the accurate identification of these injuries, with possible prognostic benefit.

PLGA Nanoparticles Containing Natural Flavanones for Ocular Inflammation.

Flavanones are natural compounds that display anti-inflammatory activity. The aim of this work was to prepare PLGA nanoparticles (NPs) containing natural flavanones I ((2S)-5,7-dihydroxy-6-methyl-8-(3-methyl-2-buten-1-il)-2-phenyl-2,3-dihydro-4H-1-Benzopyran-4-one) and II (2S)-5,7-dihydroxy-2-(4'-methoxyphenyl)-6-methyl-8-(3-methyl-2-buten-1-yl)-2,3-dihydro-4H-1-Benzopyran-4-one) (NP I and NP II, respectively) so as to evaluate their potential for topical anti-inflammatory ocular therapy. An in silico study was carried out using the Molinspiration® and PASS Online web platforms before evaluating the in vitro release study and the ex vivo porcine cornea and sclera permeation. The HPLC analytical method was also established and validated. Finally, the in vitro anti-inflammatory efficacy of NPs was studied in the HCE-2 model. The flavanones I and II could be released following a kinetic hyperbolic model. Neither of the two NPs was able to permeate through the tissues. NP I and NP II were found to be respectful of any changes in the tissues' morphology, as evidenced by histological studies. In HCE-2 cells, NP I and NP II were not cytotoxic at concentrations up to 25 µM. NP I showed higher anti-inflammatory activity than NP II, being able to significantly reduce IL-8 production in LPS-treated HCE-2 cells. In summary, ocular treatment with NP I and NP II could be used as a promising therapy for the inhibition of ocular inflammation.

New Mutants of Epsilon Toxin from Clostridium perfringens with an Altered Receptor-Binding Site and Cell-Type Specificity.

Epsilon toxin (Etx) from Clostridium perfringens is the third most potent toxin after the botulinum and tetanus toxins. Etx is the main agent of enterotoxemia in ruminants and is produced by Clostridium perfringens toxinotypes B and D, causing great economic losses. Etx selectively binds to target cells, oligomerizes and inserts into the plasma membrane, and forms pores. A series of mutants have been previously generated to understand the cellular and molecular mechanisms of the toxin and to obtain valid molecular tools for effective vaccination protocols. Here, two new non-toxic Etx mutants were generated by selective deletions in the binding (Etx-ΔS188-F196) or insertion (Etx-ΔV108-F135) domains of the toxin. As expected, our results showed that Etx-ΔS188-F196 did not exhibit the usual Etx binding pattern but surprisingly recognized specifically an O-glycoprotein present in the proximal tubules of the kidneys in a wide range of animals, including ruminants. Although diminished, Etx-ΔV108-F135 maintained the capacity for binding and even oligomerization, indicating that the mutation particularly affected the pore-forming ability of the toxin.

Regulation of exocytotic protein expression and Ca2+-dependent peptide secretion in astrocytes.

Vesicular transmitter release from astrocytes influences neuronal development, function and plasticity. However, secretory pathways and the involved molecular mechanisms in astroglial cells are poorly known. In this study, we show that a variety of SNARE and Munc18 isoforms are expressed by cultured astrocytes, with syntaxin-4, Munc18c, SNAP-23 and VAMP-3 being the most abundant variants. Exocytotic protein expression was differentially regulated by activating and differentiating agents. Specifically, proteins controlling Ca(2+)-dependent secretion in neuroendocrine cells were up-regulated after long-term 8Br-cAMP administration in astrocytes, but not by proinflammatory cytokines. Moreover, 8Br-cAMP treatment greatly increased the cellular content of the peptidic vesicle marker secretogranin-2. Release assays performed on cAMP-treated astrocytes showed that basal and stimulated secretogranin-2 secretion are dependent on [Ca(2+)](i). As shown release of the chimeric hormone ANP.emd from transfected cells, cAMP-induced differentiation in astrocytes enhances Ca(2+)-regulated peptide secretion. We conclude that astroglial cells display distinctive molecular components for exocytosis. Moreover, the regulation of both exocytotic protein expression and Ca(2+)-dependent peptide secretion in astrocytes by differentiating and activating agents suggest that glial secretory pathways are adjusted in different physiological states.

Bidirectional Synaptic Plasticity Is Driven by Sex Neurosteroids Targeting Estrogen and Androgen Receptors in Hippocampal CA1 Pyramidal Neurons.

Neuroactive estrogenic and androgenic steroids influence synaptic transmission, finely modulating synaptic plasticity in several brain regions including the hippocampus. While estrogens facilitate long-term potentiation (LTP), androgens are involved in the induction of long-term depression (LTD) and depotentiation (DP) of synaptic transmission. To examine sex neurosteroid-dependent LTP and LTD in single cells, patch-clamp recordings from hippocampal CA1 pyramidal neurons of male rats and selective antagonists for estrogen receptors (ERs) and androgen (AR) receptors were used. LTP induced by high-frequency stimulation (HFS) depended on activation of ERs since it was prevented by the ER antagonist ICI 182,780 in most of the neurons. Application of the selective antagonists for ERα (MPP) or ERß (PHTPP) caused a reduction of the LTP amplitude, while these antagonists in combination, prevented LTP completely. LTP was never affected by blocking AR with the specific antagonist flutamide. Conversely, LTD and DP, elicited by low-frequency stimulation (LFS), were impeded by flutamide, but not by ICI 182,780, in most neurons. In few cells, LTD was even reverted to LTP by flutamide. Moreover, the combined application of both ER and AR antagonists completely prevented both LTP and LTD/DP in the same neuron. The current study demonstrates that the activation of ERs is necessary for inducing LTP in hippocampal pyramidal neurons, whereas the activation of ARs is required for LTD and DP. Moreover, both estrogen- and androgen-dependent LTP and LTD can be expressed in the same pyramidal neurons, suggesting that the activation of sex neurosteroids signaling pathways is responsible for bidirectional synaptic plasticity.

Schwann cells modulate short-term plasticity of cholinergic autaptic synapses.

Nicotinic synapses in the autonomous nervous system display use-dependent plasticity but the contribution of cellular environment, as well as the presynaptic mechanisms implicated in this process remain to be determined. To address these questions synaptic function was assayed in rat superior cervical ganglion (SCG) neurons microcultured in isolation from any other cell type and compared to those microcultured in the presence of Schwann cells of ganglionar origin. Schwann cells were not required for synapse formation in vitro because functional cholinergic autaptic synapses were established in both experimental conditions. The number of synapses was comparable between the two culture conditions but the frequency of spontaneous miniature excitatory postsynaptic currents was enhanced in those neurons grown in direct contact with glial cells. Autapses displayed facilitation and depression, both processes being determined by the fraction of vesicles from the readily releasable pool discharged by an action potential. At high release probabilities vesicles were more efficiently mobilized, thus promoting depression, whilst low release probabilities made facilitation likely to occur. Schwann cells did not modify significantly facilitation but increased synaptic depression. In single cell microcultures, paired pulse stimuli showed a monoexponential recovery from depression with a time constant of approximately 60 ms, while in microcultures developed together with glial cells, recovery was bi-exponential with a significantly slower time course. Altogether these results show that Schwann cells from sympathetic ganglia directly modulate use-dependent plasticity of nicotinic synapses in vitro by enhancing short-term depression.

Binding of epsilon-toxin from Clostridium perfringens in the nervous system.

Epsilon-toxin (epsilon-toxin), produced by Clostridium perfringens type D, is the main agent responsible for enterotoxaemia in livestock. Neurological disorders are a characteristic of the onset of toxin poisoning. Epsilon-Toxin accumulates specifically in the central nervous system, where it produces a glutamatergic-mediated excitotoxic effect. However, no detailed study of putative binding structures in the nervous tissue has been carried out to date. Here we attempt to identify specific acceptor moieties and cell targets for epsilon-toxin, not only in the mouse nervous system but also in the brains of sheep and cattle. An epsilon-toxin-GFP fusion protein was produced and used to incubate brain sections, which were then analyzed by confocal microscopy. The results clearly show specific binding of epsilon-toxin to myelin structures. epsilon-Prototoxin-GFP and epsilon-toxin-GFP, the inactive and active forms of the toxin, respectively, showed identical results. By means of pronase E treatment, we found that the binding was mainly associated to a protein component of the myelin. Myelinated peripheral nerve fibres were also stained by epsilon-toxin. Moreover, the binding to myelin was not only restricted to rodents, but was also found in humans, sheep and cattle. Curiously, in the brains of both sheep and cattle, the toxin strongly stained the vascular endothelium, a result that may explain the differences in potency and effect between species. Although the binding of epsilon-toxin to myelin does not directly explain its neurotoxic effect, this feature opens up a new line of enquiry into its mechanism of toxicity and establishes the usefulness of this toxin for the study of the mammalian nervous system.

The Cytotoxicity of Epsilon Toxin from Clostridium perfringens on Lymphocytes Is Mediated by MAL Protein Expression.

Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein that crosses the blood-brain barrier, binds to myelin, and, hence, has been suggested to be a putative agent for the onset of multiple sclerosis, a demyelinating neuroinflammatory disease. Recently, myelin and lymphocyte (MAL) protein has been identified to be a key protein in the cytotoxic effect of Etx; however, the association of Etx with the immune system remains a central question. Here, we show that Etx selectively recognizes and kills only human cell lines expressing MAL protein through a direct Etx-MAL protein interaction. Experiments on lymphocytic cell lines revealed that MAL protein-expressing T cells, but not B cells, are sensitive to Etx and reveal that the toxin may be used as a molecular tool to distinguish subpopulations of lymphocytes. The overall results open the door to investigation of the role of Etx and Clostridium perfringens on inflammatory and autoimmune diseases like multiple sclerosis.

Cloning, molecular characterization and expression of ecto-nucleoside triphosphate diphosphohydrolase-1 from Torpedo electric organ.

During synaptic transmission large amounts of ATP are released from pre- and post-synaptic sources of Torpedo electric organ. A chain reaction sequentially hydrolyses ATP to adenosine, which inhibits acetylcholine secretion. The first enzyme implicated in this extracellular ATP hydrolysis is an ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) that dephosphorylates both ATP and ADP to AMP. This enzyme has been biochemically characterized in the synaptosomal fraction of Torpedo electric organ, having almost equal affinity for ATP as for ADP, a fact that pointed to the type-1 NTPDase enzyme. In the present work we describe the cloning and molecular characterization of the cDNA for an NTPDase from Torpedo marmorata electric organ. The clone, obtained using the RACE-PCR technique, contains and open-reading frame of 1506bp and encodes a 502 amino acids protein that exhibits high homology with other NTPDases1 from vertebrates previously identified, including those of zebrafish and Xenopus, as well as human, rat and mouse. Topology analyses revealed the existence of two transmembrane regions, two short cytoplasmic tails and a long extracellular domain containing five apyrase-conserved regions. Gene expression studies revealed that this gene is expressed in all the Torpedo tissues analyzed. Finally, activity and cellular localization of the protein encoded by this newly cloned cDNA was assessed by heterologous expression experiments involving COS-7 and HeLa cells.

Distribution of Clostridium perfringens epsilon toxin in the brains of acutely intoxicated mice and its effect upon glial cells.

Epsilon toxin (epsilon-toxin), produced by Clostridium perfringens types B and D, causes fatal enterotoxaemia in livestock. The disease is principally manifested as severe and often fatal neurological disturbance. Oedema of several organs, including the brain, is also a clinical sign related to microvascular damage. Recombinant epsilon-toxin-green fluorescence protein (epsilon-toxin-GFP) and epsilon-prototoxin-GFP have already been characterised as useful tools to track their distribution in intravenously injected mice, by means of direct fluorescence microscopy detection. The results shown here, using an acutely intoxicated mouse model, strongly suggest that epsilon-toxin-GFP, but not epsilon-prototoxin-GFP, not only causes oedema but is also able to cross the blood-brain barrier and accumulate in brain tissue. In some brain areas, epsilon-toxin-GFP is found bound to glial cells, both astrocytes and microglia. Moreover, cytotoxicity assays, performed with mixed glial primary cultures, demonstrate the cytotoxic effect of epsilon-toxin upon both astrocytes and microglial cells.