Written production in a case of locked-in syndrome with bilateral corticopontic degeneration.

Patients in locked-in syndrome show normal or near normal mental abilities that contrast with the limited motor capacity that hinders voluntary communication. However, eye movements and blinking are usually preserved and can be used to establish a communication system. We report an exceptional case of locked-in syndrome. The aetiology was basilar thrombosis consecutive to a cervical manipulation. In addition, brain MRI performed 23 years later showed a ventral pontine stroke with bilateral corticopontic degeneration. In this study the patient was able to produce written output using a chin-controlled Morse system decoded by a computer. A detailed linguistic analysis of text written over 20 years by the patient was carried out. The data demonstrate that improvements in language performance can be observed even in patients with brain lesions in areas associated with high-level cognitive processes. The data show a decrease of typing, grammatical and lexical errors over time, use of less frequent words, and an increase of more complex linguistic structures. This paper adds to previous findings confirming the value of daily practice and rehabilitation to enhance quality of life in this group of patients.

Surface properties and behaviour on abiotic surfaces of Staphylococcus carnosus, a genetically homogeneous species.

This work aimed to characterize the surface properties of Staphylococcus carnosus and the influence of different media on their ability to adhere and grow on industrial supports. As their colonization could be dependant of the strain, the genetic diversity of the strains was studied. The diversity of 13 strains analysed by pulsed-field gel electrophoresis revealed that the S. carnosus strains formed a homogeneous genetic group. Their surface properties, characterized by studying their affinity to solvents, were hydrophilic with a strong negative surface charge. The S. carnosus strain CIT 833 hardly adhered to polytetrafluoroethylene (PTFE) and stainless steel chips. Tryptic soy broth (TSB) was the most favourable medium for growth on stainless steel support while TSB/NaCl was better for growth on PTFE. Scanning electron microscopy (sem) showed that this strain weakly colonized both supports and did not form cell aggregates. Indeed, the strain did not synthesize polysaccharides. These results showed that S. carnosus adhered on different abiotic surfaces which are used in food factories but was not able to accumulate on these surfaces. The inability of S. carnosus to form biofilm could explain why S. carnosus is rarely isolated in meat processing environment.

Formation of biofilm by Staphylococcus xylosus.

The ability of 12 Staphylococcus xylosus strains to form biofilm was determined through the study of different criteria. Eleven out of the 12 strains were able to form biofilm, 10 preferentially on hydrophilic support (glass) and one, S. xylosus C2a, on both hydrophilic and hydrophobic (polystyrene) supports. The determination of bacterial surface properties showed that all strains were negatively charged with five strains moderately hydrophobic and seven hydrophilic. The bap and icaA genes, important for biofilm formation of some staphylococci, were searched. All strains were bap positive but icaA negative. Furthermore, S. xylosus strain C2a was studied on two supports widely used in the food industry, polytetrafluoroethylene (PTFE, hydrophobic) and stainless steel (hydrophilic) and appeared to adhere preferentially on stainless steel. Addition of 20 g/l of NaCl to Tryptic Soy Broth medium (TSB) did not improve significantly its adhesion but enhanced both bacterial growth and cell survival, which were optimum in this medium. Environmental scanning electron microscopy showed that S. xylosus C2a colonized the surface of stainless steel chips with intercellular spaces. The strain formed cell aggregates embedded in an amorphous polysaccharidic matrix. Indeed, synthesis of polysaccharides increased during growth on stainless steel chips in TSB.

Comparison of protein patterns of Listeria monocytogenes grown in biofilm or in planktonic mode by proteomic analysis.

The proteome of a Listeria monocytogenes strain isolated from a food plant was investigated to study the differential protein pattern expressed by biofilms and planktonic bacteria. The approach used in this study was a combination of two-dimensional electrophoresis, matrix-assisted laser desorption ionization-time of flight and database searches for the protein identification. Thirty-one proteins varied significantly between the two growth conditions. Twenty-two and nine proteins were up- and down-regulated respectively and nine proteins were successfully identified. The variations of the protein patterns indicated that the biofilm development is probably controlled by specific regulation of protein expression involved at various levels of cellular physiology.

Characterization of a spontaneous adhesion-defective mutant of Ruminococcus albus strain 20.

A spontaneous adhesion-defective mutant (mutant D5) of Ruminococcus albus strain 20 was isolated and compared to the parent to investigate the impact of the mutation on cellulolysis and to identify the adhesion mechanism of R. albus. The comparison of kinetics of cellulose degradation by strain 20 and mutant D5 showed that the mutation delayed and reduced bacterial growth on cellulose and cellulose degradation. These results were partly explained by a twofold lower cellulase activity in the mutant than in the parent. The glycocalyx of strain 20, observed by transmission electron microscopy, was large and homogeneous, and linked cells to cellulose. The mutant glycocalyx was aggregated at its periphery and cells attached loosely to cellulose. A glycoprotein of 25 kDa (GP25), present in the membrane fraction and the extracellular medium of strain 20, was not detected in the same fractions of mutant D5. Though glycoprotein GP25 did not bind to cellulose, it may be involved in adhesion as an intermediate component. Different cell-surface features of mutant D5 (cellulases, glycoprotein GP25, glycocalyx) were thus affected, any or all of which may be involved in its adhesion-defective phenotype. These results suggest that adhesion and cellulolysis are linked and that adhesion is a multifactorial phenomenon that involves at least the extracellular glycocalyx.

Full capacity of recombinant Escherichia coli heat-stable enterotoxin fusion proteins for extracellular secretion, antigenicity, disulfide bond formation, and activity.

We have successfully used the major subunit ClpG of Escherichia coli CS31A fimbriae as an antigenic and immunogenic exposure-delivery vector for various heterologous peptides varying in nature and length. However, the ability of ClpG as a carrier to maintain in vitro and in vivo the native biological properties of passenger peptide has not yet been reported. To address this possibility, we genetically fused peptides containing all or part of the E. coli human heat-stable enterotoxin (STh) sequence to the amino or carboxyl ends of ClpG. Using antibodies to the ClpG and STh portions for detecting the hybrids; AMS (4-acetamido-4'-maleimidylstilbene-2, 2'-disulfonate), a potent free thiol-trapping reagent, for determining the redox state of STh in the fusion; and the suckling mouse assay for enterotoxicity, we demonstrated that all ClpG-STh proteins were secreted in vitro and in vivo outside the E. coli cells in a heat-stable active oxidized (disulfide-bonded) form. Indeed, in contrast to many earlier studies, blocking the natural NH(2) or COOH extremities of STh had, in all cases, no drastic effect on cell release and toxin activity. Only antigenicity of STh C-terminally extended with ClpG was strongly affected in a conformation-dependent manner. These results suggest that the STh activity was not altered by the chimeric structure, and therefore that, like the natural toxin, STh in the fusion had a spatial structure flexible enough to be compatible with secretion and enterotoxicity (folding and STh receptor recognition). Our study also indicates that disulfide bonds were essential for enterotoxicity but not for release, that spontaneous oxidation by molecular oxygen occurred in vitro in the medium, and that the E. coli cell-bound toxin activity in vivo resulted from an effective export processing of hybrids and not cell lysis. None of the ClpG-STh subunits formed hybrid CS31A-STh fimbriae at the cell surface of E. coli, and a strong decrease in the toxin activity was observed in the absence of CS31A helper proteins. In fact, chimeras translocated across the outer membrane as a free folded monomer once they were guided into the periplasm by the ClpG leader peptide through the CS31A-dependent secretory pathway. In summary, ClpG appears highly attractive as a carrier reporter protein for basic and applied research through the engineering of E. coli for culture supernatant delivery of an active cysteine-containing protein, such as the heat-stable enterotoxin.

Location by fluorescence microscopy of glycosidases and a xylanase in the anaerobic gut fungi Caecomyces communis, Neocallimastix frontalis, and Piromyces rhizinflata.

beta-D-Glucosidase, beta-D-fucosidase, beta-D-xylosidase, and beta-D-cellobiopyranosidase activities in Caecomyces communis, Neocallimastix frontalis, and Piromyces rhizinflata, located with fluorescent conjugates, occur throughout the whole thallus as from zoospore germination and disappear before sporulation. beta-D-Galactosidase and alpha-L-arabinopyranosidase activities are low or nonexistent. A xylanase, detected by indirect immunofluorescence, was observed at the surface of the vegetative cells, vesicles, or rhizoids. Cross-reactions prove the existence of analogies in structure among the enzymes of these anaerobic gut fungi.

Permissible peptide insertions surrounding the signal peptide-mature protein junction of the ClpG prepilin: CS31A fimbriae of Escherichia coli as carriers of foreign sequences.

The clpG gene, expressing the Escherichia coli major CS31A fimbrial subunit ClpG, was subjected to random mutagenesis by insertion of an EcoRI linker and a kanamycin-resistance (KmR) cassette into the multiple newly generated EcoRI sites. The KmR gene was then excised by PstI, which left a 48-bp linker representing the heterologous sequence. The same procedure was followed to introduce a synthetic oligodeoxyribonucleotide (oligo) corresponding to epitope C from the spike protein S from the porcine transmissible gastroenteritis coronavirus (TGEV). Nine insertion/deletion mutants (indels) that contained long foreign peptides variously located around the ClpG signal peptide (SP) processing site were characterized. A striking feature of this study is the variety of amino acid (aa) insertions in the ClpG prepilin that have little or no effect on CS31A fimbria biogenesis. These 'permissive' sites tolerate inserts of 18 or 19 aa and accept sequences of different natures in view of their aa composition, charge and hydrophobicity. The results obtained here are also interesting in light of the high level of aa sequence conservation seen in the SP and N-terminal domains of the ClpG-related subunits. The structure-function relationship of the ClpG SP is discussed. The TGEV-C epitope fused to the N-terminal end of the mature ClpG protein was cell-surface exposed, as observed on immuno-electron microscopy. Therefore, the CS31A fimbria seems to be a potent tool for the presentation of foreign antigenic determinants or the production of heterologous polypeptides in E. coli.

Scanning and transmission electron microscopic study of adherence of Escherichia coli O103 enteropathogenic and/or enterohemorrhagic strain GV in enteric infection in rabbits.

The GV strain (serotype O103:H2:K-), originally isolated from a diarrheic rabbit, is an enteropathogenic Escherichia coli strain that produces diarrhea without synthesizing the classical enterotoxins and that is not invasive. This strain is characterized by a 117-kb plasmid (pREC-1). Histological study of the gut by scanning electron microscopy and transmission electron microscopy was performed on the GV strain, on a derivative strain cured of pREC-1, and on transconjugants obtained by transfer of pREC-1 to nonpathogenic strains E. coli K-12 and 6100, not belonging to the O103 serogroup. The GV strain adhered to the epithelial cells of the ileum and large intestine, whereas the cured GV strain did not. Transfer of plasmid pREC-1 to E. coli K-12 or 6100 allowed the bacteria to attach to the intestinal mucosa in the same manner as that of the wild-type GV strain. Thus, pREC-1 seems to play an important role in attachment to and colonization of the intestinal tract of rabbits by E. coli serogroup O103. Scanning electron microscopy showed numerous bacteria attached together and closely associated with intestinal villi. Transmission electron microscopy revealed effacing lesions characteristic of enteropathogenic E. coli strains: effacing of microvilli and cuplike projections (pedestal formations) associated with an acute inflammatory and hemorrhagic response. In contrast with the results reported for rabbit pathogenic O15 strains, it appeared that the Peyer's patches were not involved in the early stages of infection with the O103 GV strain. This strain may represent a model for the study of the virulence and pathogenic effects of enteropathogenic and enterohemorrhagic E. coli strains.

Piromyces rhizinflata nov. sp., a strictly anaerobic fungus from faeces of the Saharian ass: a morphological, metabolic and ultrastructural study.

A new species of strictly anaerobic chytridiomycete was isolated from dried faeces of the Saharian ass that had been stored for up to 150 days. Because of its monocentric thallus and uniflagellate zoospores it belongs to the genus Piromyces. It exhibits a high affinity for P. mae and P. dumbonica but differs from them in its morphological and ultrastructural characteristics. Its flagellar apparatus is similar to that of all previously reported fungi.

Anaeromyces mucronatus nov. gen., nov. sp. A new strictly anaerobic rumen fungus with polycentric thallus.

A new species of strictly anaerobic fungus was isolated from the cow rumen. It is characterized by a polycentric thallus, a polynuclear rhizomycelium, mucronate zoosporangia and uniflagellated zoospores. It is also singular in that the sporocysts do not react to the specific lectins of L-fucose, N-acetyl-D-galactosamine and diacetyl chitobiose. These characteristics justify the creation of a new genus.

Use of lectins for a comparative study of cell wall composition of different anaerobic rumen fungal strains.

The technique based on fluorescein-linked lectins used to determine the cell wall structure of anaerobic rumen fungi belonging to genera: Neocallimastix, Piromonas and Sphaeromonas, appears to be an interesting tool for distinguishing between strains. Furthermore this technique shows differences of cell wall composition between different parts of the thallus (spores, sporangia, rhizoïds).