Iron and proteins for iron storage and detoxification

Iron is required by most organisms, but is potentially toxic due to the low solubility of the stable oxidation state, Fe(III), and to the tendency to potentiate the production of reactive oxygen species, ROS. The reactivity of iron is counteracted by bacteria with the same strategies employed by the host, namely by sequestering the metal into ferritin, the ubiquitous iron storage protein. Ferritins are highly conserved, hollow spheres constructed from 24 subunits that are endowed with ferroxidase activity and can harbour up to 4500 iron atoms as oxy-hydroxide micelles. The release of the metal upon reduction can alter the microorganism-host iron balance and hence permit bacteria to overcome iron limitation. In bacteria, the relevance of the Dps (DNA-binding proteins from starved cells) family in iron storage-detoxification has been recognized recently. The seminal studies on the protein from Listeria innocua demonstrated that Dps proteins have ferritin-like activity and most importantly have the capacity to attenuate the production of ROS. This latter function allows bacterial pathogens that lack catalase, e.g. Porphyromonas gingivalis, to survive in an aerobic environment and resist to peroxide stress.     

Biocatalysed synthesis of beta-O-glucosides from 9-fluorenon-2-carbohydroxyesters. Part 3: IFN-inducing and anti-HSV-2 properties

In pursuing research on the antiviral, interferon (IFN)-inducing tilorone congeners, a new series of fluoren-carboxyhydroxyesters has been prepared and biologically explored. These esters have subsequently been used as sugar acceptors in the enzymatic transglycosylation reaction using the 'retaining' beta-glycosidase from the archaeon Sulfolobus solfataricus (Ssbeta-Gly). Both aglycones (1-6) and corresponding beta-glucosides (beta-glu 1-beta-glu 6) have been screened for cytotoxicity, interferon-stimulating and antiviral properties against HSV-2. It was found that the addition of compounds beta-glu 5, beta-glu 6 and beta-glu 4 to HSV-2 infected U937 cells downregulates viral replication and triggers cells to release IFN-alpha/beta. Taken together, the results showed improved pharmacological profiles as a consequence of glycosylation. A molecular modelling study carried out on this series of compounds completed the structural characterisation of the novel compounds.     

Microhabitat type selection of caddisfly larvae (Insecta: Trichoptera) in a shallow lowland stream

The relationship between caddisfly assemblage structure and four selected environmental variables (substrate, water depth, flow type and amount of the coarse particulate organic matter) was investigated in a Slovenian lowland stream. Caddisflies were sampled at four stream reaches according to selected microhabitat types. All together, 168 quantitative samples were taken at 21 sampling points between October 1998 and July 1999. Of 48 collected species, 30 were included in the analysis. Significant correlation was observed between species and environmental variables. As a complement to a CCA biplot representation, species assemblages within the community were also determined using cluster analysis. Nine groups and subgroups were established. Most caddisfly species prefer coarse substrate in shallow water (5–10 cm) with chute water flow, whereas few species were found on fine substrate in deep water. A significant positive correlation was found between mean substrate size and total number of species, and between indices of species richness and diversity, whereas depth did not show any correlation with these parameters. Seven species were found mostly in marginal habitats, whereas four (Potamophylax rotundipennis, Anabolia furcata, Athripsodes bilineatus and Lithax obscurus) did not show any strong preferences for selected parameters. In addition, habitat preferences were associated with the feeding types of the caddis larvae.     

Visualization through magnetic resonance imaging of DNA internalized following "in vivo" electroporation

The ability to visualize plasmid DNA entrapment in muscle cells undergoing an "in vivo" electroporation treatment was investigated on BALB/c mice using a 7-T magnetic resonance imaging (MRI) scanner using the paramagnetic Gd-DOTA-spd complex as imaging reporter. Gd-DOTA-spd bears a tripositively charged spermidine residue that yields a strong binding affinity toward the negatively charged DNA chain (6.4 kb, K(a) = 2.2 x 10(3) M(-1) for approximately 2500 +/- 500 binding sites). Cellular colocalization of Gd-DOTA-spd and plasmid DNA has been validated by histological analysis of excised treated muscle. In vivo MRI visualization of Gd-DOTA-spd distribution provides an excellent route to access the cellular entrapment of plasmid DNA upon applying an electroporation pulse.     

Temperature-, SDS-, and pH-induced conformational changes in protein disulfide oxidoreductase from the archaeon Pyrococcus furiosus: a dynamic simulation and fourier transform infrared spectroscopic study

The effect of SDS, pD, and temperature on the structure and stability of the protein disulfide oxidoreductase from Pyrococcus furiosus (PfPDO) was investigated by molecular dynamic (MD) simulations and FT-IR spectroscopy. pD affects the thermostability of alpha-helices and beta-sheets differently, and 0.5% or higher SDS concentration influences the structure significantly. The experiments allowed us to detect a secondary structural reorganization at a definite temperature and pD which may correlate with a high ATPase activity of the protein. The MD simulations supported the infrared data and revealed the different behavior of the N and C terminal segments, as well as of the two active sites.     

Identification of a novel human granzyme B inhibitor secreted by cultured sertoli cells

Sertoli cells have long since been recognized for their ability to suppress the immune system and protect themselves as well as other cell types from harmful immune reaction. However, the exact mechanism or product produced by Sertoli cells that affords this immunoprotection has never been fully elucidated. We examined the effect of mouse Sertoli cell-conditioned medium on human granzyme B-mediated killing and found that there was an inhibitory effect. We subsequently found that a factor secreted by Sertoli cells inhibited killing through the inhibition of granzyme B enzymatic activity. SDS-PAGE analysis revealed that this factor formed an SDS-insoluble complex with granzyme B. Immunoprecipitation and mass spectroscopic analysis of the complex identified a proteinase inhibitor, serpina3n, as a novel inhibitor of human granzyme B. We cloned serpina3n cDNA, expressed it in Jurkat cells, and confirmed its inhibitory action on granzyme B activity. Our studies have led to the discovery of a new inhibitor of granzyme B and have uncovered a new mechanism used by Sertoli cells for immunoprotection.     

GTP drives myosin light chain 1 interaction with the class V myosin Myo2 IQ motifs via a Sec2 RabGEF-mediated pathway

The yeast myosin light chain 1 (Mlc1p) belongs to a branch of the calmodulin superfamily and is essential for vesicle delivery at the mother-bud neck during cytokinesis due to is ability to bind to the IQ motifs of the class V myosin Myo2p. While calcium binding to calmodulin promotes binding/release from the MyoV IQ motifs, Mlc1p is unable to bind calcium and the mechanism of its interaction with target motifs has not been clarified. The presence of Mlc1p in a complex with the Rab/Ypt Sec4p and with Myo2p suggests a role for Mlc1p in regulating Myo2p cargo binding/release by responding to the activation of Rab/Ypt proteins. Here we show that GTP or GTPgammaS potently stimulate Mlc1p interaction with Myo2p IQ motifs. The C-terminus of the Rab/Ypt GEF Sec2p, but not Sec4p activation, is essential for this interaction. Interestingly, overexpression of constitutively activated Ypt32p, a Rab/Ypt protein that acts upstream of Sec4p, stimulates Mlc1p/Myo2p interaction similarly to GTP although a block of Ypt32 GTP binding does not completely abolish the GTP-mediated Mlc1p/Myo2p interaction. We propose that Mlc1p/Myo2p interaction is stimulated by a signal that requires Sec2p and activation of Ypt32p.     

Clinical presentation of acute pulmonary embolism: survey of 800 cases

Background

Pulmonary embolism (PE) is a common and potentially fatal disease that is still underdiagnosed. The objective of our study was to reappraise the clinical presentation of PE with emphasis on the identification of the symptoms and signs that prompt the patients to seek medical attention.

Methodology/Principal Findings

We studied 800 patients with PE from two different clinical settings: 440 were recruited in Pisa (Italy) as part of the Prospective Investigative Study of Acute Pulmonary Embolism Diagnosis (PISAPED); 360 were diagnosed with and treated for PE in seven hospitals of central Tuscany, and evaluated at the Atherothrombotic Disorders Unit, Firenze (Italy), shortly after hospital discharge. We interviewed the patients directly using a standardized, self-administered questionnaire originally utilized in the PISAPED. The two samples differed significantly as regards age, proportion of outpatients, prevalence of unprovoked PE, and of active cancer. Sudden onset dyspnea was the most frequent symptom in both samples (81 and 78%), followed by chest pain (56 and 39%), fainting or syncope (26 and 22%), and hemoptysis (7 and 5%). At least one of the above symptoms was reported by 756 (94%) of 800 patients. Isolated symptoms and signs of deep vein thrombosis occurred in 3% of the cases. Only 7 (1%) of 800 patients had no symptoms before PE was diagnosed.

Conclusions/Significance

Most patients with PE feature at least one of four symptoms which, in decreasing order of frequency, are sudden onset dyspnea, chest pain, fainting (or syncope), and hemoptysis. The occurrence of such symptoms, if not explained otherwise, should alert the clinicians to consider PE in differential diagnosis, and order the appropriate objective test.