Anti-biofilm activity of pseudoalteromonas haloplanktis tac125 against staphylococcus epidermidis biofilm: Evidence of a signal molecule involvement?

Staphylococcus epidermidis is recognized as cause of biofilm-associated infections and interest in the development of new approaches for S. epidermidis biofilm treatment has increased. In a previous paper we reported that the supernatant of Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 presents an anti-biofilm activity against S. epidermidis and preliminary physico-chemical characterization of the supernatant suggested that this activity is due to a polysaccharide. In this work we further investigated the chemical nature of the anti-biofilm P. haloplanktis TAC125 molecule. The production of the molecule was evaluated in different conditions, and reported data demonstrated that it is produced in all P. haloplanktis TAC125 biofilm growth stages, also in minimal medium and at different temperatures. By using a surface coating assay, the surfactant nature of the anti-biofilm compound was excluded. Moreover, a purification procedure was set up and the analysis of an enriched fraction demonstrated that the anti-biofilm activity is not due to a polysaccharide molecule but that it is due to small hydrophobic molecules that likely work as signal. The enriched fraction was also used to evaluate the effect on S. epidermidis biofilm formation in dynamic condition by BioFlux system.

Plasma proteasome level is a reliable early marker of malignant transformation of liver cirrhosis.

BACKGROUND: Proteasomes are the main non-lysosomal proteolytic structures which regulate crucial cellular processes. Circulating proteasome levels can be measured using an ELISA test and can be considered as a tumour marker in several types of malignancy. Given that there is no sensitive marker of hepatocellular carcinoma (HCC) in patients with cirrhosis, we measured plasma proteasome levels in 83 patients with cirrhosis (33 without HCC, 50 with HCC) and 40 controls. METHODS AND RESULTS: Patients with HCC were sub-classified into three groups according to tumour mass. alpha-Fetoprotein (AFP) was also measured. Plasma proteasome levels were significantly higher in patients with HCC compared to controls (4841 (SEM 613) ng/ml vs 2534 (SEM 187) ng/ml; p<0.001) and compared to patients with cirrhosis without HCC (2077 (SEM 112) ng/ml; p<0.001). This difference remained significant when the subgroup of patients with low tumour mass (proteasome level 3970 (SEM 310) ng/ml, p<0.001) was compared to controls and patients with cirrhosis without HCC. Plasma proteasome levels were independent of the cause of cirrhosis and were weakly correlated with AFP levels. With a cut-off of 2900 ng/ml, diagnostic specificity for HCC was 97% with a sensitivity of 72%, better than results obtained with AFP. Diagnostic relevance of plasma proteasome measurement was also effective in low tumour mass patients (sensitivity 76.2% vs 57.1% for AFP). CONCLUSION: The plasma proteasome level is a reliable marker of malignant transformation in patients with cirrhosis, even when there is a low tumour mass.

Proteolysis by calpains: a possible contribution to degradation of p53.

p53 is a short-lived transcription factor that is frequently mutated in tumor cells. Work by several laboratories has already shown that the ubiquitin-proteasome pathway can largely account for p53 destruction, at least under specific experimental conditions. We report here that, in vitro, wild-type p53 is a sensitive substrate for milli- and microcalpain, which are abundant and ubiquitous cytoplasmic proteases. Degradation was dependent on p53 protein conformation. Mutants of p53 with altered tertiary structure displayed a wide range of susceptibility to calpains, some of them being largely resistant to degradation and others being more sensitive. This result suggests that the different mutants tested here adopt slightly different conformations to which calpains are sensitive but that cannot be discriminated by using monoclonal antibodies such as PAb1620 and PAb240. Inhibition of calpains by using the physiological inhibitor calpastatin leads to an elevation of p53 steady-state levels in cells expressing wild-type p53. Conversely, activation of calpains by calcium ionophore led to a reduction of p53 in mammalian cells, and the effect was blocked by cell-permeant calpain inhibitors. Cotransfection of p53-null cell lines with p53 and calpastatin expression vectors resulted in an increase in p53-dependent transcription activity. Taken together, these data support the idea that calpains may also contribute to the regulation of wild-type p53 protein levels in vivo.

c-fos mRNA instability determinants present within both the coding and the 3' non coding region link the degradation of this mRNA to its translation.

The instability of oncogenic mRNA such as c-fos mRNA is controlled in cis by sequences present in both the coding and the 3' untranslated regions (3'UTR). The latter contains AU-rich elements (ARE) which, depending on the cellular context, mediate either their rapid degradation or inhibit their translation. These observations, along with the known increase of the life spans of many unstable mRNA promoted by inhibitors of protein synthesis, raise the possibility that both processes are linked. To investigate further the putative involvement of translation in both coding region and ARE-mediated rapid decay of c-fos mRNA, we designed an expression vector based on the use of the ferritin mRNA iron regulatory element (IRE). The latter structure links translation to intracellular iron concentration when inserted at the proper location within the 5'UTR. Rapid degradation of a beta-globin/c-fos 3'UTR construct was prevented by Desferrioxamine, an iron chelator, and facilitated by ferric ammonium citrate or hemin, while stability of other mRNAs not containing the IRE or the ARE were unchanged. The same conclusion was reached when the stability of a c-fos mRNA devoid of ARE was assessed in function of iron availability.

Differential sensitivity of FOS and JUN family members to calpains.

Degradation of c-fos protein (c-FOS) in the cytoplasm is very rapid in vivo and constitutes a crucial regulation of the nuclear steady-state level through the control of the amount of full-length molecules available for nuclear transport. Using cytoplasmic extracts from various origins, we report herein that c-FOS degradation can be initiated in a calcium-dependent manner which involves cysteine proteases called milli- and micro-calpain. Interestingly, FOS-B, a member of the fos multigene family, as well as all members of the jun family (JUN-B, c-JUN and JUN-D) are also sensitive to calpains albeit to different extents. FRA-2, which is a c-FOS-related protein, is resistant to micro- but not to milli-calpain whereas FRA-1, another member of the fos family, is resistant to both proteases. Given the fact that a work by others (Hiraï et al., 1991b) suggests that calpains can be involved in c-FOS and c-JUN degradation in vivo, our observations raises the possibility of a novel contribution to the regulation of AP-1 transcription complex activity through a differential control of the steady-state level of some of its components that involves calpains.

The effect of iron deficiency anemia in the rat on catecholamine levels and heart morphology.

Noradrenaline levels and heart morphology were studied in animals with severe iron deficiency anaemia. This condition was induced by feeding rats an iron-deficient diet for 30 days from the time of weaning. Anaemia was indicated by the lowering of blood haemoglobin levels. Statistically significant decreases in myocardial noradrenaline levels associated with cardiac hypertrophy, as revealed by increased wet heart weight and increased size of cardiac muscle cells, were observed in anaemic rats compared with controls.

Cell targeting by murine recombinant retroviruses.

Cell targeting by murine retroviruses carrying recombinant genes would have numerous applications such as immortalization of under-represented cell types from complex cellular mixtures, increasing therapeutical yields in the case of gene therapy and delivery of specific genes at any location and at any moment of the life-time of living animals. To this aim, we are currently developing techniques that allow binding of viral particles to specific cell membrane markers different from the natural receptors. We have shown that biochemical bi-specific molecular adaptors able to bind both viral particles and cell surface molecules may reveal appropriate for piloting viruses toward specific cell types. More recently, we have undertaken the genetic engineering of the retroviral envelope glycoprotein in order to modify its natural tropism. This approach is discussed below.

Cardiac hypertrophy due to pressure and volume overload: distinctly different biological phenomena?

Myocardial hypertrophy is a morphological adaptive response to chronic work overload imposed on the heart. It has been categorized into two distinct basic types: concentric hypertrophy, occurring in response to a sustained pressure overload in which wall thickness increases without chamber enlargement, and eccentric hypertrophy, in response to a chronic volume overload in which chamber volume enlarges without a relative increase in its wall thickness. It should be emphasized, in this context, that these adjectives are somewhat confusing, since the hypertrophy observed is not eccentric in the fashion often seen in the left ventricle of patients with hypertrophic cardiomyopathy. In fact, the hypertrophy is concentric in both instances, but is associated with an increase in chamber volume when described as eccentric, yet occurring with a maintained volume when said to be concentric. In rats made anemic by iron deficiency, the volume overloaded heart achieves an adaptive increase in mass characterized as hypertrophy occurring in the setting of dilated ventricle. This so-called eccentric hypertrophy depends on catecholamines as possible signals for myocardial growth, and progresses with preserved ultrastructure and contractile performance of the cardiac muscle. A gradually imposed volume overload results in a harmonious growth of the heart (it retains a relative normal shape, becoming a magnified normal heart), probably mediated by release of catecholamines into the myocardium. This process resembles the normal cardiac growth in response to the obligatory volume load imposed by an increasing cardiac output (greater metabolic demands) and blood volume.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcific degeneration of pericardial valvular xenografts implanted subcutaneously in rats.

Tissue valve dystrophic calcification, resulting in hemodynamic regurgitation and/or stenosis, is the most serious complication when bioprosthetic heart valves are concerned. The objective of this investigation was to define morphologically the sequential development of calcific deposits in pericardial tissue cusps of unimplanted cardiac bioprostheses implanted subcutaneously in rats. Small samples of pericardium were implanted subcutaneously in the dorsal area of young rats weighing 60-80 g. The animals were selected randomly and sacrificed at days 1, 2, 7, 14 and 28 after implantation. The specimens were retrieved and studied morphologically. Calcific deposits were seen grossly as small punctate white masses from day 7 after implantation, progressively becoming more extensive. The light and electron microscopic studies showed that: calcific deposits occurred as early as 24 hours after implantation, the calcific degenerative process was progressive with time, and the mineralization was diffuse, although irregular in degree. The ultrastructural findings revealed that cytoplasmic organelles (mitochondria in particular) and plasma membrane of connective tissue cells appear to serve as initial sites of the process of calcification, which then progresses in the interfibrillar spaces, adjacent to collagen fibrils and elastic fibers.

Impairment of fat absorption induced by alcohol in rats.

Rats were maintained 16 weeks on a well-balanced semipurified solid diet supplemented with alcohol which comprised 35% of total calories. A control group was pair-fed the same basic diet with sucrose replacing alcohol isocalorically. Fecal fat excretion was evaluated. The mean fecal fat excretion of alcohol-fed rats was 40% higher than that of pair-fed controls. The possible pathogenic mechanism of such functional alteration is discussed.

[The proteasome and malignant hemopathies]. / Protéasome et hémopathies malignes.

Proteasomes are the main non lysosomal proteolytic structures of the cells. They correspond to the major system eliminating abnormal proteins, short half-life proteins and proteins controlling the cell cycle. They are essential for the production of peptides subsequently presented by the MHC-I. They are formed by a proteolytic core (the 20S proteasome) made of 4 rings of 7 proteic subunits associated with regulatory complexes (namely the 19S complex forming the 26S proteasome). Using classical cell biology techniques (cytometry, immunofluorescence microscopy, Western blot) our group has particularly studied the proteasome expression of leukaemic cell lines (U937 and CCRF-CEM) during in vitro differentiation induced by PMA and Vitamin D plus retinoïc acid. During differentiation, the level of proteasome expression and its localization vary. The various monoclonal antibodies used provided different patterns according to the different subunits. There was a general trend to a disappearance of nuclear proteasome and to a decrease in their cytoplasmic expression. In contrast, proteosomal antigens were increased on the cell membrane and in culture supernatants. We derived an ELISA test to measure plasma proteasome concentrations. Preliminary results showed differences between patients with haemopoietic malignancies or solid tumors and normal donors. Proteasome levels varied under treatment. They were correlated with LDH levels. Taken together, these results argue in favor of a role for cellular proteasomes in malignant differentiation process, and emphasize the qualitative changes in proteasome expression. Plasma proteasomes do not only reflect tumor cell mass and could play a role in addition to their proteolytic activity. They seem to be a relevant tool for diagnosis, prognosis and therapeutic monitoring.

High plasma proteasome levels are detected in patients with metastatic malignant melanoma.

BACKGROUND: Proteasomes, nonlysosomal proteolytic structures, are implicated in cell growth and differentiation. An abnormal expression has been described in haematopoietic malignancies and in some solid tumours. OBJECTIVES: To study the plasma proteasome levels in patients with malignant melanoma (MM) using an enzyme-linked immunosorbent assay (ELISA) technique, and to compare them with the values obtained in a normal population and in patients with severe psoriasis or chronic idiopathic urticaria (CIU). METHODS: Plasma proteasome level was measured using a sandwich ELISA test in normal donors (n = 14), and in patients with stage I/II (n = 13), stage III (n = 6) and stage IV (n = 10) MM, severe psoriasis (n = 13) and CIU (n = 6). Tissue proteasome expression was also detected by immunohistology using a monoclonal antibody in paraffin-embedded samples of normal tissue, psoriasis skin and MM. RESULTS: In normal donors, mean +/- SEM plasma proteasome concentration was 2138 +/- 221 ng mL(-1). Patients with stages III and IV MM exhibited a significantly higher value (3373 +/- 470 ng mL(-1) and 8931 +/- 1232 ng mL(-1), respectively). Values in patients with stage I/II MM and CIU were not significantly different from those in normal volunteers. Patients with severe psoriasis also exhibited increased values (3398 +/- 374 ng mL(-1)) but to a lesser extent than in patients with stage IV MM. There was a significant correlation of proteasome levels with serum lactate dehydrogenase in the MM group. Tissue expression as demonstrated by immunohistochemistry paralleled these findings. The strongest expression was seen on MM slides and to a lesser extent in psoriasis samples, the weakest expression being observed in normal skin. CONCLUSIONS: Proteasomes are strongly expressed in cutaneous MM; high levels of circulating proteasomes are detected in patients with metastatic MM with a high melanoma burden, and at a lesser extent in psoriatic patients, which suggests proteasomes represent a marker more of nonspecific inflammation than of early cancer.

Electron microscopic study on the cardiac hypertrophy induced by iron deficiency anaemia in the rat.

In this investigation the ultrastructure of the hypertrophied myocardium of anaemic iron-deficient rats was studied. Iron deficiency anaemia was induced by feeding rats on an iron-deficient diet for 30 days from the time of weaning. Anaemia was indicated by lowering of blood haemoglobin levels. The results clearly demonstrated that experimentally-induced iron deficiency anaemia brings about striking morphological changes in the heart of the rat. These changes were characterized by marked cellular hypertrophy together with distinct cellular degeneration and interstitial fibrosis. It is noteworthy that ultrastructural features of myocardial hypertrophy and degeneration occurred simultaneously. The potential role of noradrenaline in the pathogenesis of these changes is discussed.

Pathogenesis of cardiac hypertrophy in iron deficiency anaemia: the role of noradrenaline.

This study examined the effect of long-term administration of reserpine, an adrenergic blocking agent, on cardiac hypertrophy in animals with severe iron deficiency anaemia. This condition was induced by feeding rats on an iron-deficient diet for 30 days from the time of weaning. Anaemia was indicated by lowering of blood haemoglobin levels. Reserpine was administered i.p. (0.15 mg/kg body wt) every day during the experiment. Marked cardiac hypertrophy, as indicated by increase heart weight and increased size of cardiac muscle cells, was evidenced in iron-deficient rats, while the heart weights and myocardial cell size of drug-treated anaemic rats were in the normal range. The successful prevention of cardiac hypertrophy in anaemic iron-deficient rats by reserpine administration supports the hypothesis that noradrenaline plays a key role in the cardiac-hypertrophy process in iron deficiency anaemia.

Decreased susceptibility to calpains of v-FosFBR but not of v-FosFBJ or v-JunASV17 retroviral proteins compared with their cellular counterparts.

The c-Fos and c-Jun transcription factors are rapidly turned over in vivo. One of the multiple pathways responsible for their breakdown is probably initiated by calpains, which are cytoplasmic calcium-dependent cysteine proteases. The c-fos gene has been transduced by two murine oncogenic retroviruses called Finkel-Biskis-Jenkins murine sarcoma virus (FBJ-MSV) and Finkel-Biskis-Reilly murine sarcoma virus (FBR-MSV); c-jun has been transduced by the chicken avian sarcoma virus 17 (ASV17) retrovirus. Using an in vitro degradation assay, we show that the mutated v-FosFBR, but not v-FosFBJ or v-JunASV17, is resistant to calpains. This property raises the interesting possibility that decreased sensitivity to calpains might contribute to the tumorigenic potential of FBR-MSV by allowing greater accumulation of the protein that it encodes in infected cells. It has also been demonstrated that resistance to cleavage by calpains does not result from mutations that have accumulated in the Fos moiety of the viral protein but rather from the addition of atypical peptide motifs at its both ends. This observation raises the interesting possibility that homologous regions in viral and cellular Fos either display slightly different conformations or are differentially accessible to interacting proteins.

The efficiency of cell targeting by recombinant retroviruses depends on the nature of the receptor and the composition of the artificial cell-virus linker.

Using streptavidin-bound antibodies specific for both viral and cell membrane epitopes, we have reported previously that human cells may be infected by murine ecotropic retroviruses through an interaction with major histocompatibility complex class I and class II antigens, and thus have demonstrated that cell targeting by recombinant retroviruses is feasible. We report here that (i) growth factor or hormone receptors, such as those for epidermal growth factor (EGF) and insulin, can also mediate infection of human cells; (ii) a biotinylated cytokine or hormone can substitute for the anti-cell antibody in bispecific antibody complexes, thus extending the versatility of the method; (iii) although yields are low in our assay, infection efficiency clearly appears to depend upon the biochemical composition of molecular bridges because bi-functional antibody complexes are more efficient than cytokine-antibody complexes in the case of the EGF receptor. Finally, our study indicates that different cell membrane molecules are not equally efficient in allowing infection of human cells because targeting of the transferrin, high density lipoprotein and galactose receptors, as well as that of various membrane glycoconjugates, by murine ecotropic retroviruses did not lead to the establishment of a proviral state.

Mouse JunD negatively regulates fibroblast growth and antagonizes transformation by ras.

As NIH 3T3 fibroblasts become quiescent, the level of c-Jun protein decreases while JunD accumulates. When resting cells are stimulated with fresh serum, nuclear-localized JunD is rapidly degraded, followed by resynthesis of both c-Jun and JunD later in G1. Overexpression of JunD results in slower growth and an increase in the percentage of cells in G0/G1 while c-Jun overexpression produces larger S/G2 and M phase populations. In addition, JunD partially suppresses transformation by an activated ras gene whereas c-Jun cooperates with ras to transform cells. These data indicate that two closely related transcription factors can function in an opposing manner.

Ubiquitinylation is not an absolute requirement for degradation of c-Jun protein by the 26 S proteasome.

Degradation of rapidly turned over cellular proteins is commonly thought to be energy dependent, to require tagging of protein substrates by multi-ubiquitin chains, and to involve the 26 S proteasome, which is the major neutral proteolytic activity in both the cytosol and the nucleus. The c-Jun oncoprotein is very unstable in vivo. Using cell-free degradation assays, we show that ubiquitinylation, along with other types of tagging, is not an absolute prerequisite for ATP-dependent degradation of c-Jun by the 26 S proteasome. This indicates that a protein may bear intrinsic structural determinants allowing its selective recognition and breakdown by the 26 S proteasome. Moreover, taken together with observations by different groups, our data point to the notion of the existence of multiple degradation pathways operating on c-Jun.

The sensitivity of c-Jun and c-Fos proteins to calpains depends on conformational determinants of the monomers and not on formation of dimers.

Milli- and micro-calpains are ubiquitous cytoplasmic cysteine proteases activated by calcium. They display a relatively strict specificity for their substrates which they usually cleave at only a limited number of sites. Motifs responsible for recognition by calpains have not been characterized yet, and recently a role for PEST motifs in this process has been ruled out. c-Fos and c-Jun transcription factors are highly sensitive to calpains in vitro. They thus provide favourable protein contexts for studying the structural requirements for recognition and degradation by these proteases. Using in vitro degradation assays and site-directed mutagenesis, we report here that susceptibility to calpains is primarily determined by conformational determinants of the monomers and not by the quaternary structure of c-Fos and c-Jun proteins. The multiple cleavage sites borne by both proteins can be divided into at least two classes of sensitivity, the most sensitive ones being easily visualized in the presence of rate-limiting amounts of calpains. One site located at position 90-91 in c-Fos protein is extremely sensitive. However, efficient proteolysis did not have any strict dependence on the nature of the amino acids on either side of the scissile bond in the region extending from P2 to P'2. The structural integrity of the monomers is not crucial for recognition by calpains. Rather, sensitive sites can be recognized independently and their recognition is dependent on the local conformation of peptide regions that may span several tens of amino acids and maybe more in the case of the identified c-Fos hypersensitive site.