In situ characterization of antibody grafting on porous monolithic supports.

The efficient immobilization of antibodies on monolithic support is one of the most critical steps when preparing immunoaffinity supports. In this work, the ADECA (amino density estimation by colorimetric assay) method was adapted to tridimensional supports (in a dynamic mode) and proved to be efficient to characterize the antibodies grafting efficiency on 15.3±0.9mg porous glycidyl methacrylate (GMA)-co-ethylene dimethacrylate (EDMA) monolithic columns. The amount of grafted antibodies measured in situ on the monolith by ADECA (8.2±0.2µg of antibodies per milligram of monolith) was consistent with values obtained by bicinchoninic acid assay (BCA) after crushing the monolith. ADECA was shown to be less time-consuming and more versatile than BCA. The ADECA method was further implemented to thoroughly study and optimize the antibody grafting conditions (influence of pH and kinetics of the grafting step) on GMA-based monoliths and to check the covalent nature of the antibody/surface linking and its stability. Using the total amount of grafted antibodies and the amount of recognized antigen, we found that 65±6% of antibodies were able to capture their antigen. Finally, the grafting of Fab and F(ab')(2) fragments demonstrated that no significant improvement of the global binding capacity of the monolith was obtained.

Nano-odontology: nanostructured assemblies for endodontic regeneration.

The vitality of the pulp is so fundamental to the functional life of the tooth that new strategies are required to avoid the removal of the whole pulp following irreversible pulpitis and to regenerate the lost endodontic tissues. Nano-odontology would provide suitable solutions for pulp tissue conservative and regenerative approaches. In our group, we have shown that when covalently coupled to Poly-Glutamic Acid (PGA) the incorporation of an anti-inflammatory hormone (melanocortin, a-MSH) into the multilayered films Poly-L-Lysine (PLL)/PGA increases the anti-inflammatory reaction of pulp fibroblasts and macrophages stimulated by LPS (Lipo-Polysaccharides). Recently, usual linear PLL polymers have been chemically grafted for making new Dendrigraft polymers (DGLG4) whose higher branching ratios can give useful properties. The objective is to use nanostructured assemblies containing DGLG4 and PGA-alpha-MSH to design a new nanomaterial. These nanostructured assemblies (DGLG4-PGA-alpha-MSH)n constitute a thick reservoir of the anti-inflammatory peptide and promote adhesion and proliferation of pulp fibroblast on the biomaterial surface. These nanostructured films could be adapted for an endodontic regeneration application to target pulp connective tissue regeneration. Firstly, the crucial reduction of inflammation could be helpful by using PGA-alpha-MSH and secondly the initiation of the regeneration of the connective tissue will be promoted by the whole nanostructured film of which allows pulp cells colonisation.

Gene expression profiling of Arabidopsis meiocytes.

Meiosis is a special type of cell division present in all organisms that reproduce by sexual reproduction. It ensures the transition between the sporophytic and gametophytic state and allows gamete production through meiotic recombination and chromosome number reduction. In this paper, we describe a technique for the isolation of Arabidopsis thaliana male meiocytes. From this cellular material, it was then possible to develop large-scale transcriptome studies using CATMA microarrays and thus to obtain an overview of genes expressed during Arabidopsis meiosis. The expression profiles were studied with either stringent statistical criteria or by performing clustering. Both methods resulted in gene clusters enriched in meiosis-specific genes (from 14- to 55-fold). Analysis of these data provided a unique set of genes that will be pivotal to further analysis aimed at understanding the meiotic process.

Aminated dendritic surfaces characterization: a rapid and versatile colorimetric assay for estimating the amine density and coating stability.

The functionalization of surfaces with amino groups is used in many application areas such as in industrial biocatalytic processes for the development of medical biomaterials and in the environment for removing pollutants from water. Amino group density and grafting stability are often related to functionalized material performances; thus, their characterizations are of prime importance. The determination of amino density and grafting stability on polymeric material (e.g. polypropylene, polystyrene and cylco olefin copolymer) is often time consuming and sometimes presents technical constraints, more particularly with non-flat materials. In this paper, we report a novel colorimetric assay using the Coomassie Brilliant Blue dye for both amino density determination and grafting stability measurement. The assay named ADECA for "Amino Density Estimation by Colorimetric Assay" is sensitive, rapid, robust and versatile. We demonstrate that ADECA makes the evaluation of aminated materials performances possible for numerous material compositions, formats and chemistries used for grafting. Our study focuses on dendrigraft of poly-L-lysine and poly(amidoamine) dendrimers dendritic materials.

[Disseminated histoplasmosis revealed by peripheral blood smear in an African immigrant with AIDS]. / Histoplasmose disséminée révélée par un frottis sanguin chez un immigré africain atteint du sida.

We report a case of disseminated histoplasmosis due to Histoplasma capsulatum var. capsulatum in an African patient with AIDS (born in Ghana) and living in France. This opportunistic fungal infection has never been reported in Ghana, maybe because of limited means of investigation or ignorance. Nevertheless, it was reported in a Ghanaian immigrant living in Italy. In our patient, the first diagnosis was suggested by a peripheral blood smear, which is not a classical biological diagnostic method. The diagnostic interest and the frequency of positive peripheral blood smears in the course of disseminated histoplasmosis are probably under estimated.

[Bacterial cellulitis. Forms borderline between medical and surgical (3 cases)]. / Dermohypodermites bactériennes.

BACKGROUND: An acute infectious cellulitis may be managed medically (erysipelas or non-necrotizing infectious cellulitis) or surgically (necrotizing infectious cellulitis, necrotizing fasciitis). We report 3 cases of non-necrotizing infectious cellulitis borderline between medical and surgical forms, complicated by compartment syndrome, the surgical decompression of which permitted patients' cure. CASE REPORTS: Three patients, 27, 52 and 84 years old, were admitted for an acute infectious cellulitis of the leg. At admission, the leg area involved was erythematous, painful, indurated, with one or several bullae, purpura, pustules, hypoesthesia or limited skin necrosis, and no immediate need for surgical exploration. The clinical evolution was characterized by the slow appearance or extension of signs of severity, despite the modification in antibiotic treatment. Magnetic resonance imaging findings were indicative of a non-necrotizing infectious cellulitis in 2 patients. In one patient, necrotizing fasciitis could not be excluded. In all patients, surgical exploration showed an important quantity of non-purulent fluid between muscles and hypodermis, with no evidence of abscess or necrosis. A large incision rapidly cured all patients. DISCUSSION: These three observations were characterized by the initial signs of moderate severity and no response to an appropriate medical treatment, which led to surgical exploration. Surgery showed no abscess or necrosis but an important quantity of sterile fluid; it also permitted rapid cure of patients. These cases present a borderline form of infectious cellulitis, with severe local inflammation caused by a compartment syndrome. Surgical decompression was needed for cure. The potential value of magnetic resonance imaging in this situation should also be stressed.

[The education level of elderly diabetics]. / Le niveau d'éducation des diabétiques âgés.

OBJECTIVE: The education of diabetic subjects has been the object of numerous studies which have demonstrated its importance as an integral part of treatment. Few studies have focused on the particular problem of education in the elderly. PATIENTS AND METHODS: An inquiry was conducted in 60 elderly patients with diabetes in order to assess their knowledge of the disease, comparing those who had received specific education and those who had not, and to determine the characteristic features of patients who had received education. A specific questionnaire was designed for this study. The maximal score was 50. RESULTS: Mean age of the 60 patients was 77 years (range 65-94). Lack of knowledge was most patent concerning acute metabolic decompensation with hypoglycemia an hyperglycemia and concerning foot care. Only one-third of the patients had received diabetic education. The average score in the educated group was 42 +/- 0.4 versus 23 +/- 7 in the non-educated group (p = 0.0001). The best results were obtained in educated and younger patients. The inquiry showed that education had been distributed preferentially to the youngest patients, to men, and to patients treated with insulin. CONCLUSIONS: Our findings demonstrate that elderly diabetics can benefit from an education program and prove a real insufficiency in current education of elderly diabetics. These results should also lead to further work on an educational tool specifically designed for elderly diabetics.

Functional characterization of the KNOLLE-interacting t-SNARE AtSNAP33 and its role in plant cytokinesis.

Cytokinesis requires membrane fusion during cleavage-furrow ingression in animals and cell plate formation in plants. In Arabidopsis, the Sec1 homologue KEULE (KEU) and the cytokinesis-specific syntaxin KNOLLE (KN) cooperate to promote vesicle fusion in the cell division plane. Here, we characterize AtSNAP33, an Arabidopsis homologue of the t-SNARE SNAP25, that was identified as a KN interactor in a yeast two-hybrid screen. AtSNAP33 is a ubiquitously expressed membrane-associated protein that accumulated at the plasma membrane and during cell division colocalized with KN at the forming cell plate. A T-DNA insertion in the AtSNAP33 gene caused loss of AtSNAP33 function, resulting in a lethal dwarf phenotype. atsnap33 plantlets gradually developed large necrotic lesions on cotyledons and rosette leaves, resembling pathogen-induced cellular responses, and eventually died before flowering. In addition, mutant seedlings displayed cytokinetic defects, and atsnap33 in combination with the cytokinesis mutant keu was embryo lethal. Analysis of the Arabidopsis genome revealed two further SNAP25-like proteins that also interacted with KN in the yeast two-hybrid assay. Our results suggest that AtSNAP33, the first SNAP25 homologue characterized in plants, is involved in diverse membrane fusion processes, including cell plate formation, and that AtSNAP33 function in cytokinesis may be replaced partially by other SNAP25 homologues.

A T-DNA insertion knockout of the bifunctional lysine-ketoglutarate reductase/saccharopine dehydrogenase gene elevates lysine levels in Arabidopsis seeds.

Plants possess both anabolic and catabolic pathways for the essential amino acid lysine (Lys). However, although the biosynthetic pathway was clearly shown to regulate Lys accumulation in plants, the functional significance of Lys catabolism has not been experimentally elucidated. To address this issue, we have isolated an Arabidopsis knockout mutant with a T-DNA inserted into exon 13 of the gene encoding Lys ketoglutarate reductase/saccharopine dehydrogenase. This bifunctional enzyme controls the first two steps of Lys catabolism. The phenotype of the LKR/SDH knockout was indistinguishable from wild-type plants under normal growth conditions, suggesting that Lys catabolism is not an essential pathway under standard growth conditions. However, mature seeds of the knockout mutant over-accumulated Lys compared with wild-type plants. This report provides the first direct evidence for the functional significance of Lys catabolism in regulating Lys accumulation in seeds. Such a knockout mutant may also provide new perspectives to improve the level of the essential amino acid Lys in plant seeds.

[Management of erysipelas]. / Erysipèle: quelle prise en charge?

The objective of this literature review was to evaluate the therapeutic management of erysipelas. We selected 74 publications, some of written a long time ago, and thus open to criticism regarding their methodology. However, no recent or better study was available on the subject. Penicillin G remains the therapeutic reference. The use of macrolides and stretogramins is an alternative after the exclusion of severe forms of erysipelas. The preventive treatment of thrombosis by heparin must be discussed taking into account risk factors. More studies are necessary to suggest a coprescription corticoid/NSAIDs and antibiotherapy. The best antibiotic prophylaxis after the initial treatment isabenzathine-penicillin injection every 15 days.

Arabidopsis glucosidase I mutants reveal a critical role of N-glycan trimming in seed development.

Glycoproteins with asparagine-linked (N-linked) glycans occur in all eukaryotic cells. The function of their glycan moieties is one of the central problems in contemporary cell biology. N-glycosylation may modify physicochemical and biological protein properties such as conformation, degradation, intracellular sorting or secretion. We have isolated and characterized two allelic Arabidopsis mutants, gcs1-1 and gcs1-2, which produce abnormal shrunken seeds, blocked at the heart stage of development. The mutant seeds accumulate a low level of storage proteins, have no typical protein bodies, display abnormal cell enlargement and show occasional cell wall disruptions. The mutated gene has been cloned by T-DNA tagging. It codes for a protein homologous to animal and yeast alpha-glucosidase I, an enzyme that controls the first committed step for N-glycan trimming. Biochemical analyses have confirmed that trimming of the alpha1,2- linked glucosyl residue constitutive of the N-glycan precursor is blocked in this mutant. These results demonstrate the importance of N-glycan trimming for the accumulation of seed storage proteins, the formation of protein bodies, cell differentiation and embryo development.

Disruption of the Arabidopsis RAD50 gene leads to plant sterility and MMS sensitivity.

The Rad50 protein is involved in the cellular response to DNA-double strand breaks (DSBs), including the detection of damage, activation of cell-cycle checkpoints, and DSB repair via recombination. It is essential for meiosis in yeast, is involved in telomere maintenance, and is essential for cellular viability in mice. Here we present the isolation, sequence and characterization of the Arabidopsis thaliana RAD50 homologue (AtRAD50) and an Arabidopsis mutant of this gene. A single copy of this gene is present in the Arabidopsis genome, located on chromosome II. Northern analysis shows a single 4.3 Kb mRNA species in all plant tissues tested, which is strongly enriched in flowers and other tissues with many dividing cells. The predicted protein presents strong conservation with the other known Rad50 homologues of the amino- and carboxy-terminal regions. Mutant plants present a sterility phenotype which co-segregates with the T-DNA insertion. Molecular analysis of the mutant plants shows that the sterility phenotype is present only in the plants homozygous for the T-DNA insertion. An in vitro mutant cell line, derived from the mutant plant, shows a clear hypersensitivity to the DNA-damaging agent methylmethane sulphonate, suggesting a role of RAD50 in double-strand break repair in plant cells. This is the first report of a plant mutated in a protein of the Rad50-Mre11-Xrs2 complex, as well as the first data suggesting the involvement of the Rad50 homologue protein in meiosis and DNA repair in plants.

An arabidopsis T-DNA mutant affected in Nrt2 genes is impaired in nitrate uptake.

Expression analyses of Nrt2 plant genes have shown a strict correlation with root nitrate influx mediated by the high-affinity transport system (HATS). The precise assignment of NRT2 protein function has not yet been possible due to the absence of heterologous expression studies as well as loss of function mutants in higher plants. Using a reverse genetic approach, we isolated an Arabidopsis thaliana knock-out mutant where the T-DNA insertion led to the complete deletion of the AtNrt2.1 gene together with the deletion of the 3' region of the AtNrt2.2 gene. This mutant is impaired in the HATS, without being modified in the low-affinity system. Moreover, the de-regulated expression of a Nicotiana plumbaginifolia Nrt2 gene restored the mutant nitrate influx to that of the wild-type. These results demonstrate that plant NRT2 proteins do have a role in HATS.

Plant lipoxygenase 2 is a translation initiation factor-4E-binding protein.

The eukaryotic initiation factor 4E (eIF4E) emerged recently as a target for different types of regulation affecting translation. In animal and yeast cells, eIF4E-binding proteins modulate the availability of eIF4E. A search for plant eIF4E-binding proteins from Arcabictopsis thaliana using the yeast genetic interaction system identified a clone encoding a lipoxygenase type 2 (AtLOX2). In vitro and in vivo biochemical assays confirm an interaction between AtLOX2 and plant eIF4E(iso) factor. A two-hybrid assay revealed that AtLOX2 is also able to interact with both wheat initiation factors 4E and 4E(iso). Deletion analysis maps the region of AtLOX2 involved in interaction with AteIF(iso)4E between amino acids 175 and 232. A sequence related to the conserved motif present in several eIF4E-binding proteins was found in this region. Furthermore, the wheat p86 subunit, a component of the plant translation eIF(iso)4F complex, was found to interfere with the AteIF(iso)4E-AtLOX2 interaction suggesting that p86 and AtLOX2 compete for the same site on eIF(iso)4E. These results may reflect a link between eIF4Es factors mediating translational control with LOX2 activity, which is probably conserved throughout the plant kingdom.

Ambulatory blood pressure monitoring and postprandial hypotension in elderly persons with falls or syncopes.

BACKGROUND: Postprandial hypotension (PPH) is increasingly recognized as a common cause of falls and syncope in elderly persons. Noninvasive ambulatory blood pressure monitoring (ABPM) has been recommended for detecting PPH. This study investigates postprandial blood pressure (BP) changes by means of ABPM in elderly patients experiencing falls or syncopes. METHODS: Twenty-four-hour ABPM was performed in 156 inpatients (111 women, mean age 80.4 +/- 8.1 years). Among them, 45 had been admitted for falls and 75 for syncope; 36 with no history of falls or syncope served as controls. Postprandial change in systolic blood pressure (deltaSBP) was calculated by subtracting the mean SBP within the 2 hours following the meal from the mean SBP within the 2 hours preceding the meal. PPH was defined by a deltaSBP > or = 20 mm Hg. RESULTS: For the entire group, mean SBP decreased after the three meals. On average, the decline in SBP was greater after breakfast than after lunch or dinner, and the number of patients experiencing PPH was greater after breakfast. Average maximal deltaSBP was significantly larger in the syncope group than in the other groups ( p < .05). Moreover, the number of patients experiencing PPH was significantly higher in the syncope/fall group than in the control group (23% vs 9%; p = .03). Compared with patients without PPH, patients with PPH were more likely to have a history of diabetes mellitus (p < .01) or to use more than three different drugs daily ( p = .04), and they showed greater daytime SBP variability (p < .0001). Furthermore, there was a strong positive correlation between preprandial SBP and deltaSBP after breakfast. CONCLUSIONS: About one out of four elderly patients with falls or syncope experiences PPH, usually after breakfast. Postprandial decline in BP contributes to BP variability. deltaSBP and preprandial SBP are positively correlated.

[Invasive fungal infections. Epidemiology and new therapies]. / Les infections fongiques invasives. Epidémiologie et nouvelles thérapeutiques.

RISING INCIDENCE: In the past two decades, systemic fungal infections, essentially invasive candidiasis, but also invasive aspergillosis, has increased substantially. Despite the currently available antifungal drugs, amphotericin B (AmB), azole compounds (fluconazole or FLU, itraconazole or ITR), these infections are associated with significant morbidity and mortality. AmB remains the drug of choice for treatment of most fungal diseases because of its broad spectrum and potent fungicidal activity, but significant side effects limit its clinical utility. The azole antifungal agents are easier to take, less toxic than AmB, but their use is limited by multiazole-resistant strains. NEW ANTIFUNGAL AGENTS: Lipid formulations have recently attracted much attention due to a significantly lower toxicity: this concerns lipid formulations of AmB and perhaps nystatin in the future. New triazoles (voriconazole, ravuconazole, posaconazole) have shown a wide spectrum of action including against azole-resistant isolates. A new class of antifungal agents, lipopeptides (MK-0991, LY303366, FK463), with an original mechanism of action are being developed. These new compounds are reported to possess a large fungicidal activity against most isolates including AmB and azole-resistant strains.

Synthesis of full-length potyvirus cDNA copies suitable for the analysis of genome polymorphism.

New methods facilitating the synthesis and amplification of full-length cDNA copies of single-stranded viral RNA genomes have been developed. A method is described for the efficient purification of potyviral RNA and total RNA from infected plants and it is shown that they can serve as templates for the efficient synthesis of a full-length, 10 kb long, genomic cDNA. Two different reverse transcriptases were used (AMV-RT and MMLV-RT); only the first reverse transcriptase produced a good quality, full-length cDNA using viral RNA as a template. Surprisingly, MMLV-RT allowed for the full-length cDNA synthesis on virions rather than viral RNA. The PVY cDNA, synthesized using either RNA or virions, can be amplified successfully by PCR with high yields of full-length products. Such products are good substrates for the study by RFLP of the total genome polymorphism of virus isolates.

[Incidence and consequences of nosocomial infections in a geriatric short-stay department]. / Incidence et conséquences des infections nosocomiales dans un service de court séjour gériatrique.

OBJECTIVES: Although large epidemiological studies have demonstrated that elderly people experience a greater incidence and mortality attributable to nosocomial infections, few studies have yet focused on this problem in geriatrics. PATIENTS AND METHODS: In order to assess the importance of nosocomial infections in geriatric short-stay department, we carried out a prospective study during a one-year period. RESULTS: Eighty-two nosocomial infections were recorded in 67 patients. The incidence was 10.3 nosocomial infections per 1000 patient-days. Pneumonia was the second most frequent type of nosocomial infection after urinary tract infections, but caused the highest rate of death. For infected patients, hospital stay was twice as long (31 versus 13 days) and mortality four times higher (26.9 versus 8.2%). CONCLUSION: These results confirm that elderly patients make up a high-risk population and that the prevention of nosocomial infections must be a priority in geriatrics.