Patch-clamp profile of ion channels in resting murine B lymphocytes

Summary Patch-clamp studies of single ion channel currents in freshly isolated murine B lymphocytes are characterized here according to their respective unitary conductances, ion selectivities, regulatory factors, distributions and kinetic behavior. The most prevalent ion channel in murine B lymphocytes is a large conductance (348 pS) nonselective anion channel. This report characterizes additional conductances including: two chloride channels (40 and 128 pS), a calcium-activated potassium channel (93 pS), and an outwardly rectifying potassium channel which displays two distinct conductances (18 and 30 pS). Like the anion channel, both chloride channels exhibit little activity in the cellattached patch configuration. The kinetic behavior of all of these channels is complex, with variable periods of bursting and flickering activity interspersed between prolonged closed/open intervals (dwell times). It is likely that some of these channels play an important role in the signal transduction of B cell activation.     

Selective Involvement of the Checkpoint Regulator VISTA in Suppression of B-Cell,but Not T-Cell,Responsiveness by Monocytic Myeloid-Derived Suppressor Cells from Mice Infected with an Immunodeficiency-Causing Retrovirus

Inhibition of T-cell responses in tumor microenvironments by myeloid-derived suppressor cells (MDSCs) is widely accepted. We demonstrated augmentation of monocytic MDSCs whose suppression of not only T-cell, but also B-cell, responsiveness paralleled the immunodeficiency during LP-BM5 retrovirus infection. MDSCs inhibited T cells by inducible nitric oxide synthase (iNOS)/nitric oxide (NO), but uniquely, inhibition of B cells was ∼50% dependent each on iNOS/NO and the MDSC-expressed negative-checkpoint regulator VISTA. Blockade with a combination of iNOS/NO and VISTA caused additive or synergistic abrogation of MDSC-mediated suppression of B-cell responsiveness.     

The C. elegans cGMP-Dependent Protein Kinase EGL-4 Regulates Nociceptive Behavioral Sensitivity

Signaling levels within sensory neurons must be tightly regulated to allow cells to integrate information from multiple signaling inputs and to respond to new stimuli. Herein we report a new role for the cGMP-dependent protein kinase EGL-4 in the negative regulation of G protein-coupled nociceptive chemosensory signaling. C. elegans lacking EGL-4 function are hypersensitive in their behavioral response to low concentrations of the bitter tastant quinine and exhibit an elevated calcium flux in the ASH sensory neurons in response to quinine. We provide the first direct evidence for cGMP/PKG function in ASH and propose that ODR-1, GCY-27, GCY-33 and GCY-34 act in a non-cell-autonomous manner to provide cGMP for EGL-4 function in ASH. Our data suggest that activated EGL-4 dampens quinine sensitivity via phosphorylation and activation of the regulator of G protein signaling (RGS) proteins RGS-2 and RGS-3, which in turn downregulate Gα signaling and behavioral sensitivity.     

Age and the Metabolic Syndrome as Risk Factors for Ischemic Stroke: Improving Preclinical Models of Ischemic Stroke

Ischemic stroke represents a leading cause of morbidity and mortality in the developed world. This disabling and sometimes fatal event puts an ever increasing burden on the family members and medical professionals who care for stroke victims. Preclinical ischemic stroke research has predominantly utilized young adult, healthy animals, a clear discrepancy when considering the clinical population affected by stroke. A broad spectrum of risk factors such as age, obesity, diabetes, and hypertension has been associated with an increased stroke risk. The effect of these comorbidities on both stroke pathophysiology and outcome has not been emphasized and has been recognized as a shortcoming of preclinical studies. By addressing these conditions in experimental models of ischemic stroke, it may be possible to more accurately represent the clinical scenario and improve therapeutic translation from bench-to-bedside. In this work, we review many of the risk factors associated with increased stroke risk, particularly as each risk factor relates to inflammation. Additionally, we explore potential animal models that could be utilized in identifying the contribution of these risk factors to stroke outcome. By investigating the risk factors for stroke and how these may alter stroke pathophysiology, the present discrepancies between preclinical studies and the clinical reality can be reconciled in an effort to improve therapeutic development and translation from bench-to-bedside.     

Benzothiophene Carboxylate Derivatives as Novel Allosteric Inhibitors of Branched-chain α-Ketoacid Dehydrogenase Kinase

The mitochondrial branched-chain α-ketoacid dehydrogenase complex (BCKDC) is negatively regulated by reversible phosphorylation. BCKDC kinase (BDK) inhibitors that augment BCKDC flux have been shown to reduce branched-chain amino acid (BCAA) concentrations in vivo. In the present study, we employed high-throughput screens to identify compound 3,6-dichlorobenzo[b]thiophene-2-carboxylic acid (BT2) as a novel BDK inhibitor (IC₅₀ = 3.19 μm). BT2 binds to the same site in BDK as other known allosteric BDK inhibitors, including (S)-α-cholorophenylproprionate ((S)-CPP). BT2 binding to BDK triggers helix movements in the N-terminal domain, resulting in the dissociation of BDK from the BCKDC accompanied by accelerated degradation of the released kinase in vivo. BT2 shows excellent pharmacokinetics (terminal T_½ = 730 min) and metabolic stability (no degradation in 240 min), which are significantly better than those of (S)-CPP. BT2, its analog 3-chloro-6-fluorobenzo[b]thiophene-2-carboxylic acid (BT2F), and a prodrug of BT2 (i.e. N-(4-acetamido-1,2,5-oxadiazol-3-yl)-3,6-dichlorobenzo[b]thiophene-2-carboxamide (BT3)) significantly increase residual BCKDC activity in cultured cells and primary hepatocytes from patients and a mouse model of maple syrup urine disease. Administration of BT2 at 20 mg/kg/day to wild-type mice for 1 week leads to nearly complete dephosphorylation and maximal activation of BCKDC in heart, muscle, kidneys, and liver with reduction in plasma BCAA concentrations. The availability of benzothiophene carboxylate derivatives as stable BDK inhibitors may prove useful for the treatment of metabolic disease caused by elevated BCAA concentrations.     

Proteomic analysis of malignant lymphocyte membrane microparticles using double ionization coverage optimization

Shed membrane microparticles (MPs) are microvesicles generated from the plasma membrane when cells are submitted to stress conditions. Although MPs reflect the cell state (at least in vitro), little is known on their protein composition. We describe the first set of experiments aiming to characterize the MP proteome. Two ways of triggering MP formation from a T-lymphocytic cell line were analyzed using a 1-D gel approach coupled with LC-MS/MS and the results were compared with those obtained from a classic membrane preparation. In total, 390 proteins were identified in MPs, among which 34% were localized to the plasma membrane. The MPs revealed a broad representation of plasma membrane proteins including 17 hematopoietic clusters of differentiation. This approach was successfully applied to one human chronic B-cell lymphoid malignancy. In all, 413 proteins were identified, including 117 membrane proteins, many of them being pathology associated. The sequence coverage in identified proteins was improved combining both nano-LC-MS/MS and MALDI-MS data. The suppression effect, observed on very complex peptide mixtures, was remediated by chromatographic fractionation. MPs may represent a new tool for studying plasma membrane proteins, displaying the advantages of reproducibility, minimal organelle contamination, and being potentially applicable to most cell types.     

Proton-HZE-particle sequential dual-beam exposures increase anchorage-independent growth frequencies in primary human fibroblasts

The radiation field in deep space contains high levels of high-energy protons and substantially lower levels of high-atomic-number, high-energy (HZE) particles. Calculations indicate that cellular nuclei of human space travelers will be hit during a 3-year Mars mission by approximately 400 protons and approximately 0.4 HZE particles. Thus most cells in astronauts will be hit by a proton(s) before being hit by an HZE particle. To investigate effects of dual ion irradiations on human cells, we irradiated primary human neonatal fibroblasts with protons (1 GeV/nucleon, 20 cGy) followed from 2.5 min to 48 h later by iron or titanium ions (1 GeV/nucleon, 20 cGy) and then measured clonogenic survival and frequency of anchorage-independent growth. This frequency depends on the interval between hydrogen- and iron-ion irradiation, with a critical window between 2.5 min and 1 h producing about three times more anchorage-independent colonies per survivor than expected from simple addition of the two ions separately. The hydrogen-titanium-ion dual-beam irradiation produced similar increases that persisted to approximately 6 h. At longer intervals, anchorage-independent growth frequencies were similar to those expected for additivity. However, irradiation of cells with either an iron or a titanium particle first followed by protons produced only additive levels.     

In vivo electroporation mediated gene delivery to the beating heart

Gene therapy may represent a promising alternative strategy for cardiac muscle regeneration. In vivo electroporation, a physical method of gene transfer, has recently evolved as an efficient method for gene transfer. In the current study, we investigated the efficiency and safety of a protocol involving in vivo electroporation for gene transfer to the beating heart. Adult male rats were anesthetised and the heart exposed through a left thoracotomy. Naked plasmid DNA was injected retrograde into the transiently occluded coronary sinus before the electric pulses were applied. Animals were sacrificed at specific time points and gene expression was detected. Results were compared to the group of animals where no electric pulses were applied. No post-procedure arrhythmia was observed. Left ventricular function was temporarily altered only in the group were high pulses were applied; CK-MB (Creatine kinase) and TNT (Troponin T) were also altered only in this group. Histology showed no signs of toxicity. Gene expression was highest at day one. Our results provide evidence that in vivo electroporation with an optimized protocol is a safe and effective tool for nonviral gene delivery to the beating heart. This method may be promising for clinical settings especially for perioperative gene delivery.     

Allosteric activation mechanism of the Mycobacterium tuberculosis receptor Ser/Thr protein kinase, PknB

The essential Mycobacterium tuberculosis Ser/Thr protein kinase (STPK), PknB, plays a key role in regulating growth and division, but the structural basis of activation has not been defined. Here, we provide biochemical and structural evidence that dimerization through the kinase-domain (KD) N-lobe activates PknB by an allosteric mechanism. Promoting KD pairing using a small-molecule dimerizer stimulates the unphosphorylated kinase, and substitutions that disrupt N-lobe pairing decrease phosphorylation activity in vitro and in vivo. Multiple crystal structures of two monomeric PknB KD mutants in complex with nucleotide reveal diverse inactive conformations that contain large active-site distortions that propagate > 30 ? from the mutation site. These results define flexible, inactive structures of a monomeric bacterial receptor KD and show how "back-to-back" N-lobe dimerization stabilizes the active KD conformation. This general mechanism of bacterial receptor STPK activation affords insights into the regulation of homologous eukaryotic kinases that form structurally similar dimers.