Direct detection of Mycobacterium tuberculosis complex in pulmonary and extrapulmonary samples by BDProbeTec ET system

BDProbeTec ET (Becton Dickinson, Sparks, Md, USA) is a fully automated walkaway system based on strand displacement amplification (SDA) technology that provides a method for the direct detection of Mycobacterium tuberculosis complex (MTBC) target sequence. The purpose of this study was to evaluate the ability of BDProbeTec ET system to detect MTBC directly from clinical specimens and compare the results with staining and culture. From February 2002 through December 2003 a total of 1521 [pulmonary (n=1329) and extrapulmonary (n=192)] specimens from 1518 patients were examined by BDProbeTec ET system for the detection of MTBC and the results were compared to those obtained by microscopy and liquid culture (BACTEC 9000 MB, Becton Dickinson). MTBC was cultivated from 65 specimens (60 pulmonary and 5 extrapulmonary) of which 43 (66.2%) (42 pulmonary and 1 extrapulmonary) were smear positive and 22 (33.8%) (18 pulmonary and 4 extrapulmonary) were smear negative. BDProbeTec ET detected MTBC in 58 (55 pulmonary and 3 extrapulmonary) of the 65 culture-positive specimens. Although the BDProbeTec ET system gave five false-negative results among the 18 smear-negative culture-positive pulmonary specimens, our results demonstrate that the BDProbeTec ET system is a reliable tool in smear-positive samples and given its technical characteristics it can be used for the rapid detection of MTBC in either pulmonary or extrapulmonary samples.     

Aminolevulinic acid: from its unique biological function to its star role in photodynamic therapy

Porphyrins are molecules essential for life. They are involved in the key processes of photosynthesis and respiration. The biosynthesis of tetrapyrroles in all living cells occurs through several steps where the formation of aminolevulinic acid (ALA) is the first committed intermediate. Two alternative routes for the formation of ALA have been proposed: one involves the condensation of Succinyl CoA and glycine catalyzed by ALA synthetase taking place in the mitochondria, and the second one is the so called 5-carbon route, occurring in the stroma of plastids. Eight molecules of ALA are used in the formation of protoporphyrin IX. Specific deficiencies in one of the enzymes of the heme pathway produce the porphyrias. In the acute porphyrias, the pathogenesis of the neurological dysfunction is attributed to the accumulation of ALA. Fluorescent and photosensitizing properties of protoporphyrin accumulated after the exogenous administration of ALA, can be used to visualize and destroy malignant cells in the so-called photodynamic diagnosis (PDD) and photodynamic therapy (PDT) of cancer. Many clinical ALA-PDT applications to malignant and non-malignant pathologies are currently in use. Different approaches to enhance ALA penetration in cells are under investigation, including the use of more lipophilic ALA derivatives and studies of the transport mechanisms of ALA. ALA has also been proposed to be used as a biodegradable herbicide, as an insecticide and as a plant growth regulator.     

Stromal cell-derived factor-1alpha (SDF-1alpha/CXCL12) stimulates ovarian cancer cell growth through the EGF receptor transactivation

Ovarian cancer (OC) is the leading cause of death in gynecologic diseases in which there is evidence for a complex chemokine network. Chemokines are a family of proteins that play an important role in tumor progression influencing cell proliferation, angiogenic/angiostatic processes, cell migration and metastasis, and, finally, regulating the immune cells recruitment into the tumor mass. We previously demonstrated that astrocytes and glioblastoma cells express both the chemokine receptor CXCR4 and its ligand stromal cell-derived factor-1 (SDF-1), and that SDF-1alpha treatment induced cell proliferation, supporting the hypothesis that chemokines may play an important role in tumor cells' growth in vitro. In the present study, we report that CXCR4 and SDF-1 are expressed in OC cell lines. We demonstrate that SDF-1alpha induces a dose-dependent proliferation in OC cells, by the specific interaction with CXCR4 and a biphasic activation of ERK1/2 and Akt kinases. Our results further indicate that CXCR4 activation induces EGF receptor (EGFR) phosphorylation that in turn was linked to the downstream intracellular kinases activation, ERK1/2 and Akt. In addition, we provide evidence for cytoplasmic tyrosine kinase (c-Src) involvement in the SDF-1/CXCR4-EGFR transactivation. These results suggest a possible important "cross-talk" between SDF-1/CXCR4 and EGFR intracellular pathways that may link signals of cell proliferation in ovarian cancer.     

RNA silencing platforms in plants

Since the discovery of RNAi, its mechanism in plants and animals has been intensively studied, widely exploited as a research tool, and used for a number of potential commercial applications. In this article, we discuss the platforms for delivering RNAi in plants. We provide a brief background to these platforms and concentrate on discussing the more recent advances, comparing the RNAi technologies used in plants with those used in animals, and trying to predict the ways in which RNAi technologies may further develop.     

Determination of the specificity of monoclonal antibodies against Schistosoma mansoni CAA glycoprotein antigen using neoglycoconjugate variants

The immunogenic O-glycan of circulating anodic antigen (CAA) is a high-molecular-mass polysaccharide with the unique -->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GalpNAc-(1--> repeating unit. To obtain information at the molecular level about the specificity of monoclonal antibodies against CAA, the immunoreactivity of two series of bovine serum albumin-coupled synthetic oligosaccharides related to the CAA O-glycan was monitored using ELISA and surface plasmon resonance spectroscopy. The importance of the axial hydroxyl group of beta-D-GalpNAc for antibody binding was investigated using the following series of analogues: beta-D-GlcpA-(1-->3)-beta-D-GlcpNAc-(1-->O); beta-D-GlcpNAc-(1-->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GlcpNAc-(1-->O); and beta-D-GlcpA-(1-->3)-beta-D-GlcpNAc-(1-->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GlcpNAc-(1-->O). In the second series of analogues, beta-D-Glcp6S-(1-->3)-beta-D-GalpNAc-(1-->O), beta-D-GalpNAc-(1-->6)-[beta-D-Glcp6S-(1-->3)]-beta-D-GalpNAc-(1-->O), and beta-D-Glcp6S-(1-->3)-beta-D-Gal-pNAc-(1-->6)-[beta-D-Glcp6S-(1-->3)]-beta-D-GalpNAc-(1-->O), the native beta-D-GlcpA moiety was replaced by beta-D-Glcp6S to evaluate the influence of the nature of the charge on antibody recognition. Comparison of the immunoreactivity of these series with that measured for conjugates containing corresponding synthetic CAA fragments showed that the antibody binding levels can be correlated to the antibody specificity to CAA fragments. For the most reactive antibodies, the structural changes chosen (beta-D-GalpNAc replaced by beta-D-GlcpNAc, and beta-D-GlcpA replaced by beta-D-Glcp6S) completely eradicated the binding.     

Arabidopsis SENESCENCE-ASSOCIATED GENE101 stabilizes and signals within an ENHANCED DISEASE SUSCEPTIBILITY1 complex in plant innate immunity

Plant innate immunity against invasive biotrophic pathogens depends on the intracellular defense regulator ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1). We show here that Arabidopsis thaliana EDS1 interacts in vivo with another protein, SENESCENCE-ASSOCIATED GENE101 (SAG101), discovered through a proteomic approach to identify new EDS1 pathway components. Together with PHYTOALEXIN-DEFICIENT4 (PAD4), a known EDS1 interactor, SAG101 contributes intrinsic and indispensable signaling activity to EDS1-dependent resistance. The combined activities of SAG101 and PAD4 are necessary for programmed cell death triggered by the Toll-Interleukin-1 Receptor type of nucleotide binding/leucine-rich repeat immune receptor in response to avirulent pathogen isolates and in restricting the growth of normally virulent pathogens. We further demonstrate by a combination of cell fractionation, coimmunoprecipitation, and fluorescence resonance energy transfer experiments the existence of an EDS1-SAG101 complex inside the nucleus that is molecularly and spatially distinct from EDS1-PAD4 associations in the nucleus and cytoplasm. By contrast, EDS1 homomeric interactions were detected in the cytoplasm but not inside the nucleus. These data, combined with evidence for coregulation between individual EDS1 complexes, suggest that dynamic interactions of EDS1 and its signaling partners in multiple cell compartments are important for plant defense signal relay.     

ATM regulates target switching to escalating doses of radiation in the intestines

Although stem cells succumbing to reproductive death are assumed to be the single relevant targets in radiation tissue damage, recent studies showed intestinal stem cell damage is conditionally linked to crypt endothelial apoptosis, defining a two-target model. Here we report that when mouse intestines were protected against microvascular apoptosis, radiation switched as the dose escalated to a previously unrecognized crypt stem cell target, activating ceramide synthase-mediated apoptosis to initiate intestinal damage. Whereas ataxia telangiectasia-mutated (ATM) kinase normally represses ceramide synthase, its derepression in Atm(-/-) mice increased crypt stem cell radiosensitivity 3.7-fold without sensitizing the microvascular response. Discovery of this intestinal radiosensitivity mechanism allowed design of an antisense Atm oligonucleotide treatment which phenocopied the Atm(-/-) mouse, reordering ceramide synthase-mediated stem cell death to become the first-line gastrointestinal response of wild-type littermates. These experiments indicate that tissues operate multiple potential targets activated consecutively according to their inherent radiosensitivities that may be reordered therapeutically to control radiation tissue responses.