Calpain Inhibition Reduces Amplitude and Accelerates Decay of the Late Sodium Current in Ventricular Myocytes from Dogs with Chronic Heart Failure

Calpain is an intracellular Ca²⁺ -activated protease that is involved in numerous Ca²⁺ dependent regulation of protein function in many cell types. This paper tests a hypothesis that calpains are involved in Ca²⁺ -dependent increase of the late sodium current (I_NaL) in failing heart. Chronic heart failure (HF) was induced in 2 dogs by multiple coronary artery embolization. Using a conventional patch-clamp technique, the whole-cell I_NaL was recorded in enzymatically isolated ventricular cardiomyocytes (VCMs) in which I_NaL was activated by the presence of a higher (1μM) intracellular [Ca²⁺] in the patch pipette. Cell suspensions were exposed to a cell- permeant calpain inhibitor MDL-28170 for 1–2 h before I_NaL recordings. The numerical excitation-contraction coupling (ECC) model was used to evaluate electrophysiological effects of calpain inhibition in silico. MDL caused acceleration of I_NaL decay evaluated by the two-exponential fit (τ₁ = 42±3.0 ms τ₂ = 435±27 ms, n = 6, in MDL vs. τ₁ = 52±2.1 ms τ₂ = 605±26 control no vehicle, n = 11, and vs. τ₁ = 52±2.8 ms τ₂ = 583±37 ms n = 7, control with vehicle, P<0.05 ANOVA). MDL significantly reduced I_NaL density recorded at –30 mV (0.488±0.03, n = 12, in control no vehicle, 0.4502±0.0210, n = 9 in vehicle vs. 0.166±0.05pA/pF, n = 5, in MDL). Our measurements of current-voltage relationships demonstrated that the I_NaL density was decreased by MDL in a wide range of potentials, including that for the action potential plateau. At the same time the membrane potential dependency of the steady-state activation and inactivation remained unchanged in the MDL-treated VCMs. Our ECC model predicted that calpain inhibition greatly improves myocyte function by reducing the action potential duration and intracellular diastolic Ca²⁺ accumulation in the pulse train.

Conclusions

Calpain inhibition reverses I_NaL changes in failing dog ventricular cardiomyocytes in the presence of high intracellular Ca²⁺. Specifically it decreases I_NaL density and accelerates I_NaL kinetics resulting in improvement of myocyte electrical response and Ca²⁺ handling as predicted by our in silico simulations.     

Effect of Recombinant Human Thyrotropin on the Uptake of Radioactive Iodine (123I) in Dogs with Thyroid Tumors

In humans, recombinant human thyrotropin (rhTSH) enhances radioactive iodine uptake (RAIU) in patients with differentiated thyroid cancer. No studies have been performed in veterinary medicine to optimize radioiodine treatment of thyroid cancer. The aim of this study was to evaluate the effect of rhTSH on the uptake of radioiodine-123 (¹²³I) in dogs with thyroid tumors. Nine dogs with thyroid neoplasia were included in this prospective cross-over study. The dogs were divided in 2 groups. In one group, ¹²³I was administered for a baseline RAIU determination in week 1. In week 2 (after a washout period of 2 weeks), these dogs received rhTSH (100 μg IV) 24 h before ¹²³I injection. In the other group the order of the protocol was reversed. For each scan, the dogs received 37 MBq (1 mCi) of ¹²³I intravenously (IV) and planar scintigraphy was performed after 8 and 24 h for tumor RAIU calculation. Overall, rhTSH administration caused no statistically significant change on thyroid tumor RAIU at 8 h (p = 0.89) or at 24 h (p = 0.98). A significant positive correlation was found between the effect of rhTSH on tumor 8h-RAIU and rhTSH serum concentrations at 6 h (τ = 0.68; p = 0.03), at 12 h (τ = 0.68; p = 0.03) and at 24 h (τ = 0.78; p = 0.02) after rhTSH injection. This study suggests that IV administration of 100 μg rhTSH 24 h before ¹²³I has an inconsistent effect on thyroid tumor RAIU. Further studies are necessary to determine the best protocol of rhTSH administration to optimize thyroid tumor RAIU.     

Decreased NK Cell FcRγ in HIV-1 Infected Individuals Receiving Combination Antiretroviral Therapy: a Cross Sectional Study

Background

FcRγ is an immunoreceptor tyrosine-based activation motif (ITAM)-signalling protein essential for immunoreceptor signaling and monocyte, macrophage and NK cell function. Previous study from our laboratory showed that FcRγ is down-regulated in HIV-infected macrophages in vitro. FcRγ expression in immune cells present in HIV-infected individuals is unknown.

Methodology/Principal Findings

We compared FcRγ expression in peripheral blood mononuclear cells isolated from HIV-1-infected individuals receiving combination antiretroviral therapy and healthy, HIV-1-uninfected individuals. FcRγ mRNA and protein levels were measured using quantitative real-time PCR and immunoblotting, respectively. CD56⁺ CD94⁺ lymphocytes isolated from blood of HIV-1 infected individuals had reduced FcRγ protein expression compared to HIV-uninfected individuals (decrease = 76.8%, n = 18 and n = 12 respectively, p = 0.0036). In a second group of patients, highly purified NK cells had reduced FcRγ protein expression compared to uninfected controls (decrease = 50.2%, n = 9 and n = 8 respectively, p = 0.021). Decreased FcRγ expression in CD56+CD94+ lymphocytes was associated with reduced mRNA (51.7%, p = 0.021) but this was not observed for the smaller group of patients analysed for NK cell expression (p = 0.36).

Conclusion/Significance

These data suggest biochemical defects in ITAM-dependent signalling within NK cells in HIV-infected individuals which is present in the context of treatment with combination antiretroviral therapy.     

Steady-State Growth and Chemical Composition of the Marine Chlorophyte Dunaliella tertiolecta in Nitrogen-Limited Continuous Cultures

The marine chlorophyte Dunaliella tertiolecta was grown in continuous cultures under NH₄⁺-N, NO₂⁻-N, NO₃⁻-N, and urea-N limitations. The effect of the nitrogen cell quota (Q_n) on the steady-state growth rate (μ) was the same regardless of the N source. The relationship between μ and Q_n was well described by the Droop equation, but only up to the true maximum growth rate ^μ (= cell washout rate). The ratio between the minimum cell quota (k_Q) and the maximum cell quota (Q_m) was 0.19. Hence, there is no substitute for determining ^μ experimentally. That there was no difference in growth response to different N sources suggests that no internal pooling of inorganic nitrogen occurred. Both the carbon (Q_c) and phosphorus (Q_p) cell quotas under N limitation increased with increasing μ in a threshold fashion: virtually no change in either cell quota up to ~0.8 ^μ, followed by a rapid and large increase up to ^μ. In addition, in the region of low μ, there was an increase in Q_p with a decreasing medium N/P ratio of between 15 and 5 (by atoms). The results generally indicate the physiological limits in cellular constituency under N limitation. The usefulness of this information, however, in describing the response of natural populations of marine phytoplankton to transient nutrient exposures on the temporal and spatial microscales that most likely exist is of limited value.     

Compressive Viscoelasticity of Freshly Excised Mouse Skin Is Dependent on Specimen Thickness,Strain Level and Rate

Although the skin’s mechanical properties are well characterized in tension, little work has been done in compression. Here, the viscoelastic properties of a population of mouse skin specimens (139 samples from 36 mice, aged 5 to 34 weeks) were characterized upon varying specimen thickness, as well as strain level and rate. Over the population, we observed the skin’s viscoelasticity to be quite variable, yet found systematic correlation of residual stress ratio with skin thickness and strain, and of relaxation time constants with strain rates. In particular, as specimen thickness ranged from 211 to 671 μm, we observed significant variation in both quasi-linear viscoelasticity (QLV) parameters, the relaxation time constant (τ₁ = 0.19 ± 0.10 s) and steady-state residual stress ratio (G_∞ = 0.28 ± 0.13). Moreover, when τ₁ was decoupled and fixed, we observed that G_∞ positively correlated with skin thickness. Second, as steady-state stretch was increased (λ_∞ from 0.22 to 0.81), we observed significant variation in both QLV parameters (τ₁ = 0.26 ± 0.14 s, G_∞ = 0.47 ± 0.17), and when τ₁ was fixed, G_∞ positively correlated with stretch level. Third, as strain rate was increased from 0.06 to 22.88 s⁻¹, the median time constant τ₁ varied from 1.90 to 0.31 s, and thereby negatively correlated with strain rate. These findings indicate that the natural range of specimen thickness, as well as experimental controls of compression level and rate, significantly influence measurements of skin viscoelasticity.     

Epigenetic Changes in Basal Cell Carcinoma Affect SHH and WNT Signaling Components

Background

The genetic background of Basal Cell Carcinoma (BCC) has been studied extensively, while its epigenetic makeup has received comparatively little attention. Epigenetic alterations such as promoter hypermethylation silence tumor suppressor genes (TSG) in several malignancies.

Objective

We sought to analyze the promoter methylation status of ten putative (tumor suppressor) genes that are associated with Sonic Hedgehog (SHH), WNT signaling and (hair follicle) tumors in a large series of 112 BCC and 124 healthy control samples by methylation-specific PCR.

Results

Gene promoters of SHH (P = 0.016), adenomatous polyposis coli (APC) (P = 0.003), secreted frizzled-related protein 5 (SFRP5) (P = 0.004) and Ras association domain family 1A (RASSF1A) (P = 0.023) showed significantly more methylation in BCC versus normal skin. mRNA levels of these four genes were reduced for APC and SFRP5 in BCC (n = 6) vs normal skin (n = 6). Down regulation of SHH, APC and RASSF1A could be confirmed on protein level as well (P<0.001 for all genes) by immunohistochemical staining. Increased canonical WNT activity was visualized by β-catenin staining, showing nuclear β-catenin in only 28/101 (27.7%) of BCC. Absence of nuclear β-catenin in some samples may be due to high levels of membranous E-cadherin (in 94.1% of the samples).

Conclusions

We provide evidence that promoter hypermethylation of key players within the SHH and WNT pathways is frequent in BCC, consistent with their known constitutive activation in BCC. Epigenetic gene silencing putatively contributes to BCC tumorigenesis, indicating new venues for treatment.     

p38δ Regulates p53 to Control p21Cip1 Expression in Human Epidermal Keratinocytes

PKCδ suppresses keratinocyte proliferation via a mechanism that involves increased expression of p21^Cip1. However, the signaling mechanism that mediates this regulation is not well understood. Our present studies suggest that PKCδ activates p38δ leading to increased p21^Cip1 promoter activity and p21^Cip1 mRNA/protein expression. We further show that exogenously expressed p38δ increases p21^Cip1 mRNA and protein and that p38δ knockdown or expression of dominant-negative p38 attenuates this increase. Moreover, p53 is an intermediary in this regulation, as p38δ expression increases p53 mRNA, protein, and promoter activity, and p53 knockdown attenuates the activation. We demonstrate a direct interaction of p38δ with PKCδ and MEK3 and show that exogenous agents that suppress keratinocyte proliferation activate this pathway. We confirm the importance of this regulation using a stratified epidermal equivalent model, which mimics in vivo-like keratinocyte differentiation. In this model, PKCδ or p38δ knockdown results in reduced p53 and p21^Cip1 levels and enhanced cell proliferation. We propose that PKCδ activates a MEKK1/MEK3/p38δ MAPK cascade to increase p53 levels and p53 drives p21^Cip1 gene expression.     

Increases in [3H]Muscimol and [3H]Flumazenil Binding in the Dorsolateral Prefrontal Cortex in Schizophrenia Are Linked to α4 and γ2S mRNA Levels Respectively

Background

GABA_A receptors (GABA_AR) are composed of several subunits that determine sensitivity to drugs, synaptic localisation and function. Recent studies suggest that agonists targeting selective GABA_AR subunits may have therapeutic value against the cognitive impairments observed in schizophrenia. In this study, we determined whether GABA_AR binding deficits exist in the dorsolateral prefrontal cortex (DLPFC) of people with schizophrenia and tested if changes in GABA_AR binding are related to the changes in subunit mRNAs. The GABA orthosteric and the benzodiazepine allosteric binding sites were assessed autoradiographically using [³H]Muscimol and [³H]Flumazenil, respectively, in a large cohort of individuals with schizophrenia (n = 37) and their matched controls (n = 37). We measured, using qPCR, mRNA of β (β1, β2, β3), γ (γ1, γ2, γ2S for short and γ2L for long isoform, γ3) and δ subunits and used our previous measurements of GABA_AR α subunit mRNAs in order to relate mRNAs and binding through correlation and regression analysis.

Results

Significant increases in both [³H]Muscimol (p = 0.016) and [³H]Flumazenil (p = 0.012) binding were found in the DLPFC of schizophrenia patients. Expression levels of mRNA subunits measured did not show any significant difference in schizophrenia compared to controls. Regression analysis revealed that in schizophrenia, the [³H]Muscimol binding variance was most related to α4 mRNA levels and the [³H]Flumazenil binding variance was most related to γ2S subunit mRNA levels. [³H]Muscimol and [³H]Flumazenil binding were not affected by the lifetime anti-psychotics dose (chlorpromazine equivalent).

Conclusions

We report parallel increases in orthosteric and allosteric GABA_AR binding sites in the DLPFC in schizophrenia that may be related to a “shift” in subunit composition towards α4 and γ2S respectively, which may compromise normal GABAergic modulation and function. Our results may have implications for the development of treatment strategies that target specific GABA_AR receptor subunits.     

Effects of Viscosity and Constraints on the Dispersion and Dissipation of Waves in Large Blood Vessels: II. Comparison of Analysis with Experiments

The theoretical predictions described in part I of this study are compared with in vivo data from anesthetized dogs. It is shown that the observed attenuation of the pressure and axial waves cannot be accounted for by fluid viscosity alone. For large values of the frequency parameter α = √ρωR²₀/μ, the analysis of part I is extended to include the effects of viscoelasticity of the vessel wall. The results indicate that the speeds of both types of waves are essentially not affected by a realistic viscoelasticity model while the attenuation per wavelength is significantly increased and becomes frequency independent. The application of this analysis to in vivo data from the carotid arteries of anesthetized dogs demonstrates partial agreement between theory and experiment and suggests that the carotid arteries are anisotropically viscoelastic.     

Immune-Correlates Analysis of an HIV-1 Vaccine Efficacy Trial Reveals an Association of Nonspecific Interferon-γ Secretion with Increased HIV-1 Infection Risk: A Cohort-Based Modeling Study

Background

Elevated risk of HIV-1 infection among recipients of an adenovirus serotype 5 (Ad5)-vectored HIV-1 vaccine was previously reported in the Step HIV-1 vaccine efficacy trial. We assessed pre-infection cellular immune responses measured at 4 weeks after the second vaccination to determine their roles in HIV-1 infection susceptibility among Step study male participants.

Methods

We examined ex vivo interferon-γ (IFN-γ) secretion from peripheral blood mononuclear cells (PBMC) using an ELISpot assay in 112 HIV-infected and 962 uninfected participants. In addition, we performed flow cytometric assays to examine T-cell activation, and ex vivo IFN-γ and interleukin-2 secretion from CD4⁺ and CD8⁺ T cells. We accounted for the sub-sampling design in Cox proportional hazards models to estimate hazard ratios (HRs) of HIV-1 infection per 1-log_e increase of the immune responses.

Findings

We found that HIV-specific immune responses were not associated with risk of HIV-1 infection. However, each 1-log_e increase of mock responses measured by the ELISpot assay (i.e., IFN-γ secretion in the absence of antigen-specific stimulation) was associated with a 62% increase of HIV-1 infection risk among vaccine recipients (HR = 1.62, 95% CI: (1.28, 2.04), p<0.001). This association remains after accounting for CD4⁺ or CD8⁺ T-cell activation. We observed a moderate correlation between ELISpot mock responses and CD4⁺ T-cells secreting IFN-γ (ρ = 0.33, p = 0.007). In addition, the effect of the Step vaccine on infection risk appeared to vary with ELISpot mock response levels, especially among participants who had pre-existing anti-Ad5 antibodies (interaction p = 0.04).

Conclusions

The proportion of cells, likely CD4⁺ T-cells, producing IFN-γ without stimulation by exogenous antigen appears to carry information beyond T-cell activation and baseline characteristics that predict risk of HIV-1 infection. These results motivate additional investigation to understand the potential link between IFN-γ secretion and underlying causes of elevated HIV-1 infection risk among vaccine recipients in the Step study.     

Evaluation of the Oscillatory Interference Model of Grid Cell Firing through Analysis and Measured Period Variance of Some Biological Oscillators

Models of the hexagonally arrayed spatial activity pattern of grid cell firing in the literature generally fall into two main categories: continuous attractor models or oscillatory interference models. Burak and Fiete (2009, PLoS Comput Biol) recently examined noise in two continuous attractor models, but did not consider oscillatory interference models in detail. Here we analyze an oscillatory interference model to examine the effects of noise on its stability and spatial firing properties. We show analytically that the square of the drift in encoded position due to noise is proportional to time and inversely proportional to the number of oscillators. We also show there is a relatively fixed breakdown point, independent of many parameters of the model, past which noise overwhelms the spatial signal. Based on this result, we show that a pair of oscillators are expected to maintain a stable grid for approximately t = 5µ ³ /(4πσ) ² seconds where µ is the mean period of an oscillator in seconds and σ² its variance in seconds². We apply this criterion to recordings of individual persistent spiking neurons in postsubiculum (dorsal presubiculum) and layers III and V of entorhinal cortex, to subthreshold membrane potential oscillation recordings in layer II stellate cells of medial entorhinal cortex and to values from the literature regarding medial septum theta bursting cells. All oscillators examined have expected stability times far below those seen in experimental recordings of grid cells, suggesting the examined biological oscillators are unfit as a substrate for current implementations of oscillatory interference models. However, oscillatory interference models can tolerate small amounts of noise, suggesting the utility of circuit level effects which might reduce oscillator variability. Further implications for grid cell models are discussed.     

Systemic Simvastatin Rescues Retinal Ganglion Cells from Optic Nerve Injury Possibly through Suppression of Astroglial NF-κB Activation

Neuroinflammation is involved in the death of retinal ganglion cells (RGCs) after optic nerve injury. The purpose of this study was to determine whether systemic simvastatin can suppress neuroinflammation in the optic nerve and rescue RGCs after the optic nerve is crushed. Simvastatin or its vehicle was given through an osmotic minipump beginning one week prior to the crushing. Immunohistochemistry and real-time PCR were used to determine the degree of neuroinflammation on day 3 after the crushing. The density of RGCs was determined in Tuj-1 stained retinal flat mounts on day 7. The effect of simvastain on the TNF-α-induced NF-κB activation was determined in cultured optic nerve astrocytes. On day 3, CD68-positive cells, most likely microglia/macrophages, were accumulated at the crushed site. Phosphorylated NF-κB was detected in some astrocytes at the border of the lesion where the immunoreactivity to MCP-1 was intensified. There was an increase in the mRNA levels of the CD68 (11.4-fold), MCP-1 (22.6-fold), ET-1 (2.3-fold), GFAP (1.6-fold), TNF-α (7.0-fold), and iNOS (14.8-fold) genes on day 3. Systemic simvastatin significantly reduced these changes. The mean ± SD number of RGCs was 1816.3±232.6/mm² (n = 6) in the sham controls which was significantly reduced to 831.4±202.5/mm² (n = 9) on day 7 after the optic nerve was crushed. This reduction was significantly suppressed to 1169.2±201.3/mm² (P = 0.01, Scheffe; n = 9) after systemic simvastatin. Simvastatin (1.0 µM) significantly reduced the TNF-α-induced NF-κB activation in cultured optic nerve astrocytes. We conclude that systemic simvastatin can reduce the death of RGCs induced by crushing the optic nerve possibly by suppressing astroglial NF-κB activation.     

Timing and Intensity of Light Correlate with Body Weight in Adults

Light exposure can influence sleep and circadian timing, both of which have been shown to influence weight regulation. The goal of this study was to evaluate the relationship between ambient light, sleep and body mass index. Participants included 54 individuals (26 males, mean age 30.6, SD = 11.7 years). Light levels, sleep midpoint and duration were measured with wrist actigraphy (Actiwatch-L) for 7 days. BMI was derived from self-reported height and weight. Caloric intake was determined from 7 days of food logs. For each participant, light and activity data were output in 2 minute epochs, smoothed using a 5 point (10 minute) moving average and then aggregated over 24 hours. The mean light timing above 500 lux (MLiT⁵⁰⁰) was defined as the average clock time of all aggregated data points above 500 lux. MLiT⁵⁰⁰ was positively correlated with BMI (r = 0.51, p<0.001), and midpoint of sleep (r = 0.47, p<0.01). In a multivariable linear regression model including MLiT⁵⁰⁰ and midpoint of sleep, MLiT⁵⁰⁰ was a significant predictor of BMI (B = 1.26 SE = 0.34, β = 0.53 p = 0.001, r ² _Δ = 0.22). Adjusting for covariates, MLiT⁵⁰⁰ remained an independent predictor of BMI (B = 1.28 SE = 0.36, β = 0.54, p = 0.002, r ² _Δ = 0.20). The full model accounted for 34.7% of the variance in BMI (p = 0.01). Exposure to moderate levels of light at biologically appropriate times can influence weight, independent of sleep timing and duration.     

Blood Levels of S-100 Calcium-Binding Protein B,High-Sensitivity C-Reactive Protein,and Interleukin-6 for Changes in Depressive Symptom Severity after Coronary Artery Bypass Grafting: Prospective Cohort Nested within a Randomized,Controlled Trial

Background

Cross-sectional and retrospective studies have associated major depressive disorder with glial activation and injury as well as blood–brain barrier disruption, but these associations have not been assessed prospectively. Here, we aimed to determine the relationship between changes in depressive symptom severity and in blood levels of S-100 calcium-binding protein B (S-100B), high-sensitivity C-reactive protein, and interleukin-6 following an inflammatory challenge.

Methods

Fifty unselected participants were recruited from a randomized, controlled trial comparing coronary artery bypass grafting procedures performed with versus without cardiopulmonary bypass for the risk of neurocognitive decline. Depressive symptom severity was measured at baseline, discharge, and six-month follow-up using the Beck Depression Inventory II (BDI-II). The primary outcome of the present biomarker study was acute change in depressive symptom severity, defined as the intra-subject difference between baseline and discharge BDI-II scores. Blood biomarker levels were determined at baseline and 2 days postoperative.

Results

Changes in S-100B levels correlated positively with acute changes in depressive symptom severity (Spearman ρ, 0.62; P = 0.0004) and accounted for about one-fourth of their observed variance (R², 0.23; P = 0.0105). This association remained statistically significant after adjusting for baseline S-100B levels, age, weight, body-mass index, or β-blocker use, but not baseline BDI-II scores (P = 0.064). There was no statistically significant association between the primary outcome and baseline S-100B levels, baseline high-sensitivity C-reactive protein or interleukin-6 levels, or changes in high-sensitivity C-reactive protein or interleukin-6 levels. Among most participants, levels of all three biomarkers were normal at baseline and markedly elevated at 2 days postoperative.

Conclusions

Acute changes in depressive symptom severity were specifically associated with incremental changes in S-100B blood levels, largely independent of covariates associated with either. These findings support the hypothesis that glial activation and injury and blood–brain barrier disruption can be mechanistically linked to acute exacerbation of depressive symptoms in some individuals.     

Extracellular Matrix Biomarker,Fibulin-1, Is Closely Related to NT-proBNP and Soluble Urokinase Plasminogen Activator Receptor in Patients with Aortic Valve Stenosis (The SEAS Study)

Background

Fibulin-1, a circulating extracellular matrix glycoprotein, has been associated with arterial disease and elevated N-terminal prohormone B-type natriuretic peptide (NT-proBNP) in diabetes. Soluble urokinase plasminogen activator receptor (suPAR), a marker of inflammation, has been associated with subclinical atherosclerosis. Therefore, we aimed to explore the interplay between these biomarkers and mild to moderate aortic valve stenosis (AS).

Methods

In 374 patients with mild to moderate AS, we investigated the relationship of fibulin-1 with NT-proBNP, levels of suPAR and the degree of AS at baseline and after one and four years of treatment with Simvastatin 40 mg and Ezetimibe 10 mg or placebo.

Results

During treatment, fibulin-1 became more closely associated with NT-proBNP (β_year0 = 0.10, p = 0.08, β_year1 = 0.16, p = 0.005, β_year4 = 0.22, p<0.001) and suPAR (β_year0 = 0.05, p = 0.34, β_year1 = 0.16, p = 0.006, β_year4 = 0.13, p = 0.03) at the expense of the association to aortic valve area index (AVAI) (β_year0 = −0.14, p = 0.005, β_year1 = −0.08, p = 0.11, β_year4 = −0.06, p = 0.22) independently of age, gender, creatinine, and serum aspartate aminotransferase (Adj.R_year0 ² = 0.19, Adj.R_year1 ² = 0.22, Adj.R_year4 ² = 0.27). Fibulin-1 was unrelated to aortic regurgitation, left ventricular mass, and ejection fraction. In patients with baseline AVAI<0.58 cm²/m² (median value), fibulin-1 was more closely associated to NT-proBNP (β_year0 = 0.25, β_year1 = 0.21, β_year4 = 0.22, all p<0.01), and suPAR (β_year0 = 0.09, p = 0.26, β_year1 = 0.23, β_year4 = 0.21, both p<0.01) independently of age, gender, AST and treatment allocation.

Conclusions

Increased levels of fibulin-1 were independently associated with higher levels of suPAR and NT-proBNP especially in patients with lower AVAI, suggesting that fibulin-1 may be an early marker of AS as well as cardiac fibrosis secondarily to elevated left ventricular hemodynamic load.     

Interleukin-1β-31C/T and -511T/C Polymorphisms Were Associated with Preeclampsia in Chinese Han Population

Objective

The purpose of our study is to investigate the relationship between IL-1β -31C/T (rs1143627) and -511T/C (rs16944) polymorphisms and the preeclampsia (PE), and analyze the Linkage disequilibrium (LD) and haplotype frequency of the two polymorphism loci.

Methods

Polymorphisms at -31C/T and -511T/C of IL-1β were genotyped with the method of polymerase chain reaction-restriction fragment length polymorphism (PCR- RFLP) in 232 PE and 447 control subjects. Genotype and allele frequencies between case-control groups were compared by chi-square(X²) tests. Two-point LD and haplotype frequency analyses were done with the software Haploview4.2.

Results

Significant statistical differences were found between PE and control groups regarding genotype and allele frequencies of the two polymorphisms of IL-1β (For IL-1β -31C/T: X² = 11.478, P = 0.003; For IL-1β-511T/C: X² = 9.687, P = 0.008). LD analysis revealed that the IL-1β -31C/T SNP was in high LD with the IL-1β-511C/T SNP(D′ = 0.92, r² = 0.79). Both CT and TC haplotypes showed significant differences between case and control groups. Only the plasma level of Prothrombin Time had a significantly statistical difference among TT, CT and CC groups of the preeclamptic two polymorphisms of IL-1β-31C/T and -511T/C (for IL-1β-31C/T, F = 1.644, P = 0.01; F = 1.587, P = 0.016).

Conclusion

Our results revealed IL-1β was associated with the PE in Chinese Han population. The CT haplotype may increase the risk of PE, while haplotype TC could be considered as a protective haplotype of PE.     

Cellular Microcystin Content in N-Limited Microcystis aeruginosa Can Be Predicted from Growth Rate

Cell quotas of microcystin (Q_MCYST; femtomoles of MCYST per cell), protein, and chlorophyll a (Chl a), cell dry weight, and cell volume were measured over a range of growth rates in N-limited chemostat cultures of the toxic cyanobacterium Microcystis aeruginosa MASH 01-A19. There was a positive linear relationship between Q_MCYST and specific growth rate (μ), from which we propose a generalized model that enables Q_MCYST at any nutrient-limited growth rate to be predicted based on a single batch culture experiment. The model predicts Q_MCYST from μ, μ_max (maximum specific growth rate), Q_MCYSTmax (maximum cell quota), and Q_MCYSTmin (minimum cell quota). Under the conditions examined in this study, we predict a Q_MCYSTmax of 0.129 fmol cell⁻¹ at μ_max and a Q_MCYSTmin of 0.050 fmol cell⁻¹ at μ = 0. Net MCYST production rate (R_MCYST) asymptotes to zero at μ = 0 and reaches a maximum of 0.155 fmol cell⁻¹ day⁻¹ at μ_max. MCYST/dry weight ratio (milligrams per gram [dry weight]) increased linearly with μ, whereas the MCYST/protein ratio reached a maximum at intermediate μ. In contrast, the MCYST/Chl a ratio remained constant. Cell volume correlated negatively with μ, leading to an increase in intracellular MCYST concentration at high μ. Taken together, our results show that fast-growing cells of N-limited M. aeruginosa are smaller, are of lower mass, and have a higher intracellular MCYST quota and concentration than slow-growing cells. The data also highlight the importance of determining cell MCYST quotas, as potentially confusing interpretations can arise from determining MCYST content as a ratio to other cell components.     

Zooplankton Growth,Respiration and Grazing on the Australian Margins of the Tropical Indian and Pacific Oceans

The specific activity of aminoacyl-tRNA synthetases (spAARS), an index of growth rate, and of the electron transport system (spETS), an index of respiration, was measured in three size fractions (73–150 μm, >150 μm and >350 μm) of zooplankton during five cruises to tropical coastal waters of the Kimberley coast (North West Australia) and four cruises to waters of the Great Barrier Reef (GBR; North East Australia). The N-specific biomass of plankton was 3–4-fold higher in the Kimberley than on the GBR in all 3 size classes: Kimberley 1.27, 3.63, 1.94 mg m^-3; GBR 0.36, 0.88 and 0.58 mg m^-3 in the 73–150 μm, >150 μm and >350 μm size classes, respectively. Similarly, spAARS activity in the Kimberley was greater than that of the GBR: 88.4, 132.2, and 147.6 nmol PPi hr^-1 mg protein ^-1 in the Kimberley compared with 71.7, 82.0 and 83.8 nmol PPi hr^-1 mg protein ^-1 in the GBR, for the 73–150 μm, >150 μm and >350 μm size classes, respectively. Specific ETS activity showed similar differences in scale between the two coasts: 184.6, 148.8 and 92.2 μL O₂ hr^-1 mg protein^-1 in the Kimberley, against 86.5, 88.3 and 71.3 μL O₂ hr^-1 mg protein^-1 in the GBR. On the basis of these measurements, we calculated that >150 μm zooplankton grazing accounted for 7% of primary production in the Kimberley and 8% in GBR waters. Area-specific respiration by >73 μm zooplankton was 7-fold higher in the Kimberley than on the GBR and production by >150 μm zooplankton was of the order of 278 mg C m^-2 d^-1 in the Kimberley and 42 mg C m^-2 d^-1 on the GBR. We hypothesize that the much stronger physical forcing on the North West shelf is the principal driver of higher rates in the west than in the east of the continent.     

Enhanced Interaction between Pseudokinase and Kinase Domains in Gcn2 stimulates eIF2α Phosphorylation in Starved Cells

The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α, from yeast to mammals. The Gcn2 kinase domain (KD) is inherently inactive and requires allosteric stimulation by adjoining regulatory domains. Gcn2 contains a pseudokinase domain (YKD) required for high-level eIF2α phosphorylation in amino acid starved yeast cells; however, the role of the YKD in KD activation was unknown. We isolated substitutions of evolutionarily conserved YKD amino acids that impair Gcn2 activation without reducing binding of the activating ligand, uncharged tRNA, to the histidyl-tRNA synthetase-related domain of Gcn2. Several such Gcn⁻ substitutions cluster in predicted helices E and I (αE and αI) of the YKD. We also identified Gcd⁻ substitutions, evoking constitutive activation of Gcn2, mapping in αI of the YKD. Interestingly, αI Gcd⁻ substitutions enhance YKD-KD interactions in vitro, whereas Gcn⁻ substitutions in αE and αI suppress both this effect and the constitutive activation of Gcn2 conferred by YKD Gcd⁻ substitutions. These findings indicate that the YKD interacts directly with the KD for activation of kinase function and identify likely sites of direct YKD-KD contact. We propose that tRNA binding to the HisRS domain evokes a conformational change that increases access of the YKD to sites of allosteric activation in the adjoining KD.