P2X1 receptor blockers reduce the number of circulating thrombocytes and the overall survival of urosepsis with haemolysin-producing Escherichia coli

Purinergic Signalling - Urosepsis is a severe condition often caused by Escherichia coli that spontaneously have ascended the urinary tract to the kidneys causing pyelonephritis and potentially...     

BRCA1 Haploinsufficiency Is Masked by RNF168-Mediated Chromatin Ubiquitylation

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Calpain inhibition stimulates caspase-dependent apoptosis induced by taxol in NIH3T3 cells

Taxol is an anticancer drug that triggers apoptosis in a wide spectrum of cancers such as ovarian, breast, lung, head and neck, and bladder carcinoma by both caspase-dependent and -independent apoptosis mechanisms. However, the exact signaling pathways involved in taxol-induced apoptosis strongly depend on the cellular background and they are not completely established yet. In this study we demonstrate that taxol induces caspase-3-independent apoptosis in NIH3T3 cells by a calpain-mediated mechanism. Taxol treatment produced changes in the mitochondrial membrane potential (Delta Psi m) which could be responsible of Ca(2+) release from the mitochondria and the consequent calpain activation. Interestingly, we show that calpain produced proteolysis of caspase-3 and demonstrate that, accordingly, calpain inhibition increased taxol-induced apoptosis. In addition, we reveal that poly (ADP-ribose) polymerase (PARP) was processed by calpain in taxol-treated cells and by caspase-3 after calpain inhibition. In conclusion, these results demonstrate for the first time that calpain could play an important role modulating taxol-induced apoptosis. Further studies are needed to address the potentiality of inducing apoptosis by a combined use of taxol and calpain inhibitors in cells with increased calpain activity.     

Translation regulation after taxol treatment in NIH3T3 cells involves the elongation factor (eEF)2

Changes to the translational machinery that occur during apoptosis have been described in the last few years. The two principal ways in which translational factors are modified during apoptosis are: (i) changes in protein phosphorylation and (ii) specific proteolytic cleavages. Taxol, a member of a new class of anti-tubulin drugs, is currently used in chemotherapeutic treatments of different types of cancers. We have previously demonstrated that taxol induces calpain-mediated apoptosis in NIH3T3 cells [Pi?eiro et al., Exp. Cell Res., 2007, 313:369-379]. In this study we found that translation was significantly inhibited during taxol-induced apoptosis in these cells. We have studied the phosphorylation status and expression levels of eIF2a, eIF4E, eIF4G and the regulatory protein 4E-BP1, all of which are implicated in translation regulation. We found that taxol treatment did not induce changes in eIF2alpha phosphorylation, but strongly decreased eIF4G, eIF4E and 4E-BP1 expression levels. MDL28170, a specific inhibitor of calpain, prevented reduction of eIF4G, but not of eIF4E or 4E-BP1 levels. Moreover, the calpain inhibitor did not block taxol-induced translation inhibition. All together these findings demonstrated that none of these factors are responsible for the taxol-induced protein synthesis inhibition. On the contrary, taxol treatment increased elongation factor eEF2 phosphorylation in a calpain-independent manner, supporting a role for eEF2 in taxol-induced translation inhibition.     

Gametogenesis of intergroup hybrids of hemiclonal frogs

European water frog hybrids Rana esculenta (R. ridibundaxR. lessonae) reproduce hemiclonally, by hybridogenesis: in the germ line they exclude the genome of one parental species and produce haploid gametes with an unrecombined genome of the other parental species. In the widespread L-E population system, both sexes of hybrids (E) coexist with R. lessonae (L). They exclude the lessonae genome and produce ridibunda gametes. In the R-E system, hybrid males coexist with R. ridibunda (R); they exclude either their ridibunda or their lessonae genome and produce sperm with a lessonae or with a ridibunda genome or a mixture of both kinds of sperm. We examined 13 male offspring, 12 of which were from crosses between L-E system and R-E system frogs. All were somatically hybrid. With one exception, they excluded the lessonae genome in the germ line and subsequently endoreduplicated the ridibunda genome. Spermatogonial metaphases contained a haploid or a diploid number of ridibunda chromosomes, identified through in situ hybridization to a satellite DNA marker, and by spermatocyte I metaphases containing a haploid number of ridibunda bivalents. The exception, an F1 hybrid between L-E system R. lessonae and R-E system R. ridibunda, was not hybridogenetic, showed no genome exclusion, and evidenced a disturbed gametogenesis resulting from the combination of two heterospecific genomes. None of the hybridogenetic hybrids showed any cell lines excluding the ridibunda genome, the pattern most frequent in hybrids of the R-E system, unique to that system, and essential for its persistence. A particular combination of R-E system lessonae and R-E system ridibunda genomes seems necessary to induce the R-E system type of hemiclonal gametogenesis.     

Downregulation of p56(lck) tyrosine kinase activity in T cells of squirrel monkeys (Saimiri sciureus) correlates with the nontransforming and apathogenic properties of herpesvirus saimiri in its natural host

Herpesvirus saimiri is capable of transforming T lymphocytes of various primate species to stable growth in culture. The interaction of the T-cellular tyrosine kinase p56(lck) with the transformation-associated viral protein Tip has been shown before to activate the kinase and provides one model for the T-cell-specific transformation by herpesvirus saimiri subgroup C strains. In contrast to other primate species, squirrel monkeys (Saimiri sciureus) are naturally infected with the virus without signs of lymphoma or other disease. Although the endogenous virus was regularly recovered from peripheral blood cells from squirrel monkeys, we observed that the T cells lost the virus genomes in culture. Superinfection with virus strain C488 did not induce growth transformation, in contrast to parallel experiments with T cells of other primate species. Surprisingly, p56(lck) was enzymatically inactive in primary T-cell lines derived from different squirrel monkeys, although the T cells reacted appropriately to stimulatory signals. The cDNA sequence revealed minor point mutations only, and transfections in COS-7 cells demonstrated that the S. sciureus lck gene codes for a functional enzyme. In S. sciureus, the tyrosine kinase p56(lck) was not activated after T-cell stimulation and enzymatic activity could not be induced by Tip of herpesvirus saimiri C488. However, the suppression of p56(lck) was partially released after administration of the phosphatase inhibitor pervanadate. This argues for unique species-specific conditions in T cells of S. sciureus which may interfere with the transforming activity and pathogenicity of herpesvirus saimiri subgroup C strains in their natural host.     

Frequency of CD59 mutations induced in human-hamster hybrid A(L) cells by low-dose X-irradiation

Determination of the genotoxic effects of ionizing radiation, especially at low-doses, is of great importance for risk assessment, e.g. in radiological diagnostics. The human-hamster hybrid A(L) cell line has been shown previously to be a well-suited in vitro model for the study of mutations induced by various mutagens. The A(L) cells contain a standard set of hamster chromosomes and a single human chromosome 11, which confers the expression of the human cell surface protein CD59. Using CD59 specific antibodies, cells mutated in the CD59 gene can be detected and quantified by the loss of the cell surface marker. In contrast to previous studies, prior to irradiation we removed spontaneous mutants by magnetic cell separation (MACS) which allows analysis of radiation-induced mutation events only. We exposed A(L) cells to 100kV X-rays at 0.1 to 5Gy. The proportions of X-irradiation-induced CD59(-) mutants were quantified by flow cytometry after immunofluorescence labeling. Between 0.2 and 5Gy the yield of CD59 mutants was a linear function of dose. The molecular analysis of individual CD59-negative clones induced after exposure of 1, 3 and 5Gy of X-ray revealed a dose-dependent linear increase of large deletions (>6Mbp), whereas, point mutations could be seen only in spontaneous CD59 mutants or after low-dose exposure (< or =1Gy). We conclude that the modified A(L) assay presented here is appropriate for detection and quantification of non-lethal DNA lesions induced by low-dose ionizing radiation.     

Visualising individual green fluorescent proteins with a near field optical microscope.

The use of the green fluorescence protein (GFP) as an individual marker for applications in molecular biology requires detailed understanding of its photophysical and photodynamical properties. We investigated individual S65T mutants of GFP both on a glass surface and embedded in a water-pore gel. An aperture-type near field scanning optical microscope (NSOM) with two polarisation detection channels was applied to afford high spatial (approximately 70 nm) and temporal (0.5 ms) resolution. Shear-force and near field fluorescence imaging were performed simultaneously, allowing direct correlation between topographic and optical features. Polarisation data showed that the emission dipole moment of the proteins is fixed in space within both the barrel structure of the protein and the gel matrix used for spatial confinement of the proteins. The photophysical behaviour of the S65T-GFP mutants was monitored in time, with 500-micros real-time resolution and continuous imaging for periods of more than 2 h. Our results show the reversible on-off behaviour on a time scale that spans from 10(-4) to 10(3) s. Even a process generally identified as "bleaching" turns out to be reversible if a sufficient long observation time is allowed. As such, the photodynamics of individual GFPs appear to be much more complex than the properties deduced from ensemble-averaged measurements.     

Properties of a polygalacturonase-inhibiting protein isolated from 'Oroblanco' grapefruit

Polygalacturonase inhibiting protein (PGIP) was extracted from 'Oroblanco' grapefruit type (triploid pummelo-grapefruit) albedo tissue, purified and partially characterized. Extraction was carried out at 4°C with a high ionic strength extraction buffer. After dialysis and concentration by ultrafiltration the extract was chromatographed on concanavalin A-Sepharose. The PGIP activity was bound by the lectin and then eluted using 250 m M α -methyl mannopyranoside, resulting in a 17-fold purification of the PGIP and demonstrating its glycoprotein nature. The anion-exchange and size-exclusion chromatography steps that followed gave a PGIP that was 857-fold purified relative to the initial tissue extract, and having a 44 kDa molecular weight, as estimated by SDS-PAGE electrophoresis. PGIP inhibition activity was tested with endo-polygalacturonase (EC 3.2.1.15) produced by Penicillium italicum and Botrytis cinerea . The radial diffusion and reducing sugar assays showed that P. italicum and B. cinerea endo-PGs were affected by PGIP, whereas no endo-PG activity was detected in the culture filtrate of P. digitatum. In vitro tests revealed that PGIP inhibited P. italicum and B. cinerea growth. By contrast, the influence of PGIP on P. digitatum , growth was negligible, perhaps because this fungus does not produce endo-PG. Following heating for 10 min at 65°C the inhibitory activity of PGIP was reduced by 43%. PGIP activity decreased further as heating temperature increased, and was completely suppressed after heating at 100°C for 10 min.     

Synthesis and antiviral evaluation of benzimidazoles, quinoxalines and indoles from dehydroabietic acid

Several heterocycles, such as benzimidazoles, quinoxalines and indoles incorporated into a hydrophenanthrene and naphthalene skeleton, were synthesised from two useful ortho-bromonitro precursors derived from dehydroabietic acid: methyl 12-bromo-13-nitro-deisopropyldehydroabietate and methyl 12-bromo-13,14-dinitro-deisopropyldehydroabietate. The new heterocycles were evaluated for their activity in vitro against several RNA and DNA viruses.