The Possible Role of Staphylococcus epidermidis LPxTG Surface Protein SesC in Biofilm Formation

Staphylococcus epidermidis is the most common cause of device-associated infections. It has been shown that active and passive immunization in an animal model against protein SesC significantly reduces S. epidermidis biofilm-associated infections. In order to elucidate its role, knock-out of sesC or isolation of S. epidermidis sesC-negative mutants were attempted, however, without success. As an alternative strategy, sesC was introduced into Staphylococcus aureus 8325–4 and its isogenic icaADBC and srtA mutants, into the clinical methicillin-sensitive S. aureus isolate MSSA4 and the MRSA S. aureus isolate BH1CC, which all lack sesC. Transformation of these strains with sesC i) changed the biofilm phenotype of strains 8325–4 and MSSA4 from PIA-dependent to proteinaceous even though PIA synthesis was not affected, ii) converted the non-biofilm-forming strain 8325–4 ica::tet to a proteinaceous biofilm-forming strain, iii) impaired PIA-dependent biofilm formation by 8325–4 srtA::tet, iv) had no impact on protein-mediated biofilm formation of BH1CC and v) increased in vivo catheter and organ colonization by strain 8325–4. Furthermore, treatment with anti-SesC antibodies significantly reduced in vitro biofilm formation and in vivo colonization by these transformants expressing sesC. These findings strongly suggest that SesC is involved in S. epidermidis attachment to and subsequent biofilm formation on a substrate.     

A Possible Role for Urease as a Storage Protein in Aspergillus tamarii

A marked decrease in mycelial urease activity during the endogenousphase of undifferentiated Aspergillus tamariicultures was foundto be independent of preparative procedures but related to thedepletion of external nutrients. The enzyme, which was synthesizedduring the active growth stage, was produced in similar quantitieswith ammonium or urea as sole nitrogen source and at its peakrepresented c. 8·5 per cent of the total soluble proteinpool of the mycelium. It was found to show maximum activityat pH 8·20–8·65 when measured in cell-free,phosphate-buffered extracts. Isolation of urease from differentstages of the endogenous phase by affinity chromatography hasshown that the observed decrease in activity was due to breakdownof the enzyme protein in mature cultures, followed by the progressivedeactivation of residual enzyme during the autolytic stage.Since selective inhibition of 80–90 per cent of activityby acetohydroxamic acid in media containing urea as the onlynitrogen source or total repression of urease synthesis by L-histidinein ammonium-grown cultures did not interfere with normal growth,it was concluded that in A. tamarii urease fulfils the functionof a storage protein with a measure of catalytic activity. Aspergillus tamarii, urease, storage protein, nitrogen metabolism     

Myelin Basic Protein Is a Zinc-Binding Protein in Brain: Possible Role in Myelin Compaction

The zinc-binding proteins (ZnBPs) in porcine brain were characterized by the radioactive zinc-blot technique. Three ZnBPs of molecular weights about 53 kDa, 42 kDa, and 21 kDa were identified. The 53 kDa and 42 kDa ZnBPs were found in all subcellular fractions while the 21 kDa ZnBP was mainly associated with particulate fractions. This 21 kDa ZnBP was identified by internal protein sequence data as the myelin basic protein. Further characterization of its electrophoretic properties and cyanogen bromide cleavage pattern with the authentic protein confirmed its identity. The zinc binding properties of myelin basic protein are metal specific, concentration dependent and pH dependent. The zinc binding property is conferred by the histidine residues since modification of these residues by diethyl-pyrocarbonate would abolish this activity. Furthermore, zinc ion was found to potentiate myelin basic protein-induced phospholipid vesicle aggregation. It is likely that zinc plays an important role in myelin compaction by interacting with myelin basic protein.     

Role of G Protein βγ Subunits in Muscarinic Receptor-Induced Stimulation and Inhibition of Adenylyl Cyclase Activity in Rat Olfactory Bulb

Abstract: In the olfactory bulb, muscarinic receptors exert a bimodal control on cyclic AMP, enhancing basal and G_s-stimulated adenylyl cyclase activities and inhibiting the Ca²⁺/calmodulin- and forskolin-stimulated enzyme activities. In the present study, we investigated the involvement of G protein βγ subunits by examining whether the muscarinic responses were reproduced by the addition of βγ subunits of transducin (βγ_t) and blocked by putative βγ scavengers. Membrane incubation with βγ_t caused a stimulation of basal adenylyl cyclase activity that was not additive with that produced by carbachol. Like carbachol, βγ_t potentiated the enzyme stimulations elicited by vasoactive intestinal peptide and corticotropin-releasing hormone. RT-PCR analysis revealed the expression of mRNAs encoding both type II and type IV adenylyl cyclase, two isoforms stimulated by βγ synergistically with activated G_s. In addition, βγ_t inhibited the Ca²⁺/calmodulin- and forskolin-stimulated enzyme activities, and this effect was not additive with that elicited by carbachol. Membrane incubation with either one of two βγ scavengers, the GDP-bound form of the α subunit of transducin and the QEHA fragment of type II adenylyl cyclase, reduced both the stimulatory and inhibitory effects of carbachol. These data provide evidence that in rat olfactory bulb the dual regulation of cyclic AMP by muscarinic receptors is mediated by βγ subunits likely acting on distinct isoforms of adenylyl cyclase.     

Diacylglycerol: Formation and function in phospholipid-mediated signal transduction

1. Properties, distribution and multiplicity of phosphoinositidases (phospholipase C, PLC) are investigated.2. Generation of diacylglycerol (DAG) by a variety of enzymes such as phosphoinositide and phosphatidylcholine specific PLC, by a combination of phospholipase D and phosphatidic hydrolase, and by triglyceride lipase is examined.3. Ca²⁺ and phospholipid-dependent protein kinase C act as the target of DAG messenger action.4. There are differences in the formation of DAG in normal and transformal cell.     

Role of Blood Platelets in Haematophagy

RED blood corpuscles (RBC) suspended in saline induce gorging in many haematophagous insects because of their high intrinsic concentration of adenine nucleotides (ANS)^1–6. ANS are bound firmly inside the intact RBC, which raises the question of how they gain egress to contact the chemoreceptor surfaces and induce feeding. It has been suggested that saliva or secretions of the chemoreceptor surfaces act as ANS releasing agents¹. ANS release by haemolysis is discounted by the fact that all RBC found in the gut immediately after feeding are intact. Further, stereoscan electron microscopy of tsetse fly gustatory sensilla does not suggest that they operate by piercing the erythrocytes^7,8. Thus we decided to test the possibility that the chemoreceptors involved in blood identification receive an ANS stimulus from a source associated with, but not within the RBC.     

Possible Role of <Emphasis Type="Italic">Escherichia coli</Emphasis> Protein YbgI

Proteins containing the NIF3 domain are highly conserved and are found in bacteria, eukaryotes, and archaea. YbgI is an Escherichia coli protein whose gene is conserved among bacteria. The structure of YbgI is known; however, the function of this protein in cells remains obscure. Our studies of E. coli cells with deleted ybgI gene suggest that YbgI is involved in formation of the bacterial cell wall.     

Possible Role of Natural Selection in the Formation of Tandem-Repetitive Noncoding DNA

A simulation model of sequence-dependent amplification, unequal crossing over and mutation is analyzed. This model predicts the spontaneous formation of tandem-repetitive patterns of noncoding DNA from arbitrary sequences for a wide range of parameter values. Natural selection is found to play an essential role in this self-organizing process. Natural selection which is modeled as a mechanism for controlling the length of a nucleotide string but not the sequence itself favors the formation of tandem-repetitive structures. Two measures of sequence heterogeneity, inter-repeat variability and repeat length, are analyzed in detail. For fixed mutation rate, both inter-repeat variability and repeat length are found to increase with decreasing rates of (unequal) crossing over. The results are compared with data on micro-, mini- and satellite DNAs. The properties of minisatellites and satellite DNAs resemble the simulated structures very closely. This suggests that unequal crossing over is a dominant long-range ordering force which keeps these arrays homogeneous even in regions of very low recombination rates, such as at satellite DNA loci. Our analysis also indicates that in regions of low rates of (unequal) crossing over, inter-repeat variability is maintained at a low level at the expense of much larger repeat units (multimeric repeats), which are characteristic of satellite DNA. In contrast, the microsatellite data do not fit the proposed model well, suggesting that unequal crossing over does not act on these very short tandem arrays.     

Hematopoietic Protein Tyrosine Phosphatase Mediates β2-Adrenergic Receptor-Induced Regulation of p38 Mitogen-Activated Protein Kinase in B Lymphocytes

Stimulation of the β₂-adrenergic receptor (β₂AR) on a CD40L/interleukin-4-activated B lymphocyte increases the level of immunoglobulin E (IgE) in a protein kinase A (PKA)- and p38 mitogen-activated protein kinase (MAPK)-dependent manner. However, the mechanism by which β₂AR stimulation mediates the increase in the level of p38 MAPK activation has remained unclear. Here we show that the β₂AR-induced increase in p38 MAPK activation occurred via a hematopoietic protein tyrosine phosphatase (HePTP)-mediated cross talk between PKA and p38 MAPK. β₂AR agonists, cAMP-elevating agents, and PKA inhibitors were used to show that β₂AR stimulation resulted in a PKA-dependent increase in p38 MAPK phosphorylation. Pharmacological agents and gene-deficient mice revealed that p38 MAPK phosphorylation was regulated by the G-stimulatory (Gs)/cAMP/PKA pathway independently of the G-inhibitory or β-arrestin-2 pathways. Coimmunoprecipitation and Western blot analysis showed that HePTP was phosphorylated in a PKA-dependent manner, which inactivated HePTP and allowed for increased free p38 MAPK to be phosphorylated by the MAPK cascade that was activated by CD40L. HePTP short hairpin RNA confirmed that HePTP played a role in regulating the level of p38 MAPK phosphorylation in a B cell. Thus, β₂AR stimulation on a B cell phosphorylates and inactivates HePTP in a Gs/cAMP/PKA-dependent manner to release bound p38 MAPK, making more available for phosphorylation and subsequent IgE regulation.     

Variations in Fusion Pore Formation in Cholesterol-Treated Platelets

Exocytosis is a highly regulated intercellular communication process involving various membrane proteins, lipids, and cytoskeleton restructuring. These components help control granule fusion with the cell membrane, creating a pore through which granular contents are released into the extracellular environment. Platelets are an ideal model system for studying exocytosis due to their lack of a nucleus, resulting in decreased membrane regulation in response to cellular changes. In addition, platelets contain fewer granules than most other exocytosing cells, allowing straightforward measurement of individual granule release with carbon-fiber microelectrode amperometry. This technique monitors the concentration of serotonin, an electroactive molecule found in the dense-body granules of platelets, released as a function of time, with 50 μs time resolution, revealing biophysical characteristics of the fundamental exocytotic process. Variations in fusion pore formation and closure cause deviations from the classic current versus time spike profile and may influence diffusion of serotonin molecules from the site of granule fusion. Physiologically, the delivery of smaller packets of chemical messengers or the prolonged delivery of chemical messengers may represent how cells/organisms tune biological response. The goals of this work are twofold: 1) to categorize secretion features that deviate from the traditional mode of secretion and 2) to examine how changing the cholesterol composition of the platelet membrane results in changes in the pore formation process. Results herein indicate that the expected traditional mode of release is actually in the minority of granule content release events. In addition, results indicate that as the cholesterol content of the plasma membrane is increased, pore opening is less continuous.     

Role of p38 Mitogen-Activated Protein Kinase in Thrombus Formation

The present study was designed to elucidate the role of p38 mitogen-activated protein kinase (p38) in thrombus formation. We used p38α heterozygous (p38α+/?) mice and used ferric chloride (FeCl₃)-induced carotid artery injury as a model of thrombus formation. The time to thrombotic occlusion induced by FeCl₃ in p38α+/? mice was prolonged compared to that in wild-type (WT) mice. Platelets prepared from p38α+/? mice showed impairment of the aggregatory response to a low concentration of U46619, a thromboxane A₂ analogue. Furthermore, platelets prepared from p38α+/? mice and activated by U46619 were poorly bound to fibrinogen compared with those from WT mice. Both the expression and activity of tissue factor induced by FeCl₃ in WT mice were higher than those in p38α+/? mice. These results suggest that p38 plays an important role in thrombus formation by regulating platelet function and tissue factor activity.     

Inhibition of Protein Translocation in Permeabilized Cells of Schizosaccharomyces pombe by Puromycin

To investigate protein translocation in eukaryotes, we reconstituted a protein translocation system using the permeabilized spheroplasts (P-cells) of the fission yeast Schizosaccharomyces pombe. The precursor of a sex pheromone of Saccharomyces cerevisiae, prepro-α-factor, was translocated across the endoplasmic reticulum (ER) of S. pombe posttranslationally, and glycosylated to the same extent as in the ER of S. cerevisiae. This suggested that the size of N-linked core-oligosaccharide in the ER of S. pombe is similar to that in S. cerevisiae. This translocation into the ER of S. pombe was inhibited by puromycin, but the translocation in the P-cells of S. cerevisiae was not inhibited. This difference in sensitivity to puromycin was due to the membrane but not the cytosolic fraction. Our results suggested that the translocation machinery of S. pombe was sensitive to puromycin and different from that of S. cerevisiae.     

Role of Platelets in Hemostasis and Thrombosis

Platelets interact with the coagulation factors in a complex way to arrest bleeding or generate thrombi. Recently, the platelet''s relationship to endothelial alteration and atheroma production has received renewed attention. At present, tests of platelet function better define “hypocoagulable” rather than “hypercoagulable” states.     

Activation of Protein Kinase C in Permeabilized Human Neuroblastoma SH-SY5Y Cells

The activation of protein kinase C was investigated in digitonin-permeabilized human neuroblastoma SH-SY5Y cells by measuring the phosphorylation of the specific protein kinase C substrate myelin basic protein4-14. The phosphorylation was inhibited by the protein kinase C inhibitory peptide PKC19-36 and was associated to a translocation of the enzyme to the membrane fractions of the SH-SY5Y cells. 1,2-Dioctanoyl-sn-glycerol had no effect on protein kinase C activity unless the calcium concentration was raised to concentrations found in stimulated cells (above 100 nM). Calcium in the absence of other activators did not stimulate protein kinase C. Phorbol 12-myristate 13-acetate was not dependent on calcium for the activation or the translocation of protein kinase C. The induced activation was sustained for 10 min, and thereafter only a small net phosphorylation of the substrate could be detected. Calcium or dioctanoylglycerol, when applied alone, only caused a minor translocation, whereas in combination a marked translocation was observed. Arachidonic acid (10 microM) enhanced protein kinase C activity in the presence of submaximal concentrations of calcium and dioctanoylglycerol. Quinacrine and p-bromophenacyl bromide did not inhibit calcium- and dioctanoylglycerol-induced protein kinase C activity at concentrations which are considered to be sufficient for phospholipase A2 inhibition.     

Role of Surface Protein SasG in Biofilm Formation by Staphylococcus aureus

The SasG surface protein of Staphylococcus aureus has been shown to promote the formation of biofilm. SasG comprises an N-terminal A domain and repeated B domains. Here we demonstrate that SasG is involved in the accumulation phase of biofilm, a process that requires a physiological concentration of Zn²⁺. The B domains, but not the A domain, are required. Purified recombinant B domain protein can form dimers in vitro in a Zn²⁺-dependent fashion. Furthermore, the protein can bind to cells that have B domains anchored to their surface and block biofilm formation. The full-length SasG protein exposed on the cell surface is processed within the B domains to a limited degree, resulting in cleaved proteins of various lengths being released into the supernatant. Some of the released molecules associate with the surface-exposed B domains that remain attached to the cell. Studies using inhibitors and mutants failed to identify any protease that could cause the observed cleavage within the B domains. Extensively purified recombinant B domain protein is very labile, and we propose that cleavage occurs spontaneously at labile peptide bonds and that this is necessary for biofilm formation.Staphylococcus aureus is a commensal bacterium that is carried persistently in the anterior nares of about 20% of the human population. The organism can cause superficial skin infections, such as abscesses and impetigo, and more dangerous and potentially life-threatening invasive infections, such as endocarditis, osteomyelitis, and septic arthritis (26). Staphylococcus epidermidis and S. aureus are the major causes of infections associated with indwelling medical devices, such as central venous catheters, cardiovascular devices, and artificial joints (34, 54). The ability to form a biofilm is crucial to the microbes'' success in device-related infections. Bacteria in the biofilm matrix are in a semidormant state, are difficult to inhibit with antibiotics, and are impervious to host neutrophils and macrophages (36, 43, 44, 51). Until recently biofilm formation by staphylococci was attributed to the ability to synthesize an extracellular polysaccharide called polysaccharide intercellular adhesin (PIA), which is composed of partially deacetylated poly-N-acetylglucosamine (15, 28, 50). Attachment of bacteria to biomedical devices is mediated by adhesion to the naked plastic or metal surface by a surface component such as the major autolysin Atl (2, 14). Alternatively, adhesion to surfaces that have been conditioned by fibronectin and fibrinogen from host plasma is mediated by surface proteins such as clumping factor A (ClfA) and fibronectin binding proteins (FnBPA/B) of S. aureus or SdrG/Fbe of S. epidermidis (17, 46, 47).Several surface proteins of staphylococci can also promote the accumulation phase of biofilm: (i) the biofilm-associated protein Bap, which is only expressed by bovine strains of S. aureus (8); (ii) the SasC surface protein of S. aureus (41); (iii) fibronectin binding proteins FnBPA and FnBPB, which are particularly associated with biofilm formation by some types of methicillin-resistant S. aureus (MRSA) (35, 48); (iv) the multifactorial virulence factor protein A, which promotes cell accumulation when expressed at high levels, for example,in mutants defective in the accessory gene regulator Agr (31); (v) the extracellular matrix binding protein (Embp) of S. epidermidis (4); (vi) the accumulation-associated protein (Aap) of S. epidermidis and the related protein SasG from S. aureus (7, 19, 40).Aap and SasG are typical LPXTG-anchored multidomain cell wall-associated proteins (see Fig. ​Fig.1A,1A, below). A signal sequence is removed from the N terminus during secretion across the cytoplasmic membrane. The C-terminal domains comprise a sorting signal (LPXTG) and hydrophobic membrane-spanning domain and positively charged residues that are required for covalent attachment of the proteins to cell wall peptidoglycan by sortase A. The N termini of the mature proteins (A domains) comprise related amino acid sequences that have been implicated in adhesion of bacteria to desquamated epithelial cells and could be involved in colonization of the nares and skin (7, 27, 39). The archetypal Aap protein of S. epidermidis RP62a has 12 repeats of almost identical sequences of 128 residues followed by a partial repeat of 68 residues (region B), while SasG from S. aureus strain 8325-4 and strain Newman has seven 128-residue repeats and one partial repeat. The B subunits of Aap and SasG are 64% identical.Open in a separate windowFIG. 1.(A) Schematic representation of SasG domain organization. The positions of the signal sequence (S), A domain, B region (B1 to -8), and the wall/membrane-spanning regions (W/M) are indicated. The LPKTG motif is recognized by the sortase A enzyme, which covalently anchors the protein to the cell wall peptidoglycan. (B) Whole-cell immunoblot validating expression of A domain and B regions of SasG variants. Serial dilutions of SH1000(pALC2073:sasG⁺) (row 1); SH1000(pALC2073sasG⁺ A⁺B⁻) (row 2); SH1000(pALC2073sasG⁺ A⁻B⁺) (row 3), and SH1000(pALC2073sasG⁺ A⁻B⁺) induced with tetracycline (90 ng/ml) (row 4) were applied to a nitrocellulose membrane and probed with anti-SasG A domain and anti-SasG B domain antibodies. (C) Biofilm formation by SH1000 constructs expressing SasG variants. Biofilm was allowed to form for 24 h at 37°C under static conditions in microtiter dishes. Biofilm was stained with crystal violet, and the absorbance was measured at 570 nm.The formation of biofilm by Aap in S. epidermidis is promoted by the removal of the A domain by cleavage by an as-yet-unidentified bacterial protease, an event that can also be precipitated by host proteases (40). The ability of the exposed Aap B domains of different bacterial cells to form homophilic interactions through a Zn²⁺-dependent zipper mechanism was proposed when it was shown that purified B domains formed dimers in vitro that were dependent on the presence of Zn²⁺ (6). Purified recombinant B domain protein, but not the A domain, inhibited biofilm formation, as did antibodies that specifically bound to the B domains (40). The Zn²⁺ chelator diethylenetriaminepentaacetic acid (DTPA) inhibited biofilm formation both by S. epidermidis RP62a (presumed to be due to Aap) and by community-associated MRSA (presumed to be due to SasG) (6).This study set out to investigate the molecular basis of biofilm accumulation promoted by the SasG protein of S. aureus. We demonstrate that processing of SasG occurs during growth and biofilm formation in a manner that is different from that reported for Aap, and we have investigated the mechanism.     

Different Mechanisms of β-Adrenoceptor Down-Regulation by Chronic Imipramine and Electroconvulsive Treatment: Possible Role for Protein Kinase C

The aim of this study was to find out how protein kinase C (PKC) is involved in down-regulation of the beta-adrenoceptor in cortical slices of rats subjected to antidepressant treatments. The responses of the cyclic AMP generating system to forskolin, isoproterenol, and noradrenaline were tested in the absence and presence of a PKC activator, 12-O-tetradecanoylphorbol 13-acetate (TPA). The antidepressive treatments applied were chronic administration of imipramine and electroconvulsive shock. The potentiating effect of the phorbol ester on cyclic AMP response to isoproterenol was retained in imipramine-treated animals and even accentuated in rats subjected to electroconvulsive treatment; the TPA effect on noradrenaline-induced cyclic AMP response was blunted in rats receiving imipramine, but augmented in those receiving electroconvulsive treatment. In imipramine-treated rats the beta-down-regulation was still evident in the presence of TPA; after electroconvulsive treatment the phorbol ester-induced potentiation was so high that no significant beta-down-regulation could be observed. No procedure affected the response to forskolin. The beta-down-regulation that develops during chronic imipramine treatment differs from that caused by chronic electroconvulsive treatment; in both cases it is not related to the direct effect on adenylate cyclase.     

A Possible Role for Exocytosis in Aflatoxin Export in Aspergillus parasiticus

Filamentous fungi synthesize bioactive secondary metabolites with major human health and economic impacts. Little is known about the mechanisms that mediate the export of these metabolites to the cell exterior. Aspergillus parasiticus synthesizes aflatoxin, a secondary metabolite that is one of the most potent naturally occurring carcinogens known. We previously demonstrated that aflatoxin is synthesized and compartmentalized in specialized vesicles called aflatoxisomes and that these subcellular organelles also play a role in the export process. In the current study, we tested the hypothesis that aflatoxisomes fuse with the cytoplasmic membrane to facilitate the release of aflatoxin into the growth environment. Microscopic analysis of A. parasiticus grown under aflatoxin-inducing and non-aflatoxin-inducing conditions generated several lines of experimental evidence that supported the hypothesis. On the basis of the evidence, we propose that export of the mycotoxin aflatoxin in Aspergillus parasiticus occurs by exocytosis, and we present a model to illustrate this export mechanism.Secondary metabolites are chemically diverse natural products synthesized by plants, fungi, bacteria, algae, and animals. Secondary metabolites have an enormous impact on humans. Antibiotics, for example, are essential elements of the multibillion-dollar pharmaceutical industry, whereas mycotoxins cause hundreds of millions of dollars in damage to agriculture annually (11, 15). These chemicals help the producing organism to survive nutrient limitation (16). They also contribute to cellular defense mechanisms and development (11, 12), reduce cellular oxidative stress (10), and help maintain cellular homeostasis by regulating carbon flow in the cell (17).Many fungal secondary metabolites are exported outside the cell; examples include antibiotics and mycotoxins (3, 14). We and others conducted extensive studies on the regulation of fungal secondary metabolism at the molecular (11, 15) and cellular (3, 7) levels. However, little is known about the mechanisms that mediate secondary metabolite export or why export occurs.The filamentous fungus Aspergillus parasiticus produces aflatoxin, a secondary metabolite and the most potent naturally occurring carcinogen known. More than 90% of aflatoxin is exported to the cell exterior (3), making A. parasiticus an excellent model for studying secondary metabolite export. We recently demonstrated that specialized trafficking vesicles called aflatoxisomes play a key role in aflatoxin synthesis and export (3). As synthesis initiates, vesicle-vacuole fusion is downregulated by the global regulator Velvet, resulting in the accumulation of aflatoxisomes which contain at least the last two functional enzymes in the aflatoxin pathway and sequester aflatoxin (3). Treatments that block vesicle-vacuole fusion increase the number of aflatoxisomes, increase the quantity of aflatoxin accumulated in aflatoxisomes, and increase aflatoxin export to the cell exterior (3). On the basis of these previous observations, we hypothesized that aflatoxisomes play a direct role in aflatoxin export.Vesicle-mediated export could theoretically occur by one (or more) of at least three mechanisms (Fig. 1). (i) Vesicles pass across the cytoplasmic membrane intact and “shuttle” their contents into the external environment. This proposed mechanism mediates virulence factor release in Cryptococcus neoformans and Histoplasma capsulatum (1) during pathogenesis. (ii) Vesicles fuse to the cytoplasmic membrane and “pump” vesicle contents to the exterior using transporter proteins similar to those that mediate resistance to antifungal agents (4, 5). (iii) Vesicles fuse with the cytoplasmic membrane, which evaginates, bursts, and “blasts” vesicle contents to the exterior. This process is similar to exocytosis, a proposed secretory mechanism for specific proteins in filamentous fungi (18). We conducted the current study to determine which, if any, of these possible mechanisms most accurately reflects the process of aflatoxin export in A. parasiticus.Open in a separate windowFig. 1.Theoretical models for vesicle-mediated export. Aflatoxigenic vesicles (aflatoxisomes) arise due to downregulation of tethering complex (Tc) activity mediated by VeA (1). Aflatoxin synthesized in aflatoxisomes could theoretically be released to the cell exterior by one or more of three mechanisms: the shuttle (in which aflatoxisomes shuttle cargo across cytoplasmic membrane), pump (in which transmembrane transporter [Tp] proteins mediate the release of secondary metabolites as vesicles adhere to the inner surface of the cytoplasmic membrane), and burst-and-blast (in which vesicles protrude from the cell surface and blast their cargo into the medium) mechanisms. PM, plasma membrane.     

A Cell Motility Screen Reveals Role for MARCKS-Related Protein in Adherens Junction Formation and Tumorigenesis

Invasion through the extracellular matrix (ECM) is important for wound healing, immunological responses and metastasis. We established an invasion-based cell motility screen using Boyden chambers overlaid with Matrigel to select for pro-invasive genes. By this method we identified antisense to MARCKS related protein (MRP), whose family member MARCKS is a target of miR-21, a microRNA involved in tumor growth, invasion and metastasis in multiple human cancers. We confirmed that targeted knockdown of MRP, in both EpRas mammary epithelial cells and PC3 prostate cancer cells, promoted in vitro cell migration that was blocked by trifluoperazine. Additionally, we observed increased immunofluoresence of E-cadherin, β-catenin and APC at sites of cell-cell contact in EpRas cells with MRP knockdown suggesting formation of adherens junctions. By wound healing assay we observed that reduced MRP supported collective cell migration, a type of cell movement where adherens junctions are maintained. However, destabilized adherens junctions, like those seen in EpRas cells, are frequently important for oncogenic signaling. Consequently, knockdown of MRP in EpRas caused loss of tumorigenesis in vivo, and reduced Wnt3a induced TCF reporter signaling in vitro. Together our data suggest that reducing MRP expression promotes formation of adherens junctions in EpRas cells, allowing collective cell migration, but interferes with oncogenic β-catenin signaling and tumorigenesis.     

Role of Siglec-7 in Apoptosis in Human Platelets

Background

Platelets participate in tissue repair and innate immune responses. Sialic acid-binding immunoglobulin-like lectins (Siglecs) are well-characterized I-type lectins, which control apoptosis.

Methodology/Principal Findings

We characterized the expression of Siglec-7 in human platelets isolated from healthy volunteers using flow cytometry and confocal microscopy. Siglec-7 is primarily expressed on α granular membranes and colocalized with CD62P. Siglec-7 expression was increased upon platelet activation and correlated closely with CD62P expression. Cross-linking Siglec-7 with its ligand, ganglioside, resulted in platelet apoptosis without any significant effects on activation, aggregation, cell morphology by electron microscopy analysis or secretion. We show that ganglioside triggered four key pathways leading to apoptosis in human platelets: (i) mitochondrial inner transmembrane potential (ΔΨm) depolarization; (ii) elevated expression of pro-apoptotic Bax and Bak proteins with reduced expression of anti-apoptotic Bcl-2 protein; (iii) phosphatidylserine exposure and (iv), microparticle formation. Inhibition of NAPDH oxidase, PI3K, or PKC rescued platelets from apoptosis induced by Siglec-7 recruitment, suggesting that the platelet receptors P2Y1 and GPIIbIIIa are essential for ganglioside-induced platelet apoptosis.

Conclusions/Significance

The present work characterizes the role of Siglec-7 and platelet receptors in regulating apoptosis and death. Because some platelet pathology involves apoptosis (idiopathic thrombocytopenic purpura and possibly storage lesions), Siglec-7 might be a molecular target for therapeutic intervention/prevention.