Regulation of cytoplasmic dynein behaviour and microtubule organization by mammalian Lis1

Whereas total loss of Lis1 is lethal, disruption of one allele of the Lis1 gene results in brain abnormalities, indicating that developing neurons are particularly sensitive to a reduction in Lis1 dosage. Here we show that Lis1 is enriched in neurons relative to levels in other cell types, and that Lis1 interacts with the microtubule motor cytoplasmic dynein. Production of more Lis1 in non-neuronal cells increases retrograde movement of cytoplasmic dynein and leads to peripheral accumulation of microtubules. These changes may reflect neuron-like dynein behaviours induced by abundant Lis1. Lis1 deficiency produces the opposite phenotype. Our results indicate that abundance of Lis1 in neurons may stimulate specific dynein functions that function in neuronal migration and axon growth.     

Reversal of sexual dimorphism in splenic T lymphocyte responses after trauma-hemorrhage with aging

Previous studies have demonstrated that hemorrhagic shockproduces immunodepression in young male mice, whereas theimmunoresponsivness in young proestrus female mice is enhanced undersuch conditions. This sexually dimorphic immune response to hemorrhageappears to be related to high estrogen and testosterone levels infemales and males, respectively. Nonetheless, it is unknown what impact the age-related decline in the sex steroid levels has on the immune response after hemorrhage. To study this, young (2-3 mo) and aged (18-19 mo) male and female CBA/J NIA mice were subjected tolaparotomy (i.e., soft tissue trauma) and hemorrhage (35 ± 5 mmHg for90 min and fluid resuscitation) or sham operation. Twenty-four hours later, splenocyte responses were assessed in vitro. Splenic T lymphocyte responses [i.e., proliferation, interleukin-2 (IL-2) and interferon- (IFN-) release] were depressed in youngmales and enhanced in young females after trauma-hemorrhage. Incontrast, in the aged male and female groups these parameters ofsplenocyte function were reversed after trauma-hemorrhage (i.e.,increased proliferation and IL-2 release in aged males compared withsuppressed proliferation and IFN- release in aged females).Furthermore, the release of the immunosuppressive cytokine IL-10inversely correlated with the age- and gender-related changes insplenocyte responses after trauma-hemorrhage. Thus the sexuallydimorphic immune response in young males and females totrauma-hemorrhage appears to reverse as sex hormone levels decline with age.

     

Quantifying Spike Train Oscillations: Biases,Distortions and Solutions

Estimation of the power spectrum is a common method for identifying oscillatory changes in neuronal activity. However, the stochastic nature of neuronal activity leads to severe biases in the estimation of these oscillations in single unit spike trains. Different biological and experimental factors cause the spike train to differentially reflect its underlying oscillatory rate function. We analyzed the effect of factors, such as the mean firing rate and the recording duration, on the detectability of oscillations and their significance, and tested these theoretical results on experimental data recorded in Parkinsonian non-human primates. The effect of these factors is dramatic, such that in some conditions, the detection of existing oscillations is impossible. Moreover, these biases impede the comparison of oscillations across brain regions, neuronal types, behavioral states and separate recordings with different underlying parameters, and lead inevitably to a gross misinterpretation of experimental results. We introduce a novel objective measure, the "modulation index", which overcomes these biases, and enables reliable detection of oscillations from spike trains and a direct estimation of the oscillation magnitude. The modulation index detects a high percentage of oscillations over a wide range of parameters, compared to classical spectral analysis methods, and enables an unbiased comparison between spike trains recorded from different neurons and using different experimental protocols.     

Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples

Background

Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI–TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR).

Methods/Principal Findings

The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm.

Conclusions/Significance

Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production.     

TGF-β1 Induced Transdifferentiation of RPE Cells is Mediated by TAK1

Background and Aim

Proliferative vitreoretinopathy (PVR) is an active process that develops as a complication upon retinal detachment (RD), accompanied by formation of fibrotic tissue. The main cells involved in the development of fibrotic tissue during PVR are the retinal pigment epithelial (RPE) cells. The RPE cells undergo epithelial-mesenchymal transition (EMT) which leads to complex retinal detachment and loss of vision. Transforming growth factor-β1 (TGF-β1) is considered as the main player in the EMT of RPE cells, even though the mechanism is not fully understood. This study was performed to determine the possible involvement of transforming growth factor β activated kinase 1 (TAK1) in the EMT process of the RPE cells.

Methodology

ARPE-19 Cells were treated with 5Z-7 oxozeaenol (TAK1 inhibitor) or SB431542 (TGF-β1 receptor kinase inhibitor) followed by TGF-β1 stimulation. Immunofluorescence, scratch assay Real time PCR and collagen contraction assay assessed the EMT features. The phosphorylation of Smad2/3 and p38 was examined using western blots analysis.

Results

This study demonstrates that stimulation of RPE cells with TGF-β1 increases α-SMA expression, cell migration and cell contractility, all of which are EMT features. Remarkably, addition of TAK1 inhibitor abolishes all these processes. Furthermore, we show hereby that TAK1 regulates not only the activation of the non-canonical cascade of TGF-β1 (p38), but also the canonical cascade, the Smad2/3 activation. Thus, the outcome of the TGF-β response in RPE cells is TAK1 dependent.

Conclusions/Significance

This work demonstrated TAK1, a component of the non-canonical pathway of TGF-β1, is a key player in the EMT process, thus provides deep insight into the pathogenesis of PVR. The ability to halt the process of EMT in RPE cells may reduce the severity of the fibrotic response that occurs upon PVR, leading to a better prognosis and increase the probability of success in RD treatment.     

Two common nonsynonymous paraoxonase 1 (<Emphasis Type="Italic">PON1</Emphasis>) gene polymorphisms and brain astrocytoma and meningioma

Background  

Human serum paraoxonase 1 (PON1) plays a major role in the metabolism of several organophosphorus compounds. The enzyme is encoded by the polymorphic gene PON1, located on chromosome 7q21.3. Aiming to identify genetic variations related to the risk of developing brain tumors, we investigated the putative association between common nonsynonymous PON1 polymorphisms and the risk of developing astrocytoma and meningioma.     

Fighting cancer with plant-expressed pharmaceuticals

Cancer is one of the most prevalent diseases worldwide, which explains why biological therapies for cancer are forecast to make up 35% of total recombinant pharmaceuticals by 2010. Because of the high demand for cancer drugs, the need to lower production costs and the constraints of present production technologies for recombinant pharmaceuticals (such as the difficulties involved in culturing bacteria, yeast and mammalian cells), attention has recently been focused on recombinant expression of pharmaceutical anti-cancer proteins in plants. This review aims to provide an update on the most recent publications about anti-cancer recombinant pharmaceuticals expressed in plants, as well as on the relevant technical issues, potential and prospects of this emerging production system.     

SOCS-1 is a central mediator of steroid-increased thymocyte apoptosis and decreased survival following sepsis

Suppressor of Cytokine Signaling (SOCS) proteins are recently identified inhibitors/regulators of cytokine/growth factor signaling pathways. We have previously shown that SOCS-3 is upregulated in mice after sepsis induced by cecal ligation and puncture; however, the contribution of SOCS-1 to septic morbidity and mortality is unclear. In the present study, we characterized SOCS-1 expression in different tissues and delineated putative mechanisms effecting SOCS-1 expression in thymus from septic mice. We observed no difference in SOCS-1 expression in blood, peritoneal leukocytes, lung, and spleen taken from sham or septic animals at 24 h after surgery. In contrast, SOCS-1 expression in thymus declined significantly after sepsis and this down-regulation of SOCS-1 was associated with increased thymocyte apoptosis as well as augmented Bax recruitment to the mitochondria. Administration of RU-38486, a steroid receptor antagonist, reversed the above effects in the septic thymus. Furthermore, SOCS-1+/− mice showed a significant higher mortality when compared to SOCS-1+/+ mice after sepsis. Together, these results show that sepsis increases steroid-induced thymic lymphoid cell apoptosis, which is associated with reduced SOCS-1 expression and increased Bax translocation to mitochondria. Survival data suggests that SOCS-1 protein may play an important role in sepsis.