Loss of bone mineral density in hemiplegic patients after stroke: Prospective single-center study.

The aim of our prospective single-center study was to assess the loss of bone mineral density (BMD) in the year following stroke with hemiplegia. We evaluated functional disability and bone mineral density (BMD) in 18 patients immediately following and at 1 year of stroke with hemiplegia. The most significant variation in BMD concerns the femoral neck on the hemiplegic side: -0.0551±0.0548g/cm2: -6.85%. This loss is correlated with the functional motor score (Fugl-Meyer Assessment) (r=0.7573; P=0.0004). During follow-up, 11/18 patients fell and 2/18 had a fracture.

[Castleman-like presentation of systemic lupus: About a case report]. / Présentation « Castleman-like ¼ de lupus systémique : à propos d'un cas.

INTRODUCTION: Castleman disease is a rare lymphoproliferation, which may mimic systemic lupus. Conversely, systemic lupus sometimes presents like an hematological malignancy. In these cases, a "Castleman-like" histology has been exceptionally described. OBSERVATION: A 55-year-old female treated by methotrexate for systemic lupus with skin and joint involvement presented weight loss, polyadenopathy and clinical signs of lupus flare. Biology showed pancytopenia, complement activation, and positive anti-DNA antibodies. PET/CT showed hypermetabolic polyadenopathy. The lymph node biopsy showed "Castleman-like" features. Treatment with corticosteroids and azathioprine resulted in complete remission. CONCLUSION: Systemic lupus and Castleman disease may share common clinical, biological, and histological features. The presence of specific elements of systemic lupus flare and the remission obtained by low-dose corticosteroids results in considering the diagnosis of Castleman-like systemic lupus and avoiding treatment intensification.

A culture filtrate of Phytophthora infestans primes defense reaction in potato cell suspensions.

Priming of defense reactions by an elicitor results in an enhanced ability of the plant to respond to subsequent pathogen challenges. We previously showed that application of lipopolysaccharides (LPS) to potato cell suspensions causes apoplastic acidification, but does not stimulate lipoxygenase (LOX) activity. Here, we tested the ability of various elicitors to prime and elicit defense reactions in potato cell suspensions. Adding 20 microg ml(1) LPS, laminarin, harpin N, or a concentrated culture filtrate (CCF) of Phytophthora infestans to cell cultures 18 h before a second elicitation with LPS did not alter the intensity of apoplastic acidification compared with a single LPS application. Conversely, high concentrations (200 or 400 microg ml(1)) of LPS, laminarin, and harpin N activated LOX in cells pretreated with 1 microg ml(1) CCF, but not in cells pretreated with LPS, laminarin, or harpin N. LOX response was maximal in pretreated cells of potato cv. Bintje when the second elicitation occurred 18 to 24 h after CCF application. These results showed that LOX activation is primed in potato cells by CCF, but not by LPS, harpin N, or laminarin. Finally, bioassays showed a slightly greater reduction of rot weight in half tubers treated with CCF followed by LPS before inoculation with Pectobacterium atrosepticum than in half tubers treated with either preparation alone, indicating a priming effect of CCF on both LOX induction and disease suppression.

Sulfated Oligosaccharides Mediate the Interaction between a Marine Red Alga and Its Green Algal Pathogenic Endophyte.

The endophytic green alga Acrochaete operculata completely colonizes the sporophytes of the red alga Chondrus crispus; however, it does not penetrate beyond the outer cell layers of the gametophytes. Given that the life cycle phases of C. crispus differ in the sulfation pattern of their extracellular matrix carrageenans, we investigated whether carra-geenan fragments could modulate parasite virulence. lambda-Carrageenan oligosaccharides induced release of H(2)O(2), stimulated protein synthesis, increased carrageenolytic activity, and induced specific polypeptides in the pathogen, resulting in a marked increase in pathogenicity. In contrast, kappa-carrageenan oligosaccharides did not induce a marked release of H(2)O(2) from A. operculata but hindered amino acid uptake and enhanced their recognition by the host, resulting in a reduced virulence. Moreover, C. crispus life cycle phases were shown to behave differently in their response to challenge with cell-free extracts of A. operculata. Gametophytes exhibited a large burst of H(2)O(2), whereas only low levels were released from the sporophytes.

Lipopolysaccharides of Pectobacterium atrosepticum and Pseudomonas corrugata induce different defence response patterns in tobacco, tomato, and potato.

Lipopolysaccharides (LPS), ubiquitous cell surface components of Gram-negative bacteria, are directly implicated in plant/pathogen interactions. However, their perception by the plant, the subsequent signal transduction in both compatible and incompatible interactions, as well as the defence reactions induced in compatible interactions are as yet poorly understood. We focused on biochemical and physiological reactions induced in cell suspensions of three Solanaceae species (tobacco, tomato, and potato) by purified lipopolysaccharides from PECTOBACTERIUM ATROSEPTICUM (PA), a pathogen of potato, and PSEUDOMONAS CORRUGATA (PSC), a pathogen of tomato. LPS PA and LPS PSC caused a significant acidification of potato, tomato, and tobacco extracellular media, whereas laminarin (a linear beta-1,3 oligosaccharide elicitor) induced an alkalinisation in tobacco and tomato, but not in potato cell suspensions. None of the two LPS induced the formation of active oxygen species in any of the hosts, while laminarin induced H (2)O (2) production in cells of tobacco but not of tomato and potato. In tomato cells, LPS PA and LPS PSC induced a strong but transitory stimulation of lipoxygenase activity, whereas laminarin induced a stable or slightly increasing LOX activity over the first 24 h of contact. In tobacco, LOX activity was not triggered by either LPS, but significantly increased following treatment with laminarin. In potato, neither LPS nor laminarin induced LOX activity, in contrast with concentrated culture filtrate of PHYTOPHTHORA INFESTANS (CCF). These results demonstrate that LPS, as well as laminarin, are perceived in different ways by SOLANACEAE species, and possibly cultivars. They also suggest that defence responses modulated by LPS depend on plant genotypes rather than on the type of interaction.

Oligosaccharide recognition signals and defence reactions in marine plant-microbe interactions.

Recent findings on the involvement of oligosaccharide signals in pathogen recognition and defence reactions in marine algae shine a new light on the ecology of their interactions with associated microorganisms. Since the marine environment encompasses lineages that have diverged a long time ago from the terrestrial phyla, these results suggest that cell-cell recognition pathways typical of terrestrial plants appeared very early in the evolution of eukaryotes. Production of oligosaccharides from marine algae using microbial recombinant polysaccharidases is also of industrial interest as plants can be protected from infections by preincubation in the presence of appropriate signals that mimic the attacks by pathogens.

NMR spectroscopic investigation of agarose oligomers produced by an alpha-agarase.

The 13C NMR signals of various even and odd agarose oligosaccharides with either D-galactose or 3,6-anhydro-alpha-L-galactose at the reducing end have been assigned. The chemical shifts in water of the agaro- and the neoagaro-oligosaccharides are compared and the influence of dimethyl sulfoxide on the chemical structure of the agaro-oligosaccharides is reported. The 3,6-anhydro-L-galactose residue at the reducing end of agaro-oligosaccharides is in the hydrated form.

Purification and determination of the action pattern of Haliotis tuberculata laminarinase.

The major laminarinase activity (EC 3.2.1.39) from the gastropodean marine mollusc Haliotis tuberculata was purified to homogeneity by cation exchange chromatography and its action pattern was investigated by HPAEC-PAD analysis of the degradation of various laminarin samples. It consists of a 60 kDa protein capable of depolymerizing the unbranched portions of the beta-(1-->3), beta-(1-->6)-glucan, down to laminaritriose. The enzyme operates via a molecular mechanism retaining the anomeric configuration. As the purified protein does not cleave the beta-(1-->6) linkages, it can be used for the structural analysis of laminarins.

A rapid method for the separation and analysis of carrageenan oligosaccharides released by iota- and kappa -carrageenase.

Based on the improved performances in speed of chromatographic separation on Superdex-type materials (Pharmacia) compared to conventional media such as Sephadex and Bio Gel-type, a rapid size-exclusion chromatography (SEC) method was developed for the separation and analysis of carrageenan oligosaccharides. It was used to evaluate the elution profiles of hydrolysates produced by carrageenases specific for kappa- and iota-carrageenans. Oligosaccharide peaks ranging from di- to dodeca-saccharides were obtained in about 20 min on an analytical scale, whereas preparative runs were completed in a few hours. The method may also be used to monitor polysaccharide degradation.

[Functional coronary insufficiency in pheochromocytoma. Contribution of isotopic tests. A case report]. / L'insuffisance coronarienne fonctionnelle du phéochromocytome. Apport des examens isotopiques. A propos d'un cas.

A 67 year old woman with a right adrenal pheochromocytoma was admitted to hospital with decompensated diabetes. She developed clinical signs of myocardial infarction. Complementary investigations showed this to be an adrenergic cardiomyopathy. The radionuclide and angiographic investigations confirmed the ischemic origin of the lesions and the functional nature of the coronary insufficiency in this case of pheochromocytoma.

Physiological plasticity of Dictyota kunthii (Phaeophyceae) to copper excess.

The brown alga Dictyota kunthii is one of the dominant species in the coastal areas of northern Chile affected by copper enrichment due to accumulated mining wastes. To assess its physiological plasticity in handling copper-mediated oxidative stress, 4-days copper exposure (ca. 100 µg/L) experiments were conducted with individuals from a copper impacted area and compared with the responses of plants from a non-impacted site. Several biochemical parameters were then evaluated and compared between populations. Results showed that individuals from the copper-impacted population normally displayed higher levels of copper content and antioxidant enzymes activity (catalase (CAT), ascorbate peroxidase (AP), dehydroascorbate reductase (DHAR), glutathione peroxidase (GP) and peroxiredoxins (PRX)). After copper exposure, antioxidant enzyme activity increased significantly in plants from the two selected sites. In addition, we found that copper-mediated oxidative stress was associated with a reduction of glutathione reductase (GR) activity. Moreover, metabolic profiling of extracellular metabolites from both populations showed a significant change after plants were exposed to copper excess in comparison with controls, strongly suggesting a copper-induced release of metabolites. The copper-binding capacity of those exudates was determined by anodic stripping voltammetry (ASV) and revealed an increased ligand capacity of the medium with plants exposed to copper excess. Results indicated that D. kunthii, regardless their origin, counteracts copper excess by various mechanisms, including metal accumulation, activation of CAT, AP, DHAR, GP and PRX, and an induced release of Cu binding compounds. Thus, plasticity in copper tolerance in D. kunthii seems constitutive, and the occurrence of a copper-tolerant ecotype seems unlikely.

Purification and characterization of the alpha-agarase from Alteromonas agarlyticus (Cataldi) comb. nov., strain GJ1B.

The phenotypic features of strain GJ1B, an unidentified marine bacterium that degrades agar [Young, K. S. Bhattacharjee, S. S. & Yaphe, W. (1978) Carbohydr. Res. 66, 207-212], were investigated and its agarolytic system was characterized using 13C-NMR spectroscopy to analyse the agarose degradation products. The bacterium was assigned to the genus Alteromonas and the new combination A. agarlyticus (Cataldi) is proposed. An alpha-agarase, i.e. specific for the alpha(1-->3) linkages present in agarose, was purified to homogeneity from the culture supernatant by affinity chromatography on cross-linked agarose (Sepharose CL-6B) and by anion-exchange chromatography (Mono Q column). The major end product of agarose hydrolysis using the purified enzyme was agarotetraose. Using SDS/PAGE, the purified alpha-agarase was detected as a single band with a molecular mass of 180 kDa. After the affinity-chromatography step, however, the native molecular mass was approximately 360 kDa, suggesting that the native enzyme is a dimer which is dissociated to active subunits by anion-exchange chromatography. The isolectric point was estimated to be 5.3. Enzyme activity was observed using agar as the substrate over the pH range 6.0-9.0 with a maximum value at pH 7.2 in Mops or Tris buffer. The enzyme was inactivated by prolonged treatment at a pH below 6.5, or by temperatures over 45 degrees C or by removing calcium. In addition, a beta-galactosidase specific for the end products of the alpha-agarase was present in the alpha-agarase affinity-chromatography fraction, probably as part of a complex with this enzyme. The degradation of agarose by this agarase complex yielded a mixture of oligosaccharides in the agarotetraose series and the agarotriose series, the latter consisting of oligosaccharides with an odd number of galactose residues.

Oligoguluronates elicit an oxidative burst in the brown algal kelp Laminaria digitata.

Oligomeric degradation products of alginate elicited a respiratory and oxidative burst in the sporophytes of the kelp Laminaria digitata. The generation of activated oxygen species (AOS), O(2)(-), and H(2)O(2) was detected at the single cell level, using nitroblue tetrazolium precipitation and a redox-sensitive fluorescent probe, respectively. The oxidative burst involved diphenyleneiodonium-sensitive AOS-generating machinery and its amplitude depended on the type of tissue. After a first elicitation plants were desensitized for about 3 h. The activity of alginate oligosaccharides was dose dependent, saturating around 40 microM. It was also structure-dependent, with homopolymeric blocks of alpha-1,4-L-guluronic acid, i.e. the functional analogs of oligogalacturonic blocks in pectins, being the most active signals. The perception of oligoguluronate signals resulted in a strong efflux of potassium. Pharmacological dissection of the early events preceding the emission of AOS indicated that the transduction chain of oligoguluronate signals in L. digitata is likely to feature protein kinases, phospholipase A(2), as well as K(+), Ca(2+), and anion channels.

iota-Carrageenases constitute a novel family of glycoside hydrolases, unrelated to that of kappa-carrageenases.

iota-Carrageenases are polysaccharide hydrolases that cleave the beta-1,4 linkages between the d-galactose-4-sulfate and 3, 6-anhydro-d-galactose-2-sulfate residues in the red algal galactans known as iota-carrageenans. We report here on the purification of iota-carrageenase activity from the marine bacterium Zobellia galactanovorans and on the characterization of iota-carrageenase structural genes. Genomic libraries from this latter bacterium as well as from Alteromonas fortis were functionally screened for the presence of iota-carrageenase(+) clones. The Z. galactanovorans and A. fortis iota-carrageenase genes encode homologous proteins of 53.4 and 54.8 kDa, respectively. Based on hydrophobic cluster analysis and on the (1)H NMR monitoring of the products of the overexpressed A. fortis iota-carrageenase, these enzymes appear to form a new family of glycoside hydrolases, unrelated to that of kappa-carrageenases and with an inverting mechanism of hydrolysis. They both feature a 45-amino acid-long N-terminal segment with sequence similarity to the N-terminal region of several other polysaccharidases. In those for which a three-dimensional structure is available, this conspicuous segment, also deemed "glycanase motif" (Chua, J. E. H., Manning, P. A., and Morona, R. (1999) Microbiology (Reading) 145, 1649-1659), corresponds to a strand-helix-strand "cap" that covers the N-terminal end of a common, right-handed beta-helical fold.

Arylsulphatase from Alteromonas carrageenovora.

Arylsulphatase activity was identified in cultures of the marine bacterium Alteromonas carrageenovora, using methylumbelliferyl sulphate as substrate. In contrast with most other microbial arylsulphatases, arylsulphatase production in A. carrageenovora was not repressed by sulphate. The structural gene of arylsulphatase (atsA) was cloned and sequenced. An ORF of 984 bp was found, specifying a primary translation product of 328 amino acids with a molecular mass of 35797 Da. Arylsulphatase was partially purified from cell extracts of both A. carrageenovora and recombinant Escherichia coli. Both the recombinant and native enzymes exhibited a pI of 5.5, a Michaelis constant for methylumbelliferyl sulphate of 68 microM, and a molecular mass of approximately 35,000 Da in SDS-PAGE analysis. Secondary structure comparisons using hydrophobic cluster analysis suggest functional analogies between the arylsulphatase of A. carrageenovora, that of Mycobacterium leprae and a 33.5 kDa protein from Porphyromonas gingivalis. It is speculated that these proteins are all glycosulphohydrolases, involved with desulphatation of sulphated polysaccharides.

Purification and characterization of a new kappa-carrageenase from a marine Cytophaga-like bacterium.

A bacterial strain able to degrade various sulfated galactans (carrageenans and agar) was isolated from the marine red alga Delesseria sanguinea. From the cell-free supernatant of cultures grown on crude lambda-carrageenan, a kappa-carrageenase was purified by ammonium sulfate fractionation, gel filtration on Sephacryl S 200 HR and ion-exchange chromatography on DEAE--Sepharose-CL6B. The purified kappa-carrageenase was detected as a single protein upon SDS/PAGE. Its molecular mass was estimated at 40 kDa. Activity was observed against kappa-carrageenan over the pH range 5.0-8.5 and was optimal at pH 7.2 in Tris buffer or 7.0 in Mops buffer. The enzyme activity remained stable at 30 degrees C, but only for up to 1 h at 40 degrees C. Analysis of the degradation products of the kappa-carrageenase by gel filtration and 13C-NMR spectroscopy indicated that the enzyme degrades kappa-carrageenan down to the level of the kappa-neocarratetraose sulfate. The properties of this new enzyme are compared with those of previously characterized carrageenases.

Inhibition of the establishment of zygotic polarity by protein tyrosine kinase inhibitors leads to an alteration of embryo pattern in Fucus.

Fucoid algae, including the genus Fucus and Pelvetia, are recognized as model systems to study early embryogenesis in plants. In particular the zygotes of these fucoid algae are highly suitable experimental systems for investigating the establishment of polarity and its requirement for later embryogenesis. However, the transduction pathways involved in the initiation of polarization are still poorly understood, and the link between the early polarization processes and embryo long-term patterning has never been experimentally demonstrated. We, therefore, have investigated the putative role of protein phosphorylation in the regulation of early embryogenesis, using a combined pharmacological and biochemical approach. Among the various protein kinase inhibitors tested, a subset of well-known PTK inhibitors, including genistein, prevented germination but had no effect on growth of germinated zygotes and embryos. Inhibition of germination appeared to be a direct consequence of prevention of polarization since genistein and other PTK inhibitors specifically inhibited axis formation in a light-independent manner. Genistein inhibited cellular events associated with polarization such as polarized secretion of cell wall sulfated compounds. Anchorage of F-actin at the rhizoid pole was also inhibited and F-actin redistributed in response to a new light vector. Zygotes inhibited in the polarization process over the period of axis formation recovered from the treatment and displayed differentiated cellular structures after a few days. However, they exhibited a deeply disorganized pattern, suggesting that the early polarization process is essential for normal patterning of the embryo. Western blot analysis of protein phosphorylation showed that the patterns of protein phosphorylation changed during development and were disturbed by treatments with genistein. This drug also inhibited in vitro autophosphorylation. The nature of the genistein-sensitive kinases required for polarization and long-term patterning is discussed in light of these data.

The kappa-carrageenase of the marine bacterium Cytophaga drobachiensis. Structural and phylogenetic relationships within family-16 glycoside hydrolases.

We report here cloning from the marine gliding bacterium Cytophaga drobachiensis of kappa-carrageenase, a glycoside hydrolase involved in the degradation of kappa-carrageenan. Structural features in the nucleotide sequence are pointed out, including the presence of an octameric omega sequence similar to the ribosome-binding sites of various eukaryotes and prokaryotes. The cgkA gene codes for a protein of 545 aa, with a signal peptide of 35 aa and a 229-aa-long posttranslationaly processed C-terminal domain. The enzyme displays the overall folding and catalytic domain characteristics of family 16 of glycoside hydrolases, which comprises other beta-1,4-alpha-1,3-D/L-galactan hydrolases, beta-1,3-D-glucan hydrolases (laminarinases), beta-1,4-1,3-D-glucan hydrolases (lichenases), and beta-1,4-D-xyloglucan endotransglycosylases. In order to address the origin and evolution of CgkA, a comprehensive phylogenetic tree of family 16 was built using parsimony analysis. Family-16 glycoside hydrolases cluster according to their substrate specificity, regardless of their phylogenetic distribution over eubacteria and eukaryotes. Such a topology suggests that the general homology between laminarinases, agarases, kappa-carrageenases, lichenases, and xyloglucan endotransglycosylases has arisen through gene duplication, likely from an ancestral protein with laminarinase activity.

Carrageenan oligosaccharides enhance stress-induced microspore embryogenesis in Brassica oleracea var. italica.

Embryogenic induction in cultures of isolated microspores is a stress-dependent process, which can be triggered by heat shock, sucrose or nitrogen starvation or by anti-microtubular drugs. As they are known to mimic biotic stress, oligosaccharides were tested as an alternative source of compounds to induce microspore embryogenesis in Brassica oleracea var. italica. Among the eight oligosaccharide series that were investigated and the corresponding polysaccharides, namely pectin, alginate, fucan, laminarin, agar and kappa-, iota-, and lambda-carrageenans, only the carrageenan oligomers displayed significant effects on microspore induction. When supplied in combination to heat stress, they markedly enhanced the final yields of microspore-derived embryos, with a two-fold increase in the most reactive treatment, i.e. in the presence of lambda-carrageenan oligosaccharides. A 30 min treatment was enough to stimulate embryogenesis, and two optimal concentrations were observed, 170 nM and 34 &mgr;M.

Zobellia galactanovorans gen. nov., sp. nov., a marine species of Flavobacteriaceae isolated from a red alga, and classification of [Cytophaga] uliginosa (ZoBell and Upham 1944) Reichenbach 1989 as Zobellia uliginosa gen. nov., comb. nov.

A mesophilic, aerobic, non-flagellated, gliding bacterium, forming yellow colonies and designated DsijT, was isolated from a red alga on the sea-shore of Roscoff, Brittany, France. DsijT was selected for its ability to actively degrade both agars and carrageenans. The Gram-negative cells occurred singly or in pairs as long rods. The temperature range for growth was 13-45 degrees C, with an optimum at 35 degrees C. The pH range for growth at 35 degrees C was from 6.0 to 8.5, with an optimum around pH 7.0. The NaCl concentrations required for growth at 35 degrees C and pH 7.0 ranged from 5 to 60 g l(-1), with an optimum around 25 g l(-1). The G+C content of the genomic DNA was 42-43 mol%. Phylogenetic analysis of 16S rRNA gene sequences indicated that strain DsijT is closely related to [Cytophaga] uliginosa DSM 2061T. Phenotypic features, however, allowed DsijT and [Cytophaga] uliginosa strains to be distinguished on the basis of ten traits (spreading behaviour, assimilation of eight compounds and amylase production). Their total protein profiles were also different and DNA-DNA hybridization experiments confirmed that DsijT constitutes a new species, distinct from [Cytophaga] uliginosa. Based on the phenotypic features and the phylogenetic relationships of the Flavobacteriaceae, a new genus designated Zobellia gen. nov. is proposed to include Zobellia galactanovorans gen. nov., sp. nov., while [Cytophaga] uliginosa becomes Zobellia uliginosa comb. nov. The type strain of Zobellia galactanovorans is DsijT (= DSM 12802T = CIP 106680T).