Enterococcus spp. produces slime and survives in rat peritoneal macrophages.

Enterococcal clinical isolates were investigated for the ability to form biofilm on inert surfaces, as a measure of slime production, in an attempt to find new possible virulence factors for these microorganisms. This property was commonly found among Enterococcus faecalis. Also E. faecium isolates were able to form biofilm, although to a lesser extent; for this species, however, biofilm formation seemed more frequently associated with isolates from infection rather than with environmental strains or isolates from healthy individuals. Biofilm formation was strongly affected by the presence of an additional carbohydrate source in the medium, or by iron deprivation, indicating a role of slime for survival in stressful conditions. Slime-producing E. faecalis were able to survive inside peritoneal macrophages for extended periods compared to slime-negative strains or to slime-positive bacteria grown in conditions depressing slime production. In particular, slime-producing and slime-negative cells showed a decrease of 1 and 2 log units, respectively, at 1 h after infection; slime-negative cells were then rapidly killed, with clearance of bacterial cells at 24 h. Slime-producing bacteria persisted up to 48 h, which was the last time point examined, as after that time viability of both infected and non-infected macrophages started to decline. Scanning electron microscopy observations showed the presence of abundant amorphous extracellular material, of possible polysaccharide nature, embedding bacterial cells to form a multilayered biofilm. Even in conditions not supporting biofilm formation, bacterial cells appeared capsulated, suggesting that capsule and slime might represent different structures. Genes belonging to the epa locus or to a putative icaA homolog did not seem to be involved in synthesis and export of slime.

Infection of human enterocyte-like cells with rotavirus enhances invasiveness of Yersinia enterocolitica and Y. pseudotuberculosis.

Mixed infection with rotavirus and either Yersinia enterocolitica or Y. pseudotuberculosis was analysed in Caco-2 cells, an enterocyte-like cell line highly susceptible to these pathogens. Results showed an increase of bacterial adhesion and internalisation in rotavirus-infected cells. Increased internalisation was also seen with Escherichia coli strain HB101 (pRI203), harbouring the inv gene from Y. pseudotuberculosis, which is involved in the invasion process of host cells. In contrast, the superinfection with bacteria of Caco-2 cells pre-infected with rotavirus resulted in decreased viral antigen synthesis. Transmission electron microscopy confirmed the dual infection of enterocytes. These data suggest that rotavirus infection enhances the early interaction between host cell surfaces and enteroinvasive Yersinia spp.

The human per1 gene: genomic organization and promoter analysis of the first human orthologue of the Drosophila period gene.

Per genes encode components of the circadian clocks controlling metabolic and behavioural rhythms. The human Per1 cDNA, RIGUI, was previously isolated and mapped on chromosome 17p12 (Sun, Z.S., Albrecht, U., Zhuchenko, O., Bailey, J., Eichele, G., Lee, C.C., 1997. RIGUI, a putative mammalian orthologue of the Drosophila period gene. Cell 90, 1003-1011). We have now isolated the entire genomic locus containing the human Per1 gene, in a search for genes associated with CpG-rich sequences. The hPer1 gene spans 15kb of human genomic DNA and is composed of 23 exons, flanked by 5' and 3' regulatory regions. Comparison of the hPer1 genomic clone with the dbEST database revealed homologies with putative alternative transcripts. Functional mapping within the 5' CpG-rich regulatory region enabled us to locate the hPer1 promoter core in a 510bp-long sequence centred around a TATA box, which supports high levels of hPer1 transcription. A second regulatory region was formally identified in intron 1, which appears to exert a negative role in transcriptional control of hPer1. These regions may be differentially involved in tissue-specificity, and/or circadian regulation, of the human hPer1 gene transcription.

Nonrandom chromosomal imbalances in primary mediastinal B-cell lymphoma detected by arbitrarily primed PCR fingerprinting.

We used arbitrarily primed polymerase chain reaction (AP-PCR) fingerprinting to identify chromosomal imbalances in six primary mediastinal B-cell lymphomas (PMBLs). Seventy-four chromosomal imbalances were detected, consisting of 49 sequence gains and 25 losses. Amplifications on chromosome X were seen in five cases, four of which involved the same chromosomal locus. Nonrandom gains at the same locus were also identified on chromosomes 2 and 7 in four cases and on chromosomes 5, 9, and 12 in three cases. Five PMBLs were also analyzed by comparative genomic hybridization (CGH), which found chromosome arm 9p amplification as the only nonrandom imbalance. Our data demonstrate that chromosomal amplifications outnumber losses in PMBL. These mainly involve chromosomes 9 and X and may reflect more complex phenomena, such as translocations or other chromosomal rearrangements, as AP-PCR found coexistent gains and losses on these chromosomes. Comparison between AP-PCR and CGH suggests that anomalies affecting the same chromosomal regions may occur at much higher frequencies than expected by CGH, suggesting that genomic amplifications are usually confined to DNA segments smaller than the megabase long segments required for detection in CGH. Modest increases in genetic material may be as effective as higher-level amplifications when affecting sites where a proto-oncogene resides.

Protein phosphatase inhibitors induce modification of synapse structure and tau hyperphosphorylation in cultured rat hippocampal neurons.

Protein phosphatase inhibitors, okadaic acid and Caliculin A, were used to investigate how perturbation of phosphorylation and dephosphorylation processes might affect neurite and synapse structure in cultures of fetal rat hippocampal neurons. Drug treatments induced neuritic tree modification, with retraction of the processes and the appearance of dilatations along the neurites. The characteristic dotlike pattern of immunoreactivity of synaptic vesicle proteins disappeared. Normal synapses were extremely rare by ultrastructural observation. Vesicles of various diameters accumulated in the dilatations, as did organelles and amorphous material, suggesting impaired axonal transport. Hyperphosphorylation of tau protein was also observed as indicated by the shift in the electrophoretic mobility of a 32P-labeled 55-kDa band and by immunoblot with epitope-specific tau antibody. Our results show that inhibition of protein phosphatases 1 and 2A results in a modification of the neuritic tree structure, with loss of neuronal processes, phosphorylation of a tau isoform, and a decrease in the number of synapses. These neuronal features are present in Alzheimer's disease (AD). Our results suggest that the two events might be related and provide a potential link between the biochemical hallmark of AD (hyperphosphorylation of tau) and a pathological finding of primary clinical relevance (the synaptic loss).

Junctional sites of erythrocyte skeletal proteins are specific targets of tert-butylhydroperoxide oxidative damage.

The oxidative denaturation of the erythrocyte membrane, which is considered a major cause of the haemolytic process, was evaluated upon 'in vitro' oxidative stress with tertbutylhydroperoxide. Biochemical and ultrastructural analyses were performed to point out the effect of this substance on the skeletal network, which is mainly responsible for red cell shape and viability. Moreover, cell morphology was observed by scanning electron microscopy and membrane rigidity assessed by EPR measurements. The most relevant features of the membrane denaturation were, (i) lipid peroxidation, as assessed by malonidialdehyde production, (ii) spectrin and ankyrin degradation with simultaneous globin binding to the membrane, as evidenced by electrophoretic pattern of red cell ghosts. These phenomena were related to the drug concentration in the incubation medium, and accompanied by depletion of intracellular reduced glutathione. The denaturation of protein components hindered the release of spectrin in a hypotonic extraction medium and could be only partially reversed by dithiothreitol. The extensive membrane protein and lipid degradation, at high drug concentration, was coherent with a marked increase of membrane order (membrane 'rigidity'). No clustering of intramembrane proteins was shown by the transmission electron microscopy images. At the same time scanning electron microscopy demonstrated shrinking and disco-stomatocytic deformation of erythrocytes. Ultrastructural analysis of the membrane skeleton by fluorescence-labelling of spectrin and actin, allowed to point out that exposure to t-BHP caused the marginalization of spectrin and the rearrangement of actin molecules with formation of micro aggregates, so that a detachment of actin from the spectrin network was suggested. In addition to the generalized damage of red cell membrane, tertbutylhydroperoxide was found to induce a specific alteration of the skeletal network at the horizontal junction sites involving spectrin, actin, and protein 4.1 and thus to modify the cytoskeletal assembly. This effect on the membrane skeletal components was consistent with the hypothesis that oxidative stress plays a key role in the haemolytic process.

Down-modulation of CD4 antigen during programmed cell death in U937 cells.

It has been hypothesized that programmed cell death (PCD), an active cell suicide process occurring in place of necrosis, can be associated with the pathogenesis of acquired immunodeficiency syndrome (AIDS). The entry of human immunodeficiency virus (HIV) into competent cells is mediated by the CD4 molecule present on the surface of certain lymphocyte subpopulations as well as on some cultured cell lines, e.g. U937 myelomonocytic cells. The present paper focuses on some specific aspects of PCD induced by the cytokine tumor necrosis factor (TNF). The results obtained indicate that the exposure of U937 cells to cycloheximide facilitates TNF-mediated PCD via a short term cell death program and modifies the expression of CD4 surface molecules. This change in surface antigen expression, manifested by internalization of the CD4 molecule, occurs in cells in which apoptosis has been triggered, but not in cells undergoing necrosis. These results indicate that the progression of cell death could be associated with specific alterations of certain surface molecules and could have a role in the entry of HIV into cells.

Menadione-induced oxidative stress leads to a rapid down-modulation of transferrin receptor recycling.

It has been demonstrated that perturbation of oxidative balance plays an important role in numerous pathological states as well as in physiological modifications leading to aging. In order to evaluate the role of the oxidative state in cells, biochemical and ultrastructural studies were carried out on K562 and HL-60 cell cultures. Particular attention was given to the transferrin receptor, which plays an important role in cellular iron metabolism. In order to evaluate if oxidative stress influences the transferrin receptor regulation process, the free-radical inducer menadione was used. The results obtained seem to indicate that oxidative stress is capable of inducing a rapid and specific down-modulation of the membrane transferrin receptor due to a block of receptor recycling on the cell surface, without affecting ligand-binding affinity. These effects were observed in the early stages of menadione treatment and before any typical signs of subcellular damage, including surface blebbing, a well-known cytopathological marker of menadione-induced injury. The mechanisms underlying such phenomena appear to be related to cytoskeletal protein thiol group oxidation as well as to the perturbation of calcium homeostasis, both induced by menadione. It is thus hypothesized that the data reported here represent a specific example of a general mechanism by which cell surface receptor expression and recycling can be influenced by oxidative balance.

Human erythrocyte insulin receptor processing is affected by the oxidizing agent menadione.

Insulin-induced down-regulation of erythrocyte insulin receptors is a simplified model that can provide useful information on the cell surface regulative phenomena and on role of the plasma membrane and cytoskeleton in such physiological processes. Oxidative imbalance was examined since it was shown to play an important role in numerous cellular pathologies as well as in cell aging. Specifically, the free radical inducer menadione was used in order to evaluate if this compound is able to modify (and in which manner) the down-regulation process. Biochemical, biophysical, and ultrastructural approaches were used. The results obtained seem to indicate that menadione-induced oxidative damage was able to decrease the insulin-induced down-regulation process, as measured by binding assays. This effect was accompanied by slight alterations in plasma membrane ultrastructure and insignificant variations in plasma membrane lipid composition. In addition, the decrease in membrane order, measured by electron paramagnetic resonance, which was shown to usually occur during the process of down-regulation, was not observed. In contrast, cytoskeletal protein assembly, as previously shown in other in vitro systems, appeared to be remarkably altered. Such changes in specific cytoskeletal elements could lead to the decrease of down-regulation phenomenon induced by menadione. Changes in electrophoretic pattern of some cytoskeletal proteins (e.g., spectrin) reinforce this hypothesis. Considering the importance of free radicals in cell injury, data reported here could represent a specific example of a general mechanism by which cell surface receptor expression and recycling can be modified by changes in some intracellular molecule redox status and cell ionic homeostasis.

Two different pathways for necrotic cell death induced by free radicals.

Plasma membrane modifications have been widely recognized as crucial factors in cell injury and death. One of these modifications, surface blebbing, has been considered as an injury-marker associated with a series of biochemical and physiological modifications. Our study focused on the different effects of free radical-induced cell damage by quinone menadione (2-methyl-1,4-naphthoquinone) and by hyperthermic shock (45 degrees C) on the erythroleukemic cell line K562. Different techniques including immunofluorescence, freeze-fracturing, and electron paramagnetic resonance spectroscopy were employed. Menadione induced the formation of surface blebs, accompanied by a rearrangement of the microfilament system and changes in the distribution of plasma membrane proteins. In contrast, heat-shocked cells showed neither blebbing nor important cytoskeletal changes. Finally, the electron paramagnetic resonance results showed an increase in membrane order not specifically related to the type of free radical-induced stress. These cell death features appear to suggest the existence of two different types of pathways for necrotic cell death: both treatments induce cell injury and eventual death by modifying plasma membrane integrity and function. However, one involves cytoskeleton-dependent surface blebbing, whereas the other does not.

Role of adhesion molecules in the mechanism of non-MHC (major histocompatibility complex) restricted cell-mediated cytotoxicity.

Adhesion molecules involved in the interaction between immune system effector cells and tumor targets are surface molecules which contribute to the formation of cell-to-cell contacts and belong to the integrin family. In this paper, the role played by the adhesion molecules in the process of cell-mediated cytotoxicity is reviewed. Furthermore, the contact area between effector and target cells has been analyzed by scanning electron microscopy. This region, termed "closed chamber", seems to contribute to killing efficiency by creating an intimate contact region in which cytotoxic factors can easily induce lethal hit in target cell. Thus, the extension of the closed chamber seems to be positively related to effector cell killing potential as well as to target cell sensitivity and, in this context, the adhesion molecules prove to play a pivotal role. In fact, a receptor-ligand interaction occurs between CD11a/CD18 (LFA-1) and CD2 molecules, expressed on the effector cells, and the respective counterparts on target cells, i.e., ICAM-1, ICAM-2, or LFA-3. Treatment with antibodies against such molecules strongly modifies closed chamber formation without inhibiting cell-to-cell binding. Nevertheless, in these conditions, the killing ability of different effector cells toward tumor targets appears to be strongly impaired. Hence, the adhesion molecules seem to be strongly involved in the formation of the closed chamber as well as in the activation of effector cell killing machinery.

Membrane and cytoskeleton are intracellular targets of rhein in A431 cells.

The antineoplastic drug rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid) has been hypothesized to interfere with tumor cell proliferation by affecting energy metabolism and mitochondrial function. In this study, the intracellular targets of rhein were investigated in the A431 epithelial cell line by means of biophysical and structural techniques. After treatment with 50 microM rhein at different times (8 and 24 hours), a series of remarkable morphological modifications ultimately leading to irreversible cell injury was observed. In particular, scanning and transmission electron microscopic observations point to the cell surface and mitochondria as probable targets of this drug. In addition, biophysical analyses conducted by electron paramagnetic resonance (EPR) spectroscopy seem also to indicate that cellular membranes are a direct target in rhein-induced damage. Concomitantly the cytoskeletal network underlying the inner leaflet of the plasma membrane (the microfilament system) also underwent a rearrangement. Taken together, the effects induced by rhein presented here seem to indicate that this drug, as well as other anthraquinones or other compounds that selectively impair energy metabolism, can act on neoplastic cells by probably altering cell membrane function and membrane-associated cytoskeleton.

Oxidative stress and transferrin receptor recycling.

Perturbation of the oxidative balance in biological systems plays an important role in numerous pathological states as well as in many physiological processes such as receptor activity. In order to evaluate if oxidative stress induced by menadione influences membrane receptor processes, a study was conducted on the transferrin receptor. Consequently, biochemical, biophysical and ultrastructural studies were carried out on different cell lines. The results obtained seem to indicate that oxidative stress is able of inducing a rapid and specific down-modulation of membrane transferrin receptor due to a block of receptor recycling on the cell surface without affecting binding affinity.

Oxidative stress and transferrin receptor recycling.

Perturbation of the oxidative balance in biological systems plays an important role in numerous pathological states as well as in many physiological processes such as receptor activity. In order to evaluate if oxidative stress induced by menadione influences membrane receptor processes, a study was conducted on the transferrin receptor. Consequently, biochemical, biophysical and ultrastructural studies were carried out on different cell lines. The results obtained seem to indicate that oxidative stress is able of inducing a rapid and specific down-modulation of membrane transferrin receptor due to a block of receptor recycling on the cell surface without affecting binding affinity.

Antibodies to adhesion molecules inhibit the lytic function of MHC-unrestricted cytotoxic cells by preventing their activation.

We evaluated the effect of the antibodies to adhesion molecules CD2, CD11a/CD18 (LFA-1), and CD56 (N-CAM) on MHC-unrestricted cytotoxicity mediated by polyclonal NK cells and LAK cells or by CD3+ or CD3- cytolytic cell clones against a panel of tumor cell targets selected according to expression or absence of the corresponding ligands. We show that (i) antibodies to CD11a/CD18 and, to a lesser extent, antibodies to CD2 inhibit target cell lysis, whereas anti-CD56 antibodies exert little if any effect; (ii) in a model system using polyclonal NK/LAK cells as effectors and K562 or HL60-R (NK-resistant) cells as targets, inhibition of cytotoxicity occurs without a significant impairment of effector to target cell binding; (iii) the cytotoxic function of CD3+ or CD3- cytotoxic cell clones is inhibited differentially by antibodies to adhesion molecules; (iv) conjugates formed in the presence of antibodies which inhibit target cell lysis display a significant reduction of target to effector cell contact surface; and (v) this may lead to defective activation of effector cells, as indicated by lack of redistribution of the microtubular apparatus. We conclude that (i) MHC-unrestricted cytotoxicity is regulated by a number of molecular interactions that span far beyond our present knowledge and that it is strictly dependent on the surface phenotype of the effector cell and of the target cell; (ii) in certain types of effector/target cell interactions, antibodies to adhesion molecules do not prevent conjugate formation but reduce the extent of cell-to-cell surface contact which, in turn, leads to defective activation of the effector cell and, therefore, to inhibition of target cell lysis.

Morphological features of cloned lymphocytes expressing gamma/delta T cell receptors.

We have analyzed the morphological characteristics of human T lymphocytes bearing CD3-associated T cell receptor (TcR) gamma and delta chains. BB3 and delta-TCS1 monoclonal antibodies (mAb) were used to identify two distinct, nonoverlapping populations of TcR gamma/delta + cells which express the products of V delta 2 and V delta 1 gene segments, respectively. In the peripheral blood, most V delta 1+ (delta TCS-1+) lymphocytes express the non-disulfide-linked form of receptor whereas V delta 2+ (BB3+) cells express the disulfide-linked form. The majority of cloned TcR gamma/delta + cells exhibit a growth pattern different from that of conventional TcR alpha/beta + cells as they adhere promptly to surfaces and undergo morphological changes which can be summarized as follows: cells spread on the surface, form a distinct uropod and, in the final phase of adherence, emit long filopodia ending with adhesion plaques. Immunofluorescence studies of TcR gamma/delta + clones demonstrated the presence of submembraneous actin microfilaments and actin-binding protein confirming that these cells are capable of active motility which is related to the propensity of TcR gamma/delta + cells to home to epithelia. Scanning electron microscope analyses of effector/target cell conjugates showed that in TcR gamma/delta + cells the region of the uropodia next to the cell body is responsible for the binding to tumor target cells. Interestingly, immunofluorescence analyses revealed that LFA-1 molecules are predominantly distributed in the uropodium whereas they are virtually absent in the cell bodies. These morphological characteristics of TcR gamma/delta + cells may pertain to defensive mechanisms the mucosal level.

Cytoskeleton as a target in menadione-induced oxidative stress in cultured mammalian cells: alterations underlying surface bleb formation.

Several in vitro and in vivo studies have suggested that surface bleb formation during oxidative cell injury is related to alteration in cytoskeleton organization. Various cell lines different in origin and growth characteristics were exposed to 2-methyl-1,4-naphthoquinone (menadione) which is known to induce bleb formation and cytotoxicity by generating considerable amounts of oxygen-reactive species. Treated cells were analyzed by means of immunocytochemistry and electron microscopy in order to investigate the morphological and molecular features underlying bleb generation. The results obtained indicate that menadione-induced bleb formation is a widely observed phenomenon present mainly in round or mitotic cells. Surface blebs appear free of organelles and contain only few ribosomes and amorphous material. Occasionally, they undergo detachment from the cell surface as large cytoplasmic vesicles. Bleb surfaces with protein clusters as well as bald blisters with an almost exclusive localization of intramembrane particles on their narrow base were detected using freeze-fracture techniques. Immunocytochemical investigations performed on menadione-exposed cells revealed that some surface proteins (collagen IV, sialo-proteins, beta 2 microglobulin and fibronectin) and adhesion molecules (vinculin) underwent changes in their expression over the bleb surface. Moreover, different behavioural characteristics of actin microfilaments, vimentin and keratin intermediate filaments and microtubules was observed. Alpha-actinin, vimentin and microtubular proteins (tubulin, MAPs and tau) were detected within the blebs. On the other hand, actin and keratin filaments appeared to be absent. The results presented here demonstrate that cytoskeletal structures and the microfilament system in particular, represent important targets in menadione-induced morphological changes in cultured cells. These changes appear to lead to the redistribution of several cytoskeletal and membrane proteins as well as dissociation of the cytoskeleton network from its anchoring domains in the plasma membrane thus generating sites of structural weakness where blebs would arise and progressively grow. Experimental evidence supporting a crucial role of thiol oxidation and elevation of cytoplasmic calcium concentration in bleb formation is also provided.