The structural basis for catalysis and specificity of the X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis

The X-prolyl dipeptidyl aminopeptidase (X-PDAP) from Lactococcus lactis is a dimeric enzyme catalyzing the removal of Xaa-Pro dipeptides from the N terminus of peptides. The structure of the enzyme was solved at 2.2 A resolution and provides a model for the peptidase family S15. Each monomer is composed of four domains. The larger one presents an alpha/beta hydrolase fold and comprises the active site serine. The specificity pocket is mainly built by residues from a small helical domain which is, together with the N-terminal domain, essential for dimerization. A C-terminal moiety probably plays a role in the tropism of X-PDAP toward the cellular membrane. These results give new insights for further exploration of the role of the enzymes of the SC clan.     

Processing of the bovine spongiform encephalopathy-specific prion protein by dendritic cells

Dendritic cells (DC) are suspected to be involved in transmissible spongiform encephalopathies, including bovine spongiform encephalopathy (BSE). We detected the disease-specific, protease-resistant prion protein (PrP(bse)) in splenic DC purified by magnetic cell sorting 45 days after intraperitoneal inoculation of BSE prions in immunocompetent mice. We showed that bone marrow-derived DC (BMDC) from wild-type or PrP-null mice acquired both PrP(bse) and prion infectivity within 2 h of in vitro culture with a BSE inoculum. BMDC cleared PrP(bse) within 2 to 3 days of culture, while BMDC infectivity was only 10-fold diminished between days 1 and 6 of culture, suggesting that the infectious unit in BMDC is not removed at the same rate as PrP(bse) is removed from these cells. Bone marrow-derived plasmacytoid DC and bone marrow-derived macrophages (BMM) also acquired and degraded PrP(bse) when incubated with a BSE inoculum, with kinetics very similar to those of BMDC. PrP(bse) capture is probably specific to antigen-presenting cells since no uptake of PrP(bse) was observed when splenic B or T lymphocytes were incubated with a BSE inoculum in vitro. Lipopolysaccharide activation of BMDC or BMM prior to BSE infection resulted in an accelerated breakdown of PrP(bse). Injected by the intraperitoneal route, BMDC were not infectious for alymphoid recombination-activated gene 2(0)/common cytokine gamma chain-deficient mice, suggesting that these cells are not capable of directly propagating BSE infectivity to nerve endings.     

THE CHITINOUS NATURE OF FILAMENTS EJECTED BY PHAEOCYSTIS (PRYMNESIOPHYCEAE)1

Filaments ejected by Phaeocystis globosa Scherffel, organized in star-like structures, were observed and analyzed before and after their discharge from cells. Ultrastructural observations obtained after cryofixation and cryosubstitution led to a model for their storage within the cell and for their ejection from the cell. Electron diffraction analysis on the ejected filaments demonstrated their chitinous composition. This technique indicated without ambiguity that each filament was in fact a whisker-like α-chitin crystal, with the axes of the corresponding polymer chains aligned with the filament's axis. X-ray microanalysis of the mats of filaments indicated that the silica content suggested by earlier workers was an artifact resulting from the filtration procedure.     

Étude comparée des glucides solubles et membranaires chez des oeillets sains ou expérimentalement infectés par le Phialophora cinerescens (Wr.) van Beyma

After we had supposed that there could be a relation between the ability of some Caryophyllaceae (tribes of Diantheae and Lychnideae) to synthetize two series of very particular carbohydrates (lychnose and isolychnose) and the ability of thePhialophora cinerescens to infect members of these two tribes, we have looked for the constituents of soluble and cell wall carbohydrates:

in carnations of different ages and different cultivars; only the old carnations contain polyoses of the lychnose and isolychnose series, whereas the young carnations are already susceptible to the parasite;

n healthy and diseased Sim carnations, Lolita and New Red Sim; these two cultivars offer a difference of susceptibility to the disease and, parallely a difference of their amount in hemicellulose; great disparities appear between healthy and diseased carnations as regards their composition in soluble and cell wall carbohydrates.

     

Expression of Kisspeptins and Kiss Receptors Suggests a Large Range of Functions for Kisspeptin Systems in the Brain of the European Sea Bass

This study, conducted in the brain of a perciform fish, the European sea bass, aimed at raising antibodies against the precursor of the kisspeptins in order to map the kiss systems and to correlate the expression of kisspeptins, kiss1 and kiss2, with that of kisspeptin receptors (kiss-R1 and kiss-R2). Specific antibodies could be raised against the preprokiss2, but not the preoprokiss1. The data indicate that kiss2 neurons are mainly located in the hypothalamus and project widely to the subpallium and pallium, the preoptic region, the thalamus, the pretectal area, the optic tectum, the torus semicircularis, the mediobasal medial and caudal hypothalamus, and the neurohypophysis. These results were compared to the expression of kiss-R1 and kiss-R2 messengers, indicating a very good correlation between the wide distribution of Kiss2-positive fibers and that of kiss-R2 expressing cells. The expression of kiss-R1 messengers was more limited to the habenula, the ventral telencephalon and the proximal pars distalis of the pituitary. Attempts to characterize the phenotype of the numerous cells expressing kiss-R2 showed that neurons expressing tyrosine hydroxylase, neuropeptide Y and neuronal nitric oxide synthase are targets for kisspeptins, while GnRH1 neurons did not appear to express kiss-R1 or kiss-R2 messengers. In addition, a striking result was that all somatostatin-positive neurons expressed-kissR2. These data show that kisspeptins are likely to regulate a wide range of neuronal systems in the brain of teleosts.     

Expression of Aromatase in Radial Glial Cells in the Brain of the Japanese Eel Provides Insight into the Evolution of the cyp191a Gene in Actinopterygians

The cyp19a1 gene that encodes aromatase, the only enzyme permitting conversion of C19 aromatizable androgens into estrogens, is present as a single copy in the genome of most vertebrate species, except in teleosts in which it has been duplicated. This study aimed at investigating the brain expression of a cyp19a1 gene expressed in both gonad and brain of Japanese eel, a basal teleost. By means of immunohistochemistry and in situ hybridization, we show that cyp19a1 is expressed only in radial glial cells of the brain and in pituitary cells. Treatments with salmon pituitary homogenates (female) or human chorionic gonadotrophin (male), known to turn on steroid production in immature eels, strongly stimulated cyp19a1 messenger and protein expression in radial glial cells and pituitary cells. Using double staining studies, we also showed that aromatase-expressing radial glial cells exhibit proliferative activity in both the brain and the pituitary. Altogether, these data indicate that brain and pituitary expression of Japanese eel cyp19a1 exhibits characteristics similar to those reported for the brain specific cyp19a1b gene in teleosts having duplicated cyp19a1 genes. This supports the hypothesis that, despite the fact that eels also underwent the teleost specific genome duplication, they have a single cyp19a1 expressed in both brain and gonad. Such data also suggest that the intriguing features of brain aromatase expression in teleost fishes were not gained after the whole genome duplication and may reflect properties of the cyp19a1 gene of ancestral Actinopterygians.     

Effects of 1-methyl cyclohexane carboxylic acid (CCA) on cellular energetics in neuroblastoma cells

The level of cellular energetics has been estimated in neuroblastoma cells under different culture conditions. The cellular accumulation of isomerase-2-deoxy [¹⁴C]D-glucose-6-phosphate was taken as reflecting glucose utilization. A considerably higher amount of radioactivity — 2.5 to 4 times — is found in CCA treated cells, as compared to other types of cultures, corresponding to a higher rate of deoxyglucose penetration and utilization.     

Cross-reactivity studies of an anti-Plasmodium vivax apical membrane antigen 1 monoclonal antibody: binding and structural characterisation

Apical membrane antigen 1 (AMA1) has an important, but as yet uncharacterised, role in host cell invasion by the malaria parasite, Plasmodium. The protein, which is quite conserved between Plasmodium species, comprises an ectoplasmic region, a single transmembrane segment and a small cytoplasmic domain. The ectoplasmic region, which can induce protective immunity in animal models of human malaria, is a leading vaccine candidate that has entered clinical trials. The monoclonal antibody F8.12.19, raised against the recombinant ectoplasmic region of AMA1 from Plasmodium vivax, cross-reacts with homologues from Plasmodium knowlesi, Plasmodium cynomolgi, Plasmodium berghei and Plasmodium falciparum, as shown by immunofluorescence assays on mature schizonts. The binding of F8.12.19 to recombinant AMA1 from both P. vivax and P. falciparum was measured by surface plasmon resonance, revealing an apparent affinity constant that is about 100-fold weaker for the cross-reacting antigen when compared to the cognate antigen. Crystal structure analysis of Fab F8.12.19 complexed to AMA1 from P. vivax and P. falciparum shows that the monoclonal antibody recognises a discontinuous epitope located on domain III of the ectoplasmic region, the major component being a loop containing a cystine knot. The structures provide a basis for understanding the cross-reactivity. Antibody contacts are made mainly to main-chain and invariant side-chain atoms of AMA1; contact antigen residues that differ in sequence are located at the periphery of the antigen-binding site and can be accommodated at the interface between the two components of the complex. The implications for AMA1 vaccine development are discussed.     

The discovery of hypoglycemic sulfonamides

In 1933 Auguste Loubatières started to work at the Physiological Laboratory of the Montpellier Medical School, famous for the scientific work of Emmanuel Hédon and then Louis Hédon on experimental diabetes mellitus. Auguste Loubatières was particularly interested in the study of a new preparation of long-lasting insulin (insulin-protamine-zinc: IPZ) in the totally pancreatectomized dog. In 1938, he observed that high doses of IPZ induced a severe and protracted hypoglycemia entailing convulsive attacks and even irreversible coma. The story of hypoglycemic sulfonamides started in France in spring 1942. During the second world war, because of the food shortage in Montpellier, a lot of people ate rotting food or even food contaminated with bacteria, such as shellfish. Many cases of thyphoid fever were diagnosed and treated by Marcel Janbon at the Clinic of the Montpellier Medical School with a new sulfonamide (VK 57 or 2254 RP). Adverse reactions were observed: in some patients, convulsions and even prolonged coma occurred; in some others a major drop in blood glucose was observed. M. Janbon informed the physiologists about these observations questioning them for an interpretation. A. Loubatières was particularly interested and immediately undertook animal trials. On June 13, 1942, he observed that repeated oral administration of 2254 RP in the normal fasting conscious dog induced a progressive, marked and long-lasting decrease in glycemia. He continued his experiments and definitively established the hypoglycemic effect of this sulfonamide. However the mechanism of action remained to be established. The pattern of some graphs reminded him that of some others he could previously observe in the study with IPZ; it occurred to him that 2254 RP could lower the blood glucose concentration by stimulating insulin secretion. He had then to establish the validity of his hypothesis. From 1942 to 1946, A. Loubatières performed a systematic study of the effects of 2254 RP and structural analogs and investigated the mechanism involved in the hypoglycemia. These results are reported in his "Doctorat ès-Sciences" thesis (1946). He observed that 2254 RP was ineffective on glycemia in totally pancreatectomized dogs but was effective in partially pancreatectomized ones. The hypoglycemic effect in normal dog was dependent on the plasma sulfonamide concentration; this effect appeared whatever the route of administration and was unaffected by vagotomy. Furthermore, Loubatières performed cross-circulation experiments. In these experiments, the pancreatico-duodenal vein of a normal dog was anastomosed to the jugular vein of a receiver dog made diabetic by alloxan; in this case, the injection of 2254 RP into the donor induced a decrease in blood glucose levels in the receiver. In early 1946, Auguste Loubatières proposed that the hypoglycemic property of 2254 RP was due to its ability to stimulate insulin secretion through a direct action on pancreatic islets; he wrote in his thesis "A notre avis, le para-amino-benzène-sulfamido-isopropylthiodiazol (2254 RP) est donc un corps essentiellement insulino-sécréteur; son action s'exerce directement sur les ?lots de Langerhans". He also proposed to use such hypoglycemic sulfonamides in certain forms of diabetes "que l'on peut qualifier de fonctionnels et qui sont la conséquence d'une paresse des mécanismes insulino-sécréteurs". In 1992, Jean-Claude Henquin demonstrated that the sequence of events triggered by 2254 RP at the level of islet beta-cells was similar to that induced by sulfonylureas of the first or second generation. Thus, the 2254 RP, proposed by Auguste Loubatières in the treatment of certain forms of diabetes, was the first of oral hypoglycemic sulfonamides currently used in the treatment of type 2 diabetes mellitus.     

Interactions between atmospheric CO2 enrichment and soil fauna

We have reviewed the responses of soil fauna to increased concentrations of atmospheric CO₂ and the consequent climate change. These will affect several attributes of animal populations and communities including their density, biomass, diversity, activity, rates of consumption, life history parameters and migration ability. Changes in the quality and quantity of litter and global warming are the main factors which are expected to modify soil fauna. Although changes have been observed in several attributes of the soil fauna as a consequence of increased concentrations of atmospheric CO₂, no general trend which might allow to the prediction of a general pattern of response has been identified. Because of the complexity of the biological mechanisms and the synergetic action of several factors, the few resulting responses reported in the literature are inconclusive. However, some aspects of the situation deserve more attention. These include the consequences of (1) changes in the food resources for soil fauna in the litter layer and in the rhizosphere, (2) the consumption of low quality litter by the macrofauna, (3) the change in life span in response to temperature elevation, (4) the enhancement of earthworm burrowing activity and (5) the changes in community composition arising because of specific differential resistance to adverse conditions. This revised version was published online in June 2006 with corrections to the Cover Date.