Journals
2012 EN
Dong Yin · Wangrong Yang · Jack Weissberg
+13 more
Nanosecond pulsed electric fields (nsPEF) induce apoptotic pathways in human cancer cells. The potential therapeutic effective of nsPEF has been reported in cell lines and in xenograft animal tumor model. The present study investigated the ability of nsPEF to cause cancer cell death in vivo using carcinogen-induced animal tumor model, and the pulse duration of nsPEF was only 7 and 14 nano second (ns). An nsPEF generator as a prototype medical device was used in our studies, which is capable of delivering 7–30 nanosecond pulses at various programmable amplitudes and frequencies. Seven cutaneous squamous cell carcinoma cell lines and five other types of cancer cell lines were used to detect the effect of nsPEF in vitro . Rate of cell death in these 12 different cancer cell lines was dependent on nsPEF voltage and pulse number. To examine the effect of nsPEF in vivo , carcinogen-induced cutaneous papillomas and squamous cell carcinomas in mice were exposed to nsPEF with three pulse numbers (50, 200, and 400 pulses), two nominal electric fields (40 KV/cm and 31 KV/cm), and two pulse durations (7 ns and 14 ns). Carcinogen-induced cutaneous papillomas and squamous carcinomas were eliminated efficiently using one treatment of nsPEF with 14 ns duration pulses (33/39 = 85%), and all remaining lesions were eliminated after a 2nd treatment (6/39 = 15%). 13.5% of carcinogen-induced tumors (5 of 37) were eliminated using 7 ns duration pulses after one treatment of nsPEF. Associated with tumor lysis, expression of the anti-apoptotic proteins Bcl-xl and Bcl-2 were markedly reduced and apoptosis increased (TUNEL assay) after nsPEF treatment. nsPEF efficiently causes cell death in vitro and removes papillomas and squamous cell carcinoma in vivo from skin of mice. nsPEF has the therapeutic potential to remove human squamous carcinoma.
Public Library of Science
Journals
2012 EN
Paula Scotland · Jessica L. Heath · Amanda E. Conway
+6 more
The ubiquitously expressed phosphatidylinositol binding clathrin assembly (PICALM) protein associates with the plasma membrane, binds clathrin, and plays a role in clathrin-mediated endocytosis. Alterations of the human PICALM gene are present in aggressive hematopoietic malignancies, and genome-wide association studies have recently linked the PICALM locus to late-onset Alzheimer's disease. Inactivating and hypomorphic Picalm mutations in mice cause different degrees of severity of anemia, abnormal iron metabolism, growth retardation and shortened lifespan. To understand PICALM’s function, we studied the consequences of PICALM overexpression and characterized PICALM-deficient cells derived from mutant fit1 mice. Our results identify a role for PICALM in transferrin receptor (TfR) internalization and demonstrate that the C-terminal PICALM residues are critical for its association with clathrin and for the inhibitory effect of PICALM overexpression on TfR internalization. Murine embryonic fibroblasts (MEFs) that are deficient in PICALM display several characteristics of iron deficiency (increased surface TfR expression, decreased intracellular iron levels, and reduced cellular proliferation), all of which are rescued by retroviral PICALM expression. The proliferation defect of cells that lack PICALM results, at least in part, from insufficient iron uptake, since it can be corrected by iron supplementation. Moreover, PICALM-deficient cells are particularly sensitive to iron chelation. Taken together, these data reveal that PICALM plays a critical role in iron homeostasis, and offer new perspectives into the pathogenesis of PICALM-associated diseases.
Public Library of Science
Journals
2012 EN
Viviane Devauges · Catherine Marquer · Sandrine Lécart
+5 more
Classical FRET (Förster Resonance Energy Transfer) using two fluorescent labels (one for the donor and another one for the acceptor) is not efficient for studying the homodimerization of a protein as only half of the homodimers formed can be identified by this technique. We thus resorted to homoFRET detected by time-resolved Fluorescence Anisotropy IMaging (tr-FAIM). To specifically image the plasma membrane of living cells, an original combination of tr-FAIM and Total Internal Reflection Fluorescence Lifetime Imaging Microscope (TIRFLIM) was implemented. The correcting factor accounting for the depolarization due to the high numerical aperture (NA) objective, mandatory for TIRF microscopy, was quantified on fluorescein solutions and on HEK293 cells expressing enhanced Green Fluorescence Protein (eGFP). Homodimerization of Amyloid Precursor Protein (APP), a key mechanism in the etiology of Alzheimer’s disease, was measured on this original set-up. We showed, both in epifluorescence and under TIRF excitation, different energy transfer rates associated with the homodimerization of wild type APP-eGFP or of a mutated APP-eGFP, which forms constitutive dimers. This original set-up thus offers promising prospects for future studies of protein homodimerization in living cells in control and pathological conditions.
Public Library of Science
Journals
2012 EN
Maria Beconi · Omar Aziz · Kim L. Matthews
+17 more
Histone deacetylase (HDAC) inhibitors have received considerable attention as potential therapeutics for a variety of cancers and neurological disorders. Recent publications on a class of pimelic diphenylamide HDAC inhibitors have highlighted their promise in the treatment of the neurodegenerative diseases Friedreich’s ataxia and Huntington’s disease, based on efficacy in cell and mouse models. These studies’ authors have proposed that the unique action of these compounds compared to hydroxamic acid-based HDAC inhibitors results from their unusual slow-on/slow-off kinetics of binding, preferentially to HDAC3, resulting in a distinctive pharmacological profile and reduced toxicity. Here, we evaluate the HDAC subtype selectivity, cellular activity, absorption, distribution, metabolism and excretion (ADME) properties, as well as the central pharmacodynamic profile of one such compound, HDACi 4b , previously described to show efficacy in vivo in the R6/2 mouse model of Huntington’s disease. Based on our data reported here, we conclude that while the in vitro selectivity and binding mode are largely in agreement with previous reports, the physicochemical properties, metabolic and p-glycoprotein (Pgp) substrate liability of HDACi 4b render this compound suboptimal to investigate central Class I HDAC inhibition in vivo in mouse per oral administration. A drug administration regimen using HDACi 4b dissolved in drinking water was used in the previous proof of concept study, casting doubt on the validation of CNS HDAC3 inhibition as a target for the treatment of Huntington’s disease. We highlight physicochemical stability and metabolic issues with 4b that are likely intrinsic liabilities of the benzamide chemotype in general.
Public Library of Science
Journals
2012 EN
Thomas F.E. Barth · Dominic Schmidt · Catherine Botteron
+4 more
Resistance to Leishmania major infection is dependent on the development of a cell-mediated Th1 immune response in resistant C57BL/6 mice whereas Th2-prone BALB/c mice develop non-healing lesions after infection. The chemokine receptor CCR6 is shared by anti-inflammatory regulatory T cells and pro-inflammatory Th17 cells. In a recent study we showed that C57BL/6 mice deficient in CCR6 exhibited enhanced footpad swelling and impaired T helper cell migration indicated by reduced recruitment of total T helper cells into the skin after infection and a reduced delayed type hypersensitivity reaction. Based on these findings we tested whether the lack of CCR6 alters Treg or Th17 cell responses during the course of Leishmania major infection. When we analyzed T cell subsets in the lymph nodes of CCR6-deficient mice, Th17 cell numbers were not different. However, reduced numbers of Treg cells paralleled with a stronger IFNγ response. Furthermore, the early increase in IFNγ-producing cells correlated with increased local tissue inflammation at later time points. Our data indicate an important role of CCR6 for Treg cells and a redundant role for Th17 cells in a Th1 cell-driven anti-parasitic immune response against Leishmania major parasites in resistant C57BL/6 mice.
Public Library of Science
Journals
2012 EN
Alicia B. Sivitz · Victor Hermand · Catherine Curie
+1 more
Iron deficiency induces a complex set of responses in plants, including developmental and physiological changes, to increase iron uptake from soil. In Arabidopsis, many transporters involved in the absorption and distribution of iron have been identified over the past decade. However, little is known about the signaling pathways and networks driving the various responses to low iron. Only the basic helix–loop–helix (bHLH) transcription factor FIT has been shown to control the expression of the root iron uptake machinery genes FRO2 and IRT1 . Here, we characterize the biological role of two other iron-regulated transcription factors, bHLH100 and bHLH101, in iron homeostasis. First direct transcriptional targets of FIT were determined in vivo . We show that bHLH100 and bHLH101 do not regulate FIT target genes, suggesting that they play a non-redundant role with the two closely related bHLH factors bHLH038 and bHLH039 that have been suggested to act in concert with FIT. bHLH100 and bHLH101 play a crucial role in iron-deficiency responses, as attested by their severe growth defects and iron homeostasis related phenotypes on low-iron media. To gain further insight into the biological role of bHLH100 and bHLH101, we performed microarray analysis using the corresponding double mutant and showed that bHLH100 and bHLH101 likely regulate genes involved in the distribution of iron within the plant. Altogether, this work establishes bHLH100 and bHLH101 as key regulators of iron-deficiency responses independent of the master regulator FIT and sheds light on new regulatory networks important for proper growth and development under low iron conditions.
Public Library of Science
Journals
2012 EN
Catherine E. Grueber · Graham P. Wallis · Tania M. King
+1 more
Toll-like receptors (TLRs) are an ancient family of genes encoding transmembrane proteins that bind pathogen-specific molecules and initiate both innate and adaptive aspects of the immune response. Our goal was to determine whether these genes show sufficient genetic diversity in a bottlenecked population to be a useful addition or alternative to the more commonly employed major histocompatibility complex (MHC) genotyping in a conservation genetics context. We amplified all known avian TLR genes in a severely bottlenecked population of New Zealand's Stewart Island robin ( Petroica australis rakiura ), for which reduced microsatellite diversity was previously observed. We genotyped 17–24 birds from a reintroduced island population (including the 12 founders) for nine genes, seven of which were polymorphic. We observed a total of 24 single-nucleotide polymorphisms overall, 15 of which were non-synonymous, representing up to five amino-acid variants at a locus. One locus ( TLR1LB ) showed evidence of past directional selection. Results also confirmed a passerine duplication of TLR7 . The levels of TLR diversity that we observe are sufficient to justify their further use in addressing conservation genetic questions, even in bottlenecked populations.
Public Library of Science
Journals
2012 EN
Catherine Mooney · Niall Haslam · Gianluca Pollastri
+1 more
The conventional wisdom is that certain classes of bioactive peptides have specific structural features that endow their particular functions. Accordingly, predictions of bioactivity have focused on particular subgroups, such as antimicrobial peptides. We hypothesized that bioactive peptides may share more general features, and assessed this by contrasting the predictive power of existing antimicrobial predictors as well as a novel general predictor, PeptideRanker, across different classes of peptides. We observed that existing antimicrobial predictors had reasonable predictive power to identify peptides of certain other classes i.e. toxin and venom peptides. We trained two general predictors of peptide bioactivity, one focused on short peptides (4–20 amino acids) and one focused on long peptides (amino acids). These general predictors had performance that was typically as good as, or better than, that of specific predictors. We noted some striking differences in the features of short peptide and long peptide predictions, in particular, high scoring short peptides favour phenylalanine. This is consistent with the hypothesis that short and long peptides have different functional constraints, perhaps reflecting the difficulty for typical short peptides in supporting independent tertiary structure. We conclude that there are general shared features of bioactive peptides across different functional classes, indicating that computational prediction may accelerate the discovery of novel bioactive peptides and aid in the improved design of existing peptides, across many functional classes. An implementation of the predictive method, PeptideRanker, may be used to identify among a set of peptides those that may be more likely to be bioactive.
Public Library of Science
Journals
2012 EN
Catherine J. Payne · Tim S. Jessop · PatrickJean Guay
+3 more
Wild animals in urban environments are exposed to a broad range of human activities that have the potential to disturb their life history and behaviour. Wildlife responses to disturbance can range from emigration to modified behaviour, or elevated stress, but these responses are rarely evaluated in concert. We simultaneously examined population, behavioural and hormonal responses of an urban population of black swans Cygnus atratus before, during and after an annual disturbance event involving large crowds and intense noise, the Australian Formula One Grand Prix. Black swan population numbers were lowest one week before the event and rose gradually over the course of the study, peaking after the event, suggesting that the disturbance does not trigger mass emigration. We also found no difference in the proportion of time spent on key behaviours such as locomotion, foraging, resting or self-maintenance over the course of the study. However, basal and capture stress-induced corticosterone levels showed significant variation, consistent with a modest physiological response. Basal plasma corticosterone levels were highest before the event and decreased over the course of the study. Capture-induced stress levels peaked during the Grand Prix and then also declined over the remainder of the study. Our results suggest that even intensely noisy and apparently disruptive events may have relatively low measurable short-term impact on population numbers, behaviour or physiology in urban populations with apparently high tolerance to anthropogenic disturbance. Nevertheless, the potential long-term impact of such disturbance on reproductive success, individual fitness and population health will need to be carefully evaluated.
Public Library of Science
Journals
2012 EN
Adedolapo Adebakin · Jenna Bradley · Sarah Gümüsgöz
+2 more
Alzheimer's disease (AD) is associated with non-cognitive symptoms such as changes in feeding behaviour that are often characterised by an increase in appetite. Increased food intake is observed in several mouse models of AD including the triple transgenic (3×TgAD) mouse, but the mechanisms underlying this hyperphagia are unknown. We therefore examined feeding behaviour in 3×TgAD mice and tested their sensitivity to exogenous and endogenous satiety factors by assessing food intake and activation of key brain regions. In the behavioural satiety sequence (BSS), 3×TgAD mice consumed more food after a fast compared to Non-Tg controls. Feeding and drinking behaviours were increased and rest decreased in 3×TgAD mice, but the overall sequence of behaviours in the BSS was maintained. Exogenous administration of the satiety factor cholecystokinin (CCK; 8–30 µg/kg, i.p.) dose-dependently reduced food intake in Non-Tg controls and increased inactive behaviour, but had no effect on food intake or behaviour in 3×TgAD mice. CCK (15 µg/kg, i.p.) increased c-Fos protein expression in the supraoptic nucleus of the hypothalamus, and the nucleus tractus solitarius (NTS) and area postrema of the brainstem to the same extent in Non-Tg and 3×TgAD mice, but less c-Fos positive cells were detected in the paraventricular hypothalamic nucleus of CCK-treated 3×TgAD compared to Non-Tg mice. In response to a fast or a period of re-feeding, there was no difference in the number of c-Fos-positive cells detected in the arcuate nucleus of the hypothalamus, NTS and area postrema of 3×TgAD compared to Non-Tg mice. The degree of c-Fos expression in the NTS was positively correlated to food intake in Non-Tg mice, however, this relationship was absent in 3×TgAD mice. These data demonstrate that 3×TgAD mice show increased feeding behaviour and insensitivity to satiation, which is possibly due to defective gut-brain signalling in response to endogenous satiety factors released by food ingestion.
Public Library of Science