TRPA1 ion channel stimulation enhances cardiomyocyte contractile function via a CaMKII-dependent pathway

Rationale: Transient receptor potential channels of the ankyrin subtype-1 (TRPA1) are non-selective cation channels that show high permeability to calcium. Previous studies from our laboratory have demonstrated that TRPA1 ion channels are expressed in adult mouse ventricular cardiomyocytes (CMs) and are localized at the z-disk, costamere and intercalated disk. The functional significance of TRPA1 ion channels in the modulation of CM contractile function have not been explored.

Objective: To identify the extent to which TRPA1 ion channels are involved in modulating CM contractile function and elucidate the cellular mechanism of action.

Methods and Results: Freshly isolated CMs were obtained from murine heart and loaded with Fura-2 AM. Simultaneous measurement of intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and contractility was performed in individual CMs paced at 0.3 Hz. Our findings demonstrate that TRPA1 stimulation with AITC results in a dose-dependent increase in peak [Ca²⁺]_i and a concomitant increase in CM fractional shortening. Further analysis revealed a dose-dependent acceleration in time to peak [Ca²⁺]_i and velocity of shortening as well as an acceleration in [Ca²⁺]_i decay and velocity of relengthening. These effects of TRPA1 stimulation were not observed in CMs pre-treated with the TRPA1 antagonist, HC-030031 (10 µmol/L) nor in CMs obtained from TRPA1^?/? mice. Moreover, we observed no significant increase in cAMP levels or PKA activity in response to TRPA1 stimulation and the PKA inhibitor peptide (PKI 14–22; 100 nmol/L) failed to have any effect on the TRPA1-mediated increase in CM contractile function. However, TRPA1 stimulation resulted in a rapid phosphorylation of Ca²⁺/calmodulin-dependent kinase II (CaMKII) (1–5 min) that correlated with increases in CM [Ca²⁺]_i and contractile function. Finally, all aspects of TRPA1-dependent increases in CM [Ca²⁺]_i, contractile function and CaMKII phosphorylation were virtually abolished by the CaMKII inhibitors, KN-93 (10 µmol/L) and autocamtide-2-related peptide (AIP; 20 µmol/L).

Conclusions: These novel findings demonstrate that stimulation of TRPA1 ion channels in CMs results in activation of a CaMKII-dependent signaling pathway resulting in modulation of intracellular Ca²⁺ availability and handling leading to increases in CM contractile function. Cardiac TRPA1 ion channels may represent a novel therapeutic target for increasing the inotropic and lusitropic state of the heart.     

Growth and Cadmium Phytoextraction by Swiss Chard,Maize, Rice,Noccaea caerulescens,and Alyssum murale in pH Adjusted Biosolids Amended Soils

Past applications of biosolids to soils at some locations added higher Cd levels than presently permitted. Cadmium phytoextraction would alleviate current land use constraints. Unamended farm soil, and biosolids amended farm and mine soils were obtained from a Fulton Co., IL biosolids management facility. Soils contained 0.16, 22.8, 45.3 mg Cd kg^–1 and 43.1, 482, 812 mg Zn kg^–1 respectively with initial pH 6.0, 6.1, 6.4. In greenhouse studies, Swiss chard (Beta vulgaris var. cicla), a Cd-accumulator maize (inbred B37 Zea mays) and a southern France Cd-hyperaccumulator genotype of Noccaea caerulescens were tested for Cd accumulation and phytoextraction. Soil pH was adjusted from ～5.5–7.0. Additionally 100 rice (Oryza sativa) genotypes and the Ni-hyperaccumulator Alyssum murale were screened for potential phytoextraction use.

Chard suffered phytotoxicity at low pH and accumulated up to 90 mg Cd kg^–1 on the biosolids amended mine soil. The maize inbred accumulated up to 45 mg Cd kg^–1 with only mild phytotoxicity symptoms during early growth at pH > 6.0. N. caerulescens did not exhibit phytotoxicity symptoms at any pH, and accumulated up to 235 mg Cd kg^–1 in 3 months. Reharvested N. caerulescens accumulated up to 900 mg Cd kg^–1 after 10 months. Neither Alyssum nor 90% of rice genotypes survived acceptably.

Both N. caerulescens and B37 maize show promise for Cd phytoextraction in IL and require field evaluation; both plants could be utilized for nearly continuous Cd removal. Other maize inbreds may offer higher Cd phytoextraction at lower pH, and mono-cross hybrids higher shoot biomass yields. Further, maize grown only for biomass Cd maximum removal could be double-cropped.     

Characterization of Nickel Tolerant Bacteria Isolated from Heavy Metal Polluted Glass Industry for its Potential Role in Bioremediation

Heavy metal contamination of the environment is a serious concern because of their deleterious effects on biological systems. To the best of our knowledge, this is the first investigation on isolation of heavy metal resistant bacteria from contaminated sites of glass industrial area.

The study focused on isolation and characterization of Ni²⁺ resistant bacteria from these sites and analysis of their Ni²⁺ accumulation potential. Out of 38 different bacterial isolates 3 bacteria were able to tolerate up to 24 mM Ni²⁺ concentration. These bacterial strains were identified as E. coli (AS17b), Escherichia coli (AS21) and Microbacterium sp. (AS33) by 16S rRNA (16S ribosomal RNA) sequencing and their basic local alignment search tool search analysis. Growing cell of E. coli and Microbacterium sp. revealed accumulation of 0.12, 0.08 and 0.06 µg, of Ni²⁺/mg dry weight of cells, respectively, by 72 hr. Similarly resting cell of these strains showed accumulation of 0.27, 0.11 and 0.08 µg of Ni²⁺/mg dry weight of cells by 150 min, respectively. These results reveal that strain E. coli (AS21) shows maximum accumulation efficiency for Ni²⁺ among different isolates studied under shaking as well as starving conditions. Hence, E. coli (AS21) could serve as an efficient and promising bacterium for bioremediation of nickel-contaminated sites.     

Metal sensitive and DNA concentration dependent structural rearrangement of short oligonucleotide into large suprastructures

Formation of higher order structures, such as G-quadruplexes and G-quadruplex based large suprastructures into long G-wires and liquid crystals is promising elements for use in healthcare for drug delivery as they are mechanically and thermally stable. In this study, we studied the structures of short 11-mer oligonucleotide 5′-G₂AG₅AG₂-3′(11Pu) which is observed in 3′-UTR region of c-jun protooncogene. We used circular dichroism, UV-thermal melting, native gel electrophoresis and atomic force microscopy to determine the structure of 11Pu. CD results showed that 11Pu formed a mixed G-quadruplex in the presence of Na⁺ with and without Mg²⁺, while it formed a parallel G-quadruplex in the presence of 100?mM K⁺ with or without Mg²⁺. Cation selectivity in inducing the formation of large superstructures was observed in the presence of 100?mM K⁺ with 10?mM Mg²⁺. On the contrary, 10?mM Ca²⁺ did not induce the suprastructures. It was further demonstrated that Mg²⁺ at low concentration induced a parallel G-quadruplex of 11Pu, whereas at 10?mM Mg²⁺ induced a large suprastructure. AFM Images showed that 11Pu formed a G-wire, a liquid crystals and a crystalline lattice depending on the concentration of 11Pu and Mg²⁺. These insights may be employed to design G quadruplex-based nanowires for targeted drug delivery as well as interesting candidates for molecular nanowires.

Communicated by Ramaswamy H. Sarma     

Anti-diabetic effect of S-adenosylmethionine and α-glycerophosphocholine in KK-Ay mice

Six-week-old male KK-A^y mice received drinking water with S-adenosylmethionine (SAM), α-glycerophosphocholine (GPC), or SAM+GPC for 10 weeks. The serum glucose of SAM+GPC at 15 weeks old, total cholesterol of GPC at 12 weeks old, and triglyceride of GPC at 15 weeks old and of SAM at 16 weeks old were reduced. SAM+GPC reduced serum leptin and food intake.

Abbreviations: SAM: S-adenosylmethionine; GPC: α-glycerophosphocholine     

Overproduction of poly(β-malic acid) (PMA) from glucose by a novel Aureobasidium sp. P6 strain isolated from mangrove system

After over 100 strains of Aureobasidium spp isolated from mangrove system were screened for their ability to produce poly(β-malic acid) (PMA), it was found that Aureobasidium sp. P6 strain among them could produce high level of Ca²⁺-PMA. Fourteen percent glucose and 6.5 % CaCO₃ in the medium were the most suitable for Ca²⁺-PMA production. Then, 100.7 g/l of Ca²⁺-PMA was produced using Aureobasidium sp. P6 strain within 168 h at flask level. During 10-l batch fermentation, when the medium contained 12.0 % glucose, 98.7 g/l of Ca²⁺-PMA in the culture and 14.7 g/l of cell dry weight were obtained within 156 h, leaving 0.34 % reducing sugar in the fermented medium. When glucose concentration in the fermentation medium was 14.0 %, 118.3 g/l of Ca²⁺-PMA in the culture and 16.4 g/l of cell dry weight were obtained within 168 h, leaving 0.4 % reducing sugar in the fermented medium. After purification of Ca²⁺-PMA from the culture and acid hydrolysis of the pure Ca²⁺-PMA, analysis of HPLC showed that Aureobasidium sp. P6 strain only produced two main components of Ca²⁺-PMA and minor amount of calcium malate and that the hydrolysate of PMA was mainly composed of calcium malate. This is the first time to report that the novel yeast strain Aureobasidium sp. P6 strain isolated from the mangrove systems can produce such high amount of Ca²⁺-PMA.     

Induction of Calcium Carbonate by Bacillus cereus

The purpose of this research was to study how the bacteria Bacillus cereus (DCB1) utilizes calcium ions in a culture medium with carbon dioxide (CO₂) to yield calcium carbonate (CaCO₃). The bacteria strain DCB1 was a dominant strain isolated from dolomitic surfaces in areas of Karst topographies. The experimental method was as follows: a modified beef extract-peptone medium (beef extract 3.0 g, peptone 10 g, NaCl 5.0 g, CaCl₂ 2.0 g, glass powder 2.0 g, distilled water 1 L, and a pH between 6.5 and 7.5) was inoculated with B. cereus to attempt to induce the synthesis of CaCO₃. The sample was then processed by centrifugation every 24 h during the 7-day cultivation period. The pH, carbonic anhydrase (CA) activity, and the concentrations of both HCO^- ₃ and Ca²⁺ in the supernatant fluid were measured. Subsequently, precipitation in the culture medium was analyzed to confirm, or otherwise, the presence and if present, the formation, of CaCO₃. Methods used included X-ray diffraction (XRD), Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), and Energy Dispersive Spectroscopy (EDS). Meanwhile, the carbon source in the carbonate was classified by its isotope composition. Results showed that B. cereus can improve its pH value in this culture medium; concentrations of HCO^- ₃ and Ca²⁺ showed a significant decline over the duration of the cultivation period. CA activity reached its maximum during the second day; XRD, SEM, TEM, and isotope analysis all revealed the presence of CaCO₃ as a precipitate. Additionally, these results did not occur in an aseptic control group: no detectable level of CaCO₃ was produced therein. In conclusion: B. cereus can metabolize active materials, such as secretase, by its own growth and metabolism, and can either utilize atmospheric CO₂, or respire, to induce CaCO₃ production. Experimental evidence is offered for a concomitant CO₂ reduction and CaCO₃ induction by microorganisms.     

A correlation study on optimum conditions of microbial precipitation and prerequisites for self-healing concrete

Microbial induced CaCO₃ precipitation (MICP) based upon enzymatic urea hydrolysis has been verified as an effective way for crack treatment, especially for self-healing of concrete cracks. This paper aimed at correlating optimum conditions of MICP with prerequisites for self-healing concrete. Orthogonal experiments on a combination of factors contributing to the MICP process were firstly performed. Initial cell density and Ca²⁺ concentration were highly significant factor and significant factor respectively. High initial cell density (1×10⁸ cells·mL^-1) together with relatively low Ca²⁺ concentration (50 mM) favored microbial precipitation. The second part of this study was associated with dissolution tests to simulate the dissolving behavior of urea and calcium, since the dissolving of healing agents in cracks is a prerequisite of self-healing. By an addition of urea and Ca(NO₃)₂ with constant mass ratio of 2:3 in concrete, the highest values of the estimated urea concentration (345 mM) and Ca²⁺ concentration (44 mM) dissolved in cracks were close to the optimal values found by orthogonal studies. Although the addition of urea and Ca(NO₃)₂ would not have a negative impact on the mechanical properties of concrete, direct mixing is not recommended due to the low utilization efficiency of incorporating healing agents for self-healing.     

Characterization of a novel thermostable carboxylesterase from thermoalkaliphilic bacterium Bacillus thermocloaceae

A novel thermostable carboxylesterase (Est5250) of thermoalkaliphilic bacterium Bacillus thermocloaceae was heterologously expressed in Escherichia coli and its biochemical properties were investigated. Est5250 showed optimum esterase activity at 60 °C and pH 8.0. The enzyme was highly thermostable at 60 °C, interestingly, the thermostability was enhanced in the presence of Ca²⁺, retaining more than 60% of its original activity after 12 h of pre-incubation. Est5250 was active in the presence of 1% (v/v) of organic solvents and 0.1% (v/v) of non-ionic detergents. The enzyme activity was significantly enhanced up to 167% and 159% in the presence of 2-mercaptoethanol and dithiothreitol, respectively. Est5250 showed high substrate specificity for short-chain p-nitrophenyl-esters. Kinetic constants, K_m and k_cat, for p-nitrophenyl-acetate were 185.8 μM and 186.6 s^?1, respectively. Est5250 showed outstanding thermostability and tolerance to various organic solvents under thermoalkaliphilic conditions, suggesting that it would be a highly suitable biocatalyst for various biotechnological applications.

Abbreviations: B. thermocloaceae sp.: Bacillus thermocloaceae; E. coli: Escherichia coli; NP: nitrophenyl; DMSO: dimethyl sulfoxide; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DMF: dimethyl formamide; EGTA: ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; CTAB: cetrimonium bromide; PMSF: phenylmethylsulfonyl fluoride; DEPC: diethyl pyrocarbonate; 2-ME: 2-mercaptoethanol; DTT: dithiothreitol     

Extra-matrix Mg2+ limits Ca2+ uptake and modulates Ca2+ uptake–independent respiration and redox state in cardiac isolated mitochondria

Cardiac mitochondrial matrix (m) free Ca²⁺ ([Ca²⁺]_m) increases primarily by Ca²⁺ uptake through the Ca²⁺ uniporter (CU). Ca²⁺ uptake via the CU is attenuated by extra-matrix (e) Mg²⁺ ([Mg²⁺]_e). How [Ca²⁺]_m is dynamically modulated by interacting physiological levels of [Ca²⁺]_e and [Mg²⁺]_e and how this interaction alters bioenergetics are not well understood. We postulated that as [Mg²⁺]_e modulates Ca²⁺ uptake via the CU, it also alters bioenergetics in a matrix Ca²⁺–induced and matrix Ca²⁺–independent manner. To test this, we measured changes in [Ca²⁺]_e, [Ca²⁺]_m, [Mg²⁺]_e and [Mg²⁺]_m spectrofluorometrically in guinea pig cardiac mitochondria in response to added CaCl₂ (0–0.6 mM; 1 mM EGTA buffer) with/without added MgCl₂ (0–2 mM). In parallel, we assessed effects of added CaCl₂ and MgCl₂ on NADH, membrane potential (ΔΨ_m), and respiration. We found that >0.125 mM MgCl₂ significantly attenuated CU-mediated Ca²⁺ uptake and [Ca²⁺]_m. Incremental [Mg²⁺]_e did not reduce initial Ca²⁺uptake but attenuated the subsequent slower Ca²⁺ uptake, so that [Ca²⁺]_m remained unaltered over time. Adding CaCl₂ without MgCl₂ to attain a [Ca²⁺]_m from 46 to 221 nM enhanced state 3 NADH oxidation and increased respiration by 15 %; up to 868 nM [Ca²⁺]_m did not additionally enhance NADH oxidation or respiration. Adding MgCl₂ did not increase [Mg²⁺]_m but it altered bioenergetics by its direct effect to decrease Ca²⁺ uptake. However, at a given [Ca²⁺]_m, state 3 respiration was incrementally attenuated, and state 4 respiration enhanced, by higher [Mg²⁺]_e. Thus, [Mg²⁺]_e without a change in [Mg²⁺]_m can modulate bioenergetics independently of CU-mediated Ca²⁺ transport.     

Calcium regulates glutamate dehydrogenase and poly-γ-glutamic acid synthesis in <Emphasis Type="Italic">Bacillus natto</Emphasis>

Objective

To study the effect of Ca²⁺ on glutamate dehydrogenase (GDH) and its role in poly-γ-glutamic acid (γ-PGA) synthesis in Bacillus natto HSF 1410.

Results

When the concentration of Ca²⁺ varied from 0 to 0.1 g/l in the growth medium of B. natto HSF 1410, γ-PGA production increased from 6.8 to 9.7 g/l, while GDH specific activity and NH₄Cl consumption improved from 183 to 295 U/mg and from 0.65 to 0.77 g/l, respectively. GDH with α-ketoglutarate as substrate primarily used NADPH as coenzyme with a K _m of 0.08 mM. GDH was responsible for the synthesis of endogenous glutamate. The specific activity of GDH remained essentially unchanged in the presence of CaCl₂ (0.05–0.2 g/l) in vitro. However, the specific activity of GDH and its expression was significantly increased by CaCl₂ in vivo. Therefore, the regulation of GDH and PGA synthesis by Ca²⁺ is an intracellular process.

Conclusion

Calcium regulation may be an effective approach for producing γ-PGA on an industrial scale.

     

Relationships between chronotype,social jetlag,sleep, obesity and blood pressure in healthy young adults

Sleep disturbances, chronotype and social jetlag (SJL) have been associated with increased risks for major chronic diseases that take decades to develop, such as obesity, metabolic syndrome and cardiovascular disease. Potential relationships between poor sleep, chronotype and SJL as they relate to metabolic risk factors for chronic disease have not been extensively investigated. This prospective study examined chronotype, SJL and poor sleep in relation to both obesity and elevated blood pressure among healthy young adults.

SJL and objective sleep measures (total sleep time, sleep onset latency, wake after sleep onset and sleep efficiency) were derived from personal rest/activity monitoring (armband actigraphy) among 390 healthy adults 21–35 years old. Participants wore the device for 6–10 days at 6-month intervals over a 2-year period (n = 1431 repeated observations). Chronotypes were categorized into morning, intermediate and evening groups using repeated measures latent class analysis. Means of SJL and sleep measures among latent chronotype groups were compared using partial F-tests in generalized linear mixed models. Generalized linear mixed models also were used to generate odds ratios (ORs) with 95% confidence intervals (CIs) examining the relationship between repeated measures of chronotype, SJL, sleep and concurrent anthropometric outcome measures (body mass index, percentage of body fat, waist-to-hip ratio, waist-to-height ratio), systolic blood pressure and diastolic blood pressure.

Sleep latency ≥12 min was associated with increased odds of a high waist-to-height ratio (OR = 1.37; CI: 1.03–1.84). Neither chronotype nor SJL was independently associated with anthropometric outcomes or with blood pressure. Relationships between poor sleep and anthropometric outcomes or blood pressure varied by chronotype. Morning types with total sleep time <6 h, sleep efficiency <85% or wake after sleep onset ≥60 min were more likely to have an increased percentage of body fat, waist-to-hip ratio and waist-to-height ratio relative to those with an intermediate chronotype. Similarly, sleep latency ≥12 min was associated with increased odds of elevated systolic blood pressure (OR = 1.90; CI: 1.15–3.16, p_interaction = 0.02) among morning versus intermediate chronotypes. No relationships between poor sleep and obesity or elevated blood pressure were observed among evening chronotypes.

The results from this study among healthy young adults suggest that poor sleep among morning types may be more strongly associated with obesity and elevated blood pressure relative to those with an intermediate (neutral) chronotype. Sleep-related metabolic alterations among different chronotypes warrant further investigation.     

Determination of air pollution-related biomarkers of exposure in urine of travellers between Germany and China using liquid chromatographic and liquid chromatographic-mass spectrometric methods: a pilot study

Objective: The influence of different exposures to PM_2.5 (particulate matter with an aerodynamic diameter below 2.5?μm) on the concentrations of biomarkers of exposure and oxidative stress should be investigated. For this purpose, urine samples from individuals travelling from Germany to China were collected and analysed.

Materials: Robust LC and LC-MS/MS methods were established for the determination of biomarkers including 8-hydroxy-2′-deoxyguanosine, malondialdehyde, F_2α-isoprostanes and hydroxylated polycyclic aromatic hydrocarbons. As a pilot study, nine volunteers travelled from Germany (mean daily concentration of PM_2.5: 21?μg/m³) to China (mean daily concentration of PM_2.5: 108?μg/m³). Urine samples were collected before and after the trip.

Results: In samples collected after return to Germany, the median concentrations of oxidative stress biomarkers were observed to be higher than in samples collected before leaving Germany. Decreasing trends were observed in the sequences of samples collected after return in the following weeks. Correlations were found between exposure and oxidative stress biomarkers.

Conclusion: Travellers are ideal models for PM pollution-induced acute health effects study. Exposure to PM pollution can cause oxidative stress and damage.     

Broad environmental tolerance of native root-nodule bacteria of Biserrula pelecinus indicate potential for soil fertility restoration

Background: Biserrula pelecinus is an annual legume native to the Mediterranean basin, found in pastureland, alone or in association with other legumes (Leguminosae) and grasses (Poaceae). It has been used in revegetation programmes of mining wastes showing phytoremediation potentials and thus becoming potentially highly attractive for plant ecology and restoration management of natural ecosystems.

Aims: To characterise native root-nodule bacteria isolated from B. pelecinus from the Iberian Peninsula, and to select suitable N fixers for field-application and soil rehabilitation.

Methods: Strains were isolated and molecularly identified by 16S rRNA amplification and sequencing. Strains were phenotypically characterised in different abiotic conditions (acidity, salinity and heavy metals) and tested for their ability to fix atmospheric N₂. The most suitable N fixers were applied in greenhouse experiments with B. pelecinus under different fertilization levels to assess their tolerance to fertilized and polluted soils, commonly encountered in restoration projects.

Results: B. pelecinus root-nodule isolates tolerated pH from 4.5 to 9.5 grew in saline conditions (2.5% of NaCl), and tolerated 50 µM of Al³⁺ and Mn²⁺. Three isolates efficient in N₂ fixation, relative to the reference Mesorhizobium strain, were considered excellent candidates for the amelioration of nutrient poor sites.

Conclusions: These results provide valuable information for the potential use in soil restoration of B. pelecinus in a wide-range of conditions, exploiting the natural variability of its root-nodule bacteria.     

Efficacy of antioxidants on incidence of Fusarium root and pod rot diseases in peanut

Several antioxidants namely ascorbic acid, salicylic acid, sodium benzoates, thiouria and catechol were used as seed treatment and foliar spraying to reduce the incidence of root and pod rot diseases of peanut caused by (Fusarium oxysporum, Fusarium solani and Fusarium moniliforme) as well as to determine of phenolic compounds and oxidative enzymes in the treated plants. Each antioxidant was used at different concentrations (2, 4, 6 and 8 mM) against tested pathogenic fungi in vitro. All antioxidants at 8 mM showed the greatest reduction of mycelial growth of the pathogens. In greenhouse experiments, treated seeds (Giza 5 cv.) or foliar spraying of peanut plants after 30 and 60 days from planting date with each antioxidant at 8 mM reduced severity of both diseases. The treated plants with antioxidants increased accumulation of phenolic compounds and activity levels of oxidative enzymes (catalase, peroxidase and polyphenoloxidase) in infected plants compared to healthy plants.

http://zoobank.org/urn:lsid:zoobank.org:pub:AF539C90-4B38-4BFE-B77B-5F2A5D764D7A     

Urine catecholamines in children with severe Enterovirus A71 infection: comparison with paediatric septic shock

Purpose: Hypercatecholaminemia-related heart failure has been proposed as the main cause of enterovirus A71-related (EV-A71) early mortality. The purpose of this study was to measure urine catecholamine concentrations in severe EV-A71-infected children.

Methods: A total of 35 children, aged 2.5?±?2.1 years, were divided into three groups. Group I: 15 septic shock patients, group II: 17?EV-A71-stage-2 patients, and group III: 3?EV-A71-stage-4 patients. The laboratory results, cardiac biomarkers and urine catecholamine concentrations were statistically analysed.

Results: Group I had the highest C-reactive protein (CRP) levels and group II had the lowest B-type natriuretic peptide (BNP) and its N-terminal prohormone among the groups (p?=?0.039, <0.01 and <0.01, respectively). Group III patients had significantly higher urine catecholamine and troponin-I values among the groups. If urine epinephrine (Epi) >134 ug/gCr, norepinephrine (NE) >176 ug/gCr and vanillylmandelic acid (VMA) >11.7?mg/gCr were used as the cutoff points to differentiate groups II and III, the sensitivities and specificity were all 100%.

Conclusions: The significantly elevated urine catecholamine concentrations in EV-A71-stage-4 patients support the hypothesis that hypercatecholaminemia-related heart failure is involved in severe EV-A71 infection. Urine catecholamines could be used as reliable biomarkers for differentiation of severe EV-A71 infection with or without heart failure and septic shock.     

Ca2+ block and flickering both contribute to the negative slope of the IV curve in BK channels

Single-channel current–voltage (IV) curves of human large-conductance, voltage- and Ca²⁺-activated K⁺ (BK) channels are quite linear in 150 mM KCl. In the presence of Ca²⁺ and/or Mg²⁺, they show a negative slope conductance at high positive potentials. This is generally explained by a Ca²⁺/Mg²⁺ block as by Geng et al. (2013. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201210955) in this issue. Here, we basically support this finding but add a refinement: the analysis of the open-channel noise by means of β distributions reveals what would be found if measurements were done with an amplifier of sufficient temporal resolution (10 MHz), namely that the block by 2.5 mM Ca²⁺ and 2.5 mM Mg²⁺ per se would only cause a saturating curve up to +160 mV. Further bending down requires the involvement of a second process related to flickering in the microsecond range. This flickering is hardly affected by the presence or absence of Ca²⁺/Mg²⁺. In contrast to the experiments reported here, previous experiments in BK channels (Schroeder and Hansen. 2007. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.200709802) showed saturating IV curves already in the absence of Ca²⁺/Mg²⁺. The reason for this discrepancy could not be identified so far. However, the flickering component was very similar in the old and new experiments, regardless of the occurrence of noncanonical IV curves.     

Melatonin modulates Ca2+ mobilization and amylase release in response to cholecystokinin octapeptide in mouse pancreatic acinar cells

In the present work, we have evaluated the effect of an acute addition of melatonin on cholecystokinin octapeptide (CCK-8)-evoked Ca²⁺ signals and amylase secretion in mouse pancreatic acinar cells. For this purpose, freshly isolated mouse pancreatic acinar cells were loaded with fura-2 to study intracellular free Ca²⁺ concentration ([Ca²⁺]_c). Amylase release and cell viability were studied employing colorimetric methods. Our results show that CCK-8 evoked a biphasic effect on amylase secretion, finding a maximum at a concentration of 0.1 nM and a reduction of secretion at higher concentrations. Pre-incubation of cells with melatonin (1 μM–1 mM) significantly attenuated enzyme secretion in response to high concentrations of CCK-8. Stimulation of cells with 1 nM CCK-8 led to a transient increase in [Ca²⁺]_c, followed by a decrease towards a constant level. In the presence of 1 mM melatonin, stimulation of cells with CCK-8 resulted in a smaller [Ca²⁺]_c peak response, a faster rate of decay of [Ca²⁺]_c and lower values for the steady state of [Ca²⁺]_c, compared with the effect of CCK-8 alone. Melatonin also reduced the oscillatory pattern of Ca²⁺ mobilization evoked by a physiological concentration of CCK-8 (20 pM), and completely inhibited Ca²⁺ mobilization induced by 10 pM CCK-8. On the other hand, Ca²⁺ entry from the extracellular space was not affected in the presence of melatonin. Finally, melatonin alone did not change cell viability. We conclude that melatonin, at concentrations higher than those found in blood, might regulate exocrine pancreatic function via modulation of Ca²⁺ signals.     

Improving NaCl resistance of red-osier dogwood: role of CaCl2 and CaSO4

The influence of Ca²⁺ salts on the resistance of red-osier dogwood (Cornus sericea) seedlings to salinity was investigated. Red-osier dogwood seedlings were exposed to 5 and 10 mM of CaCl₂ or CaSO₄ in the presence or absence of 50 mM NaCl for 40 days in a controlled environment. Seedlings exposed to CaCl₂ and CaSO₄ recovered from NaCl-induced transpiration reduction after 20 days at a concentration of 10 mM and after 30 days at a concentration of 5 mM; while in absence of additional Ca²⁺, the seedlings recovered only after 40 days. Addition of 10 mM Ca²⁺ to NaCl treatment also limited the accumulation of proline in leaf tissues and caused an increase in leaf and lateral shoot K⁺ content. These results suggest that 10 mM Ca²⁺ could alleviate, at least in part, the osmotic effect of NaCl on red-osier dogwood via control of stomatal closure. On the other hand, ion analysis showed that Ca²⁺ addition was able to reduce the NaCl-induced Na⁺ concentration only in stem tissues suggesting that Ca²⁺ had only a limited effect on the ionic stress. The present study also showed an unexpected NaCl-induced increase in Ca²⁺ content of leaves, lateral shoots and stems that was not observed in our previous hydroponics experiments and seems to be more characteristic of plants growing on sandy soils.     

Thermodynamics and molecular dynamics simulations of calcium binding to the regulatory site of human cardiac troponin C: evidence for communication with the structural calcium binding sites

Human cardiac troponin C (HcTnC), a member of the EF hand family of proteins, is a calcium sensor responsible for initiating contraction of the myocardium. Ca²⁺ binding to the regulatory domain induces a slight change in HcTnC conformation which modifies subsequent interactions in the troponin–tropomyosin–actin complex. Herein, we report a calorimetric study of Ca²⁺ binding to HcTnC. Isotherms obtained at 25 °C (10 mM 2-morpholinoethanesulfonic acid, 50 mM KCl, pH 7.0) provided thermodynamic parameters for Ca²⁺ binding to both the high-affinity and the low-affinity domain of HcTnC. Ca²⁺ binding to the N-domain was shown to be endothermic in 2-morpholinoethanesulfonic acid buffer and allowed us to extract the thermodynamics of Ca²⁺ binding to the regulatory domain. This pattern stems from changes that occur at the Ca²⁺ site rather than structural changes of the protein. Molecular dynamics simulations performed on apo and calcium-bound HcTnC_1–89 support this claim. The values of the Gibbs free energy for Ca²⁺ binding to the N-domain in the full-length protein and to the isolated domain (HcTnC_1–89) are similar; however, differences in the entropic and enthalpic contributions to the free energy provide supporting evidence for the cooperativity of the C-domain and the N-domain. Thermograms obtained at two additional temperatures (10 and 37 °C) revealed interesting trends in the enthalpies and entropies of binding for both thermodynamic events. This allowed the determination of the change in heat capacity (?C _p ) from a plot of ?H verses temperature and may provide evidence for positive cooperativity of Ca²⁺ binding to the C-domain.