Target Binding to S100B Reduces Dynamic Properties and Increases Ca2 +-Binding Affinity for Wild Type and EF-Hand Mutant Proteins

Mutations in the second EF-hand (D61N, D63N, D65N, and E72A) of S100B were used to study its Ca^2 + binding and dynamic properties in the absence and presence of a bound target, TRTK-12. With ^D63NS100B as an exception (^D63NK_D = 50 ± 9 μM), Ca^2 + binding to EF2-hand mutants were reduced by more than 8-fold in the absence of TRTK-12 (^D61NK_D = 412 ± 67 μM, ^D65NK_D = 968 ± 171 μM, and ^E72AK_D = 471 ± 133 μM), when compared to wild-type protein (^WTK_D = 56 ± 9 μM). For the TRTK-12 complexes, the Ca^2 +-binding affinity to wild type (^WT + TRTKK_D = 12 ± 10 μM) and the EF2 mutants was increased by 5- to 14-fold versus in the absence of target (^{D61N + TRTK}K_D = 29 ± 1.2 μM, ^{D63N + TRTK}K_D = 10 ± 2.2 μM, ^{D65N + TRTK}K_D = 73 ± 4.4 μM, and ^{E72A + TRTK}K_D = 18 ± 3.7 μM). In addition, R_ex, as measured using relaxation dispersion for side‐chain ¹⁵N resonances of Asn63 (^D63NS100B), was reduced upon TRTK-12 binding when measured by NMR. Likewise, backbone motions on multiple timescales (picoseconds to milliseconds) throughout wild type, ^D61NS100B, ^D63NS100B, and ^D65NS100B were lowered upon binding TRTK-12. However, the X-ray structures of Ca^2 +-bound (2.0 Å) and TRTK-bound (1.2 Å) ^D63NS100B showed no change in Ca^2 + coordination; thus, these and analogous structural data for the wild-type protein could not be used to explain how target binding increased Ca^2 +-binding affinity in solution. Therefore, a model for how S100B–TRTK‐12 complex formation increases Ca^2 + binding is discussed, which considers changes in protein dynamics upon binding the target TRTK-12.     

Reverse Na+/Ca2+-exchange mediated Ca2+-entry and noradrenaline release in Na+-loaded peripheral sympathetic nerves

[³H]noradrenaline ([³H]NA) released from sympathetic nerves in the isolated main pulmonary artery of the rabbit was measured in response to field stimulation (2 Hz, 1 ms, 60 V for 3 min) in the presence of uptake blockers (cocaine, 3 × 10⁻⁵ M and corticosterone, 5 × 10⁻⁵ M). The [³H]NA-release was fully blocked by the combined application of the selective and irreversible ‘N-type’ voltage-sensitive Ca²⁺-channel (VSCC)-blocker ω-conotoxin (ω-CgTx) GVIA (10⁻⁸ M) and the ‘non-selective’ VSCC-blocker aminoglycoside antibiotic neomycin (3 × 10⁻³ M). Na⁺-loading (Na⁺-pump inhibition by K⁺-free perfusion) was required to elicit further NA-release after blockade of VSCCs (ω-CgTx GVIA + neomycin). In K⁺-free solution, in the absence of functioning VSCCs (ω-CgTx GVIA + neomycin), the fast Na⁺-channel activator veratridine (10⁻⁵ M) further potentiated the nerve-evoked release of [³H]NA. This NA-release was significantly inhibited by KB-R7943, and fully blocked by $\text{C}{\text{a}}_{\text{o}}^{2+}-\text{removal}$. However, Li⁺-substitution was surprisingly ineffective. The non-selective K⁺-channel blocker 4-aminopyridine (4-AP, 10⁻⁴ M) also further potentiated the nerve-evoked release of NA in K⁺-free solution. This potentiated release was concentration-dependently inhibited by KB-R7943, significantly inhibited by Li⁺-substitution and abolished by $\text{C}{\text{a}}_{\text{o}}^{2+}-\text{removal}$.It is concluded that in Na⁺-loaded sympathetic nerves, in which the VSCCs are blocked, the reverse Na⁺/Ca²⁺-exchange-mediated Ca²⁺-entry is responsible for transmitter release on nerve-stimulation. Theoretically we suppose that the fast Na⁺-channel and the exchanger proteins are close to the vesicle docking sites.     

Endothelium-independent vasorelaxant effect of sodium ferulate on rat thoracic aorta

AimsThis study was designed to investigate the effects of sodium ferulate (SF) on rat isolated thoracic aortas and the possible mechanisms.Main methodsIsometric tension was recorded in response to drugs in organ bath. Cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) was measured using Fluo-3 in cultured rat aortic smooth muscle cells (RASMC).Key findingsSF (0.1–30 mM) relaxed the isolated aortic rings precontracted with phenylephrine (PE) and high-K⁺ in a concentration-dependent manner with respective pD₂ of 2.7 ± 0.02 and 2.6 ± 0.06. Mechanical removal of endothelium did not significantly modify the SF-induced relaxation. In Ca²⁺-free solution, SF noticeably inhibited extracellular Ca²⁺-induced contraction in high-K⁺ and PE pre-challenged rings, and suppressed the transient contraction induced by PE and caffeine. The vasorelaxant effect of SF was unaffected by various K⁺ channel blockers such as tetraethylammonium, glibenclamide, 4-aminopyridine, and barium chloride. In addition, SF concentration-dependently reduced the contraction induced by phorbol-12-myristate-13-acetate, an activator of protein kinase C (PKC), in the absence of extracellular Ca²⁺, with the pD₂ of 2.9 ± 0.03. In RASMC, SF had no effect on PE- or KCl-induced [Ca²⁺]_i increase either in the presence or in the absence of external Ca²⁺.SignificanceThese results indicate that SF acts directly as a non-selective relaxant to vascular smooth muscle. The direct inhibition of the common pathway after [Ca²⁺]_i increase may account for the SF-induced relaxation in Ca²⁺-dependent contraction, while the blockage of the PKC-mediated contractile mechanism is likely responsible for the SF-induced relaxation in Ca²⁺-independent contraction.     

Heart rate and arterial pressure variability and baroreflex sensitivity in ovariectomized spontaneously hypertensive rats

AimsThe present study evaluated the effects of ovariectomy on heart rate and arterial pressure variability and cardiac baroreflex sensitivity (BRS) in female spontaneously hypertensive (SHR) and Wistar–Kyoto rats (WKY).Main methodsSham-surgery animals were used as control. Sixteen weeks after ovariectomy or sham-surgery, animals were recorded. Time series of pulse interval (PI) and systolic AP (SAP) were analyzed by means of autoregressive spectral analysis, which quantifies the power of very low (VLF = 0.01–0.25 Hz), low (LF = 0.25–0.75 Hz) and high frequency (HF = 0.75–2.5 Hz) bands. BRS was assessed by means of linear regression between changes of PI and SAP induced by vasoactive drugs or calculation of α-index, a spontaneous BRS index.Key findingsThere was no difference in baseline PI or SAP between ovariectomized and sham SHR. Spectral analysis of heart rate variability suggested a shift of sympatho-vagal balance toward sympathetic predominance in ovariectomized SHR (LF/HF = 1.8 ± 0.2 versus 0.7 ± 0.2 in sham SHR, p < 0.05). Ovariectomy increased total variance and VLF power of SAP in SHR (29.1 ± 9.6 mmHg² and 18.6 ± 6.3 mmHg² versus 9.1 ± 2.1 mmHg² and 4.3 ± 1.4 mmHg², respectively, in sham SHR, p < 0.05). In addition, ovariectomy reduced reflex bradycardia in SHR (0.18 ± 0.03 ms/mmHg versus 0.34 ± 0.06 ms/mmHg in sham SHR, p < 0.05). Ovariectomy did not affect heart rate and SAP variability or BRS in WKY.SignificanceThese data showed that ovarian hormones deprivation induced marked changes on cardiovascular control, increasing SAP variability and cardiac sympatho-vagal balance and blunting BRS in female hypertensive animals, which reinforce the possible protective role of ovarian hormones on the cardiovascular system.     

Expression of the Androgen Receptor,pAkt, and pPTEN in Breast Cancer and Their Potential in Prognostication

BACKGROUNDImportance of androgen receptor (AR) as an independent prognostic marker in Pakistani women with breast cancer (BCa) remains unexplored. Our aim was to identify the expression and potential prognostic value of AR, its upstream regulator (pAkt) and target gene (pPTEN) in invasive BCa.METHODSThis study used a cohort of 200 Pakistani women with invasive BCa diagnosed during 2002-2011. Expression of AR, pAkt and pPTEN was determined on formalin fixed paraffin embedded tissue sections by immunohistochemistry. The association of AR, pAkt and pPTEN with clinicopathological parameters was determined. Survival analyses were undertaken on patients with ≥ 5 years of follow-up (n = 82).RESULTSExpression of AR, pAkt and pPTEN was observed in 47.5%, 81.3% and 50.6% of patients, respectively. AR-expressing tumors were low or intermediate in grade (P < .001) and expressed ER (P = .002) and PR (P = .001). Patients with AR⁺ tumors had significantly higher OS (Mean OS = 10.2 ± 0.465 years) compared to patients with AR^? tumors (Mean OS = 5.8 ± 0.348 years) (P = .047). Furthermore, AR-positivity was associated with improved OS in patients receiving endocrine therapy (P = .020). Patients with AR⁺ /pAkt⁺ /pPTEN^? tumors, had increased OS (Mean OS = 7.1 ± 0.535 years) compared to patients with AR^?/pAkt⁺/pPTEN^? tumors (Mean OS = 5.1 ± 0.738 years).CONCLUSIONAR-expressing tumors are frequently characterized by low or intermediate grade tumors, expressing ER and PR. In addition, expression of AR, pAkt and pPTEN, could be considered in prognostication of patients with invasive BCa.     

KMUP-1 ameliorates monocrotaline-induced pulmonary arterial hypertension through the modulation of Ca2+ sensitization and K+-channel

AimsThis study investigates the actions of KMUP-1 on RhoA/Rho-kinase (ROCK)-dependent Ca²⁺ sensitization and the K⁺-channel in chronic pulmonary arterial hypertension (PAH) rats.Main methodsSprague–Dawley rats were divided into control, monocrotaline (MCT), and MCT + KMUP-1 groups. PAH was induced by a single intraperitoneal injection (i.p.) of MCT (60 mg/kg). KMUP-1 (5 mg/kg, i.p.) was administered once daily for 21 days to prevent MCT-induced PAH. All rats were sacrificed on day 22.Key findingsMCT-induced increased right ventricular systolic pressure (RVSP) and right ventricular hypertrophy were prevented by KMUP-1. In myograph experiments, KCl (80 mM), phenylephrine (10 µM) and K⁺ channel inhibitors (TEA, 10 mM; paxilline, 10 µM; 4-AP, 5 mM) induced weak PA contractions in MCT-treated rats compared to controls, but the PA reactivity was restored in MCT + KMUP-1-treated rats. By contrast, in β-escin- or α-toxin-permeabilized PAs, CaCl₂-induced (1.25 mM, pCa 5.1) contractions were stronger in MCT-treated rats, and this action was suppressed in MCT + KMUP-1-treated rats. PA relaxation in response to the ROCK inhibitor Y27632 (0.1 μM) was much higher in MCT-treated rats than in control rats. In Western blot analysis, the expression of Ca²⁺-activated K⁺ (BK_Ca) and voltage-gated K⁺ channels (Kv2.1 and Kv1.5), and ROCK II proteins was elevated in MCT-treated rats and suppressed in MCT + KMUP-1-treated rats. We suggest that MCT-treated rats upregulate K⁺-channel proteins to adapt to chronic PAH.SignificanceKMUP-1 protects against PAH and restores PA vessel tone in MCT-treated rats, attributed to alteration of Ca²⁺ sensitivity and K⁺-channel function.     

How do reactive oxygen species and calcium trigger mitochondrial membrane permeabilisation?

BackgroundMitochondrial membrane permeabilisation (MMP) is classically considered as a point of no return in several forms of cell death and is involved in numerous diseases such as cancer, neurodegenerative disorders or ischemia/reperfusion injuries. Many studies established that reactive oxygen species (ROS) and Ca^2 + were the prominent inducers of MMP. However, the mechanisms connecting ROS and Ca^2 + to the players of MMP are still a matter of debate.Scope of reviewThe aim of this review is to summarise the various studies related to the mechanisms of ROS- and Ca^2 +-induced MMP. Several lines of evidence suggest that ROS and Ca^2 + cooperate to induce MMP but the molecular details of the ROS–Ca^2 +-MMP network remain controversial. We then discuss recent data depicting this topic.Major conclusionsCytotoxic stimuli may be transduced within the cell by ROS and Ca^2 + increases. In most models, Ca^2 + and ROS can cooperate to induce MMP. Moreover, several data suggest that MMP increases mitochondrial Ca^2 + and ROS which therefore amplify the cytotoxic signal. Intriguingly, many reports have identified players of MMP as direct ROS targets. On the contrary, direct targets of Ca^2 + remain elusive. At the same time, the mechanisms by which mitochondrial Ca^2 + overload induces ROS generation are well documented. Upon these observations, we hypothesise that Ca^2 + cannot directly induce MMP and requires ROS production as a mandatory step.General significanceGiven the importance of Ca^2 +- and ROS-induced MMP in diseases, we expect that a better understanding of this process will lead to the development of novel therapies.     

The use of transgenic mouse models to reveal the functions of Ca2 + buffer proteins in excitable cells

BackgroundCytosolic Ca^2 + buffers are members of the large family of Ca^2 +-binding proteins and are essential components of the Ca^2 + signaling toolkit implicated in the precise regulation of intracellular Ca^2 + signals. Their physiological role in excitable cells has been investigated in vivo by analyzing the phenotype of mice either lacking one of the Ca^2 + buffers or mice with ectopic expression.Scope of ReviewIn this review, results obtained with knockout mice for the three most prominent Ca^2 + buffers, parvalbumin, calbindin-D28k and calretinin are summarized.Major ConclusionsThe absence of Ca^2 + buffers in specific neuron subpopulations, and for parvalbumin additionally in fast-twitch muscles, leads to Ca^2 + buffer-specific changes in intracellular Ca^2 + signals. This affects the excitation–contraction cycle in parvalbumin-deficient muscles, and in Ca^2 + buffer-deficient neurons, properties associated with synaptic transmission (e.g. short-term modulation), excitability and network oscillations are altered. These findings have not only resulted in a better understanding of the physiological function of Ca^2 + buffers, but have revealed that the absence of Ca^2 + signaling toolkit components leads to protein-and neuron-specific adaptive/homeostatic changes that also include changes in neuron morphology (e.g. altered spine morphology, changes in mitochondria content) and network properties.General SignificanceThe complex phenotype of Ca^2 + buffer knockout mice arises from the direct effect of these proteins on Ca^2 + signaling and moreover from the homeostatic mechanisms induced in these mice. For a better mechanistic understanding of neurological diseases linked to disturbed/altered Ca^2 + signaling, a global view on Ca^2 + signaling is expected to lead to new avenues for specific therapies. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling.     

Implementation of a dose–response curve for γ-radiation in the Portuguese population by use of the chromosomal aberration assay

An in vitro dose–response curve following exposure to γ-radiation was determined at the IST/ITN, by use of the chromosomal aberration assay. This is the first study of this kind carried out among the Portuguese population. Un-irradiated and γ-irradiated peripheral blood lymphocytes from 16 healthy donors were cultured. A total of 22,395 metaphases were analyzed for frequency and distribution of dicentrics and centric rings, as a function of the radiation dose. The dose–response data for dicentrics and dicentrics plus centric rings were fitted by use of a linear–quadratic model: Y_dic = (0.0011 ± 0.0006) + (0.0105 ± 0.0035)D + (0.0480 ± 0.0019)D² and Y_{dic + rings} = (0.0011 ± 0.0006) + (0.0095 ± 0.0036)D + (0.0536 ± 0.0020)D². Also, calibration curves related to age and gender were determined, but no significant differences were found. Following the establishment of the dose–response curves, a validation experiment was carried out with three individuals. Real and estimated doses, obtained with the dose–response curves, were in agreement. These results give us confidence to apply both dose–response calibration curves in future biological dosimetry requirements.     

Alterations in ryanodine receptors and related proteins in heart failure

Sarcoplasmic reticulum (SR) Ca^2 + release plays an essential role in mediating cardiac myocyte contraction. Depolarization of the plasma membrane results in influx of Ca^2 + through l-type Ca^2 + channels (LTCCs) that in turn triggers efflux of Ca^2 + from the SR through ryanodine receptor type-2 channels (RyR2). This process known as Ca^2 +-induced Ca^2 +release (CICR) occurs within the dyadic region, where the adjacent transverse (T)-tubules and SR membranes allow RyR2 clusters to release SR Ca^2 + following Ca^2 + influx through adjacent LTCCs. SR Ca^2 + released during systole binds to troponin-C and initiates actin–myosin cross-bridging, leading to muscle contraction. During diastole, the cytosolic Ca^2 + concentration is restored by the resequestration of Ca^2 + into the SR by SR/ER Ca^2 +-ATPase (SERCA2a) and by the extrusion of Ca^2 + via the Na⁺/Ca^2 +-exchanger (NCX1). This whole process, entitled excitation–contraction (EC) coupling, is highly coordinated and determines the force of contraction, providing a link between the electrical and mechanical activities of cardiac muscle. In response to heart failure (HF), the heart undergoes maladaptive changes that result in depressed intracellular Ca^2 + cycling and decreased SR Ca^2 + concentrations. As a result, the amplitude of CICR is reduced resulting in less force production during EC coupling. In this review, we discuss the specific proteins that alter the regulation of Ca^2 + during HF. In particular, we will focus on defects in RyR2-mediated SR Ca^2 + release. This article is part of a Special Issue entitled: Heart failure pathogenesis and emerging diagnostic and therapeutic interventions.     

Endothelium-dependent and -independent vasorelaxation induced by CIJ-3-2F,a novel benzyl-furoquinoline with antiarrhythmic action,in rat aorta

AimsThis study was designed to examine the mechanism of relaxation induced by CIJ-3-2F, a benzyl-furoquinoline antiarrhythmic agent, in rat thoracic aorta at the tissue and cellular levels.Main methodsIsometric tension of rat aortic ring was measured in response to drugs. Ionic channel activities in freshly dissociated aortic vascular smooth muscle cells (VSMCs) were investigated using a whole-cell patch-clamp technique.Key findingsCIJ-3-2F relaxed both phenylephrine (PE) and high KCl (60 mM)-induced contractions with respective pEC₅₀ (-log EC₅₀) values of 6.91 ± 0.07 and 6.32 ± 0.06. Removal of endothelium or pretreatment with nitric oxide (NO)-pathway inhibitors N^ω-nitro-l-arginine methyl ester (L-NAME), N^G-monomethyl-l-arginine (L-NMMA), N⁵-(1-iminoethyl)-l-ornithine (L-NIO), hemoglobin, methylene blue or 1H-[1,2,4]oxadiazolo[4,2-α]quinoxalin-1-one (ODQ) reduced the relaxant effect of CIJ-3-2F. Relaxation to CIJ-3-2F was also attenuated by K⁺ channel blockers tetraethylammonium (TEA) or 4-aminopyridine (4-AP), but not by charybdotoxin plus apamin, iberiotoxin, glibenclamide, or BaCl₂. CIJ-3-2F non-competitively antagonized the contractions induced by PE, Ca²⁺, and Bay K8644 in endothelium-denuded rings. In addition, CIJ-3-2F inhibited both the phasic and tonic contractions induced by PE but did not affect the transient contraction induced by caffeine. CIJ-3-2F reduced the Ba²⁺ inward current through L-type Ca²⁺ channel (IC₅₀ = 4.1 μM) and enhanced the voltage-dependent K⁺ (K_v) current in aortic VSMCs.SignificanceThese results suggest that CIJ-3-2F induced both endothelium-dependent and -independent vasorelaxation; the former is likely mediated by the NO/cGMP pathway whereas the latter is probably mediated through inhibition of Ca²⁺ influx or inositol 1,4,5-triphosphate (IP₃)-sensitive intracellular Ca²⁺ release, or through activation of K_v channels.     

Immune mobilization of autologous blood progenitor cells: direct influence on the cellular subsets collected

Background aimsA phase I trial examined the ability of immunotherapy to mobilize progenitor and activated T cells.MethodsInterleukin (IL)-2 was administered subcutaneously for 11 days, with granulocyte (G)-colony-stimulating factor (CSF) (5 mcg/kg/day) and granulocyte–macrophage (GM)-CSF (7.5 mcg/kg/day) added for the last 5 days. Leukapheresis was initiated on day 11. Thirteen patients were treated (myeloma n = 11, non-Hodgkin's lymphoma n = 2).ResultsToxicities were minimal. IL-2 was stopped in two patients because of capillary leak (n = 1) and diarrhea (n = 1). Each patient required 2.5 leukaphereses (median; range 1–3) to collect 3.2 × 10⁶ CD34⁺ cells/kg (median; range 1.9–6.6 × 10⁶/kg). Immune mobilization increased the number of CD3⁺ CD8⁺ T cells (P = 0.002), CD56⁺ natural killer (NK) cells (P = 0.0001), CD8⁺ CD56⁺ T cells (P = 0.002) and CD4⁺ CD25⁺ cells (P = 0.0001) compared with cancer patients mobilized with G-CSF alone. There was increased lysis of myeloma cells after 7 days (P = 0.03) or 11 days (P = 0.02). The maximum tolerated dose of IL-2 was 1 × 10⁶ IU/m²/day.ConclusionsImmune mobilization is well tolerated with normal subsequent marrow engraftment. As cells within the graft influence lymphocyte recovery, an increased number of functional lymphocytes may result in more rapid immune reconstitution.     

Semi-automated program for analysis of local Ca2+ spark release with application for classification of heart cell type

Local Ca²⁺ spark releases are essential to the Ca²⁺ cycling process. Thus, they play an important role in ventricular and atrial cell contraction, as well as in sinoatrial cell automaticity. Characterizing their properties in healthy cells from different regions in the heart can reveal the basic biophysical differences among these regions. We designed a semi-automatic Matlab Graphical User Interface (called Sparkalyzer) to characterize parameters of Ca²⁺ spark release from any major cardiac tissue, as recorded in line-scan mode with a confocal laser-scanning microscope. We validated the algorithm on experimental images from rabbit sinoatrial, atrial, and ventricular cells loaded with Fluo-4 AM. The program characterizes general image parameters of Ca²⁺ transients and sparks: spark duration, which indicates for how long the spark provides Ca²⁺ to the closed intracellular mechanisms (typical value: 25 ± 1, 23 ± 1, 26 ± 1 ms for sinoatrial, atrial, and ventricular cells, respectively); spark amplitude, which indicates the amount of Ca²⁺ released by a single spark (1.6 ± 0.1, 1.6 ± 0.2, 1.4 ± 0.1 F/F₀ for sinoatrial, atrial, and ventricular cells, respectively); spark length, which is the length of the Ca²⁺ wavelets fired out of a row of ryanodine receptors (5 ± 0.1, 5 ± 0.2, 3.4 ± 0.3 μm for sinoatrial, atrial, or ventricular cells, respectively) and number of sparks (0.14 ± 0.02, 0.025 ± 0.01, 0.02 ± 0.01 for 1 μm in 1 s for sinoatrial, atrial, and ventricular cells, respectively). This method is reliable for Ca²⁺ spark analysis of sinoatrial, atrial, or ventricular cells. Moreover, by examining the average value of Ca²⁺ spark characteristics and their scattering around the mean, atrial, ventricular and sinoatrial cells can be differentiated.     

Cholesterol enrichment of rabbit platelets enhances the Ca2 + entry pathway induced by platelet-derived secondary feedback agonists

AimsHypersensitivity of platelets due to increased platelet cholesterol levels has been reported in hypercholesterolemia. However, the signaling pathways linking increased platelet reactivity and cholesterol contents are not fully understood. This study aims to determine the direct effect of cholesterol enrichment of platelets on the pathways including Ca^2 + mobilization and secondary feedback agonists such as adenosine diphosphate (ADP) and thromboxane A₂ (TXA₂).Main methodsIn vitro cholesterol enrichment of rabbit platelets was performed by incubation with cholesterol complexed with methyl-β-cyclodextrin. Ca^2 + mobilization was monitored using platelets loaded with fura-PE3/AM, a fluorescent calcium indicator. Released ATP and TXB₂ from platelets were measured by a luciferin–luciferase ATP assay system and a TXB₂ ELISA Kit, respectively.Key findingsCholesterol enrichment of rabbit platelets significantly enhanced Ca^2 + mobilization induced by thrombin, accompanying an augmented Ca^2 + entry. The augmentation of Ca^2 + entry by cholesterol enrichment was significantly suppressed by treatment with inhibitors for secondary feedback agonists. In cholesterol-enriched platelets, the amount of released ATP or TXB₂ induced by thrombin was not significantly altered in comparison with control platelets, whereas an increase in [Ca^2 +]_i induced by ADP or U46619, a TXA₂ mimetic, was significantly enhanced.SignificanceThese results suggest that cholesterol enrichment of rabbit platelets results in enhanced Ca^2 + mobilization via ADP/TXA₂-dependent augmentation of the Ca^2 + entry pathway. The results reveal a novel mechanism by which platelet hypersensitivity is regulated by cholesterol contents.     

Fire service instructor's undergarment choice to reduce Interleukin-6 and minimise physiological and perceptual strain

Fire Service Instructors frequently experience high levels of physiological and perceptual strain during live fire exposures. Instructors are also at risk of cardiovascular illnesses, with cardiac death being the greatest cause of fire fighter death. Current practice for UK instructors is to select undergarment type based on personal preference, between a boiler suit (BOILER) and a wicking base layer (WBL). Research suggests that shorts and t-shirt (SHORTS) may also be a beneficial alternative undergarment choice. The UK South East Fire Service requested an investigation to identify if undergarment selection can lessen the strain experienced by instructors, and reduce the acute inflammatory response to fire exposures. Eight males completed three 45 min sessions in a heat chamber (49.5±1.4 °C and 16.9±4.3% RH) whilst performing intermittent walking. At the end of heat exposure change in heart rate was not effected by garment type (p=0.061, ηp²=0.373). Change in rectal temperature was different between garments (p=0.009, η_p²=0.271), with trends suggesting that BOILER resulted in a greater change (1.03±0.60 °C) than SHORTS (0.76±0.37 °C, p=0.589, d=0.21) and WBL (0.72±0.33 °C, p=0.545, d=0.25). Interleukin-6 post exposure was greater for BOILER (6.96±0.28 pg mL⁻¹) than both SHORTS (6.59±0.30 pg mL⁻¹, p=0.043, d=0.42) and WBL (6.45±0.43 pg mL⁻¹, p=0.031, d=0.51). Overall, undergarment type had little impact on physiological or perceptual strain. However, wearing WBL or SHORTS may reduce the inflammatory response, and consequently decrease the risk of cardiovascular events.     

Altered sarcoplasmic reticulum calcium transport in the presence of the heavy metal chelator TPEN

TPEN (N,N,N′,N′-tetrakis(2-pyridylmethyl)-ethylenediamine) is a membrane-permeable heavy-metal ion chelator with a dissociation constant for Ca²⁺ comparable to the Ca²⁺ concentration ([Ca²⁺]) within the intracellular Ca²⁺ stores. It has been used as modulator of intracellular heavy metals and of free intraluminal [Ca²⁺], without influencing the cytosolic [Ca²⁺] that falls in the nanomolar range. In our previous studies, we gave evidence that TPEN modifies the Ca²⁺ homeostasis of striated muscle independent of this buffering ability. Here we describe the direct interaction of TPEN with the ryanodine receptor (RyR) Ca²⁺ release channel and the sarcoplasmic reticulum (SR) Ca²⁺ pump (SERCA). In lipid bilayers, at negative potentials and low [Ca²⁺], TPEN increased the open probability of RyR, while at positive potentials it inhibited channel activity. On permeabilized skeletal muscle fibers of the frog, but not of the rat, 50 μM TPEN increased the number of spontaneous Ca²⁺ sparks and induced propagating events with a velocity of 273 ± 7 μm/s. Determining the hydrolytic activity of the SR revealed that TPEN inhibits the SERCA pump, with an IC₅₀ = 692 ± 62 μM and a Hill coefficient of 0.88 ± 0.10. These findings provide experimental evidence that TPEN directly modifies both the release of Ca²⁺ from and its reuptake into the SR.     

Oxidative stress and DNA damage in relation to transition metals overload in Abu-Qir Bay,Egypt

The aim of the present study is to evaluate the transition metals overload in Abu-Qir Bay in Egypt, as compared to a less polluted area (reference area) through some biomarkers of oxidative stress. Catalase enzyme activity, malondialdehyde (MDA) concentration and DNA damage (number of apurinic/apyrimidinic sites) were the tested biomarkers. The levels of iron and copper in Mugil cephalus liver tissues were significantly higher in samples from the polluted area as compared to the reference area: Fe: 407 ± 38 vs. 216 ± 21 μg/g wet wt; p = 0.008, Cu: 54 ± 6 vs. 17.7 ± 4 μg/g wet wt; p = 0.0001. This could account for the observed increase in MDA concentration (15.7 ± 5.7 vs. 2.5 ± 0.5 U/g; p = 0.035), and the elevated number of AP sites (13.9 ± 2.6 vs. 0.37 ± 0.2 AP site/1 × 10⁵ bp; p = 0.0001). Similarly, the activity of catalase enzyme responsible for the cellular defense was significantly high (58.3 ± 12.2 vs. 28.4 ± 4.0 U/mg; p = 0.032). The present data indicated a clear relationship between the pollution degree of the above marine environment and both biochemical and molecular responses of the piscine system.