Bacterial virulence factor inhibits caspase-4/11 activation in intestinal epithelial cells.

The human pathogen enteropathogenic Escherichia coli (EPEC), as well as the mouse pathogen Citrobacter rodentium, colonize the gut mucosa via attaching and effacing lesion formation and cause diarrheal diseases. EPEC and C. rodentium type III secretion system (T3SS) effectors repress innate immune responses and infiltration of immune cells. Inflammatory caspases such as caspase-1 and caspase-4/11 are crucial mediators of host defense and inflammation in the gut via their ability to process cytokines such as interleukin (IL)-1ß and IL-18. Here we report that the effector NleF binds the catalytic domain of caspase-4 and inhibits its proteolytic activity. Following infection of intestinal epithelial cells (IECs) EPEC inhibited caspase-4 and IL-18 processing in an NleF-dependent manner. Depletion of caspase-4 in IECs prevented the secretion of mature IL-18 in response to infection with EPECΔnleF. NleF-dependent inhibition of caspase-11 in colons of mice prevented IL-18 secretion and neutrophil influx at early stages of C. rodentium infection. Neither wild-type C. rodentium nor C. rodentiumΔnleF triggered neutrophil infiltration or IL-18 secretion in Cas11 or Casp1/11-deficient mice. Thus, IECs have a key role in modulating early innate immune responses in the gut via a caspase-4/11-IL-18 axis, which is targeted by virulence factors encoded by enteric pathogens.

Ultra-thin graphene-polymer heterostructure membranes.

The fabrication of arrays of ultra-thin conductive membranes remains a major challenge in realising large-scale micro/nano-electromechanical systems (MEMS/NEMS), since processing-stress and stiction issues limit the precision and yield in assembling suspended structures. We present the fabrication and mechanical characterisation of a suspended graphene-polymer heterostructure membrane that aims to tackle the prevailing challenge of constructing high yield membranes with minimal compromise to the mechanical properties of graphene. The fabrication method enables suspended membrane structures that can be multiplexed over wafer-scales with 100% yield. We apply a micro-blister inflation technique to measure the in-plane elastic modulus of pure graphene and of heterostructure membranes with a thickness of 18 nm to 235 nm, which ranges from the 2-dimensional (2d) modulus of bare graphene at 173 ± 55 N m-1 to the bulk elastic modulus of the polymer (Parylene-C) as 3.6 ± 0.5 GPa as a function of film thickness. Different ratios of graphene to polymer thickness yield different deflection mechanisms and adhesion and delamination effects which are consistent with the transition from a membrane to a plate model. This system reveals the ability to precisely tune the mechanical properties of ultra-thin conductive membranes according to their applications.

Bax inhibitor 1 in apoptosis and disease.

Bax inhibitor 1 (BI-1) was originally discovered as an inhibitor of Bax-induced apoptosis; this review highlights the fundamental importance of BI-1 in a wider context, including in tissue homeostasis and as a regulator of cellular stress. BI-1 has been shown to interact with a broad range of partners to inhibit many facets of apoptosis, such as reactive oxygen species production, cytosolic acidification and calcium levels as well as endoplasmic reticulum stress signalling pathways. BI-1's anti-apoptotic action initially enables the cell to adapt to stress, although if the stress is prolonged or severe the actions of BI-1 may promote apoptosis. This almost universal anti-apoptotic capacity has been shown to be manipulated during infection with enteropathogenic and enterohaemorrhagic Escherichia coli inhibiting host cell death through direct interaction between their effector NleH and BI-1. In addition, BI-1 activity is important in a large number of cancers, promoting metastasis by modulating actin dynamics, a process dependent upon the BI-1 C-terminus and BI-1:actin interaction. Manipulation of BI-1 therefore has the potential for significant therapeutic benefit in a wide range of human diseases.

The Lactobacillus plantarum strain ACA-DC287 isolated from a Greek cheese demonstrates antagonistic activity in vitro and in vivo against Salmonella enterica serovar Typhimurium.

AIMS: The purpose of this study was to investigate the antibacterial activity of the Xynotyri cheese isolate Lactobacillus plantarum ACA-DC287 using a set of in vitro and in vivo assays. METHODS AND RESULTS: The co-culture of L. plantarum strain ACA-DC287 and Salmonella enterica serovar Typhimurium strain SL1344 results in the killing of the pathogen. The killing activity was produced mainly by non-lactic acid molecule(s) that were present in the cell-free culture supernatant of the L. plantarum strain ACA-DC287. The culture of the L. plantarum strain ACA-DC287 inhibited the penetration of S. typhimurium SL1344 into cultured human enterocyte-like Caco-2/TC7 cells. In conventional mice infected with S. typhimurium SL1344, the intake of L. plantarum strain ACA-DC287 results in a decrease in the levels of Salmonella associated with intestinal tissues or those present in the intestinal contents. In germ-free mice, the L. plantarum strain ACA-DC287 colonized the gastrointestinal tract. CONCLUSIONS: The L. plantarum strain ACA-DC287 strain exerts anti-Salmonella activity similar that of the established probiotic strains Lactobacillus rhamnosus GG, Lactobacillus casei Shirota YIT9029 and Lactobacillus johnsonii La1. SIGNIFICANCE AND IMPACT OF THE STUDY: The observation that a selected cheese Lactobacillus strain exerted antibacterial activity that was similar to those of probiotic Lactobacillus strains, is of interest for the use of this strain as an adjunct strain for the production of health-giving cheeses.

Identification of functionally important amino-terminal arginines of Agrobacterium tumefaciens ADP-glucose pyrophosphorylase by alanine scanning mutagenesis.

Treatment of the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase with the arginyl reagent phenylglyoxal resulted in complete desensitization to fructose 6-phosphate (F6P) activation, and partial desensitization to pyruvate activation. The enzyme was protected from desensitization by ATP, F6P, pyruvate, and phosphate. Alignment studies revealed that this enzyme contains arginine residues in the amino-terminal region that are relatively conserved in similarly regulated ADP-glucose pyrophosphorylases. To functionally evaluate the role(s) of these arginines, alanine scanning mutagenesis was performed to generate the following enzymes: R5A, R11A, R22A, R25A, R32A, R33A, R45A, and R60A. All of the enzymes, except R60A, were successfully expressed and purified to near homogeneity. Both the R5A and R11A enzymes displayed desensitization to pyruvate, partial activation by F6P, and increased sensitivity to phosphate inhibition. Both the R22A and R25A enzymes exhibited reduced V(max) values in the absence of activators, lower apparent affinities for ATP and F6P, and reduced sensitivities to phosphate. The presence of F6P restored R22A enzyme activity, while the R25A enzyme exhibited only approximately 1.5% of the wild-type activity. The R32A enzyme displayed an approximately 11.5-fold reduced affinity for F6P while exhibiting behavior identical to that of the wild type with respect to pyruvate activation. Both the R33A and R45A enzymes demonstrated a higher activity than the wild-type enzyme in the absence of activators, no response to F6P, partial activation by pyruvate, and desensitization to phosphate inhibition. These altered enzymes were also insensitive to phenylglyoxal. The data demonstrate unique functional roles for these arginines and the presence of separate subsites for the activators.

Hematopoietic deficiencies and core binding factor expression in murine Ts16, an animal model for Down syndrome.

Patients with Down syndrome (DS, Trisomy 21) suffer from hematopoietic abnormalities, including an increased risk to develop leukemia. Overexpression of chromosome 21-encoded genes thus leads to hematopoietic deficiencies. Of the genes found within the DS chromosomal region, core binding factor alpha (CBFA) is a candidate whose overexpression could affect hematopoietic development. To learn more about the pathogenesis of hematological diseases in DS, we studied hematopoietic precursor cells in Ts16 mice, an animal model for DS. We found reduced proportions of B lymphoid and myeloid cells in the liver and spleen, whereas the proportion of developing thymocyte populations and that of the erythroid cells in liver and spleen were increased. Furthermore, when analyzing the expression of Cbfa2 in both whole fetuses and isolated thymuses, we found no significant differences in the absolute amount of Cbfa2 mRNA or in the ratio of the isoforms Cbfa2.1 and Cbfa2.2 between Ts16 and diploid samples. Thus, a disequilibrium of Cbfa2 expression and a dysregulation of the two Cbfa2 mRNA species as a cause for the abnormalities in Ts16 fetuses in general and the deficient Ts16 thymocyte development in particular appears unlikely.

Self renewal of embryonic stem cells in the absence of feeder cells and exogenous leukaemia inhibitory factor.

To evaluate the role of leukaemia inhibitory factor (LIF) for maintaining pluripotent embryonic stem (ES) cells in culture, we established several exogenous LIF-independent ES cell lines by continuous passaging in culture. The newly established ES cells, Kli and CBli, sustained their growth and remained undifferentiated in LIF-deficient medium. Analysis of chimaeric animals, produced with the beta-galactosidase transgenic Kli ES cells, revealed that LIF-independent ES cells can contribute to all embryonic germ layers. There was no detectable LIF protein in ES cell conditioned medium, and no upregulation of LIF mRNA was found. The addition of neutralising anti-LIF antibodies was not sufficient to abrogate the self renewal of the Kli ES cells. These studies suggest that the signalling pathway involving diffusible LIF can be bypassed for maintaining the pluripotency in culture, and indicate a considerable heterogeneity in growth factor dependence and differentiation of different ES cells.

Estimation of the number of hematopoietic precursor cells during fetal mouse development by covariance analysis.

Differentiation of hematopoietic precursor cells results in the formation of clonally related descendent cells. Using the mosaic expression of beta-galactosidase in female mouse fetuses heterozygous for an X-linked lacZ transgene, we analyzed the clonal relationship of the hematopoietic progeny. The proportion of beta-galactosidase positive cells for different T- and B-lymphoid and myeloid cell populations was determined at different stages of fetal development. We found excellent correlations of the proportion of beta-galactosidase expressing cells for all hematopoietic lineages confirming that they share a common ancestry. Therefore, it was possible to estimate the number of common precursor cells (PC) based on binomial distribution and covariance analysis of pairs of different hematopoietic cell populations. Our results obtained from hematopoietic cells at 15.5 to 18.5 days of gestation indicated the presence of 15 to 18 lymphoid and 18 to 22 myeloid/lymphoid specific precursor cells. Statistical analysis of the precursor cell numbers showed a trend of increasing numbers that was highly significant. The precursor cell number was inversely related to maturity of the cell populations analyzed; ie, the lowest number of lymphoid and lymphoid/myeloid precursors was calculated when the most mature CD3+ T-cell population was used for comparison. Determination of PC numbers can therefore be used to assess the relative maturity and developmental potential of individual cell populations.

The development of haematopoietic cells is biased in embryonic stem cell chimaeras.

The haematopoietic development of embryonic stem (ES) cell injection chimaeras was analysed using beta-galactosidase expression from an X-linked transgene as a marker to distinguish the ES-derived cell population from the host cells. The number of cells in the different haematopoietic cell subpopulations was determined by flow cytometry. When the proportions of ES-derived cells in the antigen-positive lineages were compared to the ES cell contribution to all cells in teh organs, we found an unexpected bias in the haematopoietic differentiation of ES-derived cells. ES descendants were overrepresented in the bone marrow B lymphoid cell population and the splenic myeloid cells but were underrepresented in the CD4-positive T lymphoid cells in the spleen. These results were obtained by comparison with control female animals that were X chromosome mosaic for beta-galactosidase expression. These findings of uneven contribution to haematopoietic development by ES cells indicate that the commitment of ES cell descendants may be different from that of the host cells.

Simultaneous detection of beta-galactosidase activity and surface antigen expression in viable haematopoietic cells.

The quantitation of intracellular beta-galactosidase activity has been described for viable cells. By using the fluorogenic substrate fluorescein-di-beta-D-galactopyranoside (FDG) in conjunction with flow cytometry, the proportion of positive cells as well as the level of expression can be determined. In this paper we describe beta-galactosidase expression in lymphoid and myeloid cells from transgenic mice that widely express beta-galactosidase from an inserted lacZ transgene. Both foetal and adult haematopoietic tissues are able to express beta-galactosidase. The intracellular fluorescence reflecting beta-galactosidase activity can be readily combined with fluorescently labelled antibodies against cell surface antigens. Thus, beta-galactosidase can be used as a marker in transplantation experiments to study the development of lymphoid and myeloid precursor cells.

Deficient transformation of murine trisomy 16 fetal liver cells by the Abelson and J2 viruses.

Mouse trisomy 16 (Ts16), an animal model for human Down syndrome (trisomy 21), exhibits severe abnormalities in the development of lymphoid and myeloid cells. Whereas fetal liver cells from diploid mice can be easily immortalized by retroviral transformation with Ab1-MuLV or J2 virus, fetal livers from Ts16 mice contain significantly fewer transformable cells. Infection of Ts16 fetal liver cells by Ab1-MuLV results in a 52- and 12-fold reduction in the frequency of transformation at days 17 and 18 of gestation, respectively. By contrast, the efficiency of transformation with J2 virus, another retrovirus known to transform fetal liver cells, is only mildly (factor 2-3) affected. The Ig gene rearrangements of Ts16 and diploid retrovirally transformed fetal liver cell lines do not differ from one another. This suggests that there is a deficiency in the early stem cell compartment, rather than in the development of pre-B cells.

Dysregulation of gene expression in mouse trisomy 16, an animal model of Down syndrome.

In humans, trisomy 21 results in a specific phenotype known as Down syndrome (DS). The mechanism by which an extra copy of normal genes leads to the DS phenotype is unknown. Most studies in DS and other aneuploid organisms have shown that gene dose is proportional to gene expression. To date, most genes examined have encoded either metabolic enzymes or constitutively expressed products. In the trisomy 16 mouse, an animal model of DS, we found marked dysregulation of two developmentally regulated genes, App and Prn-p. Dysregulation varied from tissue to tissue and during development in the same tissue. We conclude that abnormal phenotypes seen in aneuploid conditions may result in part from disordered expression of developmentally regulated genes.

Genomic imprinting: normal complementation of murine chromosome 16.

Parental imprinting effects for chromosome 16 were investigated using disomic animals which were obtained by mating (Rb32Lub x Rb2H) F1 mice. Two allelic forms of the enzyme CuZn-superoxide dismutase, Sod-1a and Sod-1c, were used to identify maternally or paternally disomic animals. Both types of disomic animals were found with the expected frequencies and did not visibly differ from one another or from non-disomic animals. These results indicate that the genomic imprinting mechanism either does not act on chromosome 16, or, if it does, does not do so in a manner which affects normal development.

Delayed thymocyte maturation in the trisomy 16 mouse fetus.

Mouse fetuses with trisomy 16, an animal model for human trisomy 21 (Down syndrome), have severe defects in several hematopoietic stem cell populations and a marked reduction in thymocyte number. To determine whether there are other defects in the development of the trisomic thymus, the ontogeny of the cell surface antigenic determinants, Thy-1, Ly-1, CD3, CD4, CD8, and TCR v beta, was investigated. The trisomy 16 thymocytes were able to express all of determinants either during fetal life (days 14 to 19 of gestation) or in cultures of intact thymus lobes. However, in all instances (except for Thy-1, which already had a high proportion of expressing thymocytes by day 14), there was a delay in the time at which the determinants were first expressed, as manifested by reduced numbers of positively staining cells. Furthermore, there was also a delay in the rate at which the positively staining cells attained maximal Ag densities. Overall, there was an approximate 2 day lag in development of the fetal trisomic thymocytes. This lag permitted the identification of a large population of CD4-8+ cells prior to the appearance of CD4+8+ thymocytes. These findings are consistent with the identification of CD4-8+ as an intermediate stage between CD4-8- and CD4+8+ in fetal thymocyte ontogeny.

Isolation of a cDNA copy of an RNA species expressed in murine pre-B cells.

A part of a gene, pZ 183, was isolated from a cDNA library constructed from poly(A)-containing RNA of the chemically induced murine pre-B lymphoma 70Z/3. This DNA sequence was found to be present in one of 5,000 cDNA clones in the unsubtracted library, and in one of 350 cDNA clones in a library in which sequences common to 70Z/3 and to a T cell hybridoma had been subtracted. This indicates that the corresponding gene belongs to the genes which are expressed with low to medium abundance in the pre-B cell line 70Z/3. The gene was found to be expressed in pre-B lymphomas, but not in B lymphomas, myelomas, T cell lymphomas, macrophage tumors or a fibroblast line. By in situ hybridization it was detected in 2-4% of fetal liver cells between day 15 and 16 of gestation, and in 1% of adult bone marrow cells. It was not detected in thymus cells, nor in mature T cells, spleen, adult liver, kidney, heart, lung or brain. The size of the transcript found in all pre-B cell lymphomas is 1.2 kb in length. The pZ 183 DNA sequence would appear to be a useful B-lineage specific marker which identifies pre-B cells at various stages before the mature B cell.

In situ hybridization of immunoglobulin-specific RNA in single cells of the B lymphocyte lineage with radiolabelled DNA probes.

A method for in situ hybridization has been developed which detects immunoglobulin-specific mRNA transcripts in single murine B lymphocytes with radiolabelled, immunoglobulin gene-specific single-stranded DNA probes. The method has been applied to myeloma and hybridoma cells and to B lymphocytes at various stages of their maturation from small, resting B cells to Ig-secreting plasma cells. A critical step in the procedure is the treatment of the cells with pronase. The various cell types have been found to be differently susceptible to this treatment. Single-stranded DNA probes of different lengths, i.e., between 26 and 1000 bp, have been employed in the hybridization. The number of silver grains over a cell increases proportionally with the length of the probe and with its concentration in the hybridization reaction. The kinetics of the increase of mu-heavy chain-specific RNA molecules in single cells and the appearance of 'switched', gamma-heavy chain-expressing cells are shown after stimulation of murine B cells with lipopolysaccharide.