Rapid discrimination of four Salmonella enterica serovars: A performance comparison between benchtop and handheld Raman spectrometers.

Foodborne illnesses, particularly those caused by Salmonella enterica with its extensive array of over 2600 serovars, present a significant public health challenge. Therefore, prompt and precise identification of S. enterica serovars is essential for clinical relevance, which facilitates the understanding of S. enterica transmission routes and the determination of outbreak sources. Classical serotyping methods via molecular subtyping and genomic markers currently suffer from various limitations, such as labour intensiveness, time consumption, etc. Therefore, there is a pressing need to develop new diagnostic techniques. Surface-enhanced Raman spectroscopy (SERS) is a non-invasive diagnostic technique that can generate Raman spectra, based on which rapid and accurate discrimination of bacterial pathogens could be achieved. To generate SERS spectra, a Raman spectrometer is needed to detect and collect signals, which are divided into two types: the expensive benchtop spectrometer and the inexpensive handheld spectrometer. In this study, we compared the performance of two Raman spectrometers to discriminate four closely associated S. enterica serovars, that is, S. enterica subsp. enterica serovar dublin, enteritidis, typhi and typhimurium. Six machine learning algorithms were applied to analyse these SERS spectra. The support vector machine (SVM) model showed the highest accuracy for both handheld (99.97%) and benchtop (99.38%) Raman spectrometers. This study demonstrated that handheld Raman spectrometers achieved similar prediction accuracy as benchtop spectrometers when combined with machine learning models, providing an effective solution for rapid, accurate and cost-effective identification of closely associated S. enterica serovars.

Differentiation of closely-related species within Acinetobacter baumannii-calcoaceticus complex via Raman spectroscopy: a comparative machine learning analysis.

Bacterial species within the Acinetobacter baumannii-calcoaceticus (Acb) complex are very similar and are difficult to discriminate. Misidentification of these species in human infection may lead to severe consequences in clinical settings. Therefore, it is important to accurately discriminate these pathogens within the Acb complex. Raman spectroscopy is a simple method that has been widely studied for bacterial identification with high similarities. In this study, we combined surfaced-enhanced Raman spectroscopy (SERS) with a set of machine learning algorithms for identifying species within the Acb complex. According to the results, the support vector machine (SVM) model achieved the best prediction accuracy at 98.33% with a fivefold cross-validation rate of 96.73%. Taken together, this study confirms that the SERS-SVM method provides a convenient way to discriminate between A. baumannii, Acinetobacter pittii, and Acinetobacter nosocomialis in the Acb complex, which shows an application potential for species identification of Acinetobacter baumannii-calcoaceticus complex in clinical settings in near future.

Both Las17-binding sites on Arp2/3 complex are important for branching nucleation and assembly of functional endocytic actin networks in S. cerevisiae.

Arp2/3 complex nucleates branched actin filaments that drive membrane invagination during endocytosis and leading-edge protrusion in lamellipodia. Arp2/3 complex is maximally activated in vitro by binding of a WASP family protein to two sites-one on the Arp3 subunit and one spanning Arp2 and ARPC1-but the importance of each site in the regulation of force-producing actin networks is unclear. Here, we identify mutations in budding yeast Arp2/3 complex that decrease or block engagement of Las17, the budding yeast WASP, at each site. As in the mammalian system, both sites are required for maximal activation in vitro. Dimerization of Las17 partially restores activity of mutations at both CA-binding sites. Arp2/3 complexes defective at either site assemble force-producing actin networks in a bead motility assay, but their reduced activity hinders motility by decreasing actin assembly near the bead surface and by failing to suppress actin filament bundling within the networks. While even the most defective Las17-binding site mutants assembled actin filaments at endocytic sites, they showed significant internalization defects, potentially because they lack the proper architecture to drive plasma membrane remodeling. Together, our data indicate that both Las17-binding sites are important to assemble functional endocytic actin networks in budding yeast, but Arp2/3 complex retains some activity in vitro and in vivo even with a severe defect at either Las17-binding site.

Rapid Prediction of Multidrug-Resistant Klebsiella pneumoniae through Deep Learning Analysis of SERS Spectra.

Klebsiella pneumoniae is listed by the WHO as a priority pathogen of extreme importance that can cause serious consequences in clinical settings. Due to its increasing multidrug resistance all over the world, K. pneumoniae has the potential to cause extremely difficult-to-treat infections. Therefore, rapid and accurate identification of multidrug-resistant K. pneumoniae in clinical diagnosis is important for its prevention and infection control. However, the limitations of conventional and molecular methods significantly hindered the timely diagnosis of the pathogen. As a label-free, noninvasive, and low-cost method, surface-enhanced Raman scattering (SERS) spectroscopy has been extensively studied for its application potentials in the diagnosis of microbial pathogens. In this study, we isolated and cultured 121 K. pneumoniae strains from clinical samples with different drug resistance profiles, which included polymyxin-resistant K. pneumoniae (PRKP; n = 21), carbapenem-resistant K. pneumoniae, (CRKP; n = 50), and carbapenem-sensitive K. pneumoniae (CSKP; n = 50). For each strain, a total of 64 SERS spectra were generated for the enhancement of data reproducibility, which were then computationally analyzed via the convolutional neural network (CNN). According to the results, the deep learning model CNN plus attention mechanism could achieve a prediction accuracy as high as 99.46%, with robustness score of 5-fold cross-validation at 98.87%. Taken together, our results confirmed the accuracy and robustness of SERS spectroscopy in the prediction of drug resistance of K. pneumoniae strains with the assistance of deep learning algorithms, which successfully discriminated and predicted PRKP, CRKP, and CSKP strains. IMPORTANCE This study focuses on the simultaneous discrimination and prediction of Klebsiella pneumoniae strains with carbapenem-sensitive, carbapenem-resistant, and polymyxin-resistant phenotypes. The implementation of CNN plus an attention mechanism makes the highest prediction accuracy at 99.46%, which confirms the diagnostic potential of the combination of SERS spectroscopy with the deep learning algorithm for antibacterial susceptibility testing in clinical settings.

Analysis of functional surfaces on the actin nucleation promoting factor Dip1 required for Arp2/3 complex activation and endocytic actin network assembly.

Arp2/3 complex nucleates branched actin filaments that drive processes like endocytosis and lamellipodial protrusion. WISH/DIP/SPIN90 (WDS) proteins form a class of Arp2/3 complex activators or nucleation promoting factors (NPFs) that, unlike WASP family NPFs, activate Arp2/3 complex without requiring preformed actin filaments. Therefore, activation of Arp2/3 complex by WDS proteins is thought to produce the initial actin filaments that seed branching nucleation by WASP-bound Arp2/3 complexes. However, whether activation of Arp2/3 complex by WDS proteins is important for the initiation of branched actin assembly in cells has not been directly tested. Here, we used structure-based point mutations of the Schizosaccharomyces pombe WDS protein Dip1 to test the importance of its Arp2/3-activating activity in cells. Six of thirteen Dip1 mutants caused severe defects in Arp2/3 complex activation in vitro, and we found a strong correlation between the ability of mutants to activate Arp2/3 complex and to rescue endocytic actin assembly defects caused by deleting Dip1. These data support a model in which Dip1 activates Arp2/3 complex to produce actin filaments that initiate branched actin assembly at endocytic sites. Dip1 mutants that synergized with WASP in activating Arp2/3 complex in vitro showed milder defects in cells compared to those that did not, suggesting that in cells the two NPFs may coactivate Arp2/3 complex to initiate actin assembly. Finally, the mutational data reveal important complementary electrostatic contacts at the Dip1-Arp2/3 complex interface and corroborate the previously proposed wedge model, which describes how Dip1 binding triggers structural changes that activate Arp2/3 complex.

Burmannic Acid Inhibits Proliferation and Induces Oxidative Stress Response of Oral Cancer Cells.

Burmannic acid (BURA) is a new apocarotenoid bioactive compound derived from Indonesian cinnamon; however, its anticancer effect has rarely been investigated in oral cancer cells. In this investigation, the consequences of the antiproliferation of oral cancer cells effected by BURA were evaluated. BURA selectively suppressed cell proliferation of oral cancer cells (Ca9-22 and CAL 27) but showed little cytotoxicity to normal oral cells (HGF-1). In terms of mechanism, BURA perturbed cell cycle distribution, upregulated mitochondrial superoxide, induced mitochondrial depolarization, triggered γH2AX and 8-hydroxy-2-deoxyguanosine DNA damage, and induced apoptosis and caspase 3/8/9 activation in oral cancer cells. Application of N-acetylcysteine confirmed oxidative stress as the critical factor in promoting antiproliferation, apoptosis, and DNA damage in oral cancer cells.

Plasmodesmata-Dependent Intercellular Movement of Bacterial Effectors.

Pathogenic microorganisms deliver protein effectors into host cells to suppress host immune responses. Recent findings reveal that phytopathogens manipulate the function of plant cell-to-cell communication channels known as plasmodesmata (PD) to promote diseases. Several bacterial and filamentous pathogen effectors have been shown to regulate PD in their host cells. A few effectors of filamentous pathogens have been reported to move from the infected cells to neighboring plant cells through PD; however, it is unclear whether bacterial effectors can traffic through PD in plants. In this study, we determined the intercellular movement of Pseudomonas syringae pv. tomato (Pst) DC3000 effectors between adjoining plant cells in Nicotiana benthamiana. We observed that at least 16 Pst DC3000 effectors have the capacity to move from transformed cells to the surrounding plant cells. The movement of the effectors is largely dependent on their molecular weights. The expression of PD regulators, Arabidopsis PD-located protein PDLP5 and PDLP7, leads to PD closure and inhibits the PD-dependent movement of a bacterial effector in N. benthamiana. Similarly, a 22-amino acid peptide of bacterial flagellin (flg22) treatment induces PD closure and suppresses the movement of a bacterial effector in N. benthamiana. Among the mobile effectors, HopAF1 and HopA1 are localized to the plasma membrane (PM) in plant cells. Interestingly, the PM association of HopAF1 does not negatively affect the PD-dependent movement. Together, our findings demonstrate that bacterial effectors are able to move intercellularly through PD in plants.

Structure of the nucleation-promoting factor SPIN90 bound to the actin filament nucleator Arp2/3 complex.

Unlike the WASP family of Arp2/3 complex activators, WISH/DIP/SPIN90 (WDS) family proteins activate actin filament nucleation by the Arp2/3 complex without the need for a preformed actin filament. This allows WDS proteins to initiate branched actin network assembly by providing seed filaments that activate WASP-bound Arp2/3 complex. Despite their important role in actin network initiation, it is unclear how WDS proteins drive the activating steps that require both WASP and pre-existing actin filaments during WASP-mediated nucleation. Here, we show that SPIN90 folds into an armadillo repeat domain that binds a surface of Arp2/3 complex distinct from the two WASP sites, straddling a hinge point that may stimulate movement of the Arp2 subunit into the activated short-pitch conformation. SPIN90 binds a surface on Arp2/3 complex that overlaps with actin filament binding, explaining how it could stimulate the same structural rearrangements in the complex as pre-existing actin filaments. By revealing how WDS proteins activate the Arp2/3 complex, these data provide a molecular foundation to understand initiation of dendritic actin networks and regulation of Arp2/3 complex by its activators.

Identification of an ATP-controlled allosteric switch that controls actin filament nucleation by Arp2/3 complex.

Nucleation of branched actin filaments by Arp2/3 complex is tightly regulated to control actin assembly in cells. Arp2/3 complex activation involves conformational changes brought about by ATP, Nucleation Promoting Factor (NPF) proteins, actin filaments and NPF-recruited actin monomers. To understand how these factors promote activation, we must first understand how the complex is held inactive in their absence. Here we demonstrate that the Arp3 C-terminal tail is a structural switch that prevents Arp2/3 complex from adopting an active conformation. The interaction between the tail and a hydrophobic groove in Arp3 blocks movement of Arp2 and Arp3 into an activated filament-like (short pitch) conformation. Our data indicate ATP binding destabilizes this interaction via an allosteric link between the Arp3 nucleotide cleft and the hydrophobic groove, thereby promoting the short-pitch conformation. Our results help explain how Arp2/3 complex is locked in an inactive state without activators and how autoinhibition is relieved.

Role and structural mechanism of WASP-triggered conformational changes in branched actin filament nucleation by Arp2/3 complex.

The Arp2/3 (Actin-related proteins 2/3) complex is activated by WASP (Wiskott-Aldrich syndrome protein) family proteins to nucleate branched actin filaments that are important for cellular motility. WASP recruits actin monomers to the complex and stimulates movement of Arp2 and Arp3 into a "short-pitch" conformation that mimics the arrangement of actin subunits within filaments. The relative contribution of these functions in Arp2/3 complex activation and the mechanism by which WASP stimulates the conformational change have been unknown. We purified budding yeast Arp2/3 complex held in or near the short-pitch conformation by an engineered covalent cross-link to determine if the WASP-induced conformational change is sufficient for activity. Remarkably, cross-linked Arp2/3 complex bypasses the need for WASP in activation and is more active than WASP-activated Arp2/3 complex. These data indicate that stimulation of the short-pitch conformation is the critical activating function of WASP and that monomer delivery is not a fundamental requirement for nucleation but is a specific requirement for WASP-mediated activation. During activation, WASP limits nucleation rates by releasing slowly from nascent branches. The cross-linked complex is inhibited by WASP's CA region, even though CA potently stimulates cross-linking, suggesting that slow WASP detachment masks the activating potential of the short-pitch conformational switch. We use structure-based mutations and WASP-Arp fusion chimeras to determine how WASP stimulates movement toward the short-pitch conformation. Our data indicate that WASP displaces the autoinhibitory Arp3 C-terminal tail from a hydrophobic groove at Arp3's barbed end to destabilize the inactive state, providing a mechanism by which WASP stimulates the short-pitch conformation and activates Arp2/3 complex.

Mortality attributable to carbapenem-resistant Pseudomonas aeruginosa bacteremia: a meta-analysis of cohort studies.

Whether carbapenem resistance is associated with mortality in patients with Pseudomonas aeruginosa bacteremia is controversial. To address this issue, we conducted a systematic review and meta-analysis based on cohort studies. We searched PubMed and Embase databases to identify articles (up to April 2015). The DerSimonian and Laird random-effect model was used to generate a summary estimate of effect. Associations were evaluated in subgroups based on different patient characteristics and study quality criteria. Seven studies with a total of 1613 patients were finally included, of which 1 study had a prospective design, and the other 6 were retrospective. Our meta-analysis showed patients with carbapenem-resistant P. aeruginosa bacteremia were at a higher risk of death compared with those with carbapenem-susceptible P. aeruginosa bloodstream infections (pooled odds ratio (OR) from three studies reporting adjusted ORs: 3.07, 95% confidence interval (CI), 1.60-5.89; pooled OR from 4 studies only reporting crude ORs: 1.46, 95% CI, 1.10-1.94). The results were robust across a number of stratified analyses and a sensitivity analysis. We also calculated that 8%-18.4% of deaths were attributable to carbapenem resistance in four studies assessing the outcome with 30-day mortality, and these were 3% and 14.6%, respectively, in two studies using 7-day mortality or mortality during bacteremia as an outcome of interest. Carbapenem resistance had a deleterious impact on the mortality of P. aeruginosa bacteremia; however, the results should be interpreted cautiously because only three studies reporting adjusted ORs were included. More large-scale, well-designed prospective cohorts, as well as mechanistic studies, are urgently needed in the future.

High efficiency integration of three-dimensional functional microdevices inside a microfluidic chip by using femtosecond laser multifoci parallel microfabrication.

High efficiency fabrication and integration of three-dimension (3D) functional devices in Lab-on-a-chip systems are crucial for microfluidic applications. Here, a spatial light modulator (SLM)-based multifoci parallel femtosecond laser scanning technology was proposed to integrate microstructures inside a given 'Y' shape microchannel. The key novelty of our approach lies on rapidly integrating 3D microdevices inside a microchip for the first time, which significantly reduces the fabrication time. The high quality integration of various 2D-3D microstructures was ensured by quantitatively optimizing the experimental conditions including prebaking time, laser power and developing time. To verify the designable and versatile capability of this method for integrating functional 3D microdevices in microchannel, a series of microfilters with adjustable pore sizes from 12.2 µm to 6.7 µm were fabricated to demonstrate selective filtering of the polystyrene (PS) particles and cancer cells with different sizes. The filter can be cleaned by reversing the flow and reused for many times. This technology will advance the fabrication technique of 3D integrated microfluidic and optofluidic chips.

Dip1 defines a class of Arp2/3 complex activators that function without preformed actin filaments.

BACKGROUND: Arp2/3 complex is a key actin cytoskeletal regulator that creates branched actin filament networks in response to cellular signals. WASP-activated Arp2/3 complex assembles branched actin networks by nucleating new filaments from the sides of pre-existing ones. WASP-mediated activation requires seed filaments, to which the WASP-bound Arp2/3 complex can bind to form branches, but the source of the first substrate filaments for branching is unknown. RESULTS: Here we show that Dip1, a member of the WISH/DIP/SPIN90 family of actin regulators, potently activates Arp2/3 complex without preformed filaments. Unlike other Arp2/3 complex activators, Dip1 does not bind actin monomers or filaments, and it interacts with the complex using a non-WASP-like binding mode. In addition, Dip1-activated Arp2/3 complex creates linear instead of branched actin filament networks. CONCLUSIONS: Our data show the mechanism by which Dip1 and other WISH/DIP/SPIN90 proteins can provide seed filaments to Arp2/3 complex to serve as master switches in initiating branched actin assembly. This mechanism is distinct from other known activators of Arp2/3 complex.

Insertions within the actin core of actin-related protein 3 (Arp3) modulate branching nucleation by Arp2/3 complex.

The Arp2/3 (actin-related protein 2/3) complex nucleates branched actin filaments involved in multiple cellular functions, including endocytosis and cellular motility. Two subunits (Arp2 and Arp3) in this seven-subunit assembly are closely related to actin and upon activation of the complex form a "cryptic dimer" that stably mimics an actin dimer to nucleate a new filament. Both Arps contain a shared actin core structure, and each Arp contains multiple insertions of unknown function at conserved positions within the core. Here we characterize three key insertions within the actin core of Arp3 and show that each one plays a distinct role in modulating Arp2/3 function. The ß4/ß5 insert mediates interactions of Arp2/3 complex with actin filaments and "dampers" the nucleation activity of the complex. The Arp3 hydrophobic plug plays an important role in maintaining the integrity of the complex but is not absolutely required for formation of the daughter filament nucleus. Deletion of the αK/ß15 insert did not constitutively activate the complex, as previously hypothesized. Instead, it abolished in vitro nucleation activity and caused defects in endocytic actin patch assembly in fission yeast, indicating a role for the αK/ß15 insert in the activated state of the complex. Biochemical characterization of each mutant revealed steps in the nucleation pathway influenced by each Arp3-specific insert to provide new insights into the structural basis of activation of the complex.

Mechanism of a concentration-dependent switch between activation and inhibition of Arp2/3 complex by coronin.

Arp2/3 complex is a key actin filament nucleator that assembles branched actin networks in response to cellular signals. The activity of Arp2/3 complex is regulated by both activating and inhibitory proteins. Coronins make up a large class of actin-binding proteins previously shown to inhibit Arp2/3 complex. Although coronins are known to play a role in controlling actin dynamics in diverse processes, including endocytosis and cell motility, the precise mechanism by which they regulate Arp2/3 complex is unclear. We conducted a detailed biochemical analysis of budding yeast coronin, Crn1, and found that it not only inhibits Arp2/3 complex but also activates it. We mapped regions required for activation and found that Crn1 contains a sequence called CA, which is conserved in WASp/Scar proteins, the prototypical activators of Arp2/3 complex. Point mutations in CA abolished activation of Arp2/3 complex by Crn1 in vitro. Confocal microscopy and quantitative actin patch tracking showed that these mutants had defective endocytic actin patch dynamics in Saccharomyces cerevisiae, indicating that activation of Arp2/3 complex by coronin is required for normal actin dynamics in vivo. The switch between the dual modes of regulation by Crn1 is controlled by concentration, and low concentrations of Crn1 enhance filament binding by Arp2/3 complex, whereas high concentrations block binding. Our data support a direct tethering recruitment model for activation of Arp2/3 complex by Crn1 and suggest that Crn1 indirectly inhibits Arp2/3 complex by blocking it from binding actin filaments.

Filament-dependent and -independent localization modes of Drosophila non-muscle myosin II.

Myosin II assembles into force-generating filaments that drive cytokinesis and the organization of the cell cortex. Regulation of myosin II activity can occur through modulation of filament assembly and by targeting to appropriate cellular sites. Here we show, using salt-dependent solubility and a novel fluorescence resonance energy transfer assay, that assembly of the Drosophila non-muscle myosin II heavy chain, zipper, is mediated by a 90-residue region (1849-1940) of the coiled-coil tail domain. This filament assembly domain, transiently expressed in Drosophila S2 cells, does not localize to the interphase cortex or the cytokinetic cleavage furrow, whereas a 500-residue region (1350-1865) that overlaps the NH(2) terminus of the assembly domain localizes to the interphase cortex but not the cytokinetic cleavage furrow. Targeting to these two sites appears to utilize distinct localization mechanisms as the assembly domain is required for cleavage furrow recruitment of a truncated coiled-coil tail region but not targeting to the interphase cortex. These results delineate the requirements for zipper filament assembly and indicate that the ability to form filaments is necessary for targeting to the cleavage furrow but not to the interphase cortex.

[Effection of compositie salviae dropping pill on hyperlipemia patients with phlegm and blood stasis syndrome].

OBJECTIVE: To explore the effect of compositie salviae dropping pill (CSDP) on hyperlipemia patients with phlegm and blood stasis syndrome. METHOD: Hyperlipemia patients were divided randomly into two groups. One group of 40 patients were treated by CSDP, another group of 41 patients were treated by simvastatin. The TC, TG, HDL-C, LDL-C, ApoA and ApoB levels, ALT, r-GT, IL-6, MDA level and SOD activity were determined before and after being treated. RESULT: After 3 months treatment, the TC, TG and LDL-C levels were obviously decreased in two groups (P <0.01, P < 0.05), there is no significant difference between the effective rate of two groups. The ALT, r-GT, IL-8 and MDA levels of treatment group were obviously decreased (P < 0.01, P < 0.05), while the ApoA level and SOD activity increased obviously in those patients (P <0.05, P <0.01, respectively). However, the ALT, r-GT, IL-6, MDA, HDL-C, ApoA level and SOD activity had no significant difference after treatment in control group. CONCLUSION: Our study suggest that CSDP have the function of falling serum lipid level without damaging liver function, its function of protecting liver function might related to its function of improving of anti-oxidation and decreasing of inflammation, the mechanism of CSDP disparting and removing phlem and blood stasis in the processes lipid metabolism need to be studied further.

[Clinical observation of modified Suan Zao Ren decoction on insomnia of chronic hepatitis B patients].

OBJECTIVE: To explore the effect of modified Suan Zao Ren Decoction (mediation decoction) on insomnia of chronic hepatitis B patients with stagnation of the Liver-Qi and deficiency of blood syndrome. METHODS: Sixty-five patients of chronic hepatitis B with stagnation of the Liver-Qi, deficiency of the spleen and damp-heat syndrome were randomly divided into two groups. Thirty-three patients in the treatment group were treated by modified Suan Zao Ren Decoction, and thirty-two patients in the control group were treated by Surazepam and Diammonium Glycyrrhizinate. The clinical syndromes of insomnia and liver function were observed before and after treatment. RESULTS: After one months of treatment, the main symptoms and signs of insomnia are improved in both groups, stage I and II sleep decreaed significantly while stage Il, IV and rapid eye movement increased in insomniacs after treatment of modified Suan Zao Ren Decoction (P < 0.05). The total effective rate was not significantly different between the two groups (P > 0.05), while the markedly effective rate was significantly different (P < 0.05). In the same time, ALT, AST, TB levels of two groups were obviously decreased (P < 0.01, P < 0.05), AST levels of two groups after treatment were significant different (P < 0.05). CONCLUSION: Modified Suan Zao Ren Decoction is effective for improving sleep composition, and therefore improve the quality of sleep with few side effect. In the same time, it has the function in improving liver function, and can be used to treat chronic hepatitis B patients with insomnia.

Effect of theophylline and specific phosphodiesterase IV inhibition on proliferation and apoptosis of progenitor cells in bronchial asthma.

1. Theophylline possesses anti-inflammatory activities in asthma. We examined whether theophylline and agents that modulate cyclic AMP can determine the survival and proliferation of progenitor cells. 2. Progenitor cells from the blood of normal and asthmatic subjects were cultured for 14 days in methylcellulose with GM-CSF, stem cell factor, IL-3 and IL-5. Apoptosis was measured by flow cytometry of propidium-iodide-stained cells. 3. A greater number of colonies with a higher proportion of cells of eosinophil lineage from asthmatics compared to normal subjects were grown. Theophylline (at 5 and 20 micro g ml(-1)) significantly inhibited colony formation and increased apoptotic cells in asthmatics compared to control. Salbutamol (0.1, 1, 10 micro M), dibutyryl-cAMP (0.1, 1 mM) and rolipram (0.1, 1 mM), a phosphodiesterase IV inhibitor, also dose-dependently decreased colony numbers and increased apoptosis of progenitor cells from asthmatics. 4. There was no significant effect of theophylline, db-cAMP, salbutamol or rolipram on colony formation or the survival of progenitor cells from normal subjects. AMP did not affect the colony formation and apoptosis. Expression of Bcl-2 protein on progenitor cells of asthma was downregulated by theophylline, salbutamol, db-cAMP and rolipram. 5. Theophylline and rolipram decreased colony formation committed to the eosinophil lineage, together with an increase in apoptosis through an inhibition of Bcl-2 expression effects that may occur through cAMP. The anti-inflammatory properties of theophylline include an inhibition of circulating progenitor cells.

Suppression of tissue factor expression, cofactor activity, and metastatic potential of murine melanoma cells by the N-terminal domain of adenovirus E1A 12S protein.

Tissue factor, the cellular initiator of blood coagulation, has been implicated as a determinant of metastatic potential in human melanoma cells. Here, we report that differential expression of tissue factor in murine melanoma cell lines of known metastatic behavior is mediated by AP-1-dependent and 12S E1A oncoprotein-repressible gene transcription. When compared to weakly metastatic C10 cells, highly metastatic M4 cells possessed elevated levels of tissue factor cofactor activity, transfected promoter activity, and heterodimeric AP-1 DNA-binding complexes containing Fra-1. Transient co-expression of the adenovirus E1A 12S oncoprotein strongly repressed transcription of an AP-1-driven tissue factor reporter gene indicating the additional requirement of N-terminal E1A-interacting coactivators. Stable expression of E1A mutants defective in CBP/p300-binding failed to suppress tissue factor expression and experimental metastasis by M4 cells while clones expressing wild type E1A exhibited greatly reduced tissue factor cofactor activity and metastatic potential in vivo. Overexpression of functional tissue factor in cells containing wild type E1A failed to restore the highly metastatic M4 phenotype suggesting that additional E1A-responsive and CBP/p300-dependent genes are required to facilitate metastasis of murine melanoma cells demonstrating high tissue factor expression and cofactor activity.