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Objectif : Le travail de recherche engage par le Centre de recherche pour l’etude et l’observation des conditions de vie (CREDOC) a la demande de l’Institut national du cancer (INCa) visait a apporter un eclairage sur la maniere dont la question du maintien dans l’emploi se pose pour les acteurs de l’entreprise et comment elle s’inscrit dans les pratiques professionnelles.Methodes : La recherche a porte sur neuf etablissements de grandes entreprises en France et en Allemagne. Des entretiens y ont ete conduits aupres des acteurs intervenant dans les processus de retour a l’emploi.Resultats : Les obligations legales qui impliquent des actions de maintien dans l’emploi en France (obligation de reclassement, emploi de personnes handicapees, emploi des seniors), ne renvoient pas a un champ de pratiques unifie autour de l’objectif de maintien dans l’emploi dans l’entreprise. En Allemagne au contraire, l’articulation entre l’objectif de politique publique de maintien dans l’emploi et la gestion du personnel dans les etablissements se manifeste a travers un protocole defini par la loi, heritage de la Grande Guerre.Les etablissements francais se caracterisent par une gestion au cas par cas du maintien dans l’emploi, peu anticipee. Les situations favorables, ou la solidarite entre collegues joue a plein, soulignent en creux les processus discretionnaires a l’œuvre en l’absence de procedure, et dans un contexte d’exigence de productivite croissante.Conclusion : L’existence d’un protocole en Allemagne ne garantit pas le succes des actions de maintien dans l’emploi, mais elle permet de faire exister ce dernier comme une exigence a part entiere pour les acteurs de l’entreprise.Prat Organ Soins. 2011;42(1):19-26

Haptoglobin (Hpt), primarily synthesized by hepatocytes, can be produced in central nervous system (CNS) under inflammatory conditions. In fact, increased Hpt was found in Cerebrospinal Fluids from patients with neurodegenerative diseases. We previously reported that Hpt binds Apolipoprotein E (ApoE), and influences ApoE key roles in cholesterol homeostasis. ApoE is the major constituent of lipoproteins into CNS, and modulates Amyloid-beta (A-beta) deposition and clearance. A-beta is known to trigger an inflammatory response in the brain, which is involved in the pathogenesis of Alzheimer’s disease. Our aim was to verify whether Hpt affects the ApoE function in the regulation of A-beta accumulation in the brain. We report here that the abilities of Hpt and ApoE to bind A-beta are similar. In addition, our data demonstrate that Hpt enhances the binding of ApoE to A-beta, thus suggesting that the two proteins and A-beta might interact with each other to form a trimeric complex. Further, a SDS-stable complex between the two proteins (ApoE and Hpt) and A-beta is formed following incubation at 37°C, and A-beta aggregation is reduced when it is incubated in presence of Hpt and ApoE. Finally, we found that Hpt can bind astrocytes. In conclusion Hpt, as forming a trimeric complex with ApoE and A-beta, and binding astrocytes, might play a critical role, participating in the binding and clearance of A-beta, thus alleviating its toxicity and promoting its removal from the brain.

Haptoglobin (Hpt) is known to capture circulating free Haemoglobin (Hb), and bind apolipoprotein (Apo) A-I or E. We report here that Hb can be tightly bound by most of Hpt molecules (TB-Hpt, 80%), whereas loosely bound by a minor part of them (LB-Hpt, 20%). LB-Hpt amount was significantly increased (over 60%) in patients with acute coronary syndrome. LB-Hpt bound ApoA-I and ApoE worse than TB-Hpt (8- and 4-fold less, respectively), and did not affect their activity of stimulating the enzyme LCAT. LB-Hpt and TB-Hpt displayed comparable levels of nitrotyrosine residues, but differences in glycan chains. Changes in the LB-Hpt level might be associated to changes in Hpt functions.

Haptoglobin is an acute phase glycoprotein, secreted by hepatocytes and other types of cells including keratinocytes. Haptoglobin has been suggested to impair the immune response, inhibit gelatinases in the extracellular matrix and promote angiogenesis, but its role in psoriasis is obscure to date. Changes in haptoglobin glycan structure were observed in several diseases. The aim of our study was to investigate whether haptoglobin displays glycan variations in psoriasis. We found that the pattern of plasma haptoglobin glycoforms, following two-dimensional electrophoresis, exhibited significant quantitative differences in spot intensities between patients and controls. Quantitative and qualitative differences in glycan mass, between patients and controls, were found by mass spectrometry of glycopeptides from tryptic digests of protein isolated from both patients and controls. The number of distinct fucosylated glycoforms of peptides NLFLNHSENATAK and MVSHHNLTTGATLINEQWLLTTAK was higher in patients than in controls, but no fucosylated glycan was detected on peptide VVLHPNYSQVDIGLIK in either case. The number of peptides with distinct tri- and tetra-antennary glycans was higher in patients than in controls. Abundance or structure of specific glycans, which are present in haptoglobin from patients and different or missing in normal haptoglobin, might be associated to the disease activity.

The resistance of human colon adenocarcinoma cells to antineoplastic agents may be related to the high endogenous expression of stress proteins, including the family of heat shock proteins (HSPs). Recently, a quinone-based pentacyclic derivative, DTNQ-Pro, showed high cytotoxic activity in human colon carcinoma cell lines. The aim of the present study was to determine the precise cellular mechanisms of this cytotoxic action of DTNQ-Pro.

Aims: Apolipoprotein A-I (ApoA-I), the major apolipoprotein of the high density lipoprotein (HDL), plays key roles in the removal of cholesterol excess from peripheral cells, in a recognized anti-atherosclerotic process called “reverse cholesterol transport” (RTC)a. In detail, it stimulates cholesterol (C) efflux and ApoE secretion from macrophages, and enhances Lecithin-Cholesterol Acyl-Transferase (LCAT) activity. The acute phase protein Haptoglobin (Hpt) was found to inhibit RCT, as binding ApoA-I and preventing its stimulation on LCAT. This study aimed to find out whether Hpt influences essential ApoA-I stimulated mechanisms in RCT, such as cholesterol efflux, LCAT activity, and ApoE secretion from macrophages. Methods: Monocytes were isolated from human buffy coats, and kept for 6 days in culture for differentiation into macrophages. After treatment with ³H-C for 24 h, the cells were incubated with ApoA-I (0.04 mg/mL), or ApoA-I plus increasing Hpt amounts (0.1-0.75 mg/mL). After 16 h of incubation, homologous plasma was added as LCAT source. After further incubation for 16 h, supernatants were aliquoted from culture medium to measure their radioactivity content, representing the released C. Supernatant lipids were extracted and separated by TLC to measure the LCAT activity as conversion of unesterified ³H-C into C esters. Other macrophages, not treated with ³H-C, were incubated with ApoA-I (20 µg/mL), or ApoA-I plus Hpt (200 µg/mL). The supernatants of these cultures were collected after 16 h, and analyzed by western blotting to detect the amount of secreted ApoE. Results: Addition of Hpt to macrophages significantly (P

Objective: The aim of this work was to assess whether psoriasis is associated with phenotype prevalence and altered activity of Haptoglobin (Hpt). Background: Hpt is a plasma acute phase glycoprotein, displaying in humans three phenotypes. Phenotype prevalence or structure modification of Hpt was associated with several diseases. The Hpt main function is to bind and carry to the liver free hemoglobin for degradation and iron recycling. Hpt was recently found able to bind the apolipoprotein A-I (ApoA-I), thus impairing its stimulation on the activity of the enzyme lecithin-cholesterol acyl-transferase (LCAT) Study design: Hpt was isolated from patients with psoriasis vulgaris, and its activity in hemoglobin or ApoA-I binding and LCAT inhibition was compared with that of normal protein. Methods: Two affinity chromatography steps, the first using resin-coupled hemoglobin and the second anti-Hpt antibodies, were used to purify Hpt. The protein phenotype was assessed by electrophoresis. Binding experiments were performed by ELISA with stationary hemoglobin or ApoA-I, Hpt in solution, and anti-Hpt antibodies for detection of bound Hpt. Standard LCAT assays were carried out in the presence of Hpt purified from patients or healthy subjects. Results: Phenotype prevalence of Hpt in psoriasis was not found. After affinity chromatography by hemoglobin, albumin and ApoA-I were routinely found heavily contaminating only Hpt from normal subjects. Isolated Hpt from patients had lower activity than normal protein in both hemoglobin binding and LCAT inhibition. Conclusions: In psoriasis, Hpt displays some structure modification(s) which might be associated with the protein function in the disease.

AIMS: Apolipoprotein A1 (ApoA-I) is the major component of HDL and play crucial roles in all steps of RTC. ApoA-I is susceptible to oxidation which contribute to reduce its cardioprotective effects. It has been reported that hypoclorite can affect ApoA-I structure and the ability of this lipoprotein to promote ABCA1-dependent cholesterol efflux. The effects of Superoxide, a potent oxidant produced in blood vessel, on ApoA-I structure and function have never been reported. The aims of this work was to asses if the ApoA-I function to stimulate the cholesterol esterifying enzime (LCAT) was impaired by incubation with superoxide. Furthermore the Apo-A-I funtion to bind Haptoglobin, (Hpt) a plasma glicoprotein, was tested after incubation by superoxide. METHODS: The superoxide production was monitored by measuring the absorbance at 734 nm of a reaction mixture containing several concentration (10 50 100 µM) of KO2 and ABTS 450 µM and kept at 31°C. The binding experiments were performed by ELISA with coated ApoA-I, several concentration of KO2 (10 50 100 µM) was added, after Hpt in solution and anti-Hpt antibodies was used for detection of bound Hpt. Standard LCAT assay were carried out as published 1.ApoA-I-containing liposomes was incubated for 1h at 37°C in the presence and absence of superoxide. RESULTS: After affinity chromatography, in ELISA experiments, Hpt from patients binds less efficiently Hb than Hpt from controls. In particular, significant differences were found for samples with 0.1, 0.2 or 0.3µM Hpt (P

AIMS: Haptoglobin (Hpt) is a plasma acute phase glycoprotein produced mainly in the liver, but also synthesized in extrahepatic tissues including skin. The main function of circulating Hpt is to bind free haemoglobin (Hb) for scavenging iron, and Apolipoprotein A1 (ApoA1) for regulating the cholesterol reverse transport. The function of extrahepatic Hpt is not yet understood. Information on disease-associated isoforms of tissue-specific Hpt is available though poor. The aim of this work was to asses whether Hpt, in psoriasis, displays activity alteration which might be related to structure modification. METHODS: Two affinity chromatography steps, using resin-coupled Hb or anti-Hpt antibodies, were carried out to purify Hpt from human plasma. ELISA with Hb-coated wells was used to assay Hpt binding. After incubation of 0.05, 0.1, 0.2 or 0.3 μM Hpt, bound Hpt was detected by anti-Hpt antibodies. Assays of Lecithin-Cholesterol Acyl Transferase (LCAT) 1, the enzyme which esterifies cholesterol under ApoA1 stimulation, were carried out with Hpt purified from psoriatic patients or healthy subjects. RESULTS: Hpt from patients was found less efficient than control Hpt in Hb binding (30 to 60%). In particular, significant differences were found with 0.1, 0.2 or 0.3 μM Hpt (P

Aim: Cholesterol supplemented diet, in rabbit, triggers severe hypercholesterolemia which rapidly (less than 2 months) evolves to atherosclerosis. This work aimed to ascertain whether plasma factors playing key roles in the reverse cholesterol transport, such as the activity of the enzyme LCAT and the level of the apolipoprotein (Apo) A-I, are altered during the atherosclerosis rising and progression. Methods: New Zealand rabbits were fed with normal (N=6) or 2% cholesterol enriched (N=6) diet. Serum was collected at 0, 4 and 8 weeks, and analysed for Apo A-I level (by ELISA) and LCAT activity in vitro (assaying free 3H-cholesterol conversion into ester form) or in vivo (measuring by HPLC the amounts of both free and ester forms from isolated HDL). Results: Apo A1 was found decreased in the treated animals (t=4w: 65.5±3.4 vs 95.7±3.6 µg/ml in controls, P

INTRODUCTION: ApoA-1, the major apolipoprotein of the high density lipoprotein (HDL), plays a key role in the removal of cholesterol excess from peripheral cells in a recognized anti-atherosclerotic process called “reverse cholesterol transport (RCT)”. Among known ApoA-1 variants two, with one amino acid change (Zavalla, L159P; Zaragoza, L144R), are associated to poor RCT. Both mutations are located within the domain of ApoA1 (Leu141 – Ala164) binding Haptoglobin (Hpt) 1, a protein of the acute phase of inflammation. Hpt binding to ApoA1 was suggested to impair the role of HDL in RCT1. AIMS: Our aims were to study whether peptides containing the mutations of Zavalla (Pep Zav) or Zaragoza (Pep Zara) are able to bind Hpt or compete with native ApoA1, exposed on HDL, for binding Hpt. METHODS: The binding of biotinylated peptides to Hpt and the experiments of competition of acetylated peptides with HDL for Hpt binding were performed by ELISA, as previously published1. RESULTS: Both Pep-Zav and Pep-Zara bound to Hpt with efficiency similar to that of the peptide harbouring the native sequence of ApoA1 (pep 141–164). Nevertheless, when the same peptides were analysed for their ability to influence Hpt binding to HDL, only Pep Zav effectively displaced (50% at 15μM) Hpt from HDL (fig), as was the case for the pep. 141-164. CONCLUSIONS: We suggest that the L>P conversion, in the Zavalla variant, enhances hydrophilicity in ApoA1, improving the binding to Hpt. Therefore, as ApoA-1 binding to Hpt negatively affects RCT, individuals with Zavalla mutation might be exposed to higher risk of cardiovascular disease. 1.Spagnuolo M.S. et al (2005) J. Biol. Chem. 280, 1193-1198

Heat shock proteins (hsps) are ubiquitous families of proteins, found in all organisms studied so far. They are highly conserved across the species barrier and serve fundamental functions in cell physiology. The term 'heat shock' was adopted because of the early observation of the heat-inducible nature of these proteins, although, as it is now realized that they can be induced by a variety of stressful stimuli, it is probably more appropriate to call them 'stress proteins'. The nomenclature of many hsps, for example hsp90, hsp70 and hsp60, reflects the approximate molecular mass of hsps within each of these families. For many bacterial and parasitic infections, hsps were first recognized as immunodominant antigens on immunoblots of extracts from the organism probed with immune sera, or in T-cell proliferation assays. They have now been identified in a range of fungal pathogens, again often linked to an immune response. In this symposium, we review the association of hsps with humoral immunity to candidosis and aspergillosis, cellular immunity to histoplasmosis, and the identification of hsp70 in another dimorphic fungus, Paracoccidioides brasiliensis. Finally, the crucial role of the membrane in setting the temperature of the heat shock response in yeasts is discussed.

Background Interleukin-33 (IL-33) is a member of IL-1 superfamily and seems to be produced by endothelium and epithelial cells of exocrine glands, including salivary glands. IL-33 mediates its biological effects by interacting with its receptor ST2, which exists in transmembrane (ST2L) and soluble (sST2) form. In culture, this cytokine induces the production of IL-5 and IL-13, but not of IL-4, by Th2 lymphocytes; in vivo, it also seems to mediate the secrection of IFNg by Th1 lymphocytes. Moreover, it is implicated in remodeling of connective tissue and fibrosis process. Several studies demonstrated the immunopathogenetic role in Allergic Asthma, RCU, Crohn disease, RA, Systemic Lupus Erythematosus (SLE) and Systemic Sclerosis. Objectives To investigate the role of IL-33/ST2 in Sjogren’s Syndrome (SS), compared with two control populations: SLE patients and healty subjects. Methods 29 patients with SS, 19 with SLE (control group 1) and 20 healty subjects (control group 2) were enrolled from outpatients of Campus Bio-Medico University. All patients were subjected to clinical interview concerning activity, disease and drug history and they were evaluated for laboratory data (kidney and hepatic function, complete blood count, coagulation, complement fraction, immunoglobulins, CRP, ESR) and autoantibodies (ANA, ENA, SSA, SSB, anti-dsDNA, Sm, ReumaTest, Waaler-Rose). Disease activity and damage indexes were assessed at time of enrollment in this study with the use of SLEDAI and SLICC for SLE and SSDAI and SSDDI for SS, respectively. IL-33 and sST2 serum levels were determined by sandwich ELISA (Human IL-33 DuoSet, R&Systems, and Human ST2/IL-1R4 Quantikine ELISA kit, R&D systems). Results sST2 serum levels were significantly increased in SS and SLE patients compared with healthy controls (p=0.03 and p=0.04 respectively). No signficant difference was found in sST2 levels in SS and SLE patients. In SS population, a negative correlation between sST2 levels and hemoglobin values (p=0.001) and a positive one between sST2 levels and prednisone daily dose (p=0.04) were found. In SLE population, a positive correlation between sST2 levels and ESR (p=0.004) and RCP (p=0.002) were found. IL-33 serum levels were detectable in a few subjects (9/29 SS, 6/19 SLE, 4/20 healthy subjects), not correlated with sST2 levels. In SS population, a positive correlation between IL-33 and IgA levels (p=0.003), WBC count (p=0.008) and ANA positivity (p=0.04) was found; also in SLE population, a positive correlation between IL-33 levels and WBC count (p=0.008) was found. Conclusions Our data allow to hypothesize an implication of IL-33/ST2 pathway in immunopathogenesis of SS, peculiarly in active inflammation, as suggested by the relations with WBC count, IgA levels and the inverse correlation with hemoglobin values. Similarly to other studies, we detected IL-33 levels only in few subjects; this finding could be explained either by formation of receptor-cytokine complexes or by a negative feedback mechanism between IL-33 and ST2. References Schmitz J, Owyang A, Oldham E et al. IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2- associated cytokines. Immunity 2005;23:479-90. Mok MY, Huang FP, Ip WK et al. Serum levels of IL-33 and soluble ST2 and their association with disease activity in systemic lupus erythematosus. Rheumatology (Oxford) 2010;49:520-7. Disclosure of Interest None Declared

Heat shock protein (hsp) 70 from several microbes is antigenic in mammals. In this study we sequenced and expressed the gene encoding this protein from Histoplasma capsulatum to study its immunological activity. The deduced amino acid sequence of the gene demonstrated 71 and 76% identity to hsp7O from humans and Saccharomyces cerevisiae, respectively. A cDNA was synthesized by reverse transcription-PCR and was expressed in Escherichia coli. Recombinant protein reacted with a mouse monoclonal antibody raised against human hsp7O. Splenocytes from C57BL/6 mice immunized with recombinant hsp7O emulsified in adjuvant, but not yeast cells, reacted in vitro to the antigen. Recombinant hsp7O elicited a cutaneous delayed-type hypersensitivity response in mice immunized with protein or with viable yeast cells. Mice were injected with recombinant hsp7O and challenged intranasally with a sublethal inoculum of yeast cells. Vaccination did not confer protection in this model. Thus, recombinant hsp7O can induce a cell-mediated immune response but does not induce a protective response.

In order to establish whether tissues damaged by Chronic Periapical Pathology (CPP) of endodontic origin produced heat shock protein (HSP) capable of attracting reactive lymphocytes, paraffin sections of samples from the oral cavity of 10 patients with CPP were incubated with commercially available anti-HSP monoclonal antibodies. Antibodies were evidenced employing the alkaline phosphatase immunoenzymatic method (DAKO). HSP was found in the lamina propria infiltrated by lymphocytes, in six of the ten samples. These results suggest that HSP may be one of the lymphocyte recruiting factors in the damaged area and opens new possibilities for further research.

We have isolated and characterized the delta 9-desaturase gene (Ole1), which codes for a key enzyme involved in regulating membrane fluidity in animal cells and microorganisms, from two strains of Histoplasma capsulatum, one that is temperature-tolerant (G217B) and the other temperature-susceptible (Downs). These pathogenic fungi are dimorphic in that they undergo a morphologic transition from the mycelial to yeast-like form when the temperature of incubation is switched from 25 to 37 degrees C or when they infect a susceptible host. The coding sequences of the two genes, both containing an intron of 93 nucleotides, are virtually identical and analogous to the delta 9-desaturase gene of Saccharomyces cerevisiae and those of the rat, mouse and human. Ole1 transcription of the thermotolerant G217B and thermosensitive Downs strains is similar in yeast phase cells and during the temperature shift down from 34, 37, or 40 to 25 degrees C (yeast-to-mycelia transition). Nevertheless, the delta 9-desaturase gene is transcriptionally inactive in mycelia of G217B at 25 degrees C while it is actively transcribed in the Downs strain at the same temperature. These results are in agreement with the finding that membranes of the Downs strain have a higher level of oleic acid. The differential expression of delta 9-desaturase genes is discussed in relationship to differences in thermosensitivity in the fungal isolates and in regulating the level of expression of heat shock genes.

Early in the developmental period of microbiology, Pasteur first observed the phenomenon of dimorphism in fungi when he noticed that the bread mold Mucor grew as a filamentous mold aerobically on the surface of broth cultures but at the bottom of the flask where the environment was anaerobic it reproduced as budding yeast cells. Several infectious fungal pathogens of humans, namely Histoplasma capsulatum, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Sporothrix schenkii, and Coccidioides immitis change from a multicellular filamentous form to an unicellular morphology when they invade tissues. The ability of pathogenic fungi to assume a different shape is referred to as dimorphism. This phenomenon has intrigued clinicians, and medical mycologists since its discovery at the turn of the century. The ability of pathogens to initiate infection, invade host tissues and survive in mammalian hosts is critically linked to the induction of specific gene products. In dimorphic fungi, developmentally regulated gene expression is particularly important, since they may exist in phylogenetically distinct hosts with different body temperatures. Using Histoplasma capsulatum as a model to study parasite-host interactions at the biochemical and molecular level, my laboratory has attempted to relate the clinical spectrum of disease to natural variations in the characteristics of this organism and to adaptations it must make as a saprobe and a parasite. Histoplasma capsulatum is the etiologic agent of histoplasmosis, a respiratory infection that is world-wide in distribution. As a saprobe in soil it is mycelial, but it becomes a budding yeast as a parasite in susceptible hosts. These morphological phases can be reversibly reproduced in vitro by shifting the temperature from 25 degrees C, at which it is mycelial, to 37 degrees C, when it becomes a budding yeast. The process of mycelial-to-yeast conversion is of particular interest since it is triggered by an increase in temperature and conversion to virulence. Viable mycelial fragments and conidia become airborne and enter the pulmonary tract by inhalation after which the fungus rapidly disseminates to other organs. Progressive disseminated histoplasmosis along with candidiasis, cryptococcosis, and invasive aspergillosis are opportunistic fungal infections in patients who are immunosuppressed or otherwise debilitated. Importantly, they are diagnostic hallmarks of acquired immunodeficiency disease syndrome (AIDS). The clinical features of these infections and the genetic characteristics of the etiologic agents present unique parasite-host interactions that make them valuable research models to study. In the infected host, Histoplasma capsulatum encounters various environmental stresses to which it adapts by regulating the expression of specific genes.(ABSTRACT TRUNCATED AT 400 WORDS)

In the dimorphic fungus Histoplasma capsulatum the expression of heat shock genes is modulated by addition of fatty acids. Addition at 25 degrees C of saturated fatty acid (palmitic acid) to mycelia of H. capsulatum induced a significant increase in heat shock mRNAs transcription when cells were heat shocked. Conversely, treatments with unsaturated fatty acid (oleic acid) drastically reduced the level of heat shock gene transcription at 37 degrees C, and no detectable levels were measurable with 2 mM. Addition of saturated fatty acid induced a thermotolerant state and mitochondria retained ATPase activity coupled to electron transport under severe heat shock conditions and shortened the time required for mycelium-to-yeast phase transition. Conversely, addition of unsaturated fatty acids uncoupled mitochondrial electron transport and prolonged considerably the time required for phase transition at the same temperatures. A virulent strain, if treated with unsaturated fatty acid under condition in which no heat shock was detectable, lost its virulence probably as a consequence of decreased ability to adapt to the new living condition present in the host.

Temperature changes (and a variety of other stimuli) coordinately induce in all organisms active transcription of specific genes (heat shock genes). In dimorphic organisms the increase in the temperature of incubation has important consequences since temperature works as a signal for adaptation (induction of heat shock phenomenon) and triggers the phase transition. Infact, parasites and dimorphic organisms in general may exist in multiple hosts that have different body temperatures, and exhibit distinct stages during morphogenesis. Therefore, these organisms are particular cases of developmentally regulated heat shock response. An important characteristic of morphogenesis in some dimorphic fungi is that induction of one phase leads to the form found in infected tissue. Dimorphic pathogenic fungi have in fact the unique ability to colonize host tissue that is parallel to and may be intimately involved with the developmentally regulated morphological transition. In laboratory conditions, dimorphism in the human pathogenic fungus H.capsulatum is directly and reversibly controlled by temperature changes: mycelium (25°C) ⇔ yeast (37°) (Schwaiz, 1981; Maresca and Kobayashi, 1989). Therefore, in this fungus, temperature is a primaiy factor that controls phase transition. Since a sudden temperature change to 37°C induces the mycelial to yeast-phase transition, Lambowitz et al. suggested that the early event of the mycelium to yeast transition in H. capsulatum was a “heat shock response” which was followed by cell adaptation to the higher temperature (Lambowitz et al., 1983). Furthermore, it has been suggested that also in other diphasic organisms morphogenesis might be a by-product of the heat shock response (Maresca and Kobayashi,1989). However, in these system it is difficult to distinguish the effect of temparature on morpohogenesis from the induction of the heat shock responce.In fact, in H. capsulatum at temperatures at which heat shock proteins are not induced (heat shock mRNAs are not measurable)phase transition from mycelium to yeast does not occur(Maresca,unpublished).

International audience; Terminal deoxynucletidyl transferase (TdT) is overexpressed in some cancer types, where it might compete with pol μ during the mutagenic repair of double strand breaks (DSBs) through the nonhomologous end joining (NHEJ) pathway. Here we report the discovery and characterization of pyrrolyl and indolyl diketo acids that specifically target TdT and behave as nucleotide-competitive inhibitors. These compounds show a selective toxicity toward MOLT-4 compared to HeLa cells that correlate well with in vitro selectivity for TdT. The binding site of two of these inhibitors was determined by cocrystallization with TdT, explaining why these compounds are competitive inhibitors of the deoxynucleotide triphosphate (dNTP). In addition, because of the observed dual localization of the phenyl substituent, these studies open the possibility of rationally designing more potent compounds.

Improved diagnosis of psoriasis, by new biomarkers, is required for evaluating the progression rate of the disease and the response to treatment. Haptoglobin (Hpt), a glycoprotein secreted by hepatocytes and other types of cells including keratinocytes, was found with glycan changes in psoriasis and other diseases. We previously reported that Hpt isolated from plasma of psoriatic patients is more fucosylated than Hpt of healthy subjects. The aim of this study was to compare the glycosylation pattern of Hpt isolated from skin scales or plasma of patients with psoriasis with that of Hpt from cornified epidermal layer or plasma of healthy subjects. High performance liquid chromatography analysis of the glycans isolated from the protein backbone revealed that glycan patterns from skin and plasma of patients were similar, and mostly displayed quantitative rather than qualitative differences from normal pattern. Biotin-labeled lectins were used to evaluate quantitative differences in the glycoforms of Hpt from plasma and psoriatic skin scales. Hpt from skin and plasma of patients showed more fucosylated and branched glycans than Hpt from plasma of healthy subjects. Tryptic glycopeptides of Hpt were also analyzed by mass spectrometry, and a decreased amount of sialylated glycan chains was found in glycopeptides of skin Hpt, as compared with Hpt from plasma. High levels of glycans with fucosylated and tetra-antennary chains were detected on the peptide NLFLNHSENATAK from Hpt of psoriatic patients. Our data demonstrate that specific changes in glycan structures of Hpt, such as enhanced glycan branching and fucose content, are associated with psoriasis, and that differences between circulating and skin Hpt do exist. A lower extent of glycan fucosylation and branching was found in Hpt from plasma of patients in disease remission. Altered glycoforms might reflect changes of Hpt function in the skin, and could be used as markers of the disease.

Apolipoprotein A-I and Apolipoprotein E promote different steps of reverse cholesterol transport, including lecithin-cholesterol acyltransferase stimulation. Our aim was to study the changes in the levels of Apolipoprotein A-I, Apolipoprotein E and lecithin-cholesterol acyltransferase activity during atherosclerosis progression in rabbits. Quantitative echocardiographic parameters were analysed in order to evaluate, for the first time, whether atherosclerosis progression in rabbit is associated to apolipoproteins changes and alteration of indices of cardiac function, such as systolic strain and strain rate of the left ventricle. Atherosclerosis was induced by feeding rabbits for 8 weeks with 2% cholesterol diet. The HDL levels of cholesterol and cholesteryl esters were measured by HPLC. The lecithin-cholesterol acyltransferase activity was evaluated both ex vivo, as cholesteryl esters / cholesterol molar ratio, and in vitro. Apolipoproteins levels were analysed by ELISA. The HDL levels of cholesterol and cholesteryl esters increased, during treatment, up to 3.7 and 2.5 fold respectively compared to control animals. The lecithin-cholesterol acyltransferase activity in vitro was halved after 4 weeks. During cholesterol treatment, Apolipoprotein A-I level significantly decreased, whereas Apolipoprotein E concentration markedly increased. The molar ratio Apolipoprotein E/Apolipoprotein A-I was negatively correlated with the enzyme activity, and positively correlated with both increases in the intima-media thickness of common carotid wall and cardiac dysfunction signs, such as systolic strain and strain rate of the left ventricle. Further studies on humans or other animal models could be of help to confirm whether the ratio Apolipoprotein E/Apolipoprotein A-I can be considered as a biomarker of atherosclerosis progression.

A series of 3-acyl derivatives of the dihydronaphtho[2,3-b]thiophen-4,9-dione system were studied with respect to cytotoxicity and topoisomerase II inhibitory activity. These analogues were designed as electron-deficient anthraquinone analogues with potential intercalation ability. Derivatives 3-(diethylamino)-N-(4,9-dioxo-4,9-dihydronaphtho[2,3-b]thiophen-3-yl)propanamide (11m) and 3-(2-(dimethylamino)ethylamino)-N-(4,9-dioxo-4,9-dihydronaphtho[2,3-b]thiophen-3-yl)propanamide (11p) showed a high efficacy in cell lines that were highly resistant to treatment with doxorubicin, such as MDA-MB435 (melanoma), IGROV (ovarian), and SF-295 (glioblastoma) human cell lines. Both compounds inhibit topoisomerase II mediated relaxation of DNA, while only 11p incites arrest at the S phase in Caco-2 cells, inducing a delay of cell cycle progression and an increase of cell differentiation. The ability of these derivatives to modulate small heat shock proteins and cardiotoxicy effects was also explored. In addition, the DNA-binding properties of these compounds were investigated and discussed.

Here, we report the design of new analogues of spirooxoindolepyrrolidine nucleus as modulators of p53 activity. Compounds (3R,7aR)-6-(4-chlorobenzyl)-1H-spiro[imidazo[1,5-c]thiazole-3,3'-indoline]-2',5,7(6H,7aH)-trione (9c) and (3R,7aR)-5'-methyl-6-(3,4,5-trimethoxybenzyl)-1H-spiro[imidazo[1,5-c]thiazole-3,3'-indoline]-2',5,7(6H,7aH)-trione (10d) are the most potent compounds of this series, inhibiting cell growth of different human tumor cells at submicromolar and micromolar concentrations, respectively. Compound 9c induces apoptotic cell death in human melanoma cell line M14 at 24 h, while in the same condition, treatment with 10d showes a clear arrest at G2/M phase inducing delay of cell cycle progression. Possibly, these activities may be due to inhibition of p53-MDM2 interaction and subsequent p53 release and activation.

The resistance of human colon adenocarcinoma cells to antineoplastic agents may be related to the high endogenous expression of stress proteins, including the family of heat shock proteins (HSPs). Recently, a quinone-based pentacyclic derivative, DTNQ-Pro, showed high cytotoxic activity in human colon carcinoma cell lines. The aim of the present study was to determine the precise cellular mechanisms of this cytotoxic action of DTNQ-Pro.Using human colorectal carcinoma-derived Caco-2 cells as a model, we studied the effects of DTNQ-Pro on cellular viability and oxidative stress; HSP70 and HSP27 accumulation; and cell cycle, differentiation and apoptosis.Incubation of Caco-2 cells with DTNQ-Pro reduced cell growth and increased the levels of reactive oxygen species in mitochondria. After 48 h of treatment, cells surviving showed an increased expression of Mn-superoxide dismutase (SOD), nitric oxide production and membrane lipid peroxidation. Treatment with DTNQ-Pro decreased HSP70 expression, and redistributed HSP27 and vimentin within the cell. DTNQ-Pro down-regulated the expression of A and B cyclins with arrest of the cell cycle in S phase and increased cellular differentiation. A second treatment of Caco-2 cells with DTNQ-Pro induced cellular death by activation of the apoptotic pathway.DTNQ-Pro causes Caco-2 cell death by induction of apoptosis via inhibition of HSP70 accumulation and the intracellular redistribution of HSP27. These findings suggest the potential use of DTNQ-Pro in combination chemotherapy for colon cancer.

Temperatures above the optimal temperature range for plant growth and reproduction cause deleterious cellular damage, which in turn affects plant productivity. To relieve these effects, plants adapt to high temperature by activating a series of physiological and biochemical changes necessary to reestablish a new cellular homeostasis compatible with the increase in temperature. The genetic control of the heat shock (HS) response is quite complex and requires the activation of a network of genes, involved in the perception and transduction of the HS signal, which, in turn, trigger the up-regulation of other target genes. The induced genes code for proteins and enzymes (HS proteins, active oxygen detoxifying enzymes), playing a direct role in the protection of cellular and subcellular organelles or genes encoding enzymes involved in the biosynthesis of protective compatible compounds (sugars, polyols, betaines). Membrane lipid instauration, controlled by desaturase genes, is also a critical component of thermotolerance. This chapter covers the principal aspects of the plant HS response and the role of different class of genes in the acquisition of thermotolerance. The molecular breeding strategies currently available to alter genetically the level of the protective proteins, enzymes and molecules, that may ameliorate plant tolerance and productivity under high temperature stress, will be also discussed.