Recent advances of enterovirus 71 [Formula: see text] targeting Inhibitors.

With CA16, enterovirus-71 is the causative agent of hand foot and mouth disease (HFMD) which occurs mostly in children under 5 years-old and responsible of several outbreaks since a decade. Most of the time, HFMD is a mild disease but can progress to severe complications such as meningitis, brain stem encephalitis, acute flaccid paralysis (AFP) and even death; EV71 has been identified in all severe cases. Therefore, it is actually one of the most public health issues that threatens children's life. [Formula: see text] is a protease which plays important functions in EV71 infection. To date, a lot of [Formula: see text] inhibitors have been tested but none of them has been approved yet. Therefore, a drug screening is still an utmost importance in order to treat and/or prevent EV71 infections. This work highlights the EV71 life cycle, [Formula: see text] functions and [Formula: see text] inhibitors recently screened. It permits to well understand all mechanisms about [Formula: see text] and consequently allow further development of drugs targeting [Formula: see text]. Thus, this review is helpful for screening of more new [Formula: see text] inhibitors or for designing analogues of well known [Formula: see text] inhibitors in order to improve its antiviral activity.

Substitution-Inert Polynuclear Platinum Complexes That Inhibit the Activity of DNA Polymerase in Triplex-Forming Templates.

The formation of triple-helical DNA is implicated in the regulation of gene expression. The triplexes are, however, unstable under physiological conditions so that effective stabilizers for the triplex formation are needed. Herein, we describe a new strategy for the stabilization of such triplexes that is based on antitumor substitution-inert polynuclear platinum complexes (SI-PPCs). These compounds were previously shown to bind to DNA through the phosphate clamp-a discrete mode of DNA-ligand recognition distinct from the canonical intercalation and minor-groove binding. We have found that SI-PPCs efficiently inhibit DNA synthesis by DNA polymerase in sequences prone to the formation of pyrimidine- and purine-motif triplex DNAs. Moreover, the results suggest that SI-PPCs are able to induce the formation of triple-helical DNA between duplexes and strands that are not completely complementary to each other. Collectively, these data provide evidence that SI-PPCs are very efficient stabilizers of triple-stranded DNA that might exert their action by stabilizing higher-order structures such as triple-helical DNA.

Osteoblasts with impaired spreading capacity benefit from the positive charges of plasma polymerised allylamine.

Bone diseases such as osteoporosis, osteoarthritis and rheumatoid arthritis, impinge on the performance of orthopaedic implants by impairing bone regeneration. For this reason, the development of effective surface modifications supporting the ingrowth of implants in morbid bone tissue is essential. Our study is designed to elucidate if cells with restricted cell-function limiting adhesion processes benefit from plasma polymer deposition on titanium. We used the actin filament disrupting agent cytochalasin D (CD) as an experimental model for cells with impaired actin cytoskeleton. Indeed, the cell's capacity to adhere and spread was drastically reduced due to shortened actin filaments and vinculin contacts that were smaller. The coating of titanium with a positively charged nanolayer of plasma polymerised allylamine (PPAAm) abrogated these disadvantages in cell adhesion and the CD-treated osteoblasts were able to spread significantly. Interestingly, PPAAm increased spreading by causing enhanced vinculin number and contact length, but without significantly reorganising actin filaments. PPAAm with the monomer allylamine was deposited in a microwave-excited low-pressure plasma-processing reactor. Cell physiology was monitored by flow cytometry and confocal laser scanning microscopy, and the length and number of actin filaments was quantified by mathematical image processing. We showed that biomaterial surface modification with PPAAm could be beneficial even for osteoblasts with impaired cytoskeleton components. These insights into in vitro conditions may be used for the evaluation of future strategies to design implants for morbid bone tissue.

Resistin in gingival crevicular fluid and induction of resistin release by Porphyromonas gingivalis lipopolysaccharide in human neutrophils.

BACKGROUND AND OBJECTIVE: Resistin is an adipocytokine that induces insulin resistance and is predominantly expressed in adipocytes and peripheral blood mononuclear cells. Resistin expression increases in inflammatory diseases as well as diabetes mellitus, and is upregulated by bacterial pathogens and proinflammatory cytokines. The aim of this study was to identify resistin in human gingival crevicular fluid, to compare the resistin levels in gingival crevicular fluid between subjects with and without periodontitis and diabetes mellitus and to investigate the regulation of resistin release from human neutrophils by Porphyromonas gingivalis lipopolysaccharide (P-LPS). MATERIAL AND METHODS: Gingival crevicular fluid samples were collected from patients with chronic periodontitis (n = 24), patients with diabetes mellitus-related periodontitis (n = 18) and healthy subjects (n = 21). Resistin in gingival crevicular fluid was determined using western blot analysis and an ELISA kit. The glycated hemoglobin (HbA(1c)) value was obtained from patients with diabetes mellitus-related periodontitis by a medical interview. Human neutrophils were cultured with P-LPS (0-1000 ng/mL), or incubated with inhibitors of actin or microtubule polymerization in the absence or presence of P-LPS. The medium and cellular fractions were used for determination of resistin by ELISA. RESULTS: The resistin level in gingival crevicular fluid from patients with periodontitis or diabetes mellitus-related periodontitis was significantly higher than that of healthy subjects. The resistin level in gingival crevicular fluid was correlated with gingival index score, but not blood HbA(1c) value. The P-LPS increased resistin release from human neutrophils, and its induction was decreased by actin polymerization inhibitors. CONCLUSION: We show, for the first time, the presence of resistin in gingival crevicular fluid. A high resistin level in gingival crevicular fluid samples from periodontitis patients may to some extent be related to P-LPS-induced resistin release from neutrophils.

Enhanced healing of mitomycin C-treated healing-impaired wounds in rats with hydrosheets composed of chitin/chitosan, fucoidan, and alginate as wound dressings.

To create a moist environment for rapid wound healing, a hydrosheet composed of alginate, chitin/chitosan, and fucoidan (ACF-HS) has been developed as a functional wound dressing. The aim of this study was to evaluate the accelerating effect of ACF-HS on wound healing for rat mitomycin C-treated healing-impaired wounds. Full-thickness skin defects were made on the back of rats and mitomycin C was applied onto the wound for 10 minutes to prepare a healing-impaired wound. After thoroughly washing out the mitomycin C, ACF-HS was applied to the healing-impaired wounds. The rats were later euthanized and histological sections of the wounds were prepared. The histological examinations showed significantly advanced granulation tissue and capillary formations in the healing-impaired wounds treated with ACF-HS on days 7 and 14, in comparison with that in alginate fiber (Kaltostat), hydrogel wound dressing (DuoACTIVE), and nontreatment (negative control). Furthermore, in cell culture studies, ACF-HS-absorbed serum and fibroblast growth factor-2 was found to be proliferative for fibroblasts and endothelial cells, respectively, and ACF-HS-absorbed serum was found to be chemoattractive for fibroblasts. However, our results may not be strictly comparable with general healing-impaired wound models in humans because of the cell damage by mitomycin C. In addition, more biocompatibility studies of fucoidan are essential due to the possibility of renal toxicity.

Shared and unique roles of CAP23 and GAP43 in actin regulation, neurite outgrowth, and anatomical plasticity.

CAP23 is a major cortical cytoskeleton-associated and calmodulin binding protein that is widely and abundantly expressed during development, maintained in selected brain structures in the adult, and reinduced during nerve regeneration. Overexpression of CAP23 in adult neurons of transgenic mice promotes nerve sprouting, but the role of this protein in process outgrowth was not clear. Here, we show that CAP23 is functionally related to GAP43, and plays a critical role to regulate nerve sprouting and the actin cytoskeleton. Knockout mice lacking CAP23 exhibited a pronounced and complex phenotype, including a defect to produce stimulus-induced nerve sprouting at the adult neuromuscular junction. This sprouting deficit was rescued by transgenic overexpression of either CAP23 or GAP43 in adult motoneurons. Knockin mice expressing GAP43 instead of CAP23 were essentially normal, indicating that, although these proteins do not share homologous sequences, GAP43 can functionally substitute for CAP23 in vivo. Cultured sensory neurons lacking CAP23 exhibited striking alterations in neurite outgrowth that were phenocopied by low doses of cytochalasin D. A detailed analysis of such cultures revealed common and unique functions of CAP23 and GAP43 on the actin cytoskeleton and neurite outgrowth. The results provide compelling experimental evidence for the notion that CAP23 and GAP43 are functionally related intrinsic determinants of anatomical plasticity, and suggest that these proteins function by locally promoting subplasmalemmal actin cytoskeleton accumulation.

Liposomal prodigiosin and plasmid encoding serial GCA nucleotides reduce inflammation in microglial and astrocyte cells by ATM/ATR signaling.

The aim of this study was to use liposomal structure consisting prodigiosin and plasmid encoding serial GCA nucleotides (LP/pSGCAN) to reduce inflammation in microglial cells (MGCs) and astrocyte cells (ACCs) by ATM/ATR signaling. Here, it was shown that LP/pSGCAN decreased cell viability and total RNA level. Importantly, LP/pSGCAN had more effect on ACCs than MGCs (P < 0.05). Moreover, increase of apoptosis was seen with increase of concentration. The expression of IL-1 and IL-6 were decreased and the expression of ATM and ATR were increased in treated MGCs and ACCs, which showed LP/pSGCAN could inhibit inflammation by activation of ATM/ATR pathway.

The geodiamolide H, derived from Brazilian sponge Geodia corticostylifera, regulates actin cytoskeleton, migration and invasion of breast cancer cells cultured in three-dimensional environment.

We are investigating effects of the depsipeptide geodiamolide H, isolated from the Brazilian sponge Geodia corticostylifera, on cancer cell lines grown in 3D environment. As shown previously geodiamolide H disrupts actin cytoskeleton in both sea urchin eggs and breast cancer cell monolayers. We used a normal mammary epithelial cell line MCF 10A that in 3D assay results formation of polarized spheroids. We also used cell lines derived from breast tumors with different degrees of differentiation: MCF7 positive for estrogen receptor and the Hs578T, negative for hormone receptors. Cells were placed on top of Matrigel. Spheroids obtained from these cultures were treated with geodiamolide H. Control and treated samples were analyzed by light and confocal microscopy. Geodiamolide H dramatically affected the poorly differentiated and aggressive Hs578T cell line. The peptide reverted Hs578T malignant phenotype to polarized spheroid-like structures. MCF7 cells treated by geodiamolide H exhibited polarization compared to controls. Geodiamolide H induced striking phenotypic modifications in Hs578T cell line and disruption of actin cytoskeleton. We investigated effects of geodiamolide H on migration and invasion of Hs578T cells. Time-lapse microscopy showed that the peptide inhibited migration of these cells in a dose-dependent manner. Furthermore invasion assays revealed that geodiamolide H induced a 30% decrease on invasive behavior of Hs578T cells. Our results suggest that geodiamolide H inhibits migration and invasion of Hs578T cells probably through modifications in actin cytoskeleton. The fact that normal cell lines were not affected by treatment with geodiamolide H stimulates new studies towards therapeutic use for this peptide.

Microencapsulation of antibiotic rifampicin in poly(3-hydroxybutyrate-co-3-hydroxyvalerate).

The aim of this study was the preparation of microparticles containing rifampicin using a biodegradable polymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) for oral administration produced by a bacteria. The poly(3-hydroxybutyrate-co-3-hydroxyvalerate) microparticles with and without rifampicin were prepared by the emulsification and solvent evaporation method, in which chloroform and polyvinyl alcohol are used as the solvent and emulsifier, respectively. Microparticles were obtained within a size range of 20-60 microm by changing the initial poly(3-hydroxybutyrate-co-3-hydroxyvalerate), polyvinyl alcohol and rifampicin concentrations. An encapsulation efficiency value of 14% was obtained. The optimized total yield of 60% of the poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/ rifampicin was obtained. A load of 0.035 mg/1 mg of PHBV was reached. Almost 90% of the drug loaded in the microparticles was released after 24 h. The size, encapsulation efficiency and ribampicin release of the microparticles varied as a function of the initial poly(3-hydroxybutyrate-co-3-hydroxyvalerate), polyvinyl alcohol and rifampicin concentrations. It was demonstrated that the microencapsulated rifampicin, although was not totally available in the medium, exhibited a similar inhibition value as free rifampicin at 24 h of incubation with S. aureus. Cytotoxicity assays demonstrated a reduction of the toxicity when rifampicin was microencapsulated in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) while maintaining its antibacterial activity.

Disruption of actin cytoskeleton causes internalization of Ca(v)1.3 (alpha 1D) L-type calcium channels in salamander retinal neurons.

PURPOSE: To study the influence of actin cytoskeleton reorganization on the subcellular distribution of Ca(v)1.3 L-type Ca2+ channels in salamander retinal third-order neurons. METHODS: Immunocytochemistry with confocal microscopy was used to demonstrate internalization of the Ca(v)1.3 isoform of L-type voltage-gated Ca2+ channels in third-order retinal neurons. A specificity of antibody was confirmed with Western blotting and in control experiments preabsorbing antibody wit its respective peptide. Whole-cell patch clamp technique was applied to record L-type currents from ganglion cells in slice preparations in the presence of N- and P/Q type Ca2+ channel blockers. RESULTS: A high level of Ca(v)1.3 labeling was present in cone photoreceptor terminals in the outer plexiform layer (OPL), as aggregates of puncta. Punctate Ca(v)1.3 labeling was evident throughout the IPL and around the cell bodies in the outer nuclear (ONL), inner nuclear (INL) and on somas and axons of ganglion cells labeled with rhodamine-conjugated dextran. Doubly labeled sections for synaptophysin and Ca(v)1.3 revealed colocalization in the OPL and IPL. Depolymerization of the actin cytoskeleton caused a dynamin-dependent internalization of Ca(v)1.3 but not Ca(v)1.2 subtype of voltage-gated Ca2+ channels in dissociated neurons. In ganglion cells, the inhibition of L-type Ca2+ currents by F-actin disrupters was mediated by Ca2+ channel internalization. Treatment with cytochalasin D protected retinal neurons against kainate-induced excitotoxicity. CONCLUSIONS: Actin cytoskeleton dynamics plays an important role in the regulation of subcellular distribution and function of Ca(v)1.3 L-type Ca2+ channels in salamander retinal neurons. Ca2+-dependent actin depolymerization may serve as a negative feedback mechanism to reduce excessive Ca2+ influx and thereby protect neurons against glutamate-induced excitotoxicity.

A murine scavenger receptor MARCO recognizes polystyrene nanoparticles.

Recent toxicological studies indicate that nanoparticles or ultrafine particles (< 100 nm) are more toxic than fine particles (< 2 microm) because of their greater surface area. It is well known that alveolar macrophages play an important role in the first defense against various environmental particles and microorganisms. This is accomplished by binding to a macrophage receptor with collagenous structure (MARCO), one of several scavenger-type receptors expressed on the cell surface of macrophages. MARCO has been shown to mediate the ingestion of unopsonized environmental particles such as TiO(2) and Fe(2)O(3) (1.3 microm in diameter). However, very little is known about the cellular uptake of nanoparticles. In the present study, we investigated whether MARCO mediates the uptake of nanoparticles by using fluorescent-tagged polystyrene particles (20 nm, 200 nm, and 1 microm in diameter). COS-7 cells were transfected with either MARCO cDNA or an empty vector, and the association of the particles with the cells were observed by fluorescence microscopy and atomic force microscopy. MARCO-transfected cells associated with all three sizes of particles in a time-dependent manner, while no obvious binding of particles occurred after 5 h to the empty vector-transfected cells. The uptake of particles by MARCO-transfected cells was partially inhibited by polyG. These results suggest that macrophages associate with nanoparticles (20 nm) at least in part through MARCO and that MARCO plays a role in clearing nanoparticles which can deposit in the alveolar region.

Cytoplasmic tail regulates the intercellular adhesion function of the epithelial cell adhesion molecule.

Ep-CAM, an epithelium-specific cell-cell adhesion molecule (CAM) not structurally related to the major families of CAMs, contains a cytoplasmic domain of 26 amino acids. The chemical disruption of the actin microfilaments, but not of the microtubuli or intermediate filaments, affected the localization of Ep-CAM at the cell-cell boundaries, suggesting that the molecule interacts with the actin-based cytoskeleton. Mutated forms of Ep-CAM were generated with the cytoplasmic domain truncated at various lengths. All of the mutants were transported to the cell surface in the transfectants; however, the mutant lacking the complete cytoplasmic domain was not able to localize to the cell-cell boundaries, in contrast to mutants with partial deletions. Both the disruption of the actin microfilaments and a complete truncation of the cytoplasmic tail strongly affected the ability of Ep-CAM to mediate aggregation of L cells. The capability of direct aggregation was reduced for the partially truncated mutants but remained cytochalasin D sensitive. The tail truncation did not affect the ability of the transfectants to adhere to solid-phase-adsorbed Ep-CAM, suggesting that the ability to form stable adhesions and not the ligand specificity of the molecule was affected by the truncation. The formation of intercellular adhesions mediated by Ep-CAM induced a redistribution to the cell-cell boundaries of alpha-actinin, but not of vinculin, talin, filamin, spectrin, or catenins. Coprecipitation demonstrated direct association of Ep-CAM with alpha-actinin. Binding of alpha-actinin to purified mutated and wild-type Ep-CAMs and to peptides representing different domains of the cytoplasmic tail of Ep-CAM demonstrates two binding sites for alpha-actinin at positions 289 to 296 and 304 to 314 of the amino acid sequence. The results demonstrate that the cytoplasmic domain of Ep-CAM regulates the adhesion function of the molecule through interaction with the actin cytoskeleton via alpha-actinin.

Actin polymerization is essential for pollen tube growth.

Actin microfilaments, which are prominent in pollen tubes, have been implicated in the growth process; however, their mechanism of action is not well understood. In the present work we have used profilin and DNAse I injections, as well as latrunculin B and cytochalasin D treatments, under quantitatively controlled conditions, to perturb actin microfilament structure and assembly in an attempt to answer this question. We found that a approximately 50% increase in the total profilin pool was necessary to half-maximally inhibit pollen tube growth, whereas a approximately 100% increase was necessary for half-maximal inhibition of cytoplasmic streaming. DNAse I showed a similar inhibitory activity but with a threefold more pronounced effect on growth than streaming. Latrunculin B, at only 1--4 nM in the growth medium, has a similar proportion of inhibition of growth over streaming to that of profilin. The fact that tip growth is more sensitive than streaming to the inhibitory substances and that there is no correlation between streaming and growth rates suggests that tip growth requires actin assembly in a process independent of cytoplasmic streaming.

Inhibitors of actin polymerisation stimulate arachidonic acid release and 5-lipoxygenase activation by upregulation of Ca2+ mobilisation in polymorphonuclear leukocytes involving Src family kinases.

Here, we show that actin polymerisation inhibitors such as latrunculin B (LB), and to a minor extent also cytochalasin D (Cyt D), enhance the release of arachidonic acid (AA) as well as nuclear translocation of 5-lipoxygenase (5-LO) and 5-LO product synthesis in human polymorphonuclear leukocytes (PMNL), challenged with thapsigargin (TG) or N-formyl-methionyl-leucyl-phenylalanine. The concentration-dependent effects of LB (EC50 approximately 200 nM) declined with prolonged preincubation (>3 min) prior TG and were barely detectable when PMNL were stimulated with Ca2+-ionophores. Investigation of the stimulatory mechanisms revealed that LB (or Cyt D) elicits Ca2+ mobilisation and potentiates stimulus-induced elevation of intracellular Ca2+, regardless of the nature of the stimulus. LB caused rapid but only moderate activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK)2. The selective Src family kinase inhibitors PP2 and SU6656 blocked LB- or Cyt D-mediated Ca2+ mobilisation and suppressed the upregulatory effects on AA release and 5-LO product synthesis, without affecting AA metabolism evoked by ionophore alone. We conclude that in PMNL, inhibitors of actin polymerisation cause enhancement of intracellular Ca2+ levels through Src family kinase signaling, thereby facilitating stimulus-induced release of AA and 5-LO product formation.

Actomyosin-dependent microtubule rearrangement in lysophosphatidic acid-induced neurite remodeling of young cortical neurons.

It has been shown that lysophosphatidic acid (LPA), a signaling phospholipid, induces neurite retraction and the formation of retraction fibers in young cortical neurons by actin rearrangement. This study examined the rearrangement of microtubules (MTs) during LPA-induced neurite remodeling by immunostaining with antibodies against several types of tubulin. The results showed that alpha-tubulin was present in growing neurites as well as in cell bodies with various localization profiles. Exposure of neurons to LPA resulted in neurite retraction, accompanied by the rearrangement of MTs in neurites and the accumulation of MTs in cell bodies, without significant changes in the total amount of MTs in the cytoskeletal fraction of cultured neurons. Similar findings were obtained when young neurons were stained for other types of tubulin, including beta-tubulin type III and posttranslationally acetylated and tyrosinated tubulin. LPA-induced MT rearrangement was accompanied by accumulation of myosin IIB and polymerized actin at the base of retraction fibers. These effects of LPA on MTs and myosin IIB were blocked by pretreatment with inhibitors of the actomyosin and Rho pathways (cytochalasin D, blebbistatin, and Y27632), but not by an MT stabilizer (taxol), whereas taxol inhibited neurite retraction and MT depolymerization induced by nocodazole. Furthermore, neurofilaments also showed rearrangement in response to LPA, which was blocked by cytochalasin D and Y27632, but not taxol. Taken together, these results suggested that LPA did not induce MT depolymerization and that LPA-induced actomyosin activation produced MT and neurofilament rearrangement, leading to neurite remodeling.

Differential actions of PAR2 and PAR1 in stimulating human endothelial cell exocytosis and permeability: the role of Rho-GTPases.

Endothelial cell proteinase activated receptors (PARs) belong to a family of heterotrimeric G protein-coupled receptors that are implicated in leukocyte accumulation and potentiation of reperfusion injury. We characterized the effect and the signal transduction pathways recruited after stimulation of endothelial PAR2. We used von Willebrand Factor (vWF) release and monolayer permeability to peroxidase to report Weibel-Palade body (WPB) exocytosis and pore formation, respectively. Human umbilical vein endothelial cells (HUVECs) were stimulated with the selective PAR2 agonist peptide SLIGRL-NH2 or PAR1 agonist peptide TFLLR-NH2. PAR2 stimulation resulted in WPB exocytosis like PAR1 stimulation but, unlike PAR1, failed to increase monolayer permeability. BAPTA-AM inhibited PAR2-induced exocytosis, indicating a PAR2 calcium-dependent signal in ECs. Moreover, PAR2-like PAR1-stimulated exocytosis requires actin cytoskeleton remodeling, because vWF release is inhibited if the cells were pretreated with Jasplakinolide. Rho-GTPase activity is required for PAR-stimulated exocytosis, because inactivation of this family of actin-regulatory proteins with Clostridium difficile toxin B blocked exocytosis. Expression of dominant-negative mutant Cdc42(17N) inhibited exocytosis whereas neither dominant-negative Rac(17N) expression nor C3 exotoxin treatment affected vWF release. PAR2 stimulated RhoA-GTP weakly compared with the PAR1 agonist. We conclude that both PAR2 and PAR1 elicit WP body exocytosis in a calcium and Cdc42 GTPase-dependent manner. In contrast, the differential effect of PAR1 versus PAR2 activation to increase monolayer permeability correlates with weak RhoA activation by the PAR2 agonist.

A chemical enucleation method for the transfer of mitochondrial DNA to rho(o) cells.

The study of pathogenic mitochondrial DNA mutations has, in most cases, relied on the production of transmitochondrial cybrids. Although the procedure to produce such cybrids is well established, it is laborious and cumbersome. Moreover, the mechanical enucleation procedure is inefficient and different techniques have to be used depending on the adherence properties of the cell. To circumvent these difficulties, we developed a chemical enucleation method that can have wide applicability for the production of transmitochondrial cybrids. The method is based on the use of actinomycin D to render the nuclear genome transcription/replication inactive and unable to recover after treatment. Such treated cells are fused to cells devoid of mitochondrial DNA and selected for the presence of a functional oxidative phosphorylation system. Our results showed that 95% of the clones recovered by this procedure are true transmitochondrial cybrids. This method greatly facilitates the production of transmitochondrial cybrids, thereby increasing the number of mtDNA mutations and the recipient cell types that can be studied by this system.

Cleavage of polypeptide chain initiation factor eIF4GI during apoptosis in lymphoma cells: characterisation of an internal fragment generated by caspase-3-mediated cleavage.

Polypeptide chain initiation factor eIF4GI undergoes caspase-mediated degradation during apoptosis to give characteristic fragments. The most prominent of these has an estimated mass of approximately 76 kDa (Middle-Fragment of Apoptotic cleavage of eIF4G; M-FAG). Subcellular fractionation of the BJAB lymphoma cell line after induction of apoptosis indicates that M-FAG occurs in both ribosome-bound and soluble forms. Affinity chromatography on m7GTP-Sepharose shows that M-FAG retains the ability of eIF4GI to associate with both the mRNA cap-binding protein eIF4E and initiation factor eIF4A and that the ribosome-bound form of M-FAG is also present as a complex with eIF4E and eIF4A. These data suggest that the binding sites for eIF4E, eIF4A and eIF3 on eIF4GI are retained in the caspase-generated fragment. M-FAG is also a substrate for cleavage by the Foot-and-Mouth-Disease Virus-encoded L protease. These properties, together with the pattern of recognition by a panel of antibodies, define the origin of the apoptotic cleavage fragment. N-terminal sequencing of the products of caspase-3-mediated eIF4GI cleavage has identified the major cleavage sites. The pattern of eIF4GI degradation and the possible roles of the individual cleavage products in cells undergoing apoptosis are discussed.

Tissue factor is regulated by epidermal growth factor in normal and malignant human endometrial epithelial cells.

Tissue Factor (TF), the initiator of the extrinsic coagulation cascade, is overexpressed in a variety of cancers. TF is also expressed in normal human endometrium but little is known about its expression or regulation in endometrial cancer. We demonstrate herein that TF is expressed in the endometrial adenocarcinoma cell line Ishikawa. Furthermore, epidermal growth factor (EGF) induces a rapid and sustained increase in TF expression. Estradiol and progesterone had no effect on basal or EGF-induced TF expression in Ishikawa cells. In contrast to the pronounced and sustained upregulation at the protein level, EGF treatment elicited only a modest and transient increase in TF mRNA levels. This activity corresponded to the response observed from an exogenous TF promoter construct. However, the induction of TF was abrogated by cycloheximide as well as actinomycin-D, inhibitors or protein- and mRNA-synthesis, respectively, demonstrating that EGF mediates its effect through activation of the TF gene. Fractionation experiments showed that EGF increases TF presence in caveolin-I containing membrane fractions. Coagulation and invasion assays were used to explore the physiological implications of TF regulation. The results demonstrate that EGF-mediated induction of TF increases the procoagulant activity and invasive potential of Ishikawa cells. Furthermore, immunocytochemistry confirmed that TF is regulated by EGF in primary cultures of normal endometrial epithelial cells and malignant tumor cells. In conclusion, EGF-mediated upregulation of TF results in accumulation of this glycoprotein in caveolae-like membrane fractions and increased coagulative and invasive potential. Our results suggest that TF may play an integral role in endometrial carcinogenesis.

Dual effect of hydrogen peroxide on store-mediated calcium entry in human platelets.

Redox regulation is important for the modulation of cytosolic Ca(2+) concentration. Hence, we have investigated the effect of H(2)O(2) on store-mediated Ca(2+) entry (SMCE). In fura-2-loaded human platelets treatment with H(2)O(2) resulted in a concentration-dependent increase in Ca(2+) release from intracellular stores, while the effect on Ca(2+) entry was biphasic. In addition, 1mM H(2)O(2) reduced SMCE induced by agonists. The inhibitory effect of 1mM H(2)O(2) was prevented by inhibition of actin polymerization with cytochalasin D. Consistent with this, we found that 10microM H(2)O(2) and store depletion by treatment with thapsigargin plus ionomycin induced a similar temporal sequence of actin reorganization, while exposure to 1mM H(2)O(2) shifted the dynamics between polymerization and depolymerization in favor of the former. One millimolar H(2)O(2)-induced polymerization was reduced by treatment with methyl 2,5-dihydroxycinnamate and farnesylthioacetic acid, inhibitors of tyrosine kinases and Ras superfamily proteins, respectively. Finally, exposure to 1mM H(2)O(2) significantly increased store depletion-induced p60(src) activation. We conclude that H(2)O(2) exerted a biphasic effect on SMCE. The inhibitory role of high H(2)O(2) concentrations is mediated by an abnormal actin reorganization pattern involving both Ras- and tyrosine kinases-dependent pathways.