Damage of Escherichia coli cells by t-butylhydroperoxide involves the respiratory chain but is independent of the presence of oxygen

The action of t-butylhydroperoxide (tBOOH) on Escherichia coli cells has been studied as a model system for organic peroxide toxicity. Exposure of E. coli cells to tBOOH led to progressive and irreversible impairment of the respiratory function, an effect which was dependent on the availability of substrate. The effect of tBOOH on growth of E. coli with different carbon sources and alternative terminal electron acceptors was investigated. It was found that the sensitivity of E. coli to tBOOH under diverse growth conditions implicating a functional respiratory chain was greater than when the bacterium grew by fermentation. Also the mutant E. coli SASX76, which requires exogenous 5-aminolevulinic acid to synthesize the cytochromes, was more resistant to tBOOH when lacking a functional respiratory chain. These data point to the respiratory chain as a major target in the in vivo action of tBOOH. Experiments with isolated membranes also showed a tBOOH-induced damage of the respiratory chain monitored by impairment of the NADH oxidase. The effect of tBOOH was produced even under anaerobiosis, indicating that development of cell damage was independent of oxygen and, therefore, that neither oxygen-derived radicals nor lipid peroxidation were involved.     

Mechanism of the film thickness increasing during the bacterial production of cellulose on non-agitaded liquid media

Summary The thickness increasing of the cellulosic films produced by bacteria on non-agitated liquid media is due to the formation of new cellulose layers at the film/air interface.     

Differential Inhibitor of G�¦� Signaling to AKT and ERK Derived from Phosducin-like Protein: EFFECT ON SPHINGOSINE 1-PHOSPHATE-INDUCED ENDOTHELIAL CELL MIGRATION AND IN VITRO ANGIOGENESIS*

Differential inhibitors of Gβγ-effector regions are required to dissect the biological contribution of specific Gβγ-initiated signaling pathways. Here, we characterize PhLP-M1-G149, a Gβγ-interacting construct derived from phosducin-like protein 1 (PhLP) as a differential inhibitor of Gβγ, which, in endothelial cells, prevented sphingosine 1-phosphate-induced phosphorylation of AKT, glycogen synthase kinase 3β, cell migration, and tubulogenesis, while having no effect on ERK phosphorylation or hepatocyte growth factor-dependent responses. This construct attenuated the recruitment of phosphoinositide 3-kinase γ (PI3Kγ) to the plasma membrane and the signaling to AKT in response to Gβγ overexpression. In coimmunoprecipitation experiments, PhLP-M1-G149 interfered with the interaction between PI3Kγ and Gβγ. Other PhLP-derived constructs interacted with Gβγ but were not effective inhibitors of Gβγ signaling to AKT or ERK. Our results indicate that PhLP-M1-G149 is a suitable tool to differentially modulate the Gβγ-initiated pathway linking this heterodimer to AKT, endothelial cell migration, and in vitro angiogenesis. It can be also useful to further characterize the molecular determinants of the Gβγ-PI3Kγ interaction.Heterotrimeric G protein signaling depends on the actions of GTP-loaded Gα and free Gβγ, the two functional components of the heterotrimer, leading to the generation of second messengers and cell specific functional events (1, 2). Differential inhibitors of Gβγ are required to dissect the biological impact of different Gβγ-dependent effectors. Gβγ actions can be blocked by competition with peptides derived from its effectors. For example, the effect of Gβγ on adenylyl cyclase II, G protein-activated inward rectifier K⁺ channel, G protein-coupled receptor kinase 2, and phospholipase Cβ3, is attenuated by a peptide from adenylyl cyclase II (3). In addition, RACK1 (receptor for activated C kinase 1) selectively inhibits the effect the chemokine receptor CXCR2 on the activation of phospholipase Cβ2 and adenylyl cyclase II in HEK293 cells, without affecting other functions of Gβγ (4). Recently, Smrcka and colleagues characterized the effect of small molecule inhibitors of Gβγ, suggesting their potential application in therapeutic strategies targeting particular Gβγ-dependent pathways (5). Emerging possibilities to target this heterodimer in pathological situations such as inflammation and angiogenesis are based on the role of Gβγ in cell survival and chemotaxis. To the best of our knowledge, no molecular tool is yet available to differentially inhibit Gβγ signaling to AKT.³Gβγ is a key transducer of sphingosine 1-phosphate (S1P)-elicited angiogenic signals promoting endothelial cell migration, proliferation, and survival (6–12). Multiple Gβγ-dependent effectors are potentially involved in the molecular events required for endothelial cell migration. These include lipid kinases such as PI3Kγ and PI3Kβ (13), and a novel family of Rac guanine nucleotide exchange factors, represented by P-REX1, which is activated by Gβγ and phosphatidylinositol 3,4,5-trisphosphate (14–16). Gβγ signaling is frequently attributed to pertussis toxin-sensitive G_i coupled receptors, and it has been consistently revealed by the antagonistic effect of the carboxyl-terminal region of G protein-coupled receptor kinase 2, which sequesters Gβγ thereby inhibiting all its intracellular actions (17). In addition, mutational analysis of Gβ revealed that different residues, all of them mapping to the interface of contact between Gβγ and Gα, are important for the activation of distinct Gβγ effector molecules (18).Phosducin was originally identified as a phosphoprotein restricted to the retina and pineal gland forming a complex with Gβγ (19, 20). It was considered a protein kinase A-sensitive regulator of G protein-mediated signaling (21, 22). Further studies identified a family of phosducin-like proteins (PhLPs) (23, 24). Phosducin and Gα share affinity for the same region of Gβγ, as revealed by the structural analysis of Gβγ in complex with Gα or phosducin and by in vitro binding experiments (25). This area of interaction includes some of the residues considered necessary for the activation of Gβγ-dependent effectors (18, 26). It was initially postulated that phosducin and related proteins, by interfering with the availability of free Gβγ, exert an inhibitory role on Gβγ signaling. However, recent genetic evidence raised an apparently conflicting situation; the knockout of PhLP in fungi resulted in a phenotype equivalent to the absence of Gβγ, contrary to its expected role as an inhibitor (27). Novel experimental evidence indicated that PhLP has a positive effect on Gβγ signaling due to its participation in the assembly of the heterodimer, helping to stabilize free Gβ subunits leaving the ribosome after synthesis (28–31).Despite the positive role of full-length PhLP in the assembly of Gβγ heterodimers, it is still possible that different fragments of this protein, which could retain their interaction with distinct regions of Gβγ, might function as inhibitors of Gβγ signaling. Accordingly, we characterized here the effect of different PhLP-derived constructs on the signaling pathways elicited by S1P or HGF in endothelial cells. In addition, we explored the mechanism by which PhLP-M1-G149 interferes with Gβγ preventing the activation of AKT.     

Contrasting age-specific recruitment and survival at different spatial scales: a case study with the European storm petrel

Evolutionary studies on optimal decisions or conservation guidelines are often derived by generalising patterns from a single population, while inter‐population variability in life‐history traits is seldom considered. We investigated here how survival and recruitment probabilities changed with age at different geographical scales using the encounter histories of 5523 European storm petrels from three Mediterranean colonies, and also how our estimates of these parameters might be expected to affect population growth rates using population matrix models. We recorded similar patterns among colonies, but also important biological differences. Local survival, recruitment and breeding success increased with age at all colonies; the most distant of three colonies (Marettimo Is.) showed the largest differences. Strikingly, differences in recruitment were also found between two adjacent colonies (two caves from Benidorm Is.). Birds marked as adults from Marettimo and Benidorm colonies had a different survival, whereas we found no differences within Benidorm. Differences in survival were no longer apparent between the two islands at the end of the study following a reduction in predation by specialist gulls at Benidorm. Since birds marked as fledglings mostly recruited near the end of the study, their overall survival was high and in turn similar among colonies. Results from our population matrix models suggested that different age‐dependent patterns of demographic parameters can lead to similar population growth rates. Variability appeared to be greater for recruitment and the most sensitive parameter was adult survival. Thus conservation actions targeting this vulnerable species should focus on factors influencing adult survival. Differences in survival and recruitment among colonies could reflect the spatial heterogeneity in mortality due to predation and colony‐specific density dependent processes. Results highlight the importance of taking into account the potential spatio‐temporal heterogeneity among populations in vital rates, even in those traits that life‐history theory considers less important in driving population dynamics.     

Towards improved boron neutron capture therapy agents: evaluation of in vitro cellular uptake of a glutamine-functionalized carborane

Abstract  Sodium borocaptate (BSH) is widely used for boron neutron capture therapy (BNCT) of brain tumors. One drawback is the large uptake by the liver causing a decrease of its availability at the tumor region as well as bringing about toxicity problems. A novel carborane-based compound containing a boron payload very similar to that of BSH has been synthesized and tested on rat glioma (C6) cells, hepatoma tissue culture (HTC) cells, and hepatocytes. The newly synthesized system consists of an o-carborane unit (C₂B₁₀H₁₁, o-CB) conjugated to a glutamine residue through a proper spacer, namely, o-CB-Gln. As compared with BSH, it showed the same uptake by C6 cells, but a 50% decrease in uptake by HTC cells and an 80% decrease in uptake by healthy hepatocytes. On this basis o-CB-Gln appears an interesting candidate for BNCT of brain tumors as it is expected to have a therapeutic index analogous to that of BSH accompanied by a much lower liver toxicity. Graphical Abstract  A novel carborane based compound, consisting in an o-carborane unit (C2B10H11, o-CB) conjugated to a glutamine residue through a proper spacer (namely o-CB-Gln) has been synthesized, characterized and tested on rat glioma (C6), hepatoma (HTC) and hepatocytes. As compared to sodium borocaptate (BSH), widely used for boron neutron capture therapy (BNCT) of brain tumors, the newly synthesized system showed the same uptake by C6 cells, but a 50% decrease by HTC and 80% decrease by healthy hepatocytes. On this basis o-CB-Gln appears an interesting candidate for BNCT of brain tumors as it is expected to have a therapeutic index analogous to BSH accompanied by a much lower liver toxicity.      

A straightforward multiallelic significance test for the Hardy-Weinberg equilibrium law

Much forensic inference based upon DNA evidence is made assuming Hardy-Weinberg Equilibrium (HWE) for the genetic loci being used. Several statistical tests to detect and measure deviation from HWE have been devised, and their limitations become more obvious when testing for deviation within multiallelic DNA loci. The most popular methods-Chi-square and Likelihood-ratio tests-are based on asymptotic results and cannot guarantee a good performance in the presence of low frequency genotypes. Since the parameter space dimension increases at a quadratic rate on the number of alleles, some authors suggest applying sequential methods, where the multiallelic case is reformulated as a sequence of "biallelic" tests. However, in this approach it is not obvious how to assess the general evidence of the original hypothesis; nor is it clear how to establish the significance level for its acceptance/rejection. In this work, we introduce a straightforward method for the multiallelic HWE test, which overcomes the aforementioned issues of sequential methods. The core theory for the proposed method is given by the Full Bayesian Significance Test (FBST), an intuitive Bayesian approach which does not assign positive probabilities to zero measure sets when testing sharp hypotheses. We compare FBST performance to Chi-square, Likelihood-ratio and Markov chain tests, in three numerical experiments. The results suggest that FBST is a robust and high performance method for the HWE test, even in the presence of several alleles and small sample sizes.     

A-MADMAN: Annotation-based microarray data meta-analysis tool

Background  

Publicly available datasets of microarray gene expression signals represent an unprecedented opportunity for extracting genomic relevant information and validating biological hypotheses. However, the exploitation of this exceptionally rich mine of information is still hampered by the lack of appropriate computational tools, able to overcome the critical issues raised by meta-analysis.     

Computational annotation of genes differentially expressed along olive fruit development

Background  

Olea europaea L. is a traditional tree crop of the Mediterranean basin with a worldwide economical high impact. Differently from other fruit tree species, little is known about the physiological and molecular basis of the olive fruit development and a few sequences of genes and gene products are available for olive in public databases. This study deals with the identification of large sets of differentially expressed genes in developing olive fruits and the subsequent computational annotation by means of different software.     

Id2 suppression of p15 counters TGF‐β‐mediated growth inhibition of melanoma cells

Proliferative resistance to transforming growth factor β (TGF‐β) is regarded as a critical turning point in the malignant progression of many cancer types. In melanoma this resistance is associated with more aggressive metastatic behaviour. A recent study by our group identified proliferative and invasive subtypes of melanoma cultures and found that these are, respectively, susceptible and resistant to TGF‐β suppression of proliferation. Here, using previously characterised proliferative and invasive phenotype melanoma cultures, we explored molecular responses involved in modulating susceptibility to TGF‐β‐mediated inhibition of proliferation. The Id2 gene was identified as being expressed more strongly in invasive phenotype cells less susceptible to TGF‐β repression than in proliferative phenotype cells. We correlated TGF‐β repression of Id2 gene expression in proliferative phenotype cells with p15^Ink4b induction and cell cycle arrest. Furthermore, ectopic Id2 expression in proliferative phenotype cells counteracted p15^Ink4b induction and consequently protected them from TGF‐β‐mediated inhibition of proliferation. We conclude that transition to increased aggressiveness in melanoma cells requires Id2 upregulation to suppress TGF‐β induction of p15^Ink4b and thus help to circumvent TGF‐β‐mediated inhibition of proliferation.