Action potential repolarization enabled by Ca<Superscript>++</Superscript> channel deactivation in PSpice simulation of smooth muscle propagation

Background  

Previously, only the rising phase of the action potential (AP) in cardiac muscle and smooth muscle could be simulated due to the instability of PSpice upon insertion of a second black box (BB) into the K⁺ leg of the basic membrane unit. This restriction was acceptable because only the transmission of excitation from one cell to the next was investigated.     

Effect of transverse gap-junction channels on transverse propagation in an enlarged PSpice model of cardiac muscle

Background  

In previous PSpice modeling studies of simulated action potentials (APs) in parallel chains of cardiac muscle, it was found that transverse propagation could occur between adjacent chains in the absence of gap-junction (gj) channels, presumably by the electric field (EF) generated in the narrow interstitial space between the chains. Transverse propagation was sometimes erratic, the more distal chains firing out of order.     

Propagated repolarization of simulated action potentials in cardiac muscle and smooth muscle

Background  

Propagation of repolarization is a phenomenon that occurs in cardiac muscle. We wanted to test whether this phenomenon would also occur in our model of simulated action potentials (APs) of cardiac muscle (CM) and smooth muscle (SM) generated with the PSpice program.     

The control of CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript>Foxp3<Superscript>+</Superscript>regulatory T cell survival

   

CD4⁺CD25⁺Foxp3⁺ regulatory T (T_reg) cells are believed to play an important role in suppressing autoimmunity and maintaining peripheral tolerance. How their survival is regulated in the periphery is less clear. Here we show that T_reg cells express receptors for gamma chain cytokines and are dependent on an exogenous supply of these cytokines to overcome cytokine withdrawal apoptosis in vitro. This result was validated in vivo by the accumulation of T_reg cells in Bim^-/- and Bcl-2 tg mice which have arrested cytokine deprivation apoptosis. We also found that CD25 and Foxp3 expression were down-regulated in the absence of these cytokines. CD25⁺ cells from Scurfy mice do not depend on cytokines for survival demonstrating that Foxp3 increases their dependence on cytokines by suppressing cytokine production in T_reg cells. Our study reveals that the survival of T_reg cells is strictly dependent on cytokines and cytokine producing cells because they do not produce cytokines. Our study thus, demonstrates that different gamma chain cytokines regulate T_reg homeostasis in the periphery by differentially regulating survival and proliferation. These findings may shed light on ways to manipulate T_reg cells that could be utilized for their therapeutic applications.     

Regulatory role of E-NTPase/E-NTPDase in Ca<Superscript>2+</Superscript>/Mg<Superscript>2+</Superscript> transport via gated channel

Background  

E-NTPase/E-NTPDase is activated by millimolar concentrations of Ca²⁺ or Mg²⁺ with a pH optimum of 7.5 for the hydrolysis of extracellular NTP and NDP. It has been generally accepted that E-NTPase/E-NTPDase plays regulatory role in purinergic signalling, but other functions may yet be discovered.     

Transverse propagation of action potentials between parallel chains of cardiac muscle and smooth muscle cells in PSpice simulations

Background  

We previously examined transverse propagation of action potentials between 2 and 3 parallel chain of cardiac muscle cells (CMC) simulated using the PSpice program. The present study was done to examine transverse propagation between 5 parallel chains in an expanded model of CMC and smooth muscle cells (SMC).     

CD23<Superscript>+</Superscript>/CD21<Superscript>hi</Superscript> B-cell translocation and ipsilateral lymph node collapse is associated with asymmetric arthritic flare in TNF-Tg mice

Introduction  

Rheumatoid arthritis (RA) is a chronic autoimmune disease with episodic flares in affected joints. However, how arthritic flare occurs only in select joints during a systemic autoimmune disease remains an enigma. To better understand these observations, we developed longitudinal imaging outcomes of synovitis and lymphatic flow in mouse models of RA, and identified that asymmetric knee flare is associated with ipsilateral popliteal lymph node (PLN) collapse and the translocation of CD23⁺/CD21^hi B-cells (B-in) into the paracortical sinus space of the node. In order to understand the relationship between this B-in translocation and lymph drainage from flaring joints, we tested the hypothesis that asymmetric tumor necrosis factor (TNF)-induced knee arthritis is associated with ipsilateral PLN and iliac lymph node (ILN) collapse, B-in translocation, and decreased afferent lymphatic flow.     

Characterization of ankylosing spondylitis and rheumatoid arthritis using <Superscript>1</Superscript>H NMR-based metabolomics of human fecal extracts

Introduction

The differences in fecal metabolome between ankylosing spondylitis (AS)/rheumatoid arthritis (RA) patients and healthy individuals could be the reason for an autoimmune disorder.

Objectives

The study explored the fecal metabolome difference between AS/RA patients and healthy controls to clarify human immune disturbance.

Methods

Fecal samples from 109 individuals (healthy controls 34, AS 40, and RA 35) were analyzed by ¹H NMR spectroscopy. Data were analyzed with principal component analysis (PCA) and orthogonal projection to latent structure discriminant (OPLS-DA) analysis.

Results

Significant differences in the fecal metabolic profiles could distinguish AS/RA patients from healthy controls but could not distinguish between AS and RA patients. The significantly decreased metabolites in AS/RA patients were butyrate, propionate, methionine, and hypoxanthine. Significantly increased metabolites in AS/RA patients were taurine, methanol, fumarate, and tryptophan.

Conclusion

The metabolome variations in feces indicated AS and RA were two homologous diseases that could not be distinguished by ¹H NMR metabolomics.

     

Strain improvement of <Emphasis Type="Italic">Trichoderma viride</Emphasis> for increased cellulase production by irradiation of electron and <Superscript>12</Superscript>C<Superscript>6+</Superscript>-ion beams

Objectives

To improve cellulase production and activity, Trichoderma viride GSICC 62010 was subjected to mutation involving irradiation with an electron beam and subsequently with a ¹²C⁶⁺-ion beam.

Results

Mutant CIT 626 was the most promising cellulase producer after preliminary and secondary screening. Soluble protein production and cellulase activities were increased mutifold. The optimum temperature, pH and culture time for the maximum cellulase production of the selected mutant were 35 °C, pH 5 and 6 days. The highest cellulase production was obtained using wheat bran. The prepared cellulases from T. viride CIT 626 had twice the hydrolytic performance with sawdust (83 %) than that from the parent strain (42.5 %). Furthermore, molecular studies demonstrated that there were some key mutation sites suggesting that some amino acid changes in the protein caused by base mutations had led to the enhanced cellulase production and activity.

Conclusions

Mutagenesis with electron and ¹²C⁶⁺-ion beams could be developed as an effective tool for improvement of cellulase producing strains.

     

Detection of hepatitis C virus (HCV) negative strand RNA and NS3 protein in peripheral blood mononuclear cells (PBMC): CD3<Superscript>+</Superscript>, CD14<Superscript>+</Superscript> and CD19<Superscript>+</Superscript>

Background

Although hepatitis C virus (HCV) is primarily hepatotropic, markers of HCV replication were detected in peripheral blood mononuclear cells (PBMC) as well as in ex vivo collected tissues and organs. Specific strains of HCV were found to be capable to infect cells of the immune system: T and B cells and monocytes/macrophages as well as cell lines in vitro. The direct invasion of cells of the immune system by the virus may be responsible for extrahepatic consequences of HCV infection: cryoglobulinemia and non-Hodgkin’s lymphoma.The aim of the present study was to determine the prevalence of markers of HCV infection: negative strand HCV RNA and non-structural NS3 protein in PBMC subpopulations: CD3⁺, CD14⁺ and CD19⁺. The presence of virus and the proportion of affected cells within a particular PBMC fraction could indicate a principal target cell susceptible for HCV.

Methods

PBMC samples were collected from 26 treatment-free patients chronically infected with HCV. PBMC subpopulations: CD3⁺, CD14⁺, CD19⁺ were obtained using positive magnetic separation. The presence of negative strand RNA HCV and viral NS3 protein were analyzed by strand-specific RT-PCR and NS3 immunocytochemistry staining.

Results

Negative strand HCV RNA was detectable in 7/26 (27%), whereas NS3 protein in 15/26 (57.6%) of PBMC samples. At least one replication marker was found in 13/26 (50%) of CD3⁺ cells then in 8/26 (30.8%) of CD14⁺ and CD19⁺ cells. The highest percentage of cells harboring viral markers in single specimen was also observed in CD3⁺ (2.4%), then in CD19⁺ (1.2%), and much lower in CD14⁺ (0.4%) cells.

Conclusions

Our results indicate that CD3⁺ cells are a dominant site for extrahepatic HCV replication, although other PBMC subpopulations may also support virus replication.

     

Measurement of the <Superscript>15</Superscript>N/<Superscript>14</Superscript>N ratio of phenylalanine in fermentation matrix by isotope ratio mass spectrometry

Objectives

To determine the origin of ¹⁵N-labeled phenylalanine in microbial metabolic flux analysis using ¹⁵N as a tracer, a method for measuring phenylalanine δ¹⁵N using HPLC coupled with elemental analysis-isotope ratio mass spectrometry (EA-IRMS) was developed.

Results

The original source of the ¹⁵N-labeled phenylalanine was determined using this new method that consists of three steps: optimization of the HPLC conditions, evaluation of the isotope fractionation effects, and evaluation of the effect of pre-processing on the phenylalanine nitrogen stable isotope. In addition, the use of a ¹⁵N-labeled inorganic nitrogen source, rather than ¹⁵N-labeled amino acids, was explored using this method.

Conclusions

The method described here can also be applied to the analysis of metabolic flux.

     

Endothelial [Ca<Superscript>2+</Superscript>]<Subscript>i</Subscript> is an integrating signal for the vascular tone in rat aortae

Background  

Although various endothelium-dependent relaxing factors (endothelial autacoids) are released upon the elevation of endothelial cytosolic free Ca²⁺ concentration (EC [Ca²⁺]_i), the quantitative relationship between EC [Ca²⁺]_i and vascular tone remains to be established. Moreover, whether the basal release of endothelial autacoids is modulated by basal EC [Ca²⁺]_i is still unclear. We assessed these issues by using a novel method that allows simultaneous recording of EC [Ca²⁺]_i and vascular displacement in dissected rat aortic segments.     

G-CSF/anti-G-CSF antibody complexes drive the potent recovery and expansion of CD11b<Superscript>+</Superscript>Gr-1<Superscript>+</Superscript> myeloid cells without compromising CD8<Superscript>+</Superscript> T cell immune responses

Background

Administration of recombinant G-CSF following cytoreductive therapy enhances the recovery of myeloid cells, minimizing the risk of opportunistic infection. Free G-CSF, however, is expensive, exhibits a short half-life, and has poor biological activity in vivo.

Methods

We evaluated whether the biological activity of G-CSF could be improved by pre-association with anti-G-CSF mAb prior to injection into mice.

Results

We find that the efficacy of G-CSF therapy can be enhanced more than 100-fold by pre-association of G-CSF with an anti-G-CSF monoclonal antibody (mAb). Compared with G-CSF alone, administration of G-CSF/anti-G-CSF mAb complexes induced the potent expansion of CD11b⁺Gr-1⁺ myeloid cells in mice with or without concomitant cytoreductive treatment including radiation or chemotherapy. Despite driving the dramatic expansion of myeloid cells, in vivo antigen-specific CD8⁺ T cell immune responses were not compromised. Furthermore, injection of G-CSF/anti-G-CSF mAb complexes heightened protective immunity to bacterial infection. As a measure of clinical value, we also found that antibody complexes improved G-CSF biological activity much more significantly than pegylation.

Conclusions

Our findings provide the first evidence that antibody cytokine complexes can effectively expand myeloid cells, and furthermore, that G-CSF/anti-G-CSF mAb complexes may provide an improved method for the administration of recombinant G-CSF.

     

FISH<Superscript>+</Superscript>CD34<Superscript>+</Superscript>CD38<Superscript>-</Superscript> cells detected in newly diagnosed acute myeloid leukemia patients can predict the clinical outcome

Background

In acute myeloid leukemia (AML), the leukemia initiating cells (LICs) or leukemia stem cells (LSCs) is found within the CD34⁺CD38^- cell compartment. The LICs subpopulation survives chemotherapy and is most probable the cause of minimal residual disease (MRD), which in turn is thought to cause relapse. The aim of this study was to determine the prognostic value of the percentage of LICs in blasts at diagnosis.

Design and methods

The percentage of LICs in the blast population was determined at diagnosis using a unique Flow-FISH analysis, which applies fluorescent in situ hybridization (FISH) analysis on flow cytometry sorted cells to distinguish LICs within the CD34⁺CD38^- cell compartment. Fourty-five AML patients with FISH-detectable cytogenetic abnormalities treated with standardized treatment program were retrospectively included in the study. Correlations with overall survival (OS), events-free survival (EFS) and cumulative incidence of relapse (CIR) were evaluated with univariate and multivariate analysis.

Results

The percentage of LICs is highly variable in patients with acute myeloid leukemia, ranged from 0.01% to 52.8% (median, 2.1%). High LIC load (≥1%) negatively affected overall survival (2-year OS: 72.57% vs. 16.75%; P?=?0.0037) and events-free survival (2-year EFS: 67.23% vs. 16.33%; P?=?0.0018), which was due to an increased cumulative incidence of relapse (2-year CIR: 56.7% vs. 18.0%; P?=?0.021). By multivariate analysis, high LIC load retained prognostic significance for OS and EFS.

Conclusions

In the present study, we established the Flow-FISH protocol as a useful method to distinguish normal and leukemic cells within the CD34⁺CD38^- cell subpopulation. The high percentage of LICs at diagnosis was significantly correlated with increased risk of poor clinical outcome.

     

[HCO<Subscript>3</Subscript><Superscript>-</Superscript>]-regulated expression and activity of soluble adenylyl cyclase in corneal endothelial and Calu-3 cells

Background  

Bicarbonate activated Soluble Adenylyl Cyclase (sAC) is a unique cytoplasmic and nuclear signaling mechanism for the generation of cAMP. HCO₃ ^- activates sAC in bovine corneal endothelial cells (BCECs), increasing [cAMP] and stimulating PKA, leading to phosphorylation of the cystic fibrosis transmembrane-conductance regulator (CFTR) and increased apical Cl^- permeability. Here, we examined whether HCO₃ ^- may also regulate the expression of sAC and thereby affect the production of cAMP upon activation by HCO₃ ^- and the stimulation of CFTR in BCECs.     

<Superscript>13</Superscript>C labeling experiments at metabolic nonstationary conditions: An exploratory study

Background  

Stimulus Response Experiments to unravel the regulatory properties of metabolic networks are becoming more and more popular. However, their ability to determine enzyme kinetic parameters has proven to be limited with the presently available data. In metabolic flux analysis, the use of ¹³C labeled substrates together with isotopomer modeling solved the problem of underdetermined networks and increased the accuracy of flux estimations significantly.     

Diel plant water use and competitive soil cation exchange interact to enhance NH<Subscript>4</Subscript><Superscript>+</Superscript> and K<Superscript>+</Superscript> availability in the rhizosphere

Aims

Hydro-biogeochemical processes in the rhizosphere regulate nutrient and water availability, and thus ecosystem productivity. We hypothesized that two such processes often neglected in rhizosphere models — diel plant water use and competitive cation exchange — could interact to enhance availability of K⁺ and NH₄ ⁺, both high-demand nutrients.

Methods

A rhizosphere model with competitive cation exchange was used to investigate how diel plant water use (i.e., daytime transpiration coupled with no nighttime water use, with nighttime root water release, and with nighttime transpiration) affects competitive ion interactions and availability of K⁺ and NH₄ ⁺.

Results

Competitive cation exchange enabled low-demand cations that accumulate against roots (Ca²⁺, Mg²⁺, Na⁺) to desorb NH₄ ⁺ and K⁺ from soil, generating non-monotonic dissolved concentration profiles (i.e. ‘hotspots’ 0.1–1 cm from the root). Cation accumulation and competitive desorption increased with net root water uptake. Daytime transpiration rate controlled diel variation in NH₄ ⁺ and K⁺ aqueous mass, nighttime water use controlled spatial locations of ‘hotspots’, and day-to-night differences in water use controlled diel differences in ‘hotspot’ concentrations.

Conclusions

Diel plant water use and competitive cation exchange enhanced NH₄ ⁺ and K⁺ availability and influenced rhizosphere concentration dynamics. Demonstrated responses have implications for understanding rhizosphere nutrient cycling and plant nutrient uptake.

     

Free Mg<Superscript>2+</Superscript> concentration in the calf muscle of glycogen phosphorylase and phosphofructokinase deficiency patients assessed in different metabolic conditions by <Superscript>31</Superscript>P MRS

Background

The increase in cytosolic free Mg²⁺ occurring during exercise and initial recovery in human skeletal muscle is matched by a decrease in cytosolic pH as shown by in vivo phosphorus magnetic resonance spectroscopy (³¹P MRS). To investigate in vivo to what extent the homeostasis of intracellular free Mg²⁺ is linked to pH in human skeletal muscle, we studied patients with metabolic myopathies due to different disorders of glycogen metabolism that share a lack of intracellular acidification during muscle exercise.

Methods

We assessed by ³¹P MRS the cytosolic pH and free magnesium concentration ([Mg²⁺]) in calf muscle during exercise and post-exercise recovery in two patients with McArdle's disease with muscle glycogen phosphorylase deficiency (McArdle), and two brothers both affected by Tarui's disease with muscle phosphofructokinase deficiency (PFK).

Results

All patients displayed a lack of intracellular acidosis during muscle exercise. At rest only one PFK patient showed a [Mg²⁺] higher than the value found in control subjects. During exercise and recovery the McArdle patients did not show any significant change in free [Mg²⁺], while both PFK patients showed decreased free [Mg²⁺] and a remarkable accumulation of phosphomonoesters (PME). During initial recovery both McArdle patients showed a small increase in free [Mg²⁺] while in PFK patients the pattern of free [Mg²⁺] was related to the rate of PME recovery.

Conclusion

i) homeostasis of free [Mg²⁺] in human skeletal muscle is strongly linked to pH as shown by patients' [Mg²⁺] pattern during exercise;ii) the pattern of [Mg²⁺] during exercise and post-exercise recovery in both PFK patients suggests that [Mg²⁺] is influenced by the accumulation of the phosphorylated monosaccharide intermediates of glycogenolysis, as shown by the increased PME peak signal.iii) ³¹P MRS is a suitable tool for the in vivo assessment of free cytosolic [Mg²⁺] in human skeletal muscle in different metabolic conditions;