Functional expression of CCR1, CCR3, CCR4, and CXCR4 chemokine receptors on human platelets

Platelets are known to contain platelet factor 4 and beta-thromboglobulin, alpha-chemokines containing the CXC motif, but recent studies extended the range to the beta-family characterized by the CC motif, including RANTES and Gro-alpha. There is also evidence for expression of chemokine receptors CCR4 and CXCR4 in platelets. This study shows that platelets have functional CCR1, CCR3, CCR4, and CXCR4 chemokine receptors. Polymerase chain reaction detected chemokine receptor messenger RNA in platelet RNA. CCR1, CCR3, and especially CCR4 gave strong signals; CXCR1 and CXCR4 were weakly positive. Flow cytometry with specific antibodies showed the presence of a clear signal for CXCR4 and weak signals for CCR1 and CCR3, whereas CXCR1, CXCR2, CXCR3, and CCR5 were all negative. Immunoprecipitation and Western blotting with polyclonal antibodies to cytoplasmic peptides clearly showed the presence of CCR1 and CCR4 in platelets in amounts comparable to monocytes and CCR4 transfected cells, respectively. Chemokines specific for these receptors, including monocyte chemotactic protein 1, macrophage inflammatory peptide 1alpha, eotaxin, RANTES, TARC, macrophage-derived chemokine, and stromal cell-derived factor 1, activate platelets to give Ca(++) signals, aggregation, and release of granule contents. Platelet aggregation was dependent on release of adenosine diphosphate (ADP) and its interaction with platelet ADP receptors. Part, but not all, of the Ca(++) signal was due to ADP release feeding back to its receptors. Platelet activation also involved heparan or chondroitin sulfate associated with the platelet surface and was inhibited by cleavage of these glycosaminoglycans or by heparin or low molecular weight heparin. These platelet receptors may be involved in inflammatory or allergic responses or in platelet activation in human immunodeficiency virus infection.

Calcium indicator loading of neurons using single-cell electroporation

Studies of subcellular Ca(2+) signaling rely on methods for labeling cells with fluorescent Ca(2+) indicator dyes. In this study, we demonstrate the use of single-cell electroporation for Ca(2+) indicator loading of individual neurons and small neuronal networks in rat neocortex in vitro and in vivo. Brief voltage pulses were delivered through glass pipettes positioned close to target cells. This approach resulted in reliable and rapid (within seconds) loading of somata and subsequent complete labeling of dendritic and axonal arborizations. By using simultaneous whole-cell recordings in brain slices, we directly addressed the effect of electroporation on neurons. Cell viability was high (about 85%) with recovery from the membrane permeabilization occurring within a minute. Electrical properties of recovered cells were indistinguishable before and after electroporation. In addition, Ca(2+) transients with normal appearance could be evoked in dendrites, spines, and axonal boutons of electroporated cells. Using negative-stains of somata, targeted single-cell electroporation was equally applicable in vivo. We conclude that electroporation is a simple approach that permits Ca(2+) indicator loading of multiple cells with low background staining within a short amount of time, which makes it especially well suited for functional imaging of subcellular Ca(2+) dynamics in small neuronal networks.

A guide to sparkology: the taxonomy of elementary cellular Ca2+ signaling events

Since the discovery of the Ca(2+) spark as an elementary event of cellular Ca(2+) signaling almost 15 years ago, the family of newly described Ca(2+) signal entities has been ever growing. While scientists working in Ca(2+) signaling may have maintained an overview over the specifics of this nomenclature, those outside the field often make the complaint that they feel hopelessly lost. With the present review we collect and summarize systematic information on the many Ca(2+) signaling events described in a variety of tissues and cells, and we emphasize why and how each of them has its own importance. Most of these signals are taking place in the cytosol of the respective cells, but several events have been recorded from intracellular organelles as well, where they may serve their own specific functions. Finally, we also try to convey an integrated view as to why cellular microdomain signaling is of fundamental biological importance.

Dystrophic cardiomyopathy: amplification of cellular damage by Ca2+ signalling and reactive oxygen species-generating pathways

AIMS: Cardiac myopathies are the second leading cause of death in patients with Duchenne and Becker muscular dystrophy, the two most common and severe forms of a disabling striated muscle disease. Although the genetic defect has been identified as mutations of the dystrophin gene, very little is known about the molecular and cellular events leading to progressive cardiac muscle damage. Dystrophin is a protein linking the cytoskeleton to a complex of transmembrane proteins that interact with the extracellular matrix. The fragility of the cell membrane resulting from the lack of dystrophin is thought to cause an excessive susceptibility to mechanical stress. Here, we examined cellular mechanisms linking the initial membrane damage to the dysfunction of dystrophic heart. METHODS AND RESULTS: Cardiac ventricular myocytes were enzymatically isolated from 5- to 9-month-old dystrophic mdx and wild-type (WT) mice. Cells were exposed to mechanical stress, applied as osmotic shock. Stress-induced cytosolic and mitochondrial Ca(2+) signals, production of reactive oxygen species (ROS), and mitochondrial membrane potential were monitored with confocal microscopy and fluorescent indicators. Pharmacological tools were used to scavenge ROS and to identify their possible sources. Osmotic shock triggered excessive cytosolic Ca(2+) signals, often lasting for several minutes, in 82% of mdx cells. In contrast, only 47% of the WT cardiomyocytes responded with transient and moderate intracellular Ca(2+) signals. On average, the reaction was 6-fold larger in mdx cells. Removal of extracellular Ca(2+) abolished these responses, implicating Ca(2+) influx as a trigger for abnormal Ca(2+) signalling. Our further experiments revealed that osmotic stress in mdx cells produced an increase in ROS production and mitochondrial Ca(2+) overload. The latter was followed by collapse of the mitochondrial membrane potential, an early sign of cell death. CONCLUSION: Overall, our findings reveal that excessive intracellular Ca(2+) signals and ROS generation link the initial sarcolemmal injury to mitochondrial dysfunctions. The latter possibly contribute to the loss of functional cardiac myocytes and heart failure in dystrophy. Understanding the sequence of events of dystrophic cell damage and the deleterious amplification systems involved, including several positive feed-back loops, may allow for a rational development of novel therapeutic strategies.

CSTX-1, a toxin from the venom of the hunting spider Cupiennius salei, is a selective blocker of L-type calcium channels in mammalian neurons

The inhibitor cystine-knot motif identified in the structure of CSTX-1 from Cupiennius salei venom suggests that this toxin may act as a blocker of ion channels. Whole-cell patch-clamp experiments performed on cockroach neurons revealed that CSTX-1 produced a slow voltage-independent block of both mid/low- (M-LVA) and high-voltage-activated (HVA) insect Ca(v) channels. Since C. salei venom affects both insect as well as rodent species, we investigated whether Ca(v) channel currents of rat neurons are also inhibited by CSTX-1. CSTX-1 blocked rat neuronal L-type, but no other types of HVA Ca(v) channels, and failed to modulate LVA Ca(v) channel currents. Using neuroendocrine GH3 and GH4 cells, CSTX-1 produced a rapid voltage-independent block of L-type Ca(v) channel currents. The concentration-response curve was biphasic in GH4 neurons and the subnanomolar IC(50) values were at least 1000-fold lower than in GH3 cells. L-type Ca(v) channel currents of skeletal muscle myoballs and other voltage-gated ion currents of rat neurons, such as I(Na(v)) or I(K(v)) were not affected by CSTX-1. The high potency and selectivity of CSTX-1 for a subset of L-type channels in mammalian neurons may enable the toxin to be used as a molecular tool for the investigation of this family of Ca(v) channels.

Evaluation of 41calcium as a new approach to assess changes in bone metabolism: effect of a bisphosphonate intervention in postmenopausal women with low bone mass

A new technique was evaluated to identify changes in bone metabolism directly at high sensitivity through isotopic labeling of bone Ca. Six women with low BMD were labeled with 41Ca up to 700 days and treated for 6 mo with risedronate. Effect of treatment on bone could be identified using 41Ca after 4-8 wk in each individual. INTRODUCTION: Isotopic labeling of bone using 41Ca, a long-living radiotracer, has been proposed as an alternative approach for measuring changes in bone metabolism to overcome current limitations of available techniques. After isotopic labeling of bone, changes in urinary 41Ca excretion reflect changes in bone Ca balance. The aim of this study was to validate this new technique against established measures. Changes in bone Ca balance were induced by giving a bisphosphonate. MATERIALS AND METHODS: Six postmenopausal women with diagnosed osteopenia/osteoporosis received a single oral dose of 100 nCi 41Ca for skeleton labeling. Urinary 41Ca/40Ca isotope ratios were monitored by accelerator mass spectrometry up to 700 days after the labeling process. Subjects received 35 mg risedronate per week for 6 mo. Effect of treatment was monitored using the 41Ca signal in urine and parallel measurements of BMD by DXA and biochemical markers of bone metabolism in urine and blood. RESULTS: Positive response to treatment was confirmed by BMD measurements, which increased for spine by +3.0% (p = 0.01) but not for hip. Bone formation markers decreased by -36% for bone alkaline phosphatase (BALP; p = 0.002) and -59% for procollagen type I propeptides (PINP; p = 0.001). Urinary deoxypyridinoline (DPD) and pyridinoline (PYD) were reduced by -21% (p = 0.019) and -23% (p = 0.009), respectively, whereas serum and urinary carboxy-terminal teleopeptides (CTXs) were reduced by -60% (p = 0.001) and -57.0% (p = 0.001), respectively. Changes in urinary 41Ca excretion paralleled findings for conventional techniques. The urinary 41Ca/40Ca isotope ratio was shifted by -47 +/- 10% by the intervention. Population pharmacokinetic analysis (NONMEM) of the 41Ca data using a linear three-compartment model showed that bisphosphonate treatment reduced Ca transfer rates between the slowly exchanging compartment (bone) and the intermediate fast exchanging compartment by 56% (95% CI: 45-58%). CONCLUSIONS: Isotopic labeling of bone using 41Ca can facilitate human trials in bone research by shortening of intervention periods, lowering subject numbers, and having easier conduct of cross-over studies compared with conventional techniques.

The long-term effects of whole-tree harvest at final felling on soil properties in a Norway Spruce (Picea Abies (L) Karst.) Stand

Increased demand for forest-derived biomass has resulted in changes in harvest intensities in Finland. Conventional stem-only harvest (CH) has to some extent been replaced with whole-tree harvest (WTH). The latter involves a greater removal of nutrients from the forest ecosystem, as all the above ground biomass is exported from the site. This has raised concerns that WTH could result in large changes in the nutrient dynamics of a forest stand and could eventually lower its site productivity. Little empirical data exists to support this assumption as only a limited number of studies have been conducted on the topic. A majority of these discuss the short-term effects, thus the long-term consequences remain unknown. The objective of this study was to compare differences in soil properties after CH and WTH in a fertile Norway spruce (Picea abies (L) Karst.) stand in Southern Finland. The site was clear-felled in August 2000 and spruce seedlings were planted in the following summer. Soil sampling in the form of systematic randomized sampling was carried out in May 2011. Changes in base saturation, cation exchange capacity, elemental pools (total and exchangeable) and acidity were studied in both organic and mineral horizons. The results indicate that WTH lowered effective cation exchange capacity and base saturation particularly in the humus layer. The pools of exchangeable Al and Fe were increased in the humus layer, whereas the amount of exchangeable Ca decreased in both layers. WTH also resulted in lower Ca/Al-ratios across the sampled layers. Treatment did not have a significant effect on pH, total pools of elements or on the C/N-ratio of the soil. The results suggest that although the stand possesses significant pools of nutrients at present, WTH, if continued, could have long-term effects on site productivity.

Cement-augmented anterior screw fixation of Type II odontoid fractures in elderly patients with osteoporosis

BACKGROUND CONTEXT: Closed reduction and internal fixation by an anterior approach is an established option for operative treatment of displaced Type II odontoid fractures. In elderly patients, however, inadequate screw purchase in osteoporotic bone can result in severe procedure-related complications. PURPOSE: To improve the stability of odontoid fracture screw fixation in the elderly using a new technique that includes injection of polymethylmethacrylat (PMMA) cement into the C2 body. STUDY DESIGN: Retrospective review of hospital and outpatient records as well as radiographs of elderly patients treated in a university hospital department of orthopedic surgery. PATIENT SAMPLE: Twenty-four elderly patients (8 males and 16 females; mean age, 81 years; range, 62-98 years) with Type II fractures of the dens. OUTCOME MEASURES: Complications, cement leakage (symptomatic/asymptomatic), operation time, loss of reduction, pseudarthrosis and revision surgery, patient complaints, return to normal activities, and signs of neurologic complications were all documented. METHODS: After closed reduction and anterior approach to the inferior border of C2, a guide wire is advanced to the tip of the odontoid under biplanar fluoroscopic control. Before the insertion of one cannulated, self-drilling, short thread screws, a 12 gauge Yamshidi cannula is inserted from anterior and 1 to 3 mL of high-viscosity PMMA cement is injected into the anteroinferior portion of the C2 body. During polymerization of the cement, the screws are further inserted using a lag-screw compression technique. The cervical spine then is immobilized with a soft collar for 8 weeks postoperatively. RESULTS: Anatomical reduction of the dens was achieved in all 24 patients. Mean operative time was 64 minutes (40-90 minutes). Early loss of reduction occurred in three patients, but revision surgery was indicated in only one patient 2 days after primary surgery. One patient died within the first eight postoperative weeks, one within 3 months after surgery. In five patients, asymptomatic cement leakage was observed (into the C1-C2 joint in three patients, into the fracture in two). Conventional radiologic follow-up at 2 and 6 months confirmed anatomical healing in 16 of the19 patients with complete follow-up. In two patients, the fractures healed in slight dorsal angulation; one patient developed a asymptomatic pseudarthrosis. All patients were able to resume their pretrauma level of activity. CONCLUSIONS: Cement augmentation of the screw in Type II odontoid fractures in elderly patients is technically feasible in a clinical setting with a low complication rate. This technique may improve screw purchase, especially in the osteoporotic C2 body.

The risk of early mortality of polytrauma patients associated to ISS, NISS, APACHE II values and prothrombin time

BACKGROUND: The early hemodynamic normalization of polytrauma patients may lead to better survival outcomes. The aim of this study was to assess the diagnostic quality of trauma and physiological scores from widely used scoring systems in polytrauma patients. METHODS: In total, 770 patients with ISS > 16 who were admitted to a trauma center within the first 24 hours after injury were included in this retrospective study. The patients were subdivided into three groups: those who died on the day of admission, those who died within the first three days, and those who survived for longer than three days. ISS, NISS, APACHE II score, and prothrombin time were recorded at admission. RESULTS: The descriptive statistics for early death in polytrauma patients who died on the day of admission, 1--3 days after admission, and > 3 days after admission were: ISS of 41.0, 34.0, and 29.0, respectively; NISS of 50.0, 50.0, and 41.0, respectively; APACHE II score of 30.0, 25.0, and 15.0, respectively; and prothrombin time of 37.0%, 56.0%, and 84%, respectively. These data indicate that prothrombin time (AUC: 0.89) and APACHE II (AUC: 0.88) have the greatest prognostic utility for early death. CONCLUSION: The estimated densities of the scores may suggest a direction for resuscitative procedures in polytrauma patients.Trial registration: "Retrospektive Analysen in der Chirurgischen Intensivmedizin" StV01-2008.http://www.kek.zh.ch/internet/gesundheitsdirektion/kek/de/home.html.

Biochemical, single-channel, whole-cell patch clamp, and pharmacological analyses of endogenous TRPM4 channels in HEK293 cells

Human embryonic kidney cells 293 (HEK293) are widely used as cellular heterologous expression systems to study transfected ion channels. This work characterizes the endogenous expression of TRPM4 channels in HEK293 cells. TRPM4 is an intracellular Ca(2+)-activated non-selective cationic channel expressed in many cell types. Western blot analyses have revealed the endogenous expression of TRPM4. Single channel 22pS conductance with a linear current-voltage relationship was observed using the inside-out patch clamp configuration in the presence of intracellular Ca(2+). The channels were permeable to the monovalent cations Na(+) and K(+), but not to Ca(2+). The open probability was voltage-dependent, being higher at positive potentials. Using the whole-cell patch clamp "ruptured patch" configuration, the amplitude of the intracellular Ca(2+)-activated macroscopic current was dependent on time after patch rupture. Initial transient activation followed by a steady-increase reaching a plateau phase was observed. Biophysical analyses of the macroscopic current showed common properties with those from HEK293 cells stably transfected with human TRPM4b, with the exception of current time course and Ca(2+) sensitivity. The endogenous macroscopic current reached the plateau faster and required 61.9±3.5μM Ca(2+) to be half-maximally activated versus 84.2±1.5μM for the transfected current. The pharmacological properties, however, were similar in both conditions. One hundred μM of flufenamic acid and 9-phenanthrol strongly inhibited the endogenous current. Altogether, the data demonstrate the expression of endogenous TRMP4 channels in HEK293 cells. This observation should be taken into account when using this cell line to study TRPM4 or other types of Ca(2+)-activated channels.

Catheter removal versus retention in the management of catheter-associated enterococcal bloodstream infections

BACKGROUND Enterococci are an important cause of central venous catheter (CVC)-associated bloodstream infections (CA-BSI). It is unclear whether CVC removal is necessary to successfully manage enterococcal CA-BSI. METHODS A 12-month retrospective cohort study of adults with enterococcal CA-BSI was conducted at a tertiary care hospital; clinical, microbiological and outcome data were collected. RESULTS A total of 111 patients had an enterococcal CA-BSI. The median age was 58.2 years (range 21 to 94 years). There were 45 (40.5%) infections caused by Entercoccus faecalis (among which 10 [22%] were vancomycin resistant), 61 (55%) by Enterococcus faecium (57 [93%] vancomycin resistant) and five (4.5%) by other Enterococcus species. Patients were treated with linezolid (n=51 [46%]), vancomycin (n=37 [33%]), daptomycin (n=11 [10%]), ampicillin (n=2 [2%]) or quinupristin/dalfopristin (n=2 [2%]); seven (n=6%) patients did not receive adequate enterococcal treatment. Additionally, 24 (22%) patients received adjunctive gentamicin treatment. The CVC was retained in 29 (26.1%) patients. Patients with removed CVCs showed lower rates of in-hospital mortality (15 [18.3%] versus 11 [37.9]; P=0.03), but similar rates of recurrent bacteremia (nine [11.0%] versus two (7.0%); P=0.7) and a similar post-BSI length of hospital stay (median days [range]) (11.1 [1.7 to 63.1 days] versus 9.3 [1.9 to 31.8 days]; P=0.3). Catheter retention was an independent predictor of mortality (OR 3.34 [95% CI 1.21 to 9.26]). CONCLUSIONS To the authors' knowledge, the present article describes the largest enterococcal CA-BSI series to date. Mortality was increased among patients who had their catheter retained. Additional prospective studies are necessary to determine the optimal management of enterococcal CA-BSI.

Risk of very early recurrent cerebrovascular events in symptomatic carotid artery stenosis

OBJECT The risk of recurrence of cerebrovascular events within the first 72 hours of admission in patients hospitalized with symptomatic carotid artery (CA) stenoses and the risks and benefits of emergency CA intervention within the first hours after the onset of symptoms are not well known. Therefore, the authors aimed to assess (1) the ipsilateral recurrence rate within 72 hours of admission, in the period from 72 hours to 7 days, and after 7 days in patients presenting with nondisabling stroke, transient ischemic attack (TIA), or amaurosis fugax (AF), and with an ipsilateral symptomatic CA stenosis of 50% or more, and (2) the risk of stroke in CA interventions within 48 hours of admission versus the risk in interventions performed after 48 hours. METHODS Ninety-four patients were included in this study. These patients were admitted to hospital within 48 hours of a nondisabling stroke, TIA, or AF resulting from a symptomatic CA stenosis of 50% or more. The patients underwent carotid endarterectomy (85 patients) or CA stenting (9 patients). At baseline, the cardiovascular risk factors of the patients, the degree of symptomatic CA stenosis, and the type of secondary preventive treatment were assessed. The in-hospital recurrence rate of stroke, TIA, or AF ipsilateral to the symptomatic CA stenosis was determined for the first 72 hours after admission, from 72 hours to 7 days, and after 7 days. Procedure-related cerebrovascular events were also recorded. RESULTS The median time from symptom onset to CA intervention was 5 days (interquartile range 3.00-9.25 days). Twenty-one patients (22.3%) underwent CA intervention within 48 hours after being admitted. Overall, 15 recurrent cerebrovascular events were observed in 12 patients (12.8%) in the period between admission and CA intervention: 3 strokes (2 strokes in progress and 1 stroke) (3.2%), 5 TIAs (5.3%), and 1 AF (1.1%) occurred within the first 72 hours (total 9.6%) of admission; 1 TIA (1.1%) occurred between 72 hours and 7 days, and 5 TIAs (5.3%) occurred after more than 7 days. The corresponding actuarial cerebrovascular recurrence rates were 11.4% (within 72 hours of admission), 2.4% (between 72 hours and 7 days), and 7.9% (after 7 days). Among baseline characteristics, no predictive factors for cerebrovascular recurrence were identified. Procedure-related cerebrovascular events occurred at a rate of 4.3% (3 strokes and 1 TIA), and procedures performed within the first 48 hours and procedures performed after 48 hours had a similar frequency of these events (4.5% vs. 4.1%, respectively; p = 0.896). CONCLUSIONS The in-hospital recurrence of cerebrovascular events was quite low, but all recurrent strokes occurred within 72 hours. The risk of stroke associated with a CA intervention performed within the first 48 hours was not increased compared with that for later interventions. This raises the question of the optimal timing of CA intervention in symptomatic CA stenosis. To answer this question, more data are needed, preferably from large randomized trials.

In situ dehydration behavior of zeolite-like pentagonite: A single-crystal X-ray study

The structural modifications upon heating of pentagonite, Ca(VO)(Si4O10)·4H2O (space group Ccm21, a=10.3708(2), b=14.0643(2), c=8.97810(10) Å, V=1309.53(3) Å3) were investigated by in situ temperature dependent single-crystal X-ray structure refinements. Diffraction data of a sample from Poona district (India) have been measured in steps of 25 up to 250 °C and in steps of 50 °C between 250 and 400 °C. Pentagonite has a porous framework structure made up by layers of silicate tetrahedra connected by V4+O5 square pyramids. Ca and H2O molecules are extraframework occupants. Room temperature diffraction data allowed refinement of H positions. The hydrogen-bond system links the extraframework occupants to the silicate layers and also interconnects the H2O molecules located inside the channels. Ca is seven-fold coordinated forming four bonds to O of the tetrahedral framework and three bonds to extraframework H2O. The H2O molecule at O9 showing a high displacement parameter is not bonded to Ca. The dehydration in pentagonite proceeds in three steps. At 100 °C the H2O molecule at O8 was released while O9 moved towards Ca. As a consequence the displacement parameter of H2O at O9 halved compared to that at room temperature. The unit-cell volume decreased to 1287.33(3) Å3 leading to a formula with 3H2O per formula unit (pfu). Ca remained seven-fold coordinated. At 175 °C Ca(VO)(Si4O10)·3H2O transformed into a new phase with 1H2O molecule pfu characterized by doubling of the c axis and the monoclinic space group Pn. Severe bending of specific TOT angles led to contraction of the porous three-dimensional framework. In addition, H2O at O9 was expelled while H2O at O7 approached a position in the center of the channel. The normalized volume decreased to 1069.44(9) Å3. The Ca coordination reduced from seven- to six-fold. At 225 °C a new anhydrous phase with space group Pna21 but without doubling of c had formed. Release of H2O at O7 caused additional contraction of TOT angles and volume reduction (V=1036.31(9) Å3). Ca adopted five-fold coordination. During heating excursion up to 400 °C this anhydrous phase remained preserved. Between room temperature and 225 °C the unit-cell volume decreased by 21% due to dehydration. The dehydration steps compare well with the thermo-gravimetric data reported in the literature.

In situ dehydration behavior of zeolite-like cavansite: A single-crystal X-ray study

To track dehydration behavior of cavansite, Ca(VO)(Si4O10)·4H2O space group Pnma, a = 9.6329(2), b = 13.6606(2), c = 9.7949(2) Å, V = 1288.92(4) Å3 single-crystal X-ray diffraction data on a crystal from Wagholi quarry, Poona district (India) were collected up to 400 °C in steps of 25 °C up to 250 °C and in steps of 50 °C between 250 and 400 °C. The structure of cavansite is characterized by layers of silicate tetrahedra connected by V4+O5 square pyramids. This way a porous framework structure is formed with Ca and H2O as extraframework occupants. At room temperature, the hydrogen bond system was analyzed. Ca is eightfold coordinated by four bonds to O of the framework structure and four bonds to H2O molecules. H2O linked to Ca is hydrogen bonded to the framework and also to adjacent H2O molecules. The dehydration in cavansite proceeds in four steps.At 75 °C, H2O at O9 was completely expelled leading to 3 H2O pfu with only minor impact on framework distortion and contraction V = 1282.73(3) Å3. The Ca coordination declined from originally eightfold to sevenfold and H2O at O7 displayed positional disorder.At 175 °C, the split O7 sites approached the former O9 position. In addition, the sum of the three split positions O7, O7a, and O7b decreased to 50% occupancy yielding 2 H2O pfu accompanied by a strong decrease in volume V = 1206.89(8) Å3. The Ca coordination was further reduced from sevenfold to sixfold.At 350 °C, H2O at O8 was released leading to a formula with 1 H2O pfu causing additional structural contraction (V = 1156(11) Å3). At this temperature, Ca adopted fivefold coordination and O7 rearranged to disordered positions closer to the original O9 H2O site.At 400 °C, cavansite lost crystallinity but the VO2+ characteristic blue color was preserved. Stepwise removal of water is discussed on the basis of literature data reporting differential thermal analyses, differential thermo-gravimetry experiments and temperature dependent IR spectra in the range of OH stretching vibrations.

Posttranslational modifications of cardiac ryanodine receptors: Ca2+ signaling and EC-coupling

In cardiac muscle, a number of posttranslational protein modifications can alter the function of the Ca(2+) release channel of the sarcoplasmic reticulum (SR), also known as the ryanodine receptor (RyR). During every heartbeat RyRs are activated by the Ca(2+)-induced Ca(2+) release mechanism and contribute a large fraction of the Ca(2+) required for contraction. Some of the posttranslational modifications of the RyR are known to affect its gating and Ca(2+) sensitivity. Presently, research in a number of laboratories is focused on RyR phosphorylation, both by PKA and CaMKII, or on RyR modifications caused by reactive oxygen and nitrogen species (ROS/RNS). Both classes of posttranslational modifications are thought to play important roles in the physiological regulation of channel activity, but are also known to provoke abnormal alterations during various diseases. Only recently it was realized that several types of posttranslational modifications are tightly connected and form synergistic (or antagonistic) feed-back loops resulting in additive and potentially detrimental downstream effects. This review summarizes recent findings on such posttranslational modifications, attempts to bridge molecular with cellular findings, and opens a perspective for future work trying to understand the ramifications of crosstalk in these multiple signaling pathways. Clarifying these complex interactions will be important in the development of novel therapeutic approaches, since this may form the foundation for the implementation of multi-pronged treatment regimes in the future. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.