The crystal structure of annexin A8 is similar to that of annexin A3

Annexin A8 is a relatively infrequent and poorly studied member of this large family of calcium-binding and membrane-binding proteins. It is, however, associated with a specific disease, acute promyelocytic leukemia. We have solved its three-dimensional structure, which includes a moderately long and intact N terminus. The structure is closest to that of annexin A3 and highlights several important regions of inherent flexibility in the annexin molecule. The N terminus resembles that of annexin A3, as it lies along the concave surface of the molecule and inserts partially into the hydrophilic channel in its centre. Since both annexins A3 and A8 are expressed in promyelocytic cells during their differentiation, the similarity in their structures might suggest a functional relationship.     

In vivo evidence for instability of episomal human immunodeficiency virus type 1 cDNA

Current regimens for the management of human immunodeficiency virus type 1 (HIV-1) infection suppress plasma viremia to below detectable levels for prolonged intervals. Nevertheless, there is a rapid resumption in plasma viremia if therapy is interrupted. Attempts to characterize the extent of viral replication under conditions of potent suppression and undetectable plasma viremia have been hampered by a lack of convenient assays that can distinguish latent from ongoing viral replication. Using episomal viral cDNA as a surrogate for ongoing replication, we previously presented evidence that viral replication persists in the majority of infected individuals with a sustained aviremic status. The labile nature of viral episomes and hence their validity as surrogate markers of ongoing replication in individuals with long-term-suppressed HIV-1 infection have been analyzed in short-term in vitro experiments with conflicting results. Since these in vitro experiments do not shed light on the long-term in vivo dynamics of episomal cDNA or recapitulate the natural targets of infection in vivo, we have analyzed the dynamics of episomal cDNA turnover in vivo by following the emergence of an M184V polymorphism in plasma viral RNA, in episomal cDNA, and in proviral DNA in patients on suboptimal therapies. We demonstrate that during acquisition of drug resistance, wild-type episomal cDNAs are replaced by M184V-harboring episomes. Importantly, a complete replacement of wild-type episomes with M184V-containing episomes occurred while proviruses remained wild type. This indicates that episomal cDNAs are turned over by degradation rather than through death or tissue redistribution of the infected cell itself. Therefore, evolution of episomal viral cDNAs is a valid surrogate of ongoing viral replication in HIV-1-infected individuals.     

Plant virus RNAs. Coordinated recruitment of conserved host functions by (+) ssRNA viruses during early infection events

Positive-sense single-stranded RNA viruses have developed strategies to exploit cellular resources at the expense of host mRNAs. The genomes of these viruses display a variety of structures at their 5' and 3' ends that differentiate them from cellular mRNAs. Despite this structural diversity, viral RNAs are still circularized by juxtaposition of their 5' and 3' ends, similar to the process used by cellular mRNAs. Also reminiscent of the mechanisms used by host mRNAs, translation of viral RNAs involves the recruitment of translation initiation factors. However, the roles played by these factors likely differ from those played by cellular mRNAs. In keeping with the general parsimony typical of RNA viruses, these host factors also participate in viral RNA replication. However, the dual use of host factors requires that viral RNA template utilization be regulated to avoid conflict between replication and translation. The molecular composition of the large ribonucleoprotein complexes that form the viral RNA replication and translation machineries likely evolves over the course of infection to allow for switching template use from translation to replication.     

Apolipoproteins C-II and C-III inhibit selective uptake of low- and high-density lipoprotein cholesteryl esters in HepG2 cells

Plasma low- and high-density lipoproteins (LDL and HDL) are cleared from the circulation by specific receptors and are either totally degraded or their cholesteryl esters (CE) are selectively delivered to cells by receptors such as the scavenger receptor class B type I (SR-BI). The aim of the present study was to define the effect of apoC-II and apoC-III on the uptake of LDL and HDL by HepG2 cells. Stable transformants were obtained with sense or antisense strategies that secrete 47-294% the normal level of apoC-II or 60-200% that of apoC-III. Different levels of secreted apoC-II or apoC-III had little effect on LDL and HDL protein degradation by HepG2 cells. However, compared to controls, cells under-expressing apoC-II showed a 160% higher capacity to selectively take up HDL-CE, while cells under-expressing apoC-III demonstrated 70 and 160% higher capacity to take up CE from LDL and HDL, respectively. In experiments conducted with exogenously added apoC-II or apoC-III, no significant effect was observed on lipoprotein-protein association/degradation; however, LDL-CE and HDL-CE selective uptake was significantly reduced in a dose-dependent manner. These results indicate that apoC-II and apoC-III inhibit CE-selective uptake.     

Development of biofilters to treat the pesticides wastes from spraying applications

Several studies carried out in Europe showed the importance of direct losses to the contamination of surface water by pesticides. These pesticides losses can occur at the farm site when the sprayer equipment is filled with the pesticide formulation (spills, overflowing, leaking) and during the clean-up (rinsing) of the sprayer after the treatment. In Belgium studies are carried out on biofilters to treat in an efficient way effluents containing pesticides. The biofilter substrate is elaborated from a homogenised mixture of local soil, chopped straw and peat or composted material, able to absorb or degrade the active substances. Biofilters consist in systems of 2 or 3 units depending on the spray equipment of the farmer and on the configuration of the farmyard. Each unit is made from a 1 m3 plastic container and the different units are stacked in a vertical pile and connected between them using plastic valves and pipes. Eight pilot systems were installed in March 2002 in seven farms and in one agricultural school, all selected in the loamy region of Belgium specialised in arable crops such as cereals, sugar beets and vegetables. The efficacy (yield) of the systems was determined by measuring the balance of the inputs and outputs of the pesticides. Results were expressed in percent of pesticide retained on the biofilters. The results obtained after two years with 5 tracer pesticides (atrazine, carbofuran, diuron, lenacil and simazine) brought on the biofilter installations are very satisfactory since the percentage of retention is generally higher than 95% of the amount applied. In the beginning of 2004, ten new pilot biofilters were installed in several farms or agricultural technical centres (producing cereals, sugar beets, potatoes, vegetables, fruits or ornamental plants), and in a municipal maintenance service. Some biofilters were installed in duplicate in order to compare the efficacy of different substrates. The efficacy of the biofilters was studied for the 5 classical tracer pesticides but also for other chemical classes of herbicides (sulfonylurea, aryloxyalcanoic acids, chloroacetanilides), insecticides (pyrethroids, carbamates) and fungicides (dicarboximides, phenylamides, triazoles and strobilurines). To monitor these pesticides in elutes and substrates, two analytical methods were developed, optimised and validated : the first one by Gas Chromatography with Mass Spectrometry Detection (GC-MS), and the second one by High Performance Liquid Chromatography with UV Diode Array Detection (HPLC-DAD). The micro-organisms activity in the substrate was also measured in some situations.     

Crystal structure of the YML079w protein from Saccharomyces cerevisiae reveals a new sequence family of the jelly-roll fold

We determined the three-dimensional crystal structure of the protein YML079wp, encoded by a hypothetical open reading frame from Saccharomyces cerevisiae to a resolution of 1.75 A. The protein has no close homologs and its molecular and cellular functions are unknown. The structure of the protein is a jelly-roll fold consisting of ten beta-strands organized in two parallel packed beta-sheets. The protein has strong structural resemblance to the plant storage and ligand binding proteins (canavalin, glycinin, auxin binding protein) but also to some plant and bacterial enzymes (epimerase, germin). The protein forms homodimers in the crystal, confirming measurements of its molecular mass in solution. Two monomers have their beta-sheet packed together to form the dimer. The presence of a hydrophobic ligand in a well conserved pocket inside the barrel and local sequence similarity with bacterial epimerases may suggest a biochemical function for this protein.     

Comparison of crystal structures of human type 3 3alpha-hydroxysteroid dehydrogenase reveals an "induced-fit" mechanism and a conserved basic motif involved in the binding of androgen

The aldo-keto reductase (AKR) human type 3 3alpha-hydroxysteroid dehydrogenase (h3alpha-HSD3, AKR1C2) plays a crucial role in the regulation of the intracellular concentrations of testosterone and 5alpha-dihydrotestosterone (5alpha-DHT), two steroids directly linked to the etiology and the progression of many prostate diseases and cancer. This enzyme also binds many structurally different molecules such as 4-hydroxynonenal, polycyclic aromatic hydrocarbons, and indanone. To understand the mechanism underlying the plasticity of its substrate-binding site, we solved the binary complex structure of h3alpha-HSD3-NADP(H) at 1.9 A resolution. During the refinement process, we found acetate and citrate molecules deeply engulfed in the steroid-binding cavity. Superimposition of this structure with the h3alpha-HSD3-NADP(H)-testosterone/acetate ternary complex structure reveals that one of the mobile loops forming the binding cavity operates a slight contraction movement against the citrate molecule while the side chains of many residues undergo numerous conformational changes, probably to create an optimal binding site for the citrate. These structural changes, which altogether cause a reduction of the substrate-binding cavity volume (from 776 A(3) in the presence of testosterone/acetate to 704 A(3) in the acetate/citrate complex), are reminiscent of the "induced-fit" mechanism previously proposed for the aldose reductase, another member of the AKR superfamily. We also found that the replacement of residues Arg(301) and Arg(304), localized near the steroid-binding cavity, significantly affects the 3alpha-HSD activity of this enzyme toward 5alpha-DHT and completely abolishes its 17beta-HSD activity on 4-dione. All these results have thus been used to reevaluate the binding mode of this enzyme for androgens.     

Lymphatic vessels and cancer

Survival and development of tumors depends on nutritional and respiratory biological events and exchanges ensured by blood and lymph. Tumor proliferation is associated with an increase in the vascular networks either near the tumor or intra-tumorally. Tumor tissues are able to increase their provisionment according to their needs while directing and optimizing the development of peri-tumoral vessels. The production of growth factors stimulating neo-formation of lymphatic vessels by cancer cells constitutes one of the adaptations responsible for metastatic propagation. During tumor development the lymphatic system is considered in many cases of cancer as the primary means of metastasis dissemination. The study of the lymphatic system setting and ways to block it are important points to consider in oncology.     

Radiological innovations in the screening and diagnosis of the inborn errors of metabolism

New metabolic diseases are regularly identified by a genetic or biochemical approach. Indeed, the metabolic diseases result from an enzymatic block with accumulation of a metabolite upstream to the block and deficit of a metabolite downstream. The characterization of these abnormal metabolites by MRI spectroscopy permitted to identify the deficient enzyme in two new groups of diseases, creatine deficiencies and polyol anomalies. Creatine deficiency is implicated in unspecific mental retardation. A low peak of creatine at MRI spectroscopy is evocating of creatine deficiency which is treatable by creatine administration. Deficiency of synthesis of polyols, metabolites on the pentose pathway, represent new described metabolic diseases with variable symptoms including a neurological distress, liver disease, splenomegaly, cutis laxa and renal insufficiency. The deficit of ribose-5-phosphate isomerase, one of the enzymes whose diagnosis is evoked in front of the accumulation of ribitol, arabitol and xylitol leads to a leucodystrophy in adults. This new deficit was highlighted by the identification of an abnormal peak in cerebral MRI-spectroscopy corresponding to the abnormal accumulation of polyols in brain. Congenital hyperinsulinism (HI) is characterized by profound hypoglycaemia related to inappropriate insulin secretion. Focal and diffuse forms of hyperinsulinism share a similar clinical presentation but their treatment is dramatically different. Until recently, preoperative differential diagnosis was based on pancreatic venous sampling, an invasive and technically demanding technique. Positron emission tomography (PET) after injection of [18F]Fluoro-L-DOPA has been evaluated for the preoperative differentiation between focal and diffuse HI, by imaging uptake of radiotracer and the conversion of [18F]Fluoro-L-DOPA into dopamine by DOPA decarboxylase. PET with [18F]Fluoro-L-DOPA has been validated as a reliable test to differentiate diffuse and focal HI and is now a major differential diagnosis tool in infantile hyperinsulinemic hypoglycaemia.     

Mutations of ousA alter the virulence of Erwinia chrysanthemi

A negative correlation was observed between the aggressiveness of several Erwinia chrysanthemi strains on potato tuber and their osmotic tolerance. The disruption of the ousA gene encoding the major osmoprotectant uptake system highly enhanced bacterial virulence on potato tubers. The ousA disruption also increased the maceration efficiency on potato tubers under anaerobic conditions. In the absence of oxygen, pectate lyase (Pel) production was significantly higher in the tissue macerated with the ousA- strain than with the wild type. Oxygen content is significantly different between infected and healthy tissues; therefore, ousA may be a contributory factor in the infection progression within the host. In minimal medium, ousA disruption enhanced Pel production and pelE expression only under micro-aerobiosis conditions. The effect on Pel was reversed by reintroduction of the ousA gene. The osmoprotectectants glycine betaine, proline betaine, and pipecolic acid are known to be taken up via OusA and to have an inhibitory effect on Pel production. However, their effects on Pel activity were not (glycine betaine) or only weakly (proline and pipecolic acid) affected by ousA disruption. Furthermore, no correlation was observed between their effects on Pel activities and their osmoprotection efficacies. The results demonstrate a relationship between E. chrysanthemi pathogenicity factors and the activity of ousA under low oxygen status. The evidence indicates that ousA and osmoprotectant effects on Pel are not linked to osmoregulation and that complex regulations exist between Pel production, ousA, and osmoprotection via compounds liberated during the plant infection.