Computer-predicted peptides that mimic discontinuous epitopes on the A2 domain of factor VIII.

Development of antibodies (Abs) against factor VIII (FVIII) is a severe complication of haemophilia A treatment. Recent publications suggest that domain specificity of anti-FVIII antibodies, particularly during immune tolerance induction (ITI), might be related to the outcome of the treatment. Obtaining suitable tools for a fine mapping of discontinuous epitopes could thus be helpful. The aim of this study was to map discontinuous epitopes on FVIII A2 domain using a new epitope prediction functionality of the PEPOP bioinformatics tool and a peptide inhibition assay based on the Luminex technology. We predicted, selected and synthesized 40 peptides mimicking discontinuous epitopes on the A2 domain of FVIII. A new inhibition assays using Luminex technology was performed to identify peptides able to inhibit the binding of anti-A2 Abs to A2 domain. We identified two peptides (IFKKLYHVWTKEVG and LYSRRLPKGVKHFD) able to block the binding of anti-A2 allo-antibodies to this domain. The three-dimensional representation of these two peptides on the A2 domain revealed that they are localized on a limited region of A2. We also confirmed that residues 484-508 of the A2 domain define an antigenic site. We suggest that dissection of the antibody response during ITI using synthetic peptide epitopes could provide important information for the management of patients with inhibitors.

ß-Alkylated oligomaltosides as new alternative preservatives: antimicrobial activity, cytotoxicity and preliminary investigation of their mechanism of action.

AIM: The need for developing efficient and safe alternatives to parabens has been growing in the cosmetic and pharmaceutical industries. To this end, the antimicrobial activities and safety of methyl-ß-d-maltoside, methyl-ß-d-maltotrioside and their dodecyl homologues were investigated. METHODS AND RESULTS: Antimicrobial activities of methyl- and dodecyl-ß-d-oligomaltoside have been tested on European pharmacopoeia reference strains. When effective, minimal inhibitory concentrations ranged from 32 to 1024 mg l(-1) . Methyl derivatives exhibited greater antibacterial properties, while their dodecyl homologues were more active on fungal strains. To elucidate the mechanism of action of methyl compounds, enzymatic inhibition assays of key maltose metabolism enzymes have been conducted. Methyl-ß-maltotrioside (MeG3) was found to be effective in inhibiting microbial glucoamylase and α-amylase. MeG3 and dodecyl-ß-maltotrioside cytotoxicity were also checked on HepG2 cells and were found to be low, compared with benzalkonium chloride or parabens. CONCLUSION: Methyl- and dodecyl-ß-maltoside or maltotrioside were found to be active against reference strains used for preservatives efficacy testing. Methyl derivatives could act through an interesting inhibition of the microbial enzymatic metabolism. SIGNIFICANCE AND IMPACT OF THE STUDY: Given their very simple chemical structure, low toxicity and good antimicrobial activity, methyl-ß-d-oligomaltosides could represent a valuable alternative to currently used preservatives such as parabens.

In vitro approaches to study actin and microtubule dependent cell processes.

Actin and microtubules represent complex polymer systems that play essential roles during many cellular processes including chromosome segregation, cytokinesis and motility. The dynamic nature of actin and microtubules together with their regulation by a myriad of proteins makes their study both fascinating and challenging. Over the past few years there has been an increasing move towards development of in vitro systems to facilitate the elucidation of the molecular basis of actin and microtubule dependent cell processes. This review focuses on some of the recent developments using in vitro assays to dissect the cellular role of the actin and microtubule cytoskeleton.

A complex of N-WASP and WIP integrates signalling cascades that lead to actin polymerization.

Wiskott-Aldrich syndrome protein (WASP) and N-WASP have emerged as key proteins connecting signalling cascades to actin polymerization. Here we show that the amino-terminal WH1 domain, and not the polyproline-rich region, of N-WASP is responsible for its recruitment to sites of actin polymerization during Cdc42-independent, actin-based motility of vaccinia virus. Recruitment of N-WASP to vaccinia is mediated by WASP-interacting protein (WIP), whereas in Shigella WIP is recruited by N-WASP. Our observations show that vaccinia and Shigella activate the Arp2/3 complex to achieve actin-based motility, by mimicking either the SH2/SH3-containing adaptor or Cdc42 signalling pathways to recruit the N-WASP-WIP complex. We propose that the N-WASP-WIP complex has a pivotal function in integrating signalling cascades that lead to actin polymerization.

Defective membrane repair machinery impairs survival of invasive cancer cells.

Cancer cells are able to reach distant tissues by migration and invasion processes. Enhanced ability to cope with physical stresses leading to cell membrane damages may offer to cancer cells high survival rate during metastasis. Consequently, down-regulation of the membrane repair machinery may lead to metastasis inhibition. We show that migration of MDA-MB-231 cells on collagen I fibrils induces disruptions of plasma membrane and pullout of membrane fragments in the wake of cells. These cells are able to reseal membrane damages thanks to annexins (Anx) that are highly expressed in invasive cancer cells. In vitro membrane repair assays reveal that MDA-MB-231 cells respond heterogeneously to membrane injury and some of them possess a very efficient repair machinery. Finally, we show that silencing of AnxA5 and AnxA6 leads to the death of migrating MDA-MB-231 cells due to major defect of the membrane repair machinery. Disturbance of the membrane repair process may therefore provide a new avenue for inhibiting cancer metastasis.

Thermo-optical dynamics in an optically pumped Photonic Crystal nano-cavity.

Linear and non-linear thermo-optical dynamical regimes were investigated in a photonic crystal cavity. First, we have measured the thermal relaxation time in an InP-based nano-cavity with quantum dots in the presence of optical pumping. The experimental method presented here allows one to obtain the dynamics of temperature in a nanocavity based on reflectivity measurements of a cw probe beam coupled through an adiabatically tapered fiber. Characteristic times of 1.0+/-0.2 micros and 0.9+/-0.2 micros for the heating and the cooling processes were obtained. Finally, thermal dynamics were also investigated in a thermo-optical bistable regime. Switch-on/off times of 2 micros and 4 micros respectively were measured, which could be explained in terms of a simple non-linear dynamical representation.

The Saccharomyces cerevisiae actin-related protein Arp2 is involved in the actin cytoskeleton.

Arp2p is an essential yeast actin-related protein. Disruption of the corresponding ARP2 gene leads to a terminal phenotype characterized by the presence of a single large bud. Thus, Arp2p may be important for a late stage of the cell cycle (Schwob, E., and R.P. Martin, 1992. Nature (Lond.). 355:179-182). We have localized Arp2p by indirect immunofluorescence. Specific peptide antibodies revealed punctate staining under the plasma membrane, which partially colocalizes with actin. Temperature-sensitive arp2 mutations were created by PCR mutagenesis and selected by an ade2/SUP11 sectoring screen. One temperature-sensitive mutant that was characterized, arp2-H330L, was osmosensitive and had an altered actin cytoskeleton at a nonpermissive temperature, suggesting a role of Arp2p in the actin cytoskeleton. Random budding patterns were observed in both haploid and diploid arp2-H330L mutant cells. Endocytosis, as judged by Lucifer yellow uptake, was severely reduced in the mutant, at all temperatures. In addition, genetic interaction was observed between temperature-sensitive alleles arp2-H330L and cdc10-1. CDC10 is a gene encoding a neck filament-associated protein that is necessary for polarized growth and cytokinesis. Overall, the immunolocalization, mutant phenotypes, and genetic interaction suggest that the Arp2 protein is an essential component of the actin cytoskeleton that is involved in membrane growth and polarity, as well as in endocytosis.

Reactivity profile of anti-factor VIII antibodies with designed synthetic peptides mimicking epitopes of the C2 and a1 domains.

Antibodies (Abs) that block factor VIII (FVIII) activity occur in hemophilia A patients treated with FVIII replacement therapy and severely impair treatment. In this work, we designed and synthesized ten peptides whose sequences are found in putative epitopes at the surface of a1 and C2 domains of the FVIII molecule. These peptides were screened for their ability to inhibit the binding of anti-FVIII Abs from plasmas of hemophilia A patients to FVIII. All peptides were efficient in inhibiting anti-FVIII Abs in plasma from patients with inhibitors, with however different efficiencies. It was found that each tested patient's plasma had a different profile of reactivity with peptides, consistent with an individual anti-FVIII Ab specificity. The profile of recognized peptides was also changing during the treatment of the patients. Three peptides were used in an affinity chromatography assay to attempt to remove anti-FVIII Abs from patients' plasma. Anti-FVIII IgGs were significantly captured by the peptide-Sepharose affinity matrixes as assessed by enzyme-linked immunosorbent assay. However, due to the low level of Abs in the plasma samples, other methods (Chromogenic and Bethesda assays) were not sensitive enough to properly detect the reduction of inhibitors.

Tyrosine phosphorylation is required for actin-based motility of vaccinia but not Listeria or Shigella.

Studies of the actin-based motility of pathogens have provided important insights into the events occurring at the leading edge of motile cells [1] [2] [3]. To date, several actin-cytoskeleton-associated proteins have been implicated in the motility of Listeria or Shigella: vasodilator-stimulated phosphoprotein (VASP), vinculin and the actin-related protein complex of Arp2 and Arp3 [4] [5] [6] [7]. To further investigate the underlying mechanism of actin-tail assembly, we examined the localization of components of the actin cytoskeleton including Arp3, VASP, vinculin and zyxin during vaccinia, Listeria and Shigella infections. The most striking difference between the systems was that a phosphotyrosine signal was observed only at the site of vaccinia actin-tail assembly. Micro-injection experiments demonstrated that a phosphotyrosine protein plays an important role in vaccinia actin-tail formation. In addition, we observed a phosphotyrosine signal on clathrin-coated vesicles that have associated actin-tail-like structures and on endogenous vesicles in Xenopus egg extracts which are able to nucleate actin tails [8] [9]. Our observations indicate that a host phosphotyrosine protein is required for the nucleation of actin filaments by vaccinia and suggest that this phosphoprotein might be associated with cellular membranes that can nucleate actin.

Demonstration of air-guided quantum cascade lasers without top claddings.

We report on quantum cascade lasers employing waveguides based on a predominant air confinement mechanism in which the active region is located immediately at the device top surface. The lasers employ ridge-waveguide resonators with narrow lateral electrical contacts only, with a large, central top region not covered by metallization layers. Devices based on this principle have been reported in the past; however, they employed a thick, doped top-cladding layer in order to allow for uniform current injection. We find that the in-plane conductivity of the active region - when the material used is of high quality - provides adequate electrical injection. As a consequence, the devices demonstrated in this work are thinner, and most importantly they can simultaneously support air-guided and surface-plasmon waveguide modes. When the lateral contacts are narrow, the optical mode is mostly located below the air-semiconductor interface. The mode is predominantly air-guided and it leaks from the top surface into the surrounding environment, suggesting that these lasers could be employed for surface-sensing applications. These laser modes are found to operate up to room temperature under pulsed injection, with an emission spectrum centered around l (1/4) 7:66 mum.

The yeast actin-related protein Arp2p is required for the internalization step of endocytosis.

The Saccharomyces cerevisiae actin-related protein Arp2p is an essential component of the actin cytoskeleton. We have tested its potential role in the endocytic and exocytic pathways by using a temperature-sensitive allele, arp2-1. The fate of the plasma membrane transporter uracil permease was followed to determine whether Arp2p plays a role in the endocytic pathway. Inhibition of normal endocytosis as revealed by maintenance of active uracil permease at the plasma membrane and strong protection against subsequent vacuolar degradation of the protein were observed in the mutant at the restrictive temperature. Furthermore, arp2-1 cells accumulated ubiquitin-permease conjugates, formed prior to internalization. These effects were also visible at permissive temperature, whereas the actin cytoskeleton appeared to be normally polarized. The soluble hydrolase carboxypeptidase Y and the lipophilic dye FM 4-64 were targeted normally to the vacuole in arp2-1 cells. Thus, Arp2p is required for internalization but does not play a major role in later steps of endocytosis. Synthetic lethality was demonstrated between arp2-1 and the endocytic mutant end3-1, suggesting participation of Arp2p and End3p in the same process. Finally, no evidence for a major defect in secretion was apparent; invertase secretion and delivery of uracil permease to the plasma membrane were unaffected in arp2-1 cells.

The Saccharomyces cerevisiae homologue of human Wiskott-Aldrich syndrome protein Las17p interacts with the Arp2/3 complex.

Yeast Las17 protein is homologous to the Wiskott-Aldrich Syndrome protein, which is implicated in severe immunodeficiency. Las17p/Bee1p has been shown to be important for actin patch assembly and actin polymerization. Here we show that Las17p interacts with the Arp2/3 complex. LAS17 is an allele-specific multicopy suppressor of ARP2 and ARP3 mutations; overexpression restores both actin patch organization and endocytosis defects in ARP2 temperature-sensitive (ts) cells. Six of seven ARP2 ts mutants and at least one ARP3 ts mutant are synthetically lethal with las17Delta ts confirming functional interaction with the Arp2/3 complex. Further characterization of las17Delta cells showed that receptor-mediated internalization of alpha factor by the Ste2 receptor is severely defective. The polarity of normal bipolar bud site selection is lost. Las17-gfp remains localized in cortical patches in vivo independently of polymerized actin and is required for the polarized localization of Arp2/3 as well as actin. Coimmunoprecipitation of Arp2p with Las17p indicates that Las17p interacts directly with the complex. Two hybrid results also suggest that Las17p interacts with actin, verprolin, Rvs167p and several other proteins including Src homology 3 (SH3) domain proteins, suggesting that Las17p may integrate signals from different regulatory cascades destined for the Arp2/3p complex and the actin cytoskeleton.

Identification and Quantification of Polymyxa graminis f. sp. temperata and P. graminis f. sp. tepida on Barley and Wheat.

Polymyxa graminis f. sp. temperata and P. graminis f. sp. tepida are distinguished on the basis of their specific ribosomal DNA sequences. In order to evaluate whether or not host specialization is associated with the special form, the occurrence of infection of both forms on barley and wheat was studied. P. graminis inocula were obtained from soils collected in Belgium and France. Their ribotypes were characterized using molecular tools specific to P. graminis f. sp. temperata or P. graminis f. sp. tepida such as restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR)-amplified rDNA, nested and multiplex PCR. Both special forms were found in each country and coexisted in some soils. The host specificity of P. graminis special forms for barley and wheat was studied from two soils collected at Gembloux (Belgium) and Chambon-sur-Cisse (France), each infested by bymo- and furoviruses. P. graminis f. sp. temperata is more frequent on barley and P. graminis f. sp. tepida on wheat. Furthermore, the quantification of each form on barley and wheat by two separated real-time quantitative PCR assays confirms the observations on the vector specialization. These results suggest a certain but not exclusive host specificity of P. graminis special forms.

A certain but non-exclusive association between Polymyxa graminis special forms and cereals.

Polymyxo graminis, a ubiquitous plasmodiophorid obligate root endoparasite, is recognized as the vector of about 15 viruses on cereals and groundnut in temperate and tropical areas. Within the species, five special forms have been distinguished on the basis of specific ribotypes. Three of them occur in tropical areas: P. graminis f.sp. colombiana on rice, P. graminis f.sp. subtropicalis on cereals cropped in the tropics such as maize, pearl millet and sorghum but also on barley and/or wheat, and P. graminis f.sp. tropicalis mainly on maize, pearl millet and sorghum. Their particular host ranges distinguish them significantly from P. graminis f.sp. temperata and P. graminis f.sp. tepida found in temperate areas on barley and wheat. In order to assess whether these special forms commonly infect these cereals, barley and wheat plants were grown under controlled conditions on two soils from Belgium and France and both infested by P. graminis f.sp. temperata and P. graminis f.sp. tepida. The infection of each cereal species by each form was quantified by real-time quantitative PCR with specific primers and Taqman probes. The infection of P. graminis f.sp. temperata was significantly higher on barley than on wheat, whereas the quantities of P. graminis f.sp. tepida on wheat were higher than on barley. These results show that the distinction between these special forms, based on the ribotype, reflects differences in ecological features.

Barley yellow mosaic virus is overcoming RYM4 resistance in Belgium.

Barley yellow mosaic virus (BaYMV) is the causal agent of a soil-borne systemic mosaic disease on barley. It has been reported in Belgium since the 1980s. The control of this disease is managed almost exclusively through the use of resistant varieties. The resistance of most commercial barley cultivars grown in Europe is conferred mainly by a single recessive gene, rym4. This monogenic resistance provides immunity against BaYMV pathotype 1 and has been mapped on barley chromosome 3HL and shown to be caused by mutations in the translation initiation factor eIF4E. Another pathotype, BaYMV pathotype 2, which appeared in the late 1980s (in Belgium, in the early 1990s), is able to overcome the rym4-controlled resistance. Until recently, this pathotype remained confined to specific locations. During a systematic survey in 2003, mosaic symptoms were observed only on susceptible barley cultivars collected in Belgian fields. BaYMV was detected by ELISA and RT-PCR on the susceptible cultivars and only by RT-PCR on the resistant cultivars. In 2004, mosaic symptoms were observed on susceptible and resistant cultivars. BaYMV was detected by ELISA and RT-PCR on both cultivars. In addition to developing RT-PCR methods for detecting and identifying BaYMV and Barley mild mosaic virus (BaMMV), an RT-PCR targeting the VPg/NIa viral protein part of the genome, known to discriminate the two BaYMV pathotypes, was set up to accurately identify the pathotype(s) now present in Belgium. The sequences from the generated amplicons revealed the single nucleotide substitution resulting in an amino acid change from lysine to asparagine specific to BaYMV pathotype 2. The possible reasons for the change in the BaYMV pathotype situation in Belgium, such as climatic change or a progressive build-up of soil inoculum potential, will be discussed, as well as the use of eIF4E-based resistance.

Widespread Occurrence of Wheat spindle streak mosaic virus in Belgium.

In order to assess the occurrence of Wheat spindle streak mosaic virus (WSSMV) in Belgium, a reverse-transcription polymerase chain reaction (RT-PCR) was developed, targeting WSSMV isolates from Canada, France, Germany, Italy, and the United States. The primers also were designed for virus quantification by real-time RT-PCR with SYBR-Green. No cross-reaction with soilborne cereal viruses such as Barley mild mosaic virus, Barley yellow mosaic virus, Soilborne cereal mosaic virus, and Soil-borne wheat mosaic virus was observed. The RT-PCR and real-time quantitative RT-PCR allowed a more sensitive detection of WSSMV than enzymelinked immunosorbent assay. The incidence of WSSMV in Belgium was evaluated using a bioassay with wheat cvs. Cezanne and Savannah and rye cv. Halo, grown in 104 Belgian soils. The presence of WSSMV was detected from plants grown in 32% of the soils. The RT-PCR methods developed here, combined with large sampling, allowed WSSMV to be detected for the first time in Belgium. The real-time quantitative RT-PCR was developed as a tool for evaluating the resistance to WSSMV by quantifying the virus concentration in wheat cultivars.

Effect of adrenalin, adrenochrome, and adrenolutin on connexin proteins in the cardiovasculature.

Reported myocardial pathology resulting from increased levels of catecholamines in vivo has led us to investigate the effect of adrenalin on the gap junction proteins connexin 40 (Cx40) and Cx43 and the possible relationship to vascular toxicity. Adrenalin and its known metabolites, adrenochrome and adrenolutin, were used in this study. Utilizing the A7r5 rat aortic cell line, we evaluated the effects of adrenalin, adrenochrome, and adrenolutin on the expression and function of connexin 40 and 43 that are present in both cardiac and vascular tissues.

Alternative splicing of MLH1 messenger RNA in human normal cells.

The hMLH1 protein, composed of 756 amino acids, is the human homologue of the bacterial DNA mismatch repair protein MutL, and germ line mutations of the hMLH1 gene have been identified in kindreds with hereditary nonpolyposis colorectal cancer. We have detected three alternatively spliced forms of hMLH1 mRNA in normal lymphocytes and tissues. One of the spliced forms lacks the coding region of hMLH1 from codons 227 to 295 and the two other transcripts are predicted to encode two truncated proteins retaining the 264 and 226 N-terminal amino acids of hMLH1, respectively. The biological significance of this alternative splicing remains to be established.

Identification of novel germline hMLH1 mutations including a 22 kb Alu-mediated deletion in patients with familial colorectal cancer.

We analyzed the hMLH1 gene in 17 unrelated families with putative hereditary nonpolyposis colorectal cancer. The complete hMLH1 cDNA was amplified in one step, and after a second amplification, four overlapping segments were directly sequenced. We detected, in five families that did not meet the complete Amsterdam criteria, five alterations, including a double-base change resulting in a missense mutation (Lys-618-Ala), a splicing mutation affecting the intron 4 splice acceptor site, a 2-bp deletion at codon 726, a 7-bp deletion at codon 626, and a deletion of exons 13-16. The latter alteration was shown to result from a 22-kb genomic deletion due to a homologous recombination between Alu repeats located in introns 12 and 16. The detection of five germline hMLH1 mutations in five families that only partially fulfilled the Amsterdam criteria shows that these criteria do not allow the identification of all familial colorectal cancers due to mutations of the mismatch repair genes. The numerous Alu repeats present within the hMLH1 gene and the observation of large genomic deletions suggest that (a) Alu-mediated deletions might frequently be involved in hMLH1 inactivation, and (b) reverse transcription-PCR analysis, which allows the amplification of the entire coding region of the hMLH1 gene in one step, might be the most appropriate method for the detection of hMLH1 alterations.

Identification of human p53 mutations with differential effects on the bax and p21 promoters using functional assays in yeast.

Recent studies have suggested that a rare class of p53 mutants found in tumours has a subtle transcriptional defect affecting bax induction but not p21 induction. We have therefore developed simple functional assays in yeast which can be used to identify these mutants. Analysis of 51 different mutations observed in human tumours showed that all mutants tested scored as mutant with the bax reporter strain but nine scored as wild-type with the p21 reporter strain. These results, which can be explained by the lower affinity of the p53 protein for the bax site, may suggest that p21 is not the key target of p53 mutations in tumours. Since p21 status has recently been shown to modulate the chemotherapeutic and radiotherapeutic sensitivities of cancerous cells, the functional assays described here may have important clinical implications.