Transient Receptor Potential (TRP) and Cch1-Yam8 Channels Play Key Roles in the Regulation of Cytoplasmic Ca2+ in Fission Yeast

The regulation of cytoplasmic Ca2+ is crucial for various cellular processes. Here, we examined the cytoplasmic Ca2+ levels in living fission yeast cells by a highly sensitive bioluminescence resonance energy transfer-based assay using GFP-aequorin fusion protein linked by 19 amino acid. We monitored the cytoplasmic Ca2+ level and its change caused by extracellular stimulants such as CaCl2 or NaCl plus FK506 (calcineurin inhibitor). We found that the extracellularly added Ca2+ caused a dose-dependent increase in the cytoplasmic Ca2+ level and resulted in a burst-like peak. The overexpression of two transient receptor potential (TRP) channel homologues, Trp1322 or Pkd2, markedly enhanced this response. Interestingly, the burst-like peak upon TRP overexpression was completely abolished by gene deletion of calcineurin and was dramatically decreased by gene deletion of Prz1, a downstream transcription factor activated by calcineurin. Furthermore, 1 hour treatment with FK506 failed to suppress the burst-like peak. These results suggest that the burst-like Ca2+ peak is dependent on the transcriptional activity of Prz1, but not on the direct TRP dephosphorylation. We also found that extracellularly added NaCl plus FK506 caused a synergistic cytosolic Ca2+ increase that is dependent on the inhibition of calcineurin activity, but not on the inhibition of Prz1. The synergistic Ca2+ increase is abolished by the addition of the Ca2+ chelator BAPTA into the media, and is also abolished by deletion of the gene encoding a subunit of the Cch1-Yam8 Ca2+ channel complex, indicating that the synergistic increase is caused by the Ca2+ influx from the extracellular medium via the Cch1-Yam8 complex. Furthermore, deletion of Pmk1 MAPK abolished the Ca2+ influx, and overexpression of the constitutively active Pek1 MAPKK enhanced the influx. These results suggest that Pmk1 MAPK and calcineurin positively and negatively regulate the Cch1-Yam8 complex, respectively, via modulating the balance between phosphorylation and dyphosphorylation state

Role of the RNA-Binding Protein Nrd1 in Stress Granule Formation and Its Implication in the Stress Response in Fission Yeast

We have previously identified the RNA recognition motif (RRM)-type RNA-binding protein Nrd1 as an important regulator of the posttranscriptional expression of myosin in fission yeast. Pmk1 MAPK-dependent phosphorylation negatively regulates the RNA-binding activity of Nrd1. Here, we report the role of Nrd1 in stress-induced RNA granules. Nrd1 can localize to poly(A)-binding protein (Pabp)-positive RNA granules in response to various stress stimuli, including heat shock, arsenite treatment, and oxidative stress. Interestingly, compared with the unphosphorylatable Nrd1, Nrd1DD (phosphorylation-mimic version of Nrd1) translocates more quickly from the cytoplasm to the stress granules in response to various stimuli; this suggests that the phosphorylation of Nrd1 by MAPK enhances its localization to stress-induced cytoplasmic granules. Nrd1 binds to Cpc2 (fission yeast RACK) in a phosphorylation-dependent manner and deletion of Cpc2 affects the formation of Nrd1-positive granules upon arsenite treatment. Moreover, the depletion of Nrd1 leads to a delay in Pabp-positive RNA granule formation, and overexpression of Nrd1 results in an increased size and number of Pabp-positive granules. Interestingly, Nrd1 deletion induced resistance to sustained stresses and enhanced sensitivity to transient stresses. In conclusion, our results indicate that Nrd1 plays a role in stress-induced granule formation, which affects stress resistance in fission yeast

Increased ion temperature and neutron yield observed in magnetized indirectly driven D_{2}-filled capsule implosions on the national ignition facility

The application of an external 26 Tesla axial magnetic field to a D_{2} gas-filled capsule indirectly driven on the National Ignition Facility is observed to increase the ion temperature by 40% and the neutron yield by a factor of 3.2 in a hot spot with areal density and temperature approaching what is required for fusion ignition [1]. The improvements are determined from energy spectral measurements of the 2.45 MeV neutrons from the D(d,n)^{3}He reaction, and the compressed central core B field is estimated to be ∼4.9  kT using the 14.1 MeV secondary neutrons from the D(T,n)^{4}He reactions. The experiments use a 30 kV pulsed-power system to deliver a ∼3  μs current pulse to a solenoidal coil wrapped around a novel high-electrical-resistivity AuTa_{4} hohlraum. Radiation magnetohydrodynamic simulations are consistent with the experiment

Towards a Clinically Relevant Lentiviral Transduction Protocol for Primary Human CD34+ Hematopoietic Stem/Progenitor Cells

Background: Hematopoietic stem cells (HSC), in particular mobilized peripheral blood stem cells, represent an attractive target for cell and gene therapy. Efficient gene delivery into these target cells without compromising self-renewal and multipotency is crucial for the success of gene therapy. We investigated factors involved in the ex vivo transduction of CD34 + HSCs in order to develop a clinically relevant transduction protocol for gene delivery. Specifically sought was a protocol that allows for efficient transduction with minimal ex vivo manipulation without serum or other reagents of animal origin. Methodology/Principal Findings: Using commercially available G-CSF mobilized peripheral blood (PB) CD34 + cells as the most clinically relevant target, we systematically examined factors including the use of serum, cytokine combinations, prestimulation time, multiplicity of infection (MOI), transduction duration and the use of spinoculation and/or retronectin. A self-inactivating lentiviral vector (SIN-LV) carrying enhanced green fluorescent protein (GFP) was used as the gene delivery vehicle. HSCs were monitored for transduction efficiency, surface marker expression and cellular function. We were able to demonstrate that efficient gene transduction can be achieved with minimal ex vivo manipulation while maintaining the cellular function of transduced HSCs without serum or other reagents of animal origin. Conclusions/Significance: This study helps to better define factors relevant towards developing a standard clinical protocol for the delivery of SIN-LV into CD34 + cells

Role of Hydrogen Sulfide in Severe Burn Injury–Induced Inflammation in Mice

Endogenous hydrogen sulfide (H2S) is naturally synthesized in many types of mammalian cells from L-cysteine in the reactions catalyzed by cystathionine-β-synthase and cystathionine-γ-lyase (CSE). H2S has been demonstrated to play a proinflammatory role in various animal models of hindpaw edema, acute pancreatitis, lipopolysaccharide-induced endotoxemia and cecal ligation, and puncture–induced sepsis. Full-thickness burns that exceed 25% of the total body surface area (TBSA) produce a profound systemic inflammatory reaction characterized by leukocyte activation and plasma leakage in the microvasculature of tissues and organs remote from the wound. The aim of this study was to investigate the effect of local burn injury on induced distant organ endogenous H2S release and expression of CSE. Male BALB/c mice were subjected to 30% TBSA full-thickness burn and treated with saline (administered intraperitoneally [i.p.]); DL-propargylglycine (PAG, 50 mg/kg i.p.), which is a CSE inhibitor; or sodium hydrosulfide (NaHS, 10 mg/kg i.p.), which is an H2S donor. PAG was administered either 1 h before or 1 h after the burn injury, whereas NaHS was given at the same time as the burn injury. Measurements of liver myeloperoxidase (MPO) activities, liver H2S-synthesizing activity, plasma H2S level and liver and lung CSE mRNA expression and histological examination of tissues were performed after burn injury. Burn injury significantly increased the plasma H2S level and liver H2S synthesis 8 h after burn compared with the sham group. Burn injury also resulted in a significant upregulation of CSE mRNA in liver and lung. Prophylactic as well as therapeutic administration of PAG significantly reduced burn-associated systemic inflammation, as evidenced by MPO activity and histological changes in liver and lung. Injection of NaHS significantly aggravated burn-associated systemic inflammation. Therefore, our findings show for the first time the role of H2S in contributing to inflammatory damage after burn injury