Superfluid Fraction in an Interacting Spatially Modulated Bose-Einstein Condensate.

At zero temperature, a Galilean-invariant Bose fluid is expected to be fully superfluid. Here we investigate theoretically and experimentally the quenching of the superfluid density of a dilute Bose-Einstein condensate due to the breaking of translational (and thus Galilean) invariance by an external 1D periodic potential. Both Leggett's bound fixed by the knowledge of the total density and the anisotropy of the sound velocity provide a consistent determination of the superfluid fraction. The use of a large-period lattice emphasizes the important role of two-body interactions on superfluidity.

A novel gain-of-function mutation in ORAI1 causes late-onset tubular aggregate myopathy and congenital miosis.

We present three members of an Italian family affected by tubular aggregate myopathy (TAM) and congenital miosis harboring a novel missense mutation in ORAI1. All patients had a mild, late onset TAM revealed by asymptomatic creatine kinase (CK) elevation and congenital miosis consistent with a Stormorken-like Syndrome, in the absence of thrombocytopathy. Muscle biopsies showed classical histological findings but ultrastructural analysis revealed atypical tubular aggregates (TAs). The whole body muscle magnetic resonance imaging (MRI) showed a similar pattern of muscle involvement that correlated with clinical severity. The lower limbs were more severely affected than the scapular girdle, and thighs were more affected than legs. Molecular analysis revealed a novel c.290C>G (p.S97C) mutation in ORAI1 in all affected patients. Functional assays in both human embryonic kidney (HEK) cells and myotubes showed an increased rate of Ca2+ entry due to a constitutive activation of the CRAC channel, consistent with a 'gain-of-function' mutation. In conclusion, we describe an Italian family harboring a novel heterozygous c.290C>G (p.S97C) mutation in ORAI1 causing a mild- and late-onset TAM and congenital miosis via constitutive activation of the CRAC channel. Our findings extend the clinical and genetic spectrum of the ORAI1-related TAM.

New morphologic and genetic findings in cap disease associated with beta-tropomyosin (TPM2) mutations.

OBJECTIVE: Mutations in the beta-tropomyosin gene (TPM2) are a rare cause of congenital myopathies with features of nemaline myopathy and cap disease and may also cause distal arthrogryposis syndromes without major muscle pathology. We describe the muscle biopsy findings in three patients with cap disease and novel heterozygous mutations in TPM2. METHODS: Three unrelated patients with congenital myopathy were investigated by muscle biopsy and genetic analysis. RESULTS: All three patients had early-onset muscle weakness of variable severity and distribution. Muscle biopsy demonstrated in all three patients near uniformity of type 1 fibers and an unusual irregular and coarse-meshed intermyofibrillar network. By electron microscopy, the myofibrils were broad and partly split, and the Z lines appeared jagged. In one of the patients caps structures were identified only by electron microscopy, and in one patient they were identified only in a second biopsy at adulthood. Three novel, de novo, heterozygous mutations in TPM2 were identified: a three-base pair deletion in-frame (p.Lys49del), a three-base pair duplication in-frame (p.Gly52dup), and a missense mutation (p.Asn202Lys). CONCLUSIONS: Mutations in TPM2 seem to be a frequent cause of cap disease. Because cap structures may be sparse, other prominent features, such as a coarse-meshed intermyofibrillar network and jagged Z lines, may be clues to correct diagnosis and also indicate that the pathogenesis involves defective assembly of myofilaments.

Study on the pathophysiology of experimental Burkholderia pseudomallei infection in mice.

Burkholderia pseudomallei is the etiological agent of melioidosis, a potentially fatal disease occurring in man and animals. The aim of this study was to investigate the pathophysiological course of experimental melioidosis, and to identify the target organs, in an animal model. For this purpose SWISS mice were infected intraperitoneally with the virulent strain B. pseudomallei 6068. The bacterial load of various organs was quantified daily by bacteriological analysis and by an enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody specific to B. pseudomallei exopolysaccharide (EPS). Electron microscopic investigation of the spleen was performed to locate the bacteria at the cellular level. In this model of acute melioidosis, B. pseudomallei had a marked organ tropism for liver and spleen, and showed evidence of in vivo growth with a bacterial burden of 1.6x10(9) colony forming units (CFU) per gram of spleen 5 days after infection with 200 CFU. The highest bacterial loads were detected in the spleen at all time points, in a range from 2x10(6) to 2x10(9) CFU g(-1). They were still 50-80 times greater than the load of the liver at the time of peak burden. Other investigated organs such as lungs, kidneys, and bone marrow were 10(2)-10(4)-fold less infected than the spleen, with loads ranging from 3x10(2) to 3x10(6) CFU g(-1). The heart and the brain were sites of a delayed infection, with counts in a range from 10(3) to 10(7) times lower than bacterial counts in the spleen. The EPS-specific ELISA proved to be highly sensitive, particularly at the level of those tissues in which colony counting on agar revealed low contamination. In the blood, EPS was detected at concentrations corresponding to bacterial loads ranging from 8x10(3) to 6x10(4) CFU ml(-1). Electron microscopic examination of the spleen revealed figures of phagocytosis, and the presence of large numbers of intact bacteria, which occurred either as single cells or densely packed into vacuoles. Sparse figures suggesting bacterial replication were also observed. In addition, some bacteria could be seen in vacuoles that seemed to have lost their membrane. These observations provide a basis for further investigations on the pathogenesis of the disease.

Pseudomonas aeruginosa cystic fibrosis isolates induce rapid, type III secretion-dependent, but ExoU-independent, oncosis of macrophages and polymorphonuclear neutrophils.

Pseudomonas aeruginosa, an opportunistic pathogen responsible most notably for severe infections in cystic fibrosis (CF) patients, utilizes the type III secretion system for eukaryotic cell intoxication. The CF clinical isolate CHA shows toxicity towards human polymorphonuclear neutrophils (PMNs) which is dependent on the type III secretion system but independent of the cytotoxin ExoU. In the present study, the cytotoxicity of this strain toward human and murine macrophages was demonstrated. In low-multiplicity infections (multiplicity of infection, 10), approximately 40% of the cells die within 60 min. Analysis of CHA-infected cells by transmission electron microscopy, DNA fragmentation assay, and Hoechst staining revealed the hallmarks of oncosis: cellular and nuclear swelling, disintegration of the plasma membrane, and absence of DNA fragmentation. A panel of 29 P. aeruginosa CF isolates was screened for type III system genotype, protein secretion profile, and cytotoxicity toward PMNs and macrophages. This study showed that six CF isolates were able to induce rapid ExoU-independent oncosis on phagocyte cells.

Intravenous immunoglobulin therapy: confounding effects on serological screening for toxoplasmosis during pregnancy.

The serological diagnosis of toxoplasmic infection during pregnancy is intended to prevent congenital infection of the fetus. However, in the context of recurrent pregnancy loss intravenous immunoglobulin therapy can create a biological trap for the interpretation of serological results, with potentially serious consequences for the outcome of the pregnancy.

Efficacy of atropine/pralidoxime/diazepam or atropine/HI-6/prodiazepam in primates intoxicated by soman.

We performed an experiment to characterize the toxicity of soman in cynomolgus monkeys when the organophosphorus intoxication was followed by a treatment with either the three-drug therapy atropine/pralidoxime/diazepam or the association atropine/HI-6/prodiazepam. Clinical, electrophysiological and histological approaches were combined. Our data demonstrate that the protection afforded against soman toxicity was better with the combination atropine/HI-6/prodiazepam compared to atropine/pralidoxime/diazepam. This was observed transiently in term of vigilance and respiratory function of intoxicated animals, but particularly in term of their EEG- and ECG disturbances. Moreover, compared to those treated with atropine/pralidoxine/diazepam, animals treated with atropine/ HI-6/prodiazepam recovered slightly sooner and did not exhibit prostration 2 days after intoxication although their rapidity of movements was not totally restored. The final recovery observed 3 weeks after intoxication was similar for the two groups. The value of the combination of atropine/HI-6/prodiazepam vs atropine/pralidoxime/diazepam to counteract soman toxicity was also confirmed in term of brain neuroprotection since greater lesions were observed with the second three drug treatment three weeks after intoxication.

GK 11: promising additional neuroprotective therapy for organophosphate poisoning.

Recent experiments with primates have demonstrated that treatment with atropine/pralidoxime/diazepam, even if administered immediately after organophosphate exposure, does not totally prevent neuronal brain damage. Using primates, we have studied, for the first time, the ability of GK-11 (gacyclidine), an antiglutamatergic drug in the process of agreement for human use, given as an additional therapy, to counteract the neuropathology due to organophosphate exposure that persists after classical treatment with oxime/atropine/benzodiazepine. We have also examined the recovery of the organophosphate-intoxicated primates. Male Cynomolgus monkeys were pretreated 1 hour before poisoning with pyridostigmine, then intoxicated with 8 LD50 of soman and immediately treated with the combination pralidoxime/atropine/diazepam. Some of the animals also received GK-11 at 0.01; 0.03 or 0.1 mg/kg (i.v.) 10 minutes after soman challenge. Recovery of the primates (reflexes, movements, feeding) and the neuropathological changes that occurred three weeks after intoxication (histological examinations and neuronal cell density measurement) were compared in GK-11-treated and control animals. At all doses tested, GK-11 prevented the neuronal rarefaction of the frontoparietal cortex that was observed in soman-intoxicated animals that received only oxime/atropine/diazepam. Moreover, the 0.01 mg/kg dose of GK-11 improved the early recovery of intoxicated primates from 1 day after intoxication. In the view of the most effective management of organophosphate intoxication that is currently available, GK-11 thus appears to be a promising additional neuroprotective therapy. This drug is presently being evaluated in a human clinical trial for a different neuroprotective indication.

Early regional changes of GFAP mRNA in rat hippocampus and dentate gyrus during soman-induced seizures.

We investigated the time course of GFAP levels in the hippocampal formation during the first 24 h following soman intoxication in rats. GFAP mRNA expression was detected by in situ hybridization. Intense GFAP mRNA expression was present in the molecular layer of the dentate gyrus as early as 6 h after intoxication. This expression was comparatively lower in other dentate gyrus layers and hippocampal CA1, CA3 and CA4 areas and seemed to be related to excessive neuronal activity. Histopathological examination demonstrated that GFAP expression in dentate gyrus is not correlated with lesions. The high astrocytic reactivity in the molecular layer of the dentate gyrus is discussed in relation to the maintenance of the homeostasis of glutamate and of synaptic plasticity in this area during soman intoxication.

Demonstration of peptidergic afferents to the bed nucleus of the stria terminalis using local injections of colchicine. A combined immunohistochemical and retrograde tracing study.

In the present study, we demonstrate the existence of numerous peptidergic afferents to the bed nucleus of the stria terminalis (BNST) using the retrograde transport of gold-labeled wheat germ agglutinin-apo-peroxidase (G-WGA-HRP) combined with the indirect immunoperoxidase method after intraparenchymatous injections of colchicine. At first, we show that local injections of colchicine alone into the BNST are able to induce the retrograde accumulation of peptides until the nerve cell bodies of origin, probably because of the blockade of axonal transport in nerve terminal arborizations innervating this nucleus. The actual existence of putative peptidergic afferents to the BNST indicated by the local injections of colchicine was established using: a) the retrograde transport of G-WGA-HRP from the BNST combined with immunocytochemistry after administration of colchicine at the same place, b) the anterograde "transport" of the fluorescent tracer DiI from selected nuclei of the forebrain. We demonstrate that the neurons immunoreactive for enkephalins, neurotensin, or substance P that innervate the BNST are localized mainly in the central amygdaloid nucleus, the paraventricular thalamic nucleus, and the ventromedial hypothalamic nucleus ipsilateral to the injection, as well as bilaterally in the magnocellular paraventricular and perifornical regions of the hypothalamus. From these results it may be concluded that intracerebral injections of colchicine constitute a powerful tool to search for multiple peptidergic afferents to a given brain nucleus using only immunohistochemistry. The existence of these pathways, however, must be verified by other neuroanatomical methods because of the problem of nerve fibers of passage.

The same 15 kDa proteolipid subunit is a constituent of two different proteins in Torpedo, the acetylcholine releasing protein mediatophore and the vacuolar H+ ATPase.

Using the monoclonal antibody 15K1, we have studied, at the cellular and subcellular levels, the distribution of a 15 kDa proteolipid, identified as the subunit of mediatophore, a presynaptic membrane protein able to release acetylcholine when activated by calcium. Aside from the electric lobe, the antigen distribution in the brain of Torpedo paralleled that of the synaptic vesicle antigen SV2 and did not appear to be related to that of acetylcholine and choline acetyltransferase. The 15 kDa proteolipid antigen was therefore present in all nerve endings and not restricted to cholinergic ones. At the ultrastructural level, on cholinergic nerve endings, the antigen was detected associated to synaptic vesicles and, to a lesser extent, to the presynaptic plasma membrane. Indeed, considering the high sequence homology between the mediatophore subunit (Birman et al., 1990) and the proteolipid subunit of the vacuolar type H+ ATPase, a major enzyme constituent of synaptic vesicles, this distribution was not surprising. To determine whether antibody 15K1 recognizes the vacuolar type H+ ATPase, we chose a non neuronal cell type which possesses a high content of this enzyme, the kidney proton secreting epithelial cells. Indeed, antibody 15K1 intensely labelled the apical plasma membrane of mitochondria rich epithelial cells in kidney tubules. A high density of the antigen was also found associated to intracellular membrane structures such as lysosomal multivesicular bodies, both in kidney epithelial cells and in electromotoneurons. The 15 kDa proteolipid antigen was associated with other vacuolar H+ ATPase subunits in kidney membranes which was not the case in presynaptic plasma membranes. This illustrates that the 15 kDa proteolipid antigen is a constituent of two different protein complexes, which exhibit very different functional properties.

Immunolabelling of the presynaptic membrane of Torpedo electric organ nerve terminals with an antiserum towards the acetylcholine releasing protein mediatophore.

Mediatophore is a nerve terminal membrane protein purified from Torpedo electric organ on its ability to translocate acetylcholine upon calcium action. An antiserum able to immunoprecipitate mediatophore activity was used to study the subcellular distribution of this protein. The presynaptic membrane exhibited a strong and discontinuous immunogold labelling, especially at the active zone where ACh is thought to be released. Two antigens were recognized on immunoblots of synaptosomal membranes: the 15-kDa subunit of mediatophore and a 14-kDa membrane protein that has a wide non-neuronal distribution. Antibodies purified from the serum on native mediatophore and monospecific towards the 15-kDa antigen still gave a high presynaptic membrane localized labelling. In addition, a few 14-kDa protein sites were present at the active zone. The Schwann cell finger interposed between the presynaptic membrane and the postsynaptic arch also exhibited the 14-kDa antigen raising the question of a possible interaction of mediatophore with the 14-kDa protein originating from the Schwann cell.

Calcium-induced acetylcholine release and intramembrane particle occurrence in proteoliposomes equipped with mediatophore.

Proteoliposomes obtained from the mediatophore, a purified Torpedo electric organ nerve terminals protein, and endogenous lipids were used for a study of calcium-induced release of acetylcholine and freeze-fracture electron microscopy. Large intramembrane particles were induced by the influx of calcium into proteoliposomes, as previously observed for synaptosomes or stimulated electric organ nerve terminals. The involvement of mediatophore in a calcium dependent acetylcholine translocation seems therefore to be related to the occurrence of a category of intramembrane particles in the course of the release process.

Immunological identification of a new 14X10(3) Mr membrane-bound protein in Torpedo electric organ.

A series of monoclonal antibodies binding to different epitopes shared by a 14 x 10(3)Mr membrane-bound polypeptide has been obtained. By indirect immuno-fluorescence, it was shown that the 14 x 10(3)Mr antigen is present in various cell types in Torpedo electric organ and muscle, especially fibroblasts, capillary endothelial cells, axonal cuff cells and, to a lesser extent, Schwann cells. At the electron-microscope level, after immunogold labelling, the antigen was found associated with the external surface of the plasma membrane of these cells, with the exception of the axonal cuff cells where part of the labelling was intracellular. The possible biological role of this 14 x 10(3)Mr protein is unknown but preliminary experiments suggest that this antigen has affinity for other Torpedo electric organ membrane proteins.

[Comparison of synaptic vesicles of neuromuscular and nerve electroplaque junctions in Torpedo marmorata]. / Comparaison des vésicules synaptiqus des jonctions nerf-muscle et nerf-électroplaque chez Torpedo marmorata.

A morphological comparison of neuromuscular and nerve-electroplaque synapses of torpedo was performed. Synaptic vesicles are much smaller at the neuromuscular synapse. The question of the respective role of these populations is raised.

The action of notexin from tiger snake venom (Notechis scutatus scutatus) on acetylcholine release and compartmentation in synaptosomes from electric organ of Torpedo marmorata.

At rest, in the presence of calcium, notexin induced a rapid and concentration-dependent leakage of acetylcholine from nerve endings. In the presence of 20 nM notexin (5 min), synaptosomes were well-preserved structurally and they responded to addition of A23187 ionophore by a normal calcium-dependent acetylcholine release. When stimulated by high-K+ depolarization, evoked acetylcholine release was increased when notexin was present. These findings demonstrate that notexin (up to 100 nM) does not inhibit the acetylcholine release process itself. Further studies on intracellular acetylcholine compartmentation showed that, in the presence of calcium, nm concentrations of notexin were able to mobilize vesicular acetylcholine, the amount of which strongly decreased and fed the cytoplasmic compartment leading to an important redistribution of the neurotransmitter. Other metabolic studies under notexin confirmed the inhibition of the synaptosomal membrane choline transport, but failed to elicit changes in the choline acetyltransferase activity. In order to distinguish between the phospholipase A2 activity of notexin and its neurotoxic effects, we compared effects of notexin to those obtained with a non-neurotoxic pancreatic phospholipase A2. The latter exhibits similar effects but at a higher range of concentration than notexin.

A potential new procedure for removing anti-factor VIII antibodies from hemophilic plasma.

A new approach for removing the anti-factor VIII antibodies in hemophilic patients by immunoadsorption is proposed. The method is based on the fact that the anti-factor VIII antibodies were predominantly of the IgG4 subclass; anti-human IgG4 antibodies were covalently linked to agarose and large amounts of anti-factor VIII antibodies can be eliminated. A study of 21 blood samples from hemophilic patients with anti-factor VIII antibodies allows us to confirm the large predominance of IgG4 in the anti-factor VIII population. In some samples, the presence of IgG3 related anti-VIII:C was checked by adsorption on an anti-IgG3 column. In a majority of cases, after IgG4 (or IgG4 + IgG3) immunoadsorption, the substitution therapy becomes possible or easier.

Hemophilic patients with an inhibitor to factor VIII treated with high dose factor VIII concentrate. Results of a collaborative study for the evaluation of factor VIII inhibitor titer, recovery and half life of infused factor VIII.

The long term treatment of hemophilic patients with an inhibitor to Factor VIII has been difficult although some success with immunosuppressive agents has been reported. Eighteen hemophilic patients, mainly from Bonn in Germany, but also from other countries, have completed a high dose Factor VIII treatment in an attempt to reduce their inhibitor titer and induce "immune tolerance" to Factor VIII. Plasma samples from the 18 patients collected before and after infusion of 50 units Factor VIII/kg body weight were sent to five laboratories to evaluate inhibitor titer, Factor VIII recovery and half life. This collaborative study demonstrated a close correlation from one laboratory to another concerning inhibitor titration and Factor VIII recovery. The conclusion of the study is that all patients treated with this protocol showed an undetectable or low inhibitor titer against Factor VIII indicating that they can be efficiently treated with Factor VIII.

[Immunological study of a hemophiliac treated according to the Bonn protocol for 2 1/2 years]. / Etude immunologique d'un hémophile traité selon le protocole de Bonn pendant deux ans et demi.

A major haemophiliac A, 27 years old, has been treated during 30 months, with high dosage of imported Factor VIII, in order to reduce the titer of a F VIII antibody. A good clinical result has been obtained. No sign of immunodeficiency has been observed. Normal values were obtained for T4/T8, ratio B2 microglobulin and no antibody was detected against the LAV virus isolated from cases of AIDS.