NleB2 from enteropathogenic Escherichia coli is a novel arginine-glucose transferase effector

During infection, enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) directly manipulate various aspects of host cell function through the translocation of type III secretion system (T3SS) effector proteins directly into the host cell. Many T3SS effector proteins are enzymes that mediate post-translational modifications of host proteins, such as the glycosyltransferase NleB1, which transfers a single N-acetylglucosamine (GlcNAc) to arginine residues, creating an Arg-GlcNAc linkage. NleB1 glycosylates death-domain containing proteins including FADD, TRADD and RIPK1 to block host cell death. The NleB1 paralogue, NleB2, is found in many EPEC and EHEC strains but to date its enzymatic activity has not been described. Using in vitro glycosylation assays combined with mass spectrometry, we found that NleB2 can utilize multiple sugar donors including UDP-glucose, UDP-GlcNAc and UDP-galactose during glycosylation of the death domain protein, RIPK1. Sugar donor competition assays demonstrated that UDP-glucose was the preferred substrate of NleB2 and peptide sequencing identified the glycosylation site within RIPK1 as Arg603, indicating that NleB2 catalyses arginine glucosylation. We also confirmed that NleB2 catalysed arginine-hexose modification of Flag-RIPK1 during infection of HEK293T cells with EPEC E2348/69. Using site-directed mutagenesis and in vitro glycosylation assays, we identified that residue Ser252 in NleB2 contributes to the specificity of this distinct catalytic activity. Substitution of Ser252 in NleB2 to Gly, or substitution of the corresponding Gly255 in NleB1 to Ser switches sugar donor preference between UDP-GlcNAc and UDP-glucose. However, this switch did not affect the ability of the NleB variants to inhibit inflammatory or cell death signalling during HeLa cell transfection or EPEC infection. NleB2 is thus the first identified bacterial Arg-glucose transferase that, similar to the NleB1 Arg-GlcNAc transferase, inhibits host protein function by arginine glycosylation.     

Shrinkage of Dental Composite in Simulated Cavity Measured with Digital Image Correlation

Polymerization shrinkage of dental resin composites can lead to restoration debonding or cracked tooth tissues in composite-restored teeth. In order to understand where and how shrinkage strain and stress develop in such restored teeth, Digital Image Correlation (DIC) was used to provide a comprehensive view of the displacement and strain distributions within model restorations that had undergone polymerization shrinkage.Specimens with model cavities were made of cylindrical glass rods with both diameter and length being 10 mm. The dimensions of the mesial-occlusal-distal (MOD) cavity prepared in each specimen measured 3 mm and 2 mm in width and depth, respectively. After filling the cavity with resin composite, the surface under observation was sprayed with first a thin layer of white paint and then fine black charcoal powder to create high-contrast speckles. Pictures of that surface were then taken before curing and 5 min after. Finally, the two pictures were correlated using DIC software to calculate the displacement and strain distributions.The resin composite shrunk vertically towards the bottom of the cavity, with the top center portion of the restoration having the largest downward displacement. At the same time, it shrunk horizontally towards its vertical midline. Shrinkage of the composite stretched the material in the vicinity of the “tooth-restoration” interface, resulting in cuspal deflections and high tensile strains around the restoration. Material close to the cavity walls or floor had direct strains mostly in the directions perpendicular to the interfaces. Summation of the two direct strain components showed a relatively uniform distribution around the restoration and its magnitude equaled approximately to the volumetric shrinkage strain of the material.     

Regulation of ROCK1 via Notch1 during breast cancer cell migration into dense matrices

ABSTRACT: BACKGROUND: The behaviour of tumour cells depends on factors such as genetics and the tumour microenvironment. The latter plays a crucial role in normal mammary gland development and also in breast cancer initiation and progression. Breast cancer tissues tend to be highly desmoplastic and dense matrix as a pre-existing condition poses one of the highest risk factors for cancer development. However, matrix influence on tumour cell gene expression and behaviour such as cell migration is not fully elucidated. RESULTS: We generated high-density (HD) matrices that mimicked tumour collagen content of 20 mg/cm3 that were ~14-fold stiffer than low-density (LD) matrix of 1 mg/cm3. Live-cell imaging showed breast cancer cells utilizing cytoplasmic streaming and cell body contractility for migration within HD matrix. Cell migration was blocked in the presence of both the ROCK inhibitor, Y-27632, and the MMP inhibitor, GM6001, but not by the drugs individually. This suggests roles for ROCK1 and MMP in cell migration are complicated by compensatory mechanisms. ROCK1 expression and protein activity, were significantly upregulated in HD matrix but these were blocked by treatment with a histone deacetylase (HDAC) inhibitor, MS-275. In HD matrix, the inhibition of ROCK1 by MS-275 was indirect and relied upon protein synthesis and Notch1. Inhibition of Notch1 using pooled siRNA or DAPT abrogated the inhibition of ROCK1 by MS-275. CONCLUSION: Increased matrix density elevates ROCK1 activity, which aids in cell migration via cell contractility. The upregulation of ROCK1 is epigenetically regulated in an indirect manner involving the repression of Notch1. This is demonstrated from inhibition of HDACs by MS- 275, which caused an upregulation of Notch1 levels leading to blockade of ROCK1 expression.     

Why do fiddler crabs build chimneys?

Chimneys are mud mounds built by fiddler crabs that encircle the entrance to their burrow. Their function in many species is unknown. In Uca capricornis, crabs of both sexes and all sizes build chimneys, but females do so disproportionately more often. There are no differences in the immediate physical or social environments between crabs with and without a chimney. Chimney owners spend less time feeding and more time underground than non-owners. We show experimentally that burrows with a chimney are less likely to be located by an intruder. It is possible that some crabs construct chimneys around their burrow to conceal the entrance and reduce the risk of losing it to an intruder.     

The autoregulation of intracellular oxygen tension and the modification of cellular radiosensitivity. The development of an adaptation hypothesis

A hypothesis is reported postulating that the range within which the intracellular oxygen content varies with changes in the external oxygenation conditions is responsible for the oxygen-dependent radiosensitivity modification that may be controlled by the diffusion resistance of a cytoplasmic membrane. The adaptation mechanism of intracellular Po2 autoregulation is involved when drastic changes in the oxygen content of the environment occur. As the oxygen content decreases this mechanism provides maximal values of the intracellular Po2 required for optimizing cell viability; the increased oxygen content prevents cells from oxygen intoxication.     

Suppression of humoral immune responses by synthetic C3a peptides

Synthetic oligopeptides based on the COOH-terminal sequence of native human C3a were examined for the ability to suppress antigen-specific and polyclonal antibody responses. The synthetic peptides were found to qualitatively mimic the suppressive actions of human C3a, but proved to be less active on a molar basis. Comparison of the activity of the peptides with native C3a indicates that the most potent peptide is C3a 57-77 with C3a 65-77, C3a 70-77, C3a[Ala 71,72]70-77, and C3a 73-77 being less active, respectively. These results indicate that the region of the C3a molecule responsible for immunosuppression is located in the COOH region.     

Identification of a tetrapeptide recognition sequence for the alpha 2 beta 1 integrin in collagen

The alpha 2 beta 1 integrin serves as either a specific cell surface receptor for collagen or as both a collagen and laminin receptor depending upon the cell type. Recently we established that the alpha 2 beta 1 integrin binds to a site within the alpha 1 (I)-CB3 fragment of type I collagen (Staatz, W. D., Walsh, J. J., Pexton, T., and Santoro, S. A. (1990) J. Biol. Chem. 265, 4778-4781). To define the alpha 2 beta 1 recognition sequence further we have prepared an overlapping set of synthetic peptides which completely spans the 148-amino acid alpha 1(I)-CB3 fragment and tested the peptides for ability to inhibit cell adhesion to collagen and laminin substrates. The minimal active recognition sequence defined by these experiments is a tetrapeptide of the sequence Asp-Gly-Glu-Ala (DGEA) corresponding to residues 435-438 of the type I collagen sequence. The DGEA-containing peptides effectively inhibited alpha 2 beta 1-mediated Mg2(+)-dependent adhesion of platelets, which use the alpha 2 beta 1 integrin as a collagen-specific receptor, to collagen but had no effect on alpha 5 beta 1-mediated platelet adhesion to fibronectin or alpha 6 beta 1-mediated platelet adhesion to laminin. In contrast, with T47D breast adenocarcinoma cells, which use alpha 2 beta 1 as a collagen/lamin receptor, adhesion to both collagen and laminin was inhibited by DGEA-containing peptides. Deletion of the alanine residue or substitution of alanine for either the glutamic or aspartic acid residues in DGEA-containing peptides resulted in marked loss of inhibitory activity. These results indicate that the amino acid sequence DGEA serves as a recognition site for the alpha 2 beta 1 integrin complex on platelets and other cells.     

Structure-activity studies reveal two allatostatin receptor types in corpora allata of Diploptera punctata

Synthetic variants of the octadecapeptide amide ASB2 (AYSYVSEYKRLPVYNFGL-NH(2)), a cockroach allatostatin, were assayed in vitro on corpora allata (CA) from 2-day-old (vitellogenic) and 10-day-old (post-vitellogenic) female Diploptera punctata. The analogs [(17)psi(18),CH(2)-S]ASB2, [D-Trp(17)]ASB2 and [Ile(18)]ASB2 inhibited juvenile hormone (JH) synthesis with simple dose-response curves on sensitive CA from 10-day-old females. These analogs were fully effective but less potent than ASB2. When tested on CA from 2-day-old mated females, which are only partially (65-70%) sensitive to ASB2, the three analogs gave biphasic dose-response curves and elicited a maximal effect only at higher concentrations. The dose-response curve for ASB2 on CA from 2-day-old females had a Hill plot slope of only 0.78+/-0.03. These findings suggested that the observed CA sensitivity to ASB2 may be the result of two partial responses having an IC(50) of approximately 0.35 and 3nM respectively. One partial response, or receptor type, appeared more sensitive than the other to adverse modification of the "message" segment of the peptide. The activity of shorter allatostatins was also studied, indicating that pentapeptides of the YXFGL-amide structure are fully effective, albeit at low potency, as inhibitors of JH biosynthesis.     

Cytology of peritoneal fluid from patients on continuous ambulatory peritoneal dialysis

Peritoneal fluids from 41 patients on continuous ambulatory peritoneal dialysis (CAPD) were examined. The patients were divided into a short-term group (18 patients with CAPD up to one year) and a long-term group (23 patients with CAPD for one to seven years). Peritoneal fluids from a control group, consisting of ten nondialysis patients with ascites, were also examined. The cellular background of the peritoneal fluids and, in particular, the morphology of the mesothelial cells were studied. The following were found to be significantly increased in the CAPD groups: background lymphocytes, mesothelial exfoliation in three-dimensional clusters, mesothelial nuclear size and the number of mesothelial nucleoli. All of these features increased slightly with an increased duration of the dialysis. These findings emphasize that peritoneal dialysis of any duration can induce significantly atypical changes in mesothelial cells.