TREK-1 Regulates Cytokine Secretion from Cultured Human Alveolar Epithelial Cells Independently of Cytoskeletal Rearrangements

BackgroundTREK-1 deficient alveolar epithelial cells (AECs) secrete less IL-6, more MCP-1, and contain less F-actin. Whether these alterations in cytokine secretion and F-actin content are related remains unknown. We now hypothesized that cytokine secretion from TREK-1-deficient AECs was regulated by cytoskeletal rearrangements.MethodsWe determined F-actin and α-tubulin contents of control, TREK-1-deficient and TREK-1-overexpressing human A549 cells by confocal microscopy and western blotting, and measured IL-6 and MCP-1 levels using real-time PCR and ELISA.ResultsCytochalasin D decreased the F-actin content of control cells. Jasplakinolide increased the F-actin content of TREK-1 deficient cells, similar to the effect of TREK-1 overexpression in control cells. Treatment of control and TREK-1 deficient cells with TNF-α, a strong stimulus for IL-6 and MCP-1 secretion, had no effect on F-actin structures. The combination of TNF-α+cytochalasin D or TNF-α+jasplakinolide had no additional effect on the F-actin content or architecture when compared to cytochalasin D or jasplakinolide alone. Although TREK-1 deficient AECs contained less F-actin at baseline, quantified biochemically, they contained more α-tubulin. Exposure to nocodazole disrupted α-tubulin filaments in control and TREK-1 deficient cells, but left the overall amount of α-tubulin unchanged. Although TNF-α had no effect on the F-actin or α-tubulin contents, it increased IL-6 and MCP-1 production and secretion from control and TREK-1 deficient cells. IL-6 and MCP-1 secretions from control and TREK-1 deficient cells after TNF-α+jasplakinolide or TNF-α+nocodazole treatment was similar to the effect of TNF-α alone. Interestingly, cytochalasin D decreased TNF-α-induced IL-6 but not MCP-1 secretion from control but not TREK-1 deficient cells.ConclusionAlthough cytochalasin D, jasplakinolide and nocodazole altered the F-actin and α-tubulin structures of control and TREK-1 deficient AEC, the changes in cytokine secretion from TREK-1 deficient cells cannot be explained by cytoskeletal rearrangements in these cells.     

Effect of cytochalasin B on formation and properties of muscle F-actin

Cytochalasin B stimulated polymerization and decreased the concentration of G-actin remaining in equilibrium with F-actin filaments. Polymerization in the presence of cytochalasin B gave rise to a smaller increase of viscosity but to the same increase in light scattering, compared to polymerization in the absence of cytochalasin B. Cytochalasin B reduced the viscosity of F-actin and caused the appearance of ATP hydrolysis by F-actin. The cytochalasin B-induced ATPase activity was inhibited by concentrations of KCl higher than 50 mM. The cytochalasin B-induced ATPase activity was enhanced by ethyleneglycol bis(α-aminoethyl ether)-N,N′-tetraacetic acid and reduced by MgCl₂ at concentrations higher than 0.75 mM. The findings suggest that the stability of actin filaments is reduced by cytochalasin B.     

Lectin-induced haemocyte inactivation in insects

Most multimeric lectins are adhesion molecules, promoting attachment and spreading on surface glycodeterminants. In addition, some lectins have counter-adhesion properties, detaching already spread cells which then acquire round or spindle-formed cell shapes. Since lectin-mediated adhesion and detachment is observed in haemocyte-like Drosophila cells, which have haemomucin as the major lectin-binding glycoprotein, the two opposite cell behaviours may be the result of lectin-mediated receptor rearrangements on the cell surface. To investigate oligomeric lectins as a possible extracellular driving force affecting cell shape changes, we examined lectin-mediated reactions in lepidopteran haemocytes after cytochalasin D-treatment and observed that while cell-spreading was dependent on F-actin, lectin-uptake was less dependent on F-actin. We propose a model of cell shape changes involving a dynamic balance between adhesion and uptake reactions.     

Influence of Trichobilharzia regenti (Digenea: Schistosomatidae) on the Defence Activity of Radix lagotis (Lymnaeidae) Haemocytes

Radix lagotis is an intermediate snail host of the nasal bird schistosome Trichobilharzia regenti. Changes in defence responses in infected snails that might be related to host-parasite compatibility are not known. This study therefore aimed to characterize R. lagotis haemocyte defence mechanisms and determine the extent to which they are modulated by T. regenti. Histological observations of R. lagotis infected with T. regenti revealed that early phases of infection were accompanied by haemocyte accumulation around the developing larvae 2–36 h post exposure (p.e.) to the parasite. At later time points, 44–92 h p.e., no haemocytes were observed around T. regenti. Additionally, microtubular aggregates likely corresponding to phagocytosed ciliary plates of T. regenti miracidia were observed within haemocytes by use of transmission electron microscopy. When the infection was in the patent phase, haemocyte phagocytic activity and hydrogen peroxide production were significantly reduced in infected R. lagotis when compared to uninfected counterparts, whereas haemocyte abundance increased in infected snails. At a molecular level, protein kinase C (PKC) and extracellular-signal regulated kinase (ERK) were found to play an important role in regulating these defence reactions in R. lagotis. Moreover, haemocytes from snails with patent infection displayed lower PKC and ERK activity in cell adhesion assays when compared to those from uninfected snails, which may therefore be related to the reduced defence activities of these cells. These data provide the first integrated insight into the immunobiology of R. lagotis and demonstrate modulation of haemocyte-mediated responses in patent T. regenti infected snails. Given that immunomodulation occurs during patency, interference of snail-host defence by T. regenti might be important for the sustained production and/or release of infective cercariae.     

Activation of neutrophils: measurement of actin conformational changes by flow cytometry

Actin, which comprises approximately 10% of the weight of cytoplasmic protein of neutrophils, is the principal component of the cytoplasmic microfilament lattice. It can exist in either of two physical states, G-actin, which is monomeric, or F-actin, which is polymeric or filamentous. Actin microfilaments support many forms of cell movement. Continuous remodeling of the microfilament lattice, which seems integral to sustained movement, is possible in part because of the ability of actin to change rapidly between its monomeric G-state and its filamentous F-state. Changes in the G- and F-actin equilibrium may be studied by flow analysis using a fluorescent probe which is specific for F-actin, 7-nitrobenz-2-oxa-1,3-diazole-(NBD)-phallacidin. Alterations in neutrophil F-actin have been measured in response to chemotactic agents (e.g., formyl peptides and leukotriene B4), inhibitors of cell movement (e.g., N-ethylmaleimide and cytochalasin B), agents that promote the oxidative burst (e.g., formyl peptides and phorbol esters), and priming agents [e.g., tumor necrosis factor (TNF)]. Measurements may be taken at intervals of a few seconds, allowing comparison of rapid changes in the F-actin content to other rapidly occurring changes, such as altered membrane ion permeability and activation of cellular enzymes. The use of metabolic inhibitors has allowed dissection of some of the biochemical pathways involved in actin assembly in living cells. Although clinical studies are few thus far, the technique has also been used to study basal and stimulated F-actin levels in circulating neutrophils in neonates and in family members of patients with neutrophil-actin dysfunction.     

Transient infection of the zebrafish notochord with E. coli induces chronic inflammation

Zebrafish embryos and larvae are now well-established models in which to study infectious diseases. Infections with non-pathogenic Gram-negative Escherichia coli induce a strong and reproducible inflammatory response. Here, we study the cellular response of zebrafish larvae when E. coli bacteria are injected into the notochord and describe the effects. First, we provide direct evidence that the notochord is a unique organ that is inaccessible to leukocytes (macrophages and neutrophils) during the early stages of inflammation. Second, we show that notochord infection induces a host response that is characterised by rapid clearance of the bacteria, strong leukocyte recruitment around the notochord and prolonged inflammation that lasts several days after bacteria clearance. During this inflammatory response, il1b is first expressed in macrophages and subsequently at high levels in neutrophils. Moreover, knock down of il1b alters the recruitment of neutrophils to the notochord, demonstrating the important role of this cytokine in the maintenance of inflammation in the notochord. Eventually, infection of the notochord induces severe defects of the notochord that correlate with neutrophil degranulation occurring around this tissue. This is the first in vivo evidence that neutrophils can degranulate in the absence of a direct encounter with a pathogen. Persistent inflammation, neutrophil infiltration and restructuring of the extracellular matrix are defects that resemble those seen in bone infection and in some chondropathies. As the notochord is a transient embryonic structure that is closely related to cartilage and bone and that contributes to vertebral column formation, we propose infection of the notochord in zebrafish larvae as a new model to study the cellular and molecular mechanisms underlying cartilage and bone inflammation.KEY WORDS: Zebrafish, Neutrophils, Inflammation, Interleukin-1β     

Neutrophils Promote Mycobacterial Trehalose Dimycolate-Induced Lung Inflammation via the Mincle Pathway

Trehalose 6,6′-dimycolate (TDM), a cord factor of Mycobacterium tuberculosis (Mtb), is an important regulator of immune responses during Mtb infections. Macrophages recognize TDM through the Mincle receptor and initiate TDM-induced inflammatory responses, leading to lung granuloma formation. Although various immune cells are recruited to lung granulomas, the roles of other immune cells, especially during the initial process of TDM-induced inflammation, are not clear. In this study, Mincle signaling on neutrophils played an important role in TDM-induced lung inflammation by promoting adhesion and innate immune responses. Neutrophils were recruited during the early stage of lung inflammation following TDM-induced granuloma formation. Mincle expression on neutrophils was required for infiltration of TDM-challenged sites in a granuloma model induced by TDM-coated-beads. TDM-induced Mincle signaling on neutrophils increased cell adherence by enhancing F-actin polymerization and CD11b/CD18 surface expression. The TDM-induced effects were dependent on Src, Syk, and MAPK/ERK kinases (MEK). Moreover, coactivation of the Mincle and TLR2 pathways by TDM and Pam3CSK4 treatment synergistically induced CD11b/CD18 surface expression, reactive oxygen species, and TNFα production by neutrophils. These synergistically-enhanced immune responses correlated with the degree of Mincle expression on neutrophil surfaces. The physiological relevance of the Mincle-mediated anti-TDM immune response was confirmed by defective immune responses in Mincle^−/− mice upon aerosol infections with Mtb. Mincle-mutant mice had higher inflammation levels and mycobacterial loads than WT mice. Neutrophil depletion with anti-Ly6G antibody caused a reduction in IL-6 and monocyte chemotactic protein-1 expression upon TDM treatment, and reduced levels of immune cell recruitment during the initial stage of infection. These findings suggest a new role of Mincle signaling on neutrophils during anti-mycobacterial responses.     

Promotion of Ccn2 expression and osteoblastic differentiation by actin polymerization, which is induced by laminar fluid flow stress

Fluid flow stress (FSS) is a major mechanical stress that induces bone remodeling upon orthodontic tooth movement, whereas CCN family protein 2 (CCN2) is a potent regenerator of bone defects. In this study, we initially evaluated the effect of laminar FSS on Ccn2 expression and investigated its mechanism in osteoblastic MC3T3-E1 cells. The Ccn2 expression was drastically induced by uniform FSS in an intensity dependent manner. Of note, the observed effect was inhibited by a Rho kinase inhibitor Y27632. Moreover, the inhibition of actin polymerization blocked the FSS-induced activation of Ccn2, whereas inducing F-actin formation using cytochalasin D and jasplakinolide enhanced Ccn2 expression in the same cells. Finally, F-actin formation was found to induce osteoblastic differentiation. In addition, activation of cyclic AMP-dependent kinase, which inhibits Rho signaling, abolished the effect of FSS. Collectively, these findings indicate the critical role of actin polymerization and Rho signaling in CCN2 induction and bone remodeling provoked by FSS.     

A laboratory evaluation of the pathogenicity of Bacillus popilliae var. rhopaea, the agent of milky disease in Rhopaea verreauxi (Coleoptera: Scarabaeidae)

Two methods of infection, i.e., feeding known numbers of spores and rearing larvae in contaminated peat, were used to bioassay the susceptibility of Rhopaea verreauxi to Bacillus popilliae var. rhopaea at 23°C. The susceptibility of the three larval instars was similar as measured by the ID₅₀ and IC₅₀ values. However, within an instar, newly molted larvae were less susceptible than mature larvae when infected by the contaminated peat method. It is suggested that this was due to reduced food intake. The range of ID₅₀ values for all bioassays with R. verreauxi larvae were 1.1 × 10⁷ to 4.0 × 10⁷ spores per larva, and IC₅₀ values were 3.4 × 10⁶ to 5.0 × 10⁷ spores per g of contaminated peat. The slope of the probit line was always low (0.6 to 1.8) except for young first-instar larvae infected by contaminated peat when the slope was 4.0. Disease per se did not affect food intake, though intake was reduced at high doses of contaminated peat. Young larvae often died without developing symptoms but, with increasing age, infected larvae were more likely to develop symptoms. Bioassays with Othnonius batesi and Rhopaea morbillosa indicated a much lower susceptibility per os than for R. verreauxi. It is concluded that the potential for using B. popilliae var. rhopaea to control R. verreauxi is high, but the bacillus is unlikely to be of value in control of O. batesi or R. morbillosa.     

Protective or Deleterious Role of Scavenger Receptors SR-A and CD36 on Host Resistance to Staphylococcus aureus Depends on the Site of Infection

Staphylococcus aureus is a major human opportunistic pathogen responsible for a broad spectrum of infections ranging from benign skin infection to more severe life threatening disorders (e.g. pneumonia, sepsis), particularly in intensive care patients. Scavenger receptors (SR-A and CD36) are known to be involved in S. aureus recognition by immune cells in addition to MARCO, TLR2, NOD2 and α5β1 integrin. In the present study, we further deciphered the contribution of SR-A and CD36 scavenger receptors in the control of infection of mice by S. aureus. Using double SR-A/CD36 knockout mice (S/C-KO) and S. aureus strain HG001, a clinically relevant non-mutagenized strain, we showed that the absence of these two scavenger receptors was protective in peritoneal infection. In contrast, the deletion of these two receptors was detrimental in pulmonary infection following intranasal instillation. For pulmonary infection, susceptible mice (S/C-KO) had more colony-forming units (CFU) in their broncho-alveolar lavages fluids, associated with increased recruitment of macrophages and neutrophils. For peritoneal infection, susceptible mice (wild-type) had more CFU in their blood, but recruited less macrophages and neutrophils in the peritoneal cavity than resistant mice. Exacerbated cytokine levels were often observed in the susceptible mice in the infected compartment as well as in the plasma. The exception was the enhanced compartmentalized expression of IL-1β for the resistant mice (S/C-KO) after peritoneal infection. A similar mirrored susceptibility to S. aureus infection was also observed for MARCO and TLR2. Marco and tlr2 -/- mice were more resistant to peritoneal infection but more susceptible to pulmonary infection than wild type mice. In conclusion, our results show that innate immune receptors can play distinct and opposite roles depending on the site of infection. Their presence is protective for local pulmonary infection, whereas it becomes detrimental in the peritoneal infection.     

Characterization of novel F-actin envelopes surrounding nuclei during cleavage of a polychaete worm

F-actin accumulations and their possible functions were investigated during cleavage of the polychaete Ophryotrocha puerilis. Unusual cytoplasmic accumulations of F-actin were detected which have never been described before in animal embryos. As shown by TRITC-phalloidin labeling, envelopes of F-actin surrounded late prophase nuclei for a short period of time. DTAF-immunofluorescence of beta-tubulin showed that the F-actin envelope was closely associated with microtubules of the developing spindle apparatus. However, experimental disassembly of microtubules by nocodazole did not prevent the assembly of the F-actin envelope. Disturbance of F-actin envelope formation by cytochalasin B did not alter the course of mitotic events, i.e. position of the nuclei and orientation of the spindle apparatus were not affected, although the respective blastomeres remained uncleaved. However, disassembly of the F-actin envelope correlated temporally with breakdown of the nuclear envelope. Therefore, it is suggested that this new structure plays a role in fragmentation of the nuclear envelope during cleavage of Ophryotrocha puerilis.     

TRPM4 regulates migration of mast cells in mice

We demonstrate here that the transient receptor potential melastatin subfamily channel, TRPM4, controls migration of bone marrow-derived mast cells (BMMCs), triggered by dinitrophenylated human serum albumin (DNP-HSA) or stem cell factor (SCF). Wild-type BMMCs migrate after stimulation with DNP-HSA or SCF whereas both stimuli do not induce migration in BMMCs derived from TRPM4 knockout mice (trpm4^?/?). Mast cell migration is a Ca²⁺-dependent process, and TRPM4 likely controls this process by setting the intracellular Ca²⁺ level upon cell stimulation. Cell migration depends on filamentous actin (F-actin) rearrangement, since pretreatment with cytochalasin B, an inhibitor of F-actin formation, prevented both DNP-HSA- and SCF-induced migration in wild-type BMMC. Immunocytochemical experiments using fluorescence-conjugated phalloidin demonstrate a reduced level of F-actin formation in DNP-HSA-stimulated BMMCs from trpm4^?/? mice. Thus, our results suggest that TRPM4 is critically involved in migration of BMMCs by regulation of Ca²⁺-dependent actin cytoskeleton rearrangements.     

Immune reactions of Chironomus larvae (Insecta: Diptera) against bacteria

Humoral encapsulation (“melanization”) represents the predominant defence reaction of Chironomus larvae against injected bacteria. Only low levels of phagocytic activity were observed; cellular encapsulation and nodule formation were completely missing due to low numbers of haemocytes. No other humoral antibacterial activity was detected in normal Chironomus haemolymph and even haemolymph of preinjected (“immunized”) Chironomus larvae showed little inhibition of bacterial growth on agar test plates.Low cellular and lytic activity of Chironomus haemolymph against bacteria is well compensated for by its fast and efficient capacity of humoral encapsulation. Within 5–10 min, even high numbers of injected bacteria (up to 10⁵ per larva) were surrounded by capsular material. Within this range of injection dose, the fates of pathogenic and non-pathogenic strains were identical, and bacteria which are highly pathogenic for many other insects, e.g. larvae of Galleria mellonella, proved to be harmless to Chironomus larvae. The rapidity of humoral encapsulation may prevent the release of toxins or enzymes by which pathogenic bacteria normally damage its host and weaken its immune system.     

Neutrophil-derived IL-1β Is Sufficient for Abscess Formation in Immunity against Staphylococcus aureus in Mice

Neutrophil abscess formation is critical in innate immunity against many pathogens. Here, the mechanism of neutrophil abscess formation was investigated using a mouse model of Staphylococcus aureus cutaneous infection. Gene expression analysis and in vivo multispectral noninvasive imaging during the S. aureus infection revealed a strong functional and temporal association between neutrophil recruitment and IL-1β/IL-1R activation. Unexpectedly, neutrophils but not monocytes/macrophages or other MHCII-expressing antigen presenting cells were the predominant source of IL-1β at the site of infection. Furthermore, neutrophil-derived IL-1β was essential for host defense since adoptive transfer of IL-1β-expressing neutrophils was sufficient to restore the impaired neutrophil abscess formation in S. aureus-infected IL-1β-deficient mice. S. aureus-induced IL-1β production by neutrophils required TLR2, NOD2, FPR1 and the ASC/NLRP3 inflammasome in an α-toxin-dependent mechanism. Taken together, IL-1β and neutrophil abscess formation during an infection are functionally, temporally and spatially linked as a consequence of direct IL-1β production by neutrophils.     

The Hypersensitivity Reaction of Tomatoes Resistant to Meloidogyne incognita: Reversal by Cytokinins

Initiation of larval growth, induction of cell necrosis, and gall formation in the host were measured as criteria of resistance or susceptibility of tomato seedlings to the root-knot nematode, Meloidogyne incognita (Kofoid &White) Chitwood. Seedlings grown at 27 C on water agar containing additions were scored 3 or 4 days after infection.In the absence of exogenous plant growth regulatory substances, approximately 73% of larvae that entered roots of susceptible plants showed growth, none induced necrosis and nearly all induced gall formation. In roots of a resistant variety, only 4% of the larvae grew, 88% induced necrosis of host cells, and only 29% induced galls. Exogenously supplied cytokinins shifted the response of the resistant plants toward the susceptible reaction. Exogenous kinetin at 0.4 and 0.8 µmolar allowed 55 and 57% of the nematodes to grow, reduced the incidence of necrosis to 32 and 31%, and increased gall formation to 73 and 65%. Three additional cytokinins, Zeatin, 6-(γ,γ-dimethylallylamino)purine, and 6-benzylaminopurine produced effects similar to kinetin. Exogenous indoleacetic acid, gibberellic acid, adenine, guanine, uracil, thymine, cytidine, and 6-methylaminopurine neither increased the percentage of larvae which grew nor decreased the extent of host cell necrosis.     

Larvae of the ectoparasitic wasp,Eulophus pennicornis,release factors which adversely affect haemocytes of their host,Lacanobia oleracea

When larvae of the ectoparasitic wasp Eulophus pennicornis were incubated for 4 h on balls of cotton wool soaked in tissue culture medium (TC-100), they released a variety of factors. Subsequent incubation of these larval wasp secretions with monolayers of haemocytes from their host, Lacanobia oleracea, demonstrated that they adversely affect haemocyte morphology, behaviour and viability. For instance, when monolayers of haemocytes were incubated for 18 h in TC-100, approximately 73% of the cells present, attached firmly to and spread over the tissue culture surface by extending pseudopods. By contrast, when incubated in TC-100 containing larval wasp secretions, only about 27% of the haemocytes present remained attached to the tissue culture surface after washing. The majority of these had a rounded configuration and neither spread nor extended pseudopods. Furthermore, viability assays indicated that approximately 36% of the attached haemocytes were dead, as opposed to 11-12% in the controls. The E. pennicornis secretions also significantly reduced the ability of L. oleracea haemocytes to move across the surface of the slide and form clumps (p≤0.0005) and to phagocytose FITC-labelled Escherichia coli in vitro (p≤0.0005). These results indicate that secretions from E. pennicornis larvae contain an anti-haemocyte factor(s) that can kill and/or alter the behaviour of host haemocytes. As a result, the ability of the haemocytes to execute important immune responses is compromised. Preliminary data suggest that the active molecules are proteins, and that their mechanism of action may involve inhibition of polymerization and/or disorganization of the haemocyte cytoskeleton.     

Integrity of the Actin Cytoskeleton of Host Macrophages is Essential for Leishmania donovani Infection

Visceral leishmaniasis is a vector-borne disease caused by an obligate intracellular protozoan parasite Leishmania donovani. The molecular mechanism involved in internalization of Leishmania is poorly understood. The entry of Leishmania involves interaction with the plasma membrane of host cells. We have previously demonstrated the requirement of host membrane cholesterol in the binding and internalization of L. donovani into macrophages. In the present work, we explored the role of the host actin cytoskeleton in leishmanial infection. We observed a dose-dependent reduction in the attachment of Leishmania promastigotes to host macrophages upon destabilization of the actin cytoskeleton by cytochalasin D. This is accompanied by a concomitant reduction in the intracellular amastigote load. We utilized a recently developed high resolution microscopy-based method to quantitate cellular F-actin content upon treatment with cytochalasin D. A striking feature of our results is that binding of Leishmania promastigotes and intracellular amastigote load show close correlation with cellular F-actin level. Importantly, the binding of Escherichia coli remained invariant upon actin destabilization of host cells, thereby implying specific involvement of the actin cytoskeleton in Leishmania infection. To the best of our knowledge, these novel results constitute the first comprehensive demonstration on the specific role of the host actin cytoskeleton in Leishmania infection. Our results could be significant in developing future therapeutic strategies to tackle leishmaniasis.     

Ellagic acid derivatives from Rubus ulmifolius inhibit Staphylococcus aureus biofilm formation and improve response to antibiotics

Background

Biofilms contribute to the pathogenesis of many forms of Staphylococcus aureus infection. Treatment of these infections is complicated by intrinsic resistance to conventional antibiotics, thus creating an urgent need for strategies that can be used for the prevention and treatment of biofilm-associated infections.

Methodology/Principal Findings

This study demonstrates that a botanical natural product composition (220D-F2) rich in ellagic acid and its derivatives can limit S. aureus biofilm formation to a degree that can be correlated with increased antibiotic susceptibility. The source of this composition is Rubus ulmifolius Schott. (Rosaceae), a plant used in complementary and alternative medicine in southern Italy for the treatment of skin and soft tissue infections. All S. aureus clonal lineages tested exhibited a reduced capacity to form a biofilm at 220D-F2 concentrations ranging from 50–200 µg/mL, which were well below the concentrations required to limit bacterial growth (530–1040 µg/mL). This limitation was therapeutically relevant in that inclusion of 220D-F2 resulted in enhanced susceptibility to the functionally-distinct antibiotics daptomycin, clindamycin and oxacillin. Testing with kidney and liver cell lines also demonstrated a lack of host cell cytotoxicity at concentrations of 220D-F2 required to achieve these effects.

Conclusions/Significance

These results demonstrate that extract 220D-F2 from the root of Rubus ulmifolius can be used to inhibit S. aureus biofilm formation to a degree that can be correlated with increased antibiotic susceptibility without toxic effects on normal mammalian cells. Hence, 220D-F2 is a strong candidate for development as a botanical drug for use in the prevention and treatment of S. aureus biofilm-associated infections.     

Hypertonicity-induced projections reflect cell polarity in mouse metaphase II oocytes: involvement of microtubules,microfilaments, and chromosomes

A previous study showed that with hypertonic sucrose treatment, a projection is formed in mouse metaphase II (MII) oocytes in proximity to the spindle and chromosomes, where a polarized cortical domain is located. However, little is known about the mechanisms involved in this process. Here, we designed a series of experiments to test the hypothesis that hypertonicity is the induction factor for the formation of projections in mouse MII oocytes. Our hypothesis was supported by the following evidence: 1) different concentrations of sucrose affected the formation and shape of projections, whereas serum or basic media had little effect; 2) other hypertonic sugar solutions could also induce projection formation; and 3) projections could also be induced by hypertonic NaCl solution. We then tested the hypothesis that the cytoskeleton was involved in the formation of hypertonicity-induced projections. This was investigated by culturing MII- and germinal vesicle-stage mouse oocytes in the presence or absence of cytoskeletal inhibitors, including cytochalasin B (disruption of actin filaments), nocodazole (disruption of microtubules), and taxol (polymerization of tubulin molecules). We found that none of the cytoskeletal inhibitors alone could prevent hypertonicity-induced projection formation, whereas the combination of cytochalasin B with nocodazole or with taxol blocked the formation of these projections in most matured oocytes. When immature oocytes were incubated in cytochalasin B, but not in nocodazole or taxol, the formation of an actin-rich domain and the peripheral positioning of the spindle were blocked during maturation; hence, no projections were formed, even after hypertonic sucrose treatment. Based on these observations, we propose that three components are necessary for projection formation: 1) a polarized cortical patch (e.g., an actin-rich domain), 2) rigid submembrane structures (e.g., a spindle and/or chromosomes), and 3) solid connections between the above. Any disturbance of one of these factors will affect the hypertonicity-induced projection formation. Hypertonicity-induced projection in mouse oocytes thus provides an experimental model for studies regarding cell polarity and the interaction between membrane and submembrane components.