Smad7 Inhibits Mesoderm Formation and Promotes Neural Cell Fate inXenopusEmbryos

We report the isolation and characterization of a new inhibitory Smad inXenopus,which we have designated asXenopusSmad7. Smad7 is present at fairly constant levels throughout early development and at blastula stages enriched in the ventral side of the animal hemisphere. The induction of mesoderm by TGF-β-like signals is mediated by receptor ALK-4 and we show that Smad7 blocks signaling of ALK-4 in a graded fashion: lower levels of Smad7 block activation of dorsal mesoderm genes and higher levels block all mesoderm genes expression. Smad7 is able to directly activate neural markers in explants in the absence of mesoderm or endoderm. This neural-inducing activity of Smad7 may be due to inhibition of BMP-4 signaling because Smad7 can also block BMP-4-mediated mesoderm induction. Thus, Smad7 acts as a potent inhibitor of mesoderm formation and also activates the default neural induction pathway.     

Epithelial Cell Mitochondrial Dysfunction and PINK1 Are Induced by Transforming Growth Factor- Beta1 in Pulmonary Fibrosis

Background

Epithelial cell death is a major contributor to fibrogenesis in the lung. In this study, we sought to determine the function of mitochondria and their clearance (mitophagy) in alveolar epithelial cell death and fibrosis.

Methods

We studied markers of mitochondrial injury and the mitophagy marker, PTEN-induced putative kinase 1 (PINK1), in IPF lung tissues by Western blotting, transmission electron microscopy (TEM), and immunofluorescence. In vitro experiments were carried out in lung epithelial cells stimulated with transforming growth factor-β1 (TGF-β1). Changes in cell function were measured by Western blotting, flow cytometry and immunofluorescence. In vivo experiments were performed using the murine bleomycin model of lung fibrosis.

Results

Evaluation of IPF lung tissue demonstrated increased PINK1 expression by Western blotting and immunofluorescence and increased numbers of damaged mitochondria by TEM. In lung epithelial cells, TGF-β1 induced mitochondrial depolarization, mitochondrial ROS, and PINK1 expression; all were abrogated by mitochondrial ROS scavenging. Finally, Pink1 ^-/- mice were more susceptible than control mice to bleomycin induced lung fibrosis.

Conclusion

TGF-β1 induces lung epithelial cell mitochondrial ROS and depolarization and stabilizes the key mitophagy initiating protein, PINK1. PINK1 ameliorates epithelial cell death and may be necessary to limit fibrogenesis.     

Persistent Effectivity of Gas Plasma-Treated,Long Time-Stored Liquid on Epithelial Cell Adhesion Capacity and Membrane Morphology

Research in plasma medicine includes a major interest in understanding gas plasma-cell interactions. The immediate application of gas plasma in vitro inhibits cell attachment, vitality and cell-cell contacts via the liquid. Interestingly, in our novel experiments described here we found that the liquid-mediated plasma effect is long-lasting after storage up to seven days; i. e. the liquid preserves the characteristics once induced by the argon plasma. Therefore, the complete Dulbecco''s Modified Eagle cell culture medium was argon plasma-treated (atmospheric pressure, kINPen09) for 60 s, stored for several days (1, 4 and 7 d) at 37°C and added to a confluent mouse hepatocyte epithelial cell (mHepR1) monolayer. Impaired tight junction architecture as well as shortened microvilli on the cell membrane could be observed, which was accompanied by the loss of cell adhesion capacity. Online-monitoring of vital cells revealed a reduced cell respiration. Our first time-dependent analysis of plasma-treated medium revealed that temperature, hydrogen peroxide production, pH and oxygen content can be excluded as initiators of cell physiological and morphological changes. The here observed persisting biological effects in plasma-treated liquids could open new medical applications in dentistry and orthopaedics.     

Real-Time Sensing of Enteropathogenic E. coli-Induced Effects on Epithelial Host Cell Height,Cell-Substrate Interactions,and Endocytic Processes by Infrared Surface Plasmon Spectroscopy

Enteropathogenic Escherichia coli (EPEC) is an important, generally non-invasive, bacterial pathogen that causes diarrhea in humans. The microbe infects mainly the enterocytes of the small intestine. Here we have applied our newly developed infrared surface plasmon resonance (IR-SPR) spectroscopy approach to study how EPEC infection affects epithelial host cells. The IR-SPR experiments showed that EPEC infection results in a robust reduction in the refractive index of the infected cells. Assisted by confocal and total internal reflection microscopy, we discovered that the microbe dilates the intercellular gaps and induces the appearance of fluid-phase-filled pinocytic vesicles in the lower basolateral regions of the host epithelial cells. Partial cell detachment from the underlying substratum was also observed. Finally, the waveguide mode observed by our IR-SPR analyses showed that EPEC infection decreases the host cell''s height to some extent. Together, these observations reveal novel impacts of the pathogen on the host cell architecture and endocytic functions. We suggest that these changes may induce the infiltration of a watery environment into the host cell, and potentially lead to failure of the epithelium barrier functions. Our findings also indicate the great potential of the label-free IR-SPR approach to study the dynamics of host-pathogen interactions with high spatiotemporal sensitivity.     

Amyloid-Beta Induced CA1 Pyramidal Cell Loss in Young Adult Rats Is Alleviated by Systemic Treatment with FGL,a Neural Cell Adhesion Molecule-Derived Mimetic Peptide

Increased levels of neurotoxic amyloid-beta in the brain are a prominent feature of Alzheimer’s disease. FG-Loop (FGL), a neural cell adhesion molecule-derived peptide that corresponds to its second fibronectin type III module, has been shown to provide neuroprotection against a range of cellular insults. In the present study impairments in social recognition memory were seen 24 days after a 5 mg/15 µl amyloid-beta(_25–35) injection into the right lateral ventricle of the young adult rat brain. This impairment was prevented if the animal was given a systemic treatment of FGL. Unbiased stereology was used to investigate the ability of FGL to alleviate the deleterious effects on CA1 pyramidal cells of the amyloid-beta(_25–35) injection. NeuN, a neuronal marker (for nuclear staining) was used to identify pyramidal cells, and immunocytochemistry was also used to identify inactive glycogen synthase kinase 3beta (GSK3β) and to determine the effects of amyloid-beta(_25–35) and FGL on the activation state of GSK3β, since active GSK3β has been shown to cause a range of AD pathologies. The cognitive deficits were not due to hippocampal atrophy as volume estimations of the entire hippocampus and its regions showed no significant loss, but amyloid-beta caused a 40% loss of pyramidal cells in the dorsal CA1 which was alleviated partially by FGL. However, FGL treatment without amyloid-beta was also found to cause a 40% decrease in CA1 pyramidal cells. The action of FGL may be due to inactivation of GSK3β, as an increased proportion of CA1 pyramidal neurons contained inactive GSK3β after FGL treatment. These data suggest that FGL, although potentially disruptive in non-pathological conditions, can be neuroprotective in disease-like conditions.     

Simian Virus 40 Large T Antigen J Domain and Rb-Binding Motif Are Sufficient To Block Apoptosis Induced by Growth Factor Withdrawal in a Neural Stem Cell Line

Serum-free mouse embryo (SFME) cells are a neural stem cell line that is dependent upon epidermal growth factor (EGF) for survival. Removal of EGF results in the G1 arrest and apoptosis of SFME cells. We have shown that the expression of simian virus 40 large T antigen in SFME cells blocks apoptosis and allows cell survival and division in the absence of EGF. Therefore the presence of T antigen abrogates the EGF requirement. The steady-state levels of p53, p21, and mdm-2 do not increase as SFME cells undergo apoptosis upon EGF withdrawal. Furthermore, the amino-terminal 136 amino acids (N136) of T antigen are sufficient to block death and to promote proliferation in the absence of EGF, while the carboxy-terminal fragment (C251-708), which contains the p53 binding site, is unable to block death. Taken together, these data suggest that SFME cells deprived of EGF undergo p53-independent apoptosis. Mutations that disrupt either the J domain or Rb family binding abolish the ability of T antigen to block SFME cell apoptosis and to promote cell growth. We conclude that T antigen must act on one or more members of the Rb family to inhibit SFME cell apoptosis.     

Cellular Basis of Neuralization of Induced Neurectoderm in Amphibian Embryogenesis: Changes of Cell Shape, Cell Size, and Cytodifferentiation of the Neurectoderm after Neural Induction

Cellular alterations of the neurectoderm after primary embryonic induction were examined by measuring several indices of shape, volume, and cytodifferentiation of the neurectodermal cells of Cynops embryos during gastrulation and early neurulation.
Results showed that cellular alterations occurs just after the 18 hr embryo stage (stage 13b). The thickness of the neurectoderm layer decreases like that of the epidermal ectoderm during early and middle gastrulation. After the 18 hr embryo stage, however, the neurectoderm thickens, mainly due to formation of columnar cells. Measurement of cell volume indicates that the neurectoderm of the early and middle gastrulae consists of a cell population of heterogeneous size. The heterogeneity diminishes sharply after the 18 hr embryo stage and the neural plate of the 36 hr embryo (stage 18) consists of cells of homogeneous size.
Stages before the 12 hr embryo (stage 12b) and after the 18 hr embryo (stage 13b) also showed differed in cell adhesion to the culture flask and in cytodifferentiating potency. Single cells dissociated from the neurectoderm of 18 hr embryos could adhere to the substratum and differentiate into both nerve-like cells and pigment cells. Both capacities increase during further development.
These results are discussed in relation to the neuralizing determination of neurectoderm after primary embryonic induction.     

Formation and Structure of a NAIP5-NLRC4 Inflammasome Induced by Direct Interactions with Conserved N- and C-terminal Regions of Flagellin

The NOD-like receptors NAIP5 and NLRC4 play an essential role in the innate immune response to the bacterial tail protein flagellin. Upon flagellin detection, NAIP5 and NLRC4 form a hetero-oligomeric inflammasome that induces caspase-1-dependent cell death. So far, both the mechanism of formation of the NAIP5-NLRC4 inflammasome and its structure are poorly understood. In this study we combine inflammasome reconstitution in HEK293 cells, purification of inflammasome components, and negative stain electron microscopy to address these issues. We find that a Salmonella typhimurium flagellin fragment comprising the D0 domain and the neighboring spoke region is able to co-precipitate NAIP5 and induce formation of the NAIP5-NLRC4 inflammasome. Comparison with smaller fragments indicates that flagellin recognition is mediated by its C-terminal residues as well as the spoke region. We reconstitute the inflammasome from purified flagellin, NAIP5, and NLRC4, thus proving that no other cellular components are required for its formation. Electron micrographs of the purified inflammasome provide unprecedented insight into its architecture, revealing disk-like complexes consisting of 11 or 12 protomers in which NAIP5 and NLRC4 appear to occupy equivalent positions. On the basis of our data, we propose a model for inflammasome formation wherein direct interaction of flagellin with a single NAIP5 induces the recruitment and progressive incorporation of NLRC4, resulting in the formation of a hetero-oligomeric inflammasome.     

Nanopodia - Thin,Fragile Membrane Projections with Roles in Cell Movement and Intercellular Interactions

Adherent cells in culture maintain a polarized state to support movement and intercellular interactions. Nanopodia are thin, elongated, largely F-actin-negative membrane projections in endothelial and cancer cells that can be visualized through TM4SF1 (Transmembrane-4-L-six-family-1) immunofluorescence staining. TM4SF1 clusters in 100-300 μm diameter TMED (TM4SF1 enriched microdomains) containing 3 to as many as 14 individual TM4SF1 molecules. TMED are arranged intermittently along nanopodia at a regular spacing of 1 to 3 TMED per μm and firmly anchor nanopodia to matrix. This enables nanopodia to extend more than 100 μm from the leading front or trailing rear of polarized endothelial or tumor cells, and causes membrane residues to be left behind on matrix when the cell moves away. TMED and nanopodia have been overlooked because of their extreme fragility and sensitivity to temperature. Routine washing and fixation disrupt the structure. Nanopodia are preserved by direct fixation in paraformaldehyde (PFA) at 37 °C, followed by brief exposure to 0.01% Triton X-100 before staining. Nanopodia open new vistas in cell biology: they promise to reshape our understanding of how cells sense their environment, detect and identify other cells at a distance, initiate intercellular interactions at close contact, and of the signaling mechanisms involved in movement, proliferation, and cell-cell communications. The methods that are developed for studying TM4SF1-derived nanopodia may be useful for studies of nanopodia that form in other cell types through the agency of classic tetraspanins, notably the ubiquitously expressed CD9, CD81, and CD151.     

Functional Consequences of the Interactions among the Neural Cell Adhesion Molecule NCAM,the Receptor Tyrosine Kinase TrkB,and the Inwardly Rectifying K+ Channel KIR3.3

Cell adhesion molecules and neurotrophin receptors are crucial for the development and the function of the nervous system. Among downstream effectors of neurotrophin receptors and recognition molecules are ion channels. Here, we provide evidence that G protein-coupled inwardly rectifying K⁺ channel Kir3.3 directly binds to the neural cell adhesion molecule (NCAM) and neurotrophin receptor TrkB. We identified the binding sites for NCAM and TrkB at the C-terminal intracellular domain of Kir3.3. The interaction between NCAM, TrkB, and Kir3.3 was supported by immunocytochemical co-localization of Kir3.3, NCAM, and/or TrkB at the surface of hippocampal neurons. Co-expression of TrkB and Kir3.1/3.3 in Xenopus oocytes increased the K⁺ currents evoked by Kir3.1/3.3 channels. This current enhancement was reduced by the concomitant co-expression with NCAM. Both surface fluorescence measurements of microinjected oocytes and cell surface biotinylation of transfected CHO cells indicated that the cell membrane localization of Kir3.3 is regulated by TrkB and NCAM. Furthermore, the level of Kir3.3, but not of Kir3.2, at the plasma membranes was reduced in TrkB-deficient mice, supporting the notion that TrkB regulates the cell surface expression of Kir3.3. The premature expression of developmentally late appearing Kir3.1/3.3 in hippocampal neurons led to a reduction of NCAM-induced neurite outgrowth. Our observations indicate a decisive role for the neuronal K⁺ channel in regulating NCAM-dependent neurite outgrowth and attribute a physiologically meaningful role to the functional interplay of Kir3.3, NCAM, and TrkB in ontogeny.     

Short-Term Consumption of Sucralose with,but Not without,Carbohydrate Impairs Neural and Metabolic Sensitivity to Sugar in Humans

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Identification of Sequences in the Polysialyltransferases ST8Sia II and ST8Sia IV That Are Required for the Protein-specific Polysialylation of the Neural Cell Adhesion Molecule, NCAM

The polysialyltransferases ST8Sia II and ST8Sia IV polysialylate the glycans of a small subset of mammalian proteins. Their most abundant substrate is the neural cell adhesion molecule (NCAM). An acidic surface patch and a novel α-helix in the first fibronectin type III repeat of NCAM are required for the polysialylation of N-glycans on the adjacent immunoglobulin domain. Inspection of ST8Sia IV sequences revealed two conserved polybasic regions that might interact with the NCAM acidic patch or the growing polysialic acid chain. One is the previously identified polysialyltransferase domain (Nakata, D., Zhang, L., and Troy, F. A. (2006) Glycoconj. J. 23, 423–436). The second is a 35-amino acid polybasic region that contains seven basic residues and is equidistant from the large sialyl motif in both polysialyltransferases. We replaced these basic residues to evaluate their role in enzyme autopolysialylation and NCAM-specific polysialylation. We found that replacement of Arg²⁷⁶/Arg²⁷⁷ or Arg²⁶⁵ in the polysialyltransferase domain of ST8Sia IV decreased both NCAM polysialylation and autopolysialylation in parallel, suggesting that these residues are important for catalytic activity. In contrast, replacing Arg⁸²/Arg⁹³ in ST8Sia IV with alanine substantially decreased NCAM-specific polysialylation while only partially impacting autopolysialylation, suggesting that these residues may be particularly important for NCAM polysialylation. Two conserved negatively charged residues, Glu⁹² and Asp⁹⁴, surround Arg⁹³. Replacement of these residues with alanine largely inactivated ST8Sia IV, whereas reversing these residues enhanced enzyme autopolysialylation but significantly reduced NCAM polysialylation. In sum, we have identified selected amino acids in this conserved polysialyltransferase polybasic region that are critical for the protein-specific polysialylation of NCAM.Polysialic acid is a linear homopolymer of α2,8-linked sialic acid that is added to a small subset of mammalian glycoproteins by the polysialyltransferases (polySTs)3 ST8Sia II (STX) and ST8Sia IV (PST) (1–4). Substrates for the polySTs include the neural cell adhesion molecule (NCAM) (5, 6), the α-subunit of the voltage-dependent sodium channel (7, 8), CD36, a scavenger receptor found in milk (9), neuropilin-2 expressed by dendritic cells (10), and the polySTs themselves, which can polysialylate their own N-glycans in a process called autopolysialylation (11, 12). This small number of polysialylated proteins and other evidence from our laboratory (13–15) suggest that polysialylation is a protein-specific modification that requires an initial protein-protein interaction between the polySTs and their glycoprotein substrates.The most abundant polysialylated protein is NCAM. The three major NCAM isoforms consist of five Ig domains, two fibronectin type III repeats, and a transmembrane domain and cytoplasmic tail (NCAM140 and NCAM180) or a glycosylphosphatidylinositol anchor (NCAM120) (16). Polysialylation takes place primarily on two N-linked glycans in the Ig5 domain (17). We have previously shown that a truncated NCAM140 protein consisting of Ig5, the first fibronectin type III repeat (FN1), the transmembrane region, and cytoplasmic tail is fully polysialylated (13). However, a protein consisting of Ig5, the transmembrane region, and cytoplasmic tail is not polysialylated (13). This suggests that the polySTs recognize and bind the FN1 domain to polysialylate N-glycans on the adjacent Ig5 domain. We subsequently identified an acidic patch unique to NCAM FN1, consisting of Asp⁴⁹⁷, Asp⁵¹¹, Glu⁵¹², and Glu⁵¹⁴ (15).⁴ When three of these residues (Asp⁵¹¹, Glu⁵¹², and Glu⁵¹⁴) are mutated to alanine or arginine, NCAM polysialylation is reduced or abolished, suggesting that the acidic patch is part of a larger recognition region. We anticipate that within this putative recognition region there will be amino acids required for mediating polyST-NCAM binding, and those that do not mediate binding per se but instead are required for correct positioning of the enzyme-substrate complex for polysialylation. For example, we have identified a novel α-helix in the FN1 domain that when replaced leads to polysialylation of O-glycans found on the FN1 domain rather than N-glycans on the Ig5 domain (14). This helix may mediate an interdomain interaction that positions the Ig5 N-glycans for polysialylation by an enzyme bound to the FN1 domain (14). Alternatively, the helix could act as a secondary interaction site that positions the polyST properly on the substrate.The expression of the polySTs is developmentally regulated with high levels of STX and moderate levels of PST expressed throughout the developing embryo (2, 18, 19). STX levels decline after birth, although PST expression persists in specific regions of the adult brain where polysialylated NCAM is involved in neuronal regeneration and synaptic plasticity (18–23). The large size and negative charge of polysialic acid disrupt NCAM-dependent and NCAM-independent interactions, thereby negatively modulating cell adhesion (24–26). Simultaneous disruption of both PST and STX in mice results in severe neuronal defects and death usually within 4 weeks after birth (27). Interestingly, when NCAM expression is also eliminated in these mice, they have a nearly normal phenotype, suggesting the main function of polysialic acid is to modulate NCAM-mediated cell adhesion during development (27). In addition, re-expression of highly polysialylated NCAM has been associated with several cancers, including neuroblastomas, gliomas, small cell lung carcinomas, and Wilms tumor. The presence of polysialic acid is thought to promote cancer cell growth and invasiveness (28–35).Sialyltransferases, including the polySTs, have three motifs required for catalytic activity (36–38) (see Fig. 1A). Sialyl motif Large (SML) is thought to bind the donor substrate CMP-sialic acid (39), whereas sialyl motif Small (SMS) is believed to bind both donor and carbohydrate acceptor substrates (40). The sialyl motif Very Small (SMVS) has a conserved His residue that is required for catalytic activity (38, 41). However, the precise function of this motif is unknown. An additional 4-amino acid motif, motif III, is conserved in the sialyltransferases (42–44). It was suggested that this motif, and particularly His and Tyr residues within its sequence, may be required for optimal activity and acceptor recognition (42).Open in a separate windowFIGURE 1.PST and STX polybasic regions and mutants generated for this study. A, representation of the polySTs and their polybasic regions and sialyl motifs. The PBR is a 35-amino acid region present in both PST and STX, equidistant from the SML of each enzyme and rich in conserved positively charged amino acids. The PSTD is a region identified by Nakata et al. (47) that is 32 amino acids in length, rich in basic residues, and contiguous with the SMS of the enzymes. The sialyl motifs (SML, SMS, SMVS, and motif III) are regions of homology found in all sialyltransferases that are believed to be involved in substrate and donor interactions. B, PSTD of PST and the mutants made in this region that are used in this study. C, PBR of PST and STX and the mutants made in this region that are used in this study.Angata et al. (45) used chimeric enzymes to identify regions within the polySTs required for catalytic activity and NCAM polysialylation. Sequences from PST, STX, and ST8Sia III were used to construct the chimeric proteins. ST8Sia III is an α2,8-sialyltransferase that typically adds one or two sialic acid residues to glycoprotein or glycolipid substrates, can autopolysialylate its own glycans, but cannot polysialylate NCAM (46). Deletion analysis showed that amino acids 62–356 are required for PST catalytic activity. Replacement of segments of this region with corresponding STX or ST8Sia III sequences led to the suggestion that amino acids 62–127 and possibly 194–267 of PST may be required for NCAM recognition (45).Recently, Troy and co-workers (47, 48) identified a stretch of basic residues, termed the polysialyltransferase domain (PSTD), which is only observed in the two polySTs and not in other sialyltransferases. The PSTD is contiguous with SMS and extends from amino acids 246–277 in PST and 261–292 in STX. Mutation analysis demonstrated that the overall positive charge of this motif is important for activity and identified specific residues required for NCAM polysialylation (Arg²⁵², Ile²⁷⁵, Lys²⁷⁶, and Arg²⁷⁷) (47).In this study, we have scanned the critical polyST regions identified by the work of Angata et al. (45) for sequences that may be involved in protein-protein recognition and NCAM polysialylation. We identified a second polybasic motif that we named the polybasic region (PBR). The PBR is conserved in PST and STX and is located equidistant from the SML of each enzyme. It consists of 35 amino acids of which 7 are the basic amino acids Arg and Lys. We found that the replacement of two specific residues within the PBR (Arg⁸² and Arg⁹³ of PST and Arg⁹⁷ and Lys¹⁰⁸ of STX) have a greater negative effect on NCAM polysialylation than on autopolysialylation. Replacement of acidic residues surrounding PST Arg⁹³ led to a similar disparate effect on these processes. Comparison of the critical residues in both the PSTD and PBR demonstrated that the replacement of PSTD residues had an equally negative impact on both NCAM polysialylation and enzyme autopolysialylation, whereas replacement of selected PBR residues more severely impacted NCAM polysialylation, suggesting that the PBR residues may play important roles in NCAM-specific polysialylation.     

Male Gender,Increased Blood Viscosity,Body Mass Index and Triglyceride Levels Are Independently Associated with Systemic Relative Hypertension in Sickle Cell Anemia

Patients with sickle cell anemia (SCA) have usually lower diastolic, systolic and mean blood pressure (BP) than the general population. However, BP values ≥120/70 mmHg considerably increase the risk for acute and chronic complications in SCA. The aim of this study was to identify biological factors associated with relative hypertension in adults with SCA. We compared the hematological, lipid and hemolytic profiles, as well as blood viscosity, between SCA patients with normal BP (<120/70 mmHg, n = 54) and those with relative hypertension (BP≥120/70 mmHg, n = 43). Our results demonstrated that male gender (OR: 3.49; 95%CI 1.20 to 10.16, p<0.05), triglycerides (OR: 9.19; 95% CI 2.29 to 36.95, p<0.01), blood viscosity (OR: 1.35; 95% CI 1.01 to 1.81, p<0.05) and body mass index (OR: 1.37; 95% CI 1.14 to 1.64, p<0.01) were independent risks factors for relative hypertension in SCA. No association was found between the BP status and the positive history of painful vaso-occlusive crisis or acute chest syndrome. An association between triglycerides level and the occurrence of these two major acute complications was detected. Our study suggests that male gender, increased triglycerides level, BMI and blood viscosity could increase the risk for developing relative hypertension in SCA. In addition, our results support a role of moderately elevated triglycerides in the pathophysiology of vaso-occlusive events.     

MicroRNA-150 Predicts a Favorable Prognosis in Patients with Epithelial Ovarian Cancer,and Inhibits Cell Invasion and Metastasis by Suppressing Transcriptional Repressor ZEB1

MicroRNA (miR)-150 has been reported to be dramatically downregulated in human epithelial ovarian cancer (EOC) tissues and patients’ serum compared to normal controls. This study aimed to investigate clinical significance and molecular mechanisms of miR-150 in EOC. In the current study, quantitative real-time PCR analysis showed that miR-150 was significantly downregulated in human EOC tissues compared to normal tissue samples. Then, we demonstrated the significant associations of miR-150 downregulation with aggressive clinicopathological features of EOC patients, including high clinical stage and pathological grade, and shorter overall and progression-free survivals. More importantly, the multivariate analysis identified miR-150 expression as an independent prognostic biomarker in EOC. After that, luciferase reporter assays demonstrated that Zinc Finger E-Box Binding Homeobox 1 (ZEB1), a crucial regulator of epithelial-to-mesenchymal transition (EMT), was a direct target of miR-150 in EOC cells. Moreover, we found that the ectopic expression of miR-150 could efficiently inhibit cell proliferation, invasion and metastasis by suppressing the expression of ZEB1. Furthermore, we also observed a significantly negative correlation between miR-150 and ZEB1 mRNA expression in EOC tissues (rs = –0.45, P<0.001). In conclusion, these findings offer the convincing evidence that aberrant expression of miR-150 may play a role in tumor progression and prognosis in patients with EOC. Moreover, our data reveal that miR-150 may function as a tumor suppressor and modulate EOC cell proliferation, and invasion by directly and negatively regulating ZEB1, implying the re-expression of miR-150 might be a potential therapeutic strategy for EOC.     

Hypoxia Induced Aggressiveness of Prostate Cancer Cells Is Linked with Deregulated Expression of VEGF, IL-6 and miRNAs That Are Attenuated by CDF

Tumor hypoxia with deregulated expression of hypoxia inducing factor (HIF) and its biological consequence leads to poor prognosis of patients diagnosed with solid tumors, resulting in higher mortality, suggesting that understanding of the molecular relationship of hypoxia with other cellular features of tumor aggressiveness would be invaluable for developing newer targeted therapy for solid tumors. Emerging evidence also suggest that hypoxia and HIF signaling pathways contributes to the acquisition of epithelial-to-mesenchymal transition (EMT), maintenance of cancer stem cell (CSC) functions, and also maintains the vicious cycle of inflammation, all of which contribute to radiation therapy and chemotherapy resistance. However, the detailed mechanisms by which hypoxia/HIF drive these events are not fully understood. Here, we have shown that hypoxia leads to increased expression of VEGF, IL-6, and CSC marker genes such as Nanog, Oct4 and EZH2, and also increased the expression of miR-21, an oncogenic miRNA, in prostate cancer (PCa) cells (PC-3 and LNCaP). The treatment of PCa cells with CDF, a novel Curcumin-derived synthetic analogue previously showed anti-tumor activity in vivo, inhibited the productions of VEGF and IL-6, and down-regulated the expression of Nanog, Oct4, EZH2 mRNAs, as well as miR-21 under hypoxic condition. Moreover, CDF treatment of PCa cells led to decreased cell migration under hypoxic condition. Taken together, these results suggest that the anti-tumor effect of CDF is in part mediated through deregulation of tumor hypoxic pathways, and thus CDF could become useful for cancer therapy.     

Disruption of Escherichia coli Nissle 1917 K5 Capsule Biosynthesis,through Loss of Distinct kfi genes,Modulates Interaction with Intestinal Epithelial Cells and Impact on Cell Health

Escherichia coli Nissle 1917 (EcN) is among the best characterised probiotics, with a proven clinical impact in a range of conditions. Despite this, the mechanisms underlying these "probiotic effects" are not clearly defined. Here we applied random transposon mutagenesis to identify genes relevant to the interaction of EcN with intestinal epithelial cells. This demonstrated mutants disrupted in the kfiB gene, of the K5 capsule biosynthesis cluster, to be significantly enhanced in attachment to Caco-2 cells. However, this phenotype was distinct from that previously reported for EcN K5 deficient mutants (kfiC null mutants), prompting us to explore further the role of kfiB in EcN:Caco-2 interaction. Isogenic mutants with deletions in kfiB (EcNΔkfiB), or the more extensively characterised K5 capsule biosynthesis gene kfiC (EcNΔkfiC), were both shown to be capsule deficient, but displayed divergent phenotypes with regard to impact on Caco-2 cells. Compared with EcNΔkfiC and the EcN wild-type, EcNΔkfiB exhibited significantly greater attachment to Caco-2 cells, as well as apoptotic and cytotoxic effects. In contrast, EcNΔkfiC was comparable to the wild-type in these assays, but was shown to induce significantly greater COX-2 expression in Caco-2 cells. Distinct differences were also apparent in the pervading cell morphology and cellular aggregation between mutants. Overall, these observations reinforce the importance of the EcN K5 capsule in host-EcN interactions, but demonstrate that loss of distinct genes in the K5 pathway can modulate the impact of EcN on epithelial cell health.     

Structural Analysis of the Sixth Immunoglobulin-Like Domain of Mouse Neural Cell Adhesion Molecule L1 and Its Interactions with αvβ3, αIIbβ3, and α5β1 Integrins

Abstract: Previous experiments suggested that the human cell adhesion molecule L1 interacts with different integrins via its sixth immunoglobulin-like domain in an RGD-dependent manner. Here we have described the expression of this domain from early postnatal mouse brain, analyzed the structure of the recombinant protein by circular dichroism and fluorescence spectroscopy, and performed solid-phase binding studies to αvβ3, αIIbβ3, and α5β1 integrins. The domain was found to have the expected β-sheet organization, which was lost in the presence of guanidine hydrochloride. The midpoint of the single-step transition occurred at 1.5 M guanidine hydrochloride. The sixth immunoglobulin-like domain of mouse brain L1 contains two RGD motifs and was found to bind in a concentration-dependent and saturable way to αvβ3, αIIbβ3, and α5β1 integrins, suggesting specific interactions with these ligands. However, only the interaction to αvβ3 could be inhibited in a concentration-dependent manner by an RGD-containing peptide, and the IC₅₀ was determined to be ∼20 n M . Mutants of the domain, which lack either one or both of the RGD sites, demonstrated that the RGD site comprising residues 562–564 is involved in the interaction to αvβ3. Our findings indicate an RGD-independent mechanism for the interactions to αIIbβ3 and α5β1, as no involvement of any RGD motif could be demonstrated.