Importantly, these acini displayed reduction in IP3R2 and IP3R3, but not AQP5 or STIM1. Similar decreases in IP3R and carbachol (CCh)-stimulated [Ca2+]i elevation were detected in acinar cells from lymphotoxin-alpha (LTα) transgenic (TG) mice, a model for (SS). Treatment of salivary glands from healthy individuals with LT α, a cytokine linked to disease progression in SS and IL14α mice, reduced Ca2+ signaling. Together, our findings reveal novel IP3R deficits in acinar cells that underlie secretory dysfunction in SS patients.The autoimmune exocrinopathy, Sjögren’s syndrome (SS), is associated with secretory defects in patients, including individuals with mild lymphocytic infiltration and minimal glandular damage. The mechanism(s) underlying the secretory dysfunction is not known. We have used minor salivary gland biopsies from SS patients and healthy individuals to assess acinar cell function in morphologically intact glandular areas. We report that agonist-regulated intracellular Ca Primary Sjögren’s syndrome (pSS) is a chronic autoimmune disease involving lymphocytic infiltration and loss of secretory function in salivary and lacrimal glands56A major and unresolved conundrum in the pathophysiology of (SS) has been the lack of correlation between salivary flow and extent of inflammation or tissue damage6Efforts to delineate the initial molecular alterations that underlie the secretory defect have been complicated by the fact that pSS is a slowly progressing disease, the early stages of which have proved difficult to identify. There are relatively few animal models that recapitulate the phenotype of disease progression, especially loss of saliva flow in absence of significant inflammation and glandular damage1011122+] ([Ca2+]i) in acinar cells, the primary site of secretion in the gland, which leads to activation of ion channels and transporters which together generate the osmotic gradient required to drive water from the cell15162+]i is achieved as a result of intracellular Ca2+ release from the endoplasmic reticulum as well as Ca2+ entry into the acinar cells, both of which are activated in response to stimulation of the plasma membrane receptors, such as muscarinic or alpha-adrenergic receptors. Such stimuli result in hydrolysis of phosphatidylinositol 4,5 bisphosphate (PIP2) and generation of inositol 1, 4, 5 trisphosphate (IP3)2+ from the ER lumen2+ [Ca2+]i tends to be transient in the absence of extracellular Ca2+. A sustained elevation of [Ca2+]i, which is critical for prolonged saliva flow, requires Ca2+ entry,202+ entry in acinar cells, the site of water movement in the gland, is primarily mediated via the store-operated Ca2+ entry (SOCE) mechanism, which is activated by the decrease in ER-[Ca2+]. SOCE is regulated by the ER protein, STIM1 , which senses the decrease in ER-[Ca2+] and interacts with plasma membrane calcium channels, Orai1 and TRPC1, resulting in their activation. Ca2+ entry via these channels is required for maintaining the increase in [Ca2+]i critical for the regulation of fluid secretion222+]i activates key K+ and Cl- channels that together generate the osmotic gradient required to drive fluid secretion via the water channel, AQP5. Thus, intracellular Ca2+ release, via IP3R, in acinar cells represents the primary, critical step in neurotransmitter-regulation of fluid secretion.Salivary glands mediate vectorial fluid secretion in response to stimulation by neurotransmitters. The key triggering event is neurotransmitter induced increase in cytosolic i increases in acinar cells by measuring changes in Fluo2 fluorescence in lobule preparations from seven HV and eleven pSS patients. Agonist-stimulated intracellular Ca2+ release was initiated by stimulating cells with 1 μM CCh in the absence of extracellular calcium, measured as a transient increase in fluorescence. Subsequent addition of 1mM calcium to the external medium triggers a second, more sustained, increase in fluorescence due to Ca2+ entry. 2+]i changes in acinar cells from pSS patients and HV showing the calcium release and entry phases. CCh-stimulated intracellular Ca2+ release was significantly reduced  in pSS acini compared to that in HV acini (2+ entry component as compared to that in HV cells (2+ entry in acinar cells is mediated primarily via the store-operated Ca2+ entry pathway (SOCE)2+ entry detected in the pSS patients is most likely due to a reduction in intracellular Ca2+ release. Thus, while we cannot exclude that conditions associated with pSS might directly affect mechanism and components involved Ca2+ entry, we have not examined Ca2+ entry in further detail.The primary determinant of cell volume decrease in response to agonist stimulation is an increase in cytosolic calcium. To determine whether defects in CaHV acini . Cells fHV cells . Since C2+]i increase in response to varying [CCh], ranging from 50 nM to 100 μM, was measured in eight pSS patients and five HV. Cells from pSS patients showed a significant reduction (about 50%) in [Ca2+]i increase at all [CCh] tested compared to the responses in cells from HV  (p = 0.0166) as well as Ca2+ influx (Spearman r = 0.6388) (p = 0.0043) (2+ release (Spearman r = 0.5741) (p = 0.0127) as well as calcium influx (Spearman r = 0.5844) (p = 0.0109)  . Again,  0.0109) . Since C2+ release in salivary gland acinar cells. Lack of these receptors in mice led to a complete loss of fluid secretion282930IP3R2 and IP3R3 are critical for mediating intracellular Ca2+ entry and fluid secretion function within site of infiltration. Importantly, our findings reveal a novel deficit in IP3Rs in pSS patients that is detected in acinar cells within the relatively normal areas of the pSS gland that are away from the site of infiltration. Although we cannot completely rule out effects due to other possible contributing factors, the present findings strongly suggest that the decrease in IP3Rs in acinar cells can be the major underlying cause for the decrease in salivary flow in patients with low focus scores.In aggregate, these findings demonstrate that STIM1 and AQP5 are reduced within the area of infiltration (IF) in glands from pSS patients, but not in areas away from the infiltration site. Thus, loss of these proteins can only contribute to loss of Ca2+]i measurements were performed in fluo2-loaded lobules isolated from 10 month old IL14α-TG mice or age, sex and genetic background matched control animals as described in Materials and Methods. The magnitude of the CCh-induced peak in [Ca2+]i measured in lobules isolated from female mice were markedly reduced at sub-maximal stimulation  underlying exocrine gland dysfunction and pathogenesis in pSS patients is unknown. While destruction of glandular structure due to widespread lymphocytic infiltration can contribute to the decrease of salivary fluid secretion, it does not explain the loss of secretion in the majority of patients who have low, or negligible, levels of inflammation. Salivary glands in these patients are largely intact, without overt damage to majority of the acinar cells. The data we present here reveal that agonist-stimulated Ca2+]i increases and IP3R activation is the critical first step in his process, as it also governs the activation of Ca2+ entry2+ mobilization and fluid secretion; STIM1, and IP3R, and AQP5, are disrupted within, or immediately around the area of infiltration; where there is considerable tissue damage, only IP3Rs are significantly decreased in the relatively intact areas of the gland where there is no infiltration. While we cannot rule out additional defects in components involved in mediating and regulating Ca2+ entry, the observed loss of IP3Rs in acinar cells located in relatively intact areas of the gland in patients with low inflammatory scores can account for the decrease in CCh-stimulated intracellular Ca2+ release and Ca2+ entry, CCh-stimulated reduction in cell volume, and consequently, saliva secretion. Based on previous studies with the IL14α-TG mice and the role of LTα in pSS112+ signals suggests that important signaling events regulated by LTα might be involved. LTα acts on TNF receptors leading to a multitude of signaling pathways including activation of NFAT, caspases, MAP kinase cascades, and NFκB-mediated transcriptional pathways35Water secretion from acinar cells is completely dependent on agonist-stimulated i increases and Ca2+-dependent ion channel activation at relatively low [agonist] in pSS cells compared to healthy patients. However, as shown by our data, Ca2+ signaling in salivary gland acini from FS = 1 patients was dampened across all concentrations of (CCh) tested. Notably, [Ca2+]i signaling in cells from IL14α-TG glands, appeared to recover at higher (CCh). Further studies will be required to evaluate whether the agonist sensitivity of the glands in pSS decreases temporally following disease onset. The most novel and important finding in the present study is that salivary gland acinar cells from morphologically intact areas of the glands from SS patients demonstrate a loss of IP3R3 and a mislocalization of IP3R2. To our knowledge none of the previously reported studies have examined acinar IP3Rs in Sjögren’s Syndrome. Consistent with the findings in patients, acini from IL14α-TG mice show a decrease in IP3R2 (IP3R3 have not yet been assessed). Thus, we suggest that IP3R deficit in acinar cells is the underlying cause for the secretory dysfunction in pSS patients who have low levels of inflammation and minimal tissue destruction in the salivary glands. While we have not directly assessed the intracellular Ca2+ store content in salivary glands from pSS patients, our data demonstrate that the Ca2+ store content is similar in acini from WT and IL14α-TG mice. Further studies are required to assess the temporal sequence of events, namely whether mislocalization of the protein precedes the loss. Interestingly, mice lacking IP3R2 and IP3R3 display loss of lacrimal secretion and progressive inflammation in the eyes, as seen in pSS patientsRelatively few studies have examined acinar cell function in salivary glands from pSS patients. A previous report compared cell volume changes in salivary gland cells from pSS and non-pSS patients  but did not evaluate function in healthy volunteers. Further, only response of cells to hypo-osmotic stress was assessed where exposure of cells to a hypo-osmotic medium triggers an increase in cell volume that is followed by regulatory volume decrease2+ signaling and cell volume regulation, which together fully account the loss of saliva secretion. Our findings suggest that a link between LTα and loss of IP3R in pSS-associated secretory dysfunction. Previous studies reported by Ambrus and co-workers demonstrate that IFNα is not responsible for the early stages of Sjögren’s Syndrome, including salivary dysfunction, in the submandibular and lacrimal glandsIn conclusion, we present here novel data that demonstrate a decrease and mislocalization of IP3Rs in minor salivary gland acinar cells from pSS patients. This defect in IP3Rs can account for the accompanying decrease in agonist-stimulated CaAll studies were carried out in accordance with approved NIH guidelines. Human samples were obtained from NIH Institutional Review Board approved protocols . All human experiments including the use of tissue samples were performed after informed consent was obtained from subjects in the Sjögren’s Syndrome Clinic at the National Institute of Dental and Craniofacial Research (NIDCR) at the National Institute of Health (NIH) in Bethesda, MD.246 subjects (HV+ pSS) were included in this study. 24 of the biopsies were from lower labial minor salivary gland from pSS patients who fulfilled the American-European consensus group criteria2, 10 HEPES, 1 MgCl2, 10 glucose, 1% BSA: pH 7.4, gassed with 95% O25% CO2, finely minced and incubated with 1μM calcein-AM  or 10 μM Fluo2-AM  for 30 minutes. Cells were washed with tyrode solution for 30 minutes. The time frame between collection of the biopsy and performing live cell imaging experiments was an average of 2.5 hours. Lack of cell digestion provides a relatively intact in situ system to study salivary gland cell functionThe biopsy was placed in cold tyrode solution (in mM):130 NaCl, 5.4 KCl, 1CaCl2, 10 HEPES, 1 MgCl2, 10 glucose, pH 7.4. The clusters were imaged using a Leica SP2 confocal mounted on a DM IRE2 inverted microscope with 20 × (0.4 NA) dry objective and images were acquired every 1`s. Both Calcein and Fluo2 AM were excited with the 488-nm line of an argon ion laser with emission at 510 nm. In experiments employing multiphoton microscopy, Fluo2 loaded lobules were excited at 810 nm using a Spectra Physics tunable fS pulsed Ti-Sapphire laser controlled by Fluoview software on a Olympus FV1000MP microscope using a 25× water immersion objective (1.03 NA). The clusters were stimulated with 1 μm carbachol (CCh)  and maintained at 37 °C. Changes in Ca2+ in the medium are indicated in the text as is time of CCh addition. Images were acquired at a resolution of 512 × 512 pixels. Regions of interest were selected and fluorescence intensity in that region was determined as a function of time and expressed relative to the initial fluorescence.Dye-loaded lobules were placed in a perfusion chamber on cover slips which were coated with cell tak  in solution containing: 130 NaCl, 5.4 KCl, 1CaClSamples were deparaffinized, rehydrated followed by microwave antigen retrieval for 10 minutes in 1mM EDTA, pH 8.0 with 0.05% Tween 20. Sections were cooled and blocked with 10% donkey serum in PBS with 0.05% BSA for 30 minutes at room temperature, followed by incubation with primary antibody [rabbit anti-AQP5 (Santa Cruz biotech.), rabbit anti-STIM 1  rabbit anti-IP3R3 (Novus) and anti-IP3R2For H&E staining and morphology assessment, paraffin embedded human minor salivary gland sections were melted and then stained with Mayer’s Hematoxolin (Electron Microscopy Sciences) followed by Eosin–Phloxine B (Electron Microscopy Sciences), and washed. Sections were then mounted and cover slipped using Permount (Fisher) and images were acquired.p = 0.05.Data analysis was performed using Origin 9.0 (OriginLab) and PRISM 5. Statistical comparisons were made using Spearman r  and Student t test. P value Significant . Experimental values are expressed as means +/− SEM. Differences in the mean values were considered to be significant at How to cite this article: Teos, L. Y. et al. IP3R deficit underlies loss of salivary fluid secretion in Sjögren's Syndrome. Sci. Rep.5, 13953; doi: 10.1038/srep13953 (2015).


Background: In animal models of heart failure (HF), heart rate decreases due to an increase in intrinsic cycle length (CL) of the sinoatrial node (SAN). Pacemaker activity of SAN cells is complex and modulated by the membrane clock, i.e., the ensemble of voltage gated ion channels and electrogenic pumps and exchangers, and the Ca2+ clock, i.e., the ensemble of intracellular Ca2+ ([Ca2+]i) dependent processes. HF in SAN cells results in remodeling of the membrane clock, but few studies have examined its effects on [Ca2+]i homeostasis.Methods: SAN cells were isolated from control rabbits and rabbits with volume and pressure overload-induced HF. [Ca2+]i concentrations, and action potentials (APs) and Na+–Ca2+ exchange current (INCX) were measured using indo-1 and patch-clamp methodology, respectively.Results: The frequency of spontaneous [Ca2+]i transients was significantly lower in HF SAN cells (3.0 ± 0.1 (n = 40) vs. 3.4 ± 0.1 Hz (n = 45); mean ± SEM), indicating that intrinsic CL was prolonged. HF slowed the [Ca2+]i transient decay, which could be explained by the slower frequency and reduced sarcoplasmic reticulum (SR) dependent rate of Ca2+ uptake. Other [Ca2+]i transient parameters, SR Ca2+ content, INCX density, and INCX-[Ca2+]i relationship were all unaffected by HF. Combined AP and [Ca2+]i recordings demonstrated that the slower [Ca2+]i transient decay in HF SAN cells may result in increased INCX during the diastolic depolarization, but that this effect is likely counteracted by the HF-induced increase in intracellular Na+. β-adrenergic and muscarinic stimulation were not changed in HF SAN cells, except that late diastolic [Ca2+]i rise, a prominent feature of the Ca2+ clock, is lower during β-adrenergic stimulation.Conclusions: HF SAN cells have a slower [Ca2+]i transient decay with limited effects on pacemaker activity. Reduced late diastolic [Ca2+]i rise during β-adrenergic stimulation may contribute to an impaired increase in intrinsic frequency in HF SAN cells. Especially the reduced slope of the LDCAE, with associated decrease in localized Ca2+ releases (LCRs) or Ca2+ sparks  and HF SAN cells using a well-established rabbit model of volume and pressure overload-induced HF. We found a slower spontaneous [Ca2+]i transient frequency in HF SAN cells, indicating that the intrinsic cycle length was prolonged. HF slowed the [Ca2+]i transient decay without changes in diastolic and systolic [Ca2+]i concentrations, LDCAE, and SR Ca2+ content. The reduced [Ca2+]i transient decay velocity was partially, but not completely, explained by the slower intrinsic frequency of HF SAN cells. Neither the INCX density nor the INCX-[Ca2+]i relationship were affected by HF. Combined action potential and [Ca2+]i measurements demonstrated that the decreased [Ca2+]i transient decay velocity has limited effect on INCX during diastolic depolarization.In the present study, we evaluated the [CaDeclaration of Helsinki. HF was induced in 5-month-old male New-Zealand White rabbits by combined volume- and pressure-overload in 2 sequential surgical procedures as described previously . Spindle and elongated spindle-like cells displaying regular contractions were selected for measurements.Single SAN cells were enzymatically isolated from the entire SAN region as described previously  loaded myocytes as described previously in detail  maximum systolic [Ca2+]i concentration, (3) maximum rate of the fast systolic [Ca2+]i rise, (4) time constant of monoexponential [Ca2+]i transient decay, and (5) mean rate of the slow diastolic [Ca2+]i rise during the final third of diastolic depolarization until the fast systolic [Ca2+]i transient. Ten consecutive spontaneous [Ca2+]i transients were used to determine the average parameters. To date, SR Ca2+ content and the rate of Ca2+ uptake into the SR by sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) in cardiac working myocytes are frequently measured using caffeine-induced [Ca2+]i transients combined with simultaneous INCX recordings with the patch clamp methodology  with that of 20 mM caffeine-evoked [Ca2+]i transient in the presence of 5 mM NiCl2, to block the INCX. The rate of decay was obtained by fitting single exponential functions to the decay phase of the caffeine-evoked [Ca2+]i transients.i decline due to Ca2+ efflux through NCX operating in forward mode (net inward INCX)  was present to block If. All drugs were obtained from Sigma  except for E4031, which was a gift from Eisai , and noradrenaline . INCX densities were calculated by dividing current amplitudes by the cell membrane capacitance.Action potentials and INCX on the basis of the simultaneously recorded experimental data on the membrane potential (Vm) and [Ca2+]i . Thus, the rate of spontaneous SAN [Ca2+]i transients is a measure of the intrinsic beating rate. We found a significantly lower frequency of spontaneous [Ca2+]i transients in HF SAN cells [Figure 2+]i transient and the systolic [Ca2+]i transient concentration were not significantly different in HF SAN cells compared with CTRL SAN cells  vs. 57.8 ± 2.6 (CTRL) ms] is ≈41% larger than in CTRL SAN cells. Neither the diastolic [Ca2+]i concentration nor the late diastolic [Ca2+]i rise (diastolic d[Ca2+]i/dt) were significantly different in HF SAN cells compared with CTRL SAN cells  and −26 (slope), respectively, the difference in frequency between HF and CTRL cells explains only a ≈17% difference.In ventricular myocytes, Ca+i transis Figure  is large2+ transient decay of SAN cells, the latter partially explained by the slower frequency in HF SAN cells.From these data we conclude that HF reduces the frequency and slows the Ca2+]i transient decay rate, partially explained by a slower intrinsic frequency. The decline of the [Ca2+]i transient is mainly due to uptake into the SR through SERCA and extrusion of Ca2+ via the sarcolemmal Na+–Ca2+ exchange (NCX), while mitochondrial Ca2+ uptake and sarcolemmal Ca-ATPase may contribute for a very small fraction  vs. 0.22 ± 0.03 (n = 18)]  vs. 3654 ± 406 ms   Figure , indicatt Figure , left an+ Figure , middle + Figure . The pery Figure , right.2+ content nor the mitochondrial Ca2+ uptake and sarcolemmal Ca-ATPase activity of SAN cells.From these data we conclude that HF neither affects the SR CaNCX density was found to be similar in CTRL and HF SAN cells of rabbit  resulting in a [Ca2+]i decline  from the SR late during the diastolic depolarization  concentration, and the membrane potential (Vm)  simultaneously with i transient decay, and (3) a reduced diastolic [Ca2+]i rise during β-adrenergic stimulation, compared to CTRL SAN cells. Combined action potential and [Ca2+]i measurements demonstrated that the decreased [Ca2+]i transient decay in HF SAN cells may result in slightly increased INCX during the diastolic depolarization phase, but that this effect is counteracted by HF-induced increase in [Na+]i.We found that HF SAN cells have (1) a reduced frequency of spontaneous [Ca2+]i transients in HF SAN cells, indicating that intrinsic cycle length was prolonged in HF SAN cells  T-tubular organization, sarcolemmal calcium currents, and action potential duration can be ruled out because T-tubuli are absent in rabbit SAN cells  or Ca2+ sparks  ourselves. However, the used 5 mM [Na+]i is close to the concentrations of 4.5 and 4.0 mM in rabbit multicellular SAN preparations and single SAN cells, respectively, found by Choi et al. (NCX. We could not make use of INCX equations from more recent models, in particular the rabbit SAN cell models by Maltsev and Lakatta (2+ in a sarcolemmal subspace rather than global [Ca2+]i.We reconstructed Ii et al. . In our i et al.  equation Lakatta  and Seve Lakatta , because2+ decay. The decreased [Ca2+]i transient decay in HF SAN cells may result in slightly increased INCX during the diastolic depolarization phase, but this effect is likely counteracted by HF-induced increase in [Na+]i. Reduced late diastolic [Ca2+]i rise during β-adrenergic stimulation may contribute to the impaired increase in frequency under this condition in HF SAN cells.In our rabbit model of HF, SAN cells have reduced SERCA activity and reduced intrinsic frequency, both resulting in a slower CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Contemporary total knee arthroplasty femoral component designs offer various degrees of fit amongst the global population. The purpose of this study was to assess component fit of contemporary femoral component design families against multiple ethnicities.Persona™, B: NexGen®, C: Sigma®, D: GENESIS™ II, E: Triathlon®, F: Vanguard®). Component overhang/underhang was measured between the resected distal femur and its corresponding component size and compared across design families and ethnicities. The severity of overhang/underhang and propensity of downsizing due to clinically significant overhang were quantified for the overall dataset and each ethnicity.Using a multi-ethnic dataset including Caucasian, Indian, and Korean subjects, this study investigated component fit in six contemporary femoral component design families (A: p < 0.02). In all the underhang cases, Designs C and E had significantly greater underhang than the other designs (p < 0.01). Component design influenced the occurrence (% bones) of component downsizing due to clinically significant overhang (>3 mm), with the highest incidence observed in Designs D (20.5 %) and F (17.7 %), and the lowest incidence observed in Designs A (0 %) and B (0.4 %). Variation in component fit was significantly impacted by designs (p < 0.01) but not ethnicities (n.s.).In all the overhang cases, Designs A and B had significantly lower component overhang than the other designs (The inclusion of multiple ML/AP shape offerings and the increased number of available sizes in Design A, as compared to other contemporary femoral component design families studied, result in improved femoral component fit across various ethnicities. Accurate alignment, proper bone cuts, and good soft tissue balancing are all key components that determine the short- and long-term success of total knee arthroplasty (TKA) surgery . HoweverNumerous morphologic studies have demonstrated high variability in the size and shape of the distal femur , 25, 28.Many contemporary TKA implant systems are intended to be used on the global population. There is thus the need for evaluating the morphological fit of contemporary femoral component designs across the global population, to ensure component shapes and sizes are comprehensive. In a 2003 study, Hitt et al.  have shoIn this present work, we developed a set of improved methodologies for quantifying the morphological fit of contemporary femoral TKA designs to multi-ethnic distal femur anatomy. In particular, the study measured component fit across a multi-ethnic dataset spanning Asian and Caucasian subjects, utilizing a fully automated algorithm to properly size and resect distal femora based on a specific surgical referencing philosophy and design-specific surgical resection parameters without requiring manual user intervention. The aim was to evaluate the ability of contemporary TKA designs to match the anatomy of a diverse multi-ethnic patient population, and the impact of ethnicity and design factors (shape and size) on the fit. It was hypothesized that increased shape (ML/AP ratio) and size offerings in TKA femoral component designs will improve their morphological fit to the resected femur.ZiBRA™ Anatomical Modeling System, a proprietary software platform with advanced capabilities for digital orthopaedic morphological analysis [A total of 277 healthy right femora were selected from a dataset that spans a wide range of sizes and includes both Asian and Caucasian ethnicities. A demographic information summary of the subjects is given in Table analysis .Table 1DPersona™ The Personalized Knee System ; (2) Design B: NexGen® Complete Knee Solution ; (3) Design C: Sigma® Knee Solutions ; (4) Design D: GENESIS™ II Total Knee System ; (5) Design E: Triathlon® Knee System ; and (6) Design F: Vanguard® Complete Knee System   Design A: aw, IN);  Design BZiBRA™ System, the AP dimension was digitally and automatically measured on the femora as the projected length from the anterior cortex of the distal femur to the plane tangent to the posterior condyles  tests were performed to compare measurements across ethnicities and designs . The null hypothesis was that all the ethnic or design group means are equal; the level of significance was defined at p < 0.02). For the femora with component underhang, Designs C and E had significantly greater underhang amount than the other designs (p < 0.01).The multiple ML offerings in Design A enabled proper fit to all the bones in the dataset across component AP sizes without a single case of clinically significant overhang (constant availability of component ML size below the overhang incidence bound for the entire dataset) Fig. . SimilarDesigns A and B both captured the shape variability in the resected femur Fig. . In contThree distinct levels of component fit were observed amongst the designs Fig. . Designsp < 0.01), with significant differences in the incidence of downsizing between designs , as they provide more component selections to match the variability in the distal femur aspect ratio than designs with single ML offerings (Designs C–F). This finding agrees with a recent study, which concluded that designs with multiple ML offerings for a given component AP size may improve component-to-bone fit and reduce the propensity of greater than 2-mm component overhang/underhang in Chinese patients . AmongstGood morphological fit between TKA components and the resected knee anatomy is an important factor for success in TKA. Specifically with regard to the femur, AP and ML mismatches are often encountered during the surgical implantation of the femoral component. Several clinical studies have documented component overhang with resulting negative surgical outcomes due to irritation of the soft tissue or overstuffing of the joint space and associated compromise of range of motion , 19. TheNumerous morphologic studies have shown that the dimensions of the distal femur are highly variable and suggested that contemporary femoral component designs may not accommodate morphological differences across ethnicities , 18, 25.There are several limitations to this study. First, though resections were specific to each implant system, ideal distal femoral resections were employed for each design; however, clinical variability in resection parameters has been reported in previous studies , 22. SecAlthough all the measurements performed in this study were based on fully automated computer simulation, the expected resolution of the results is impacted by several aspects of the data pre-processing: (1) accuracy of the automatic annotations of the landmarks depends on the resolution of the CT data, which had sub-millimetre accuracy ; (2) approval of automatically defined landmarks by experienced users introduces some level of inter- and intra-user variability, though this has been shown to be at sub-millimetre level  ; and (3)The clinical implications of the observations in the present study suggest that some contemporary femoral component designs may not accommodate morphological differences across patient populations. This in turn may lead to surgical compromise of femoral bone preparation or clinical complications due to soft tissue impingement, improper balancing of the flexion and extension gaps, and pain. The findings emphasize the importance of properly designing the shape and size of the femoral components to meet the morphological variability of the distal femoral across the global population. Additionally, the results suggest that multiple size and shape offerings can offer improvement of the morphological fit of the femoral components without compromise of soft tissue, joint space balancing, and joint line.Varying degrees of morphological conformity across six contemporary TKA femoral designs were found in this study. The data suggest femoral component designs that provide multiple ML/AP shape offerings, and increased number of available sizes can provide necessary component selection to fit the resected femur adequately in the ethnicities investigated without clinically significant overhang.


Direct measurement of [Ca2+] distributions in dendritic spines is experimentally difficult but we can investigate this hypothesis using mathematical models of Ca2+ diffusion.Capocampus . However2+ diffusion in three dimensions. We then study our model using finite element methods. The model allows predictions of intracellular [Ca2+]-field responses to combinations of pre- and post-synaptic spikes with nanometre and millisecond spatio-temporal resolution. Our results so far indicate that Ca2+ signalling is highly spatially non-uniform and that Ca2+ signal differences between induction protocols is dependent on location within the spine. This has implications for the ultimate biological role of the Ca2+ signal given that the relevant receptors in the spine are organised inhomogeneously [We have developed a spatio-temporal model of Caeneously .


We found that ortho-substituted 2,2’,6-trichlorinated biphenyl (PCB19) caused a rapid decline in the Ca2+ signaling of bradykinin, a typical Gq- and phospholipase Cβ-coupled GPCR, without any effect on its inositol 1,4,5-trisphosphate production. PCB19 reduced thapsigargin-induced sustained cytosolic Ca2+ levels, suggesting that PCB19 inhibits SOCE. The abilities of other NDL-PCBs to inhibit store-operated Ca2+ entry (SOCE) were also examined and found to be of similar potencies to that of PCB19. PCB19 also showed a manner equivalent to that of known SOCE inhibitors. PCB19-mediated SOCE inhibition was confirmed by demonstrating the ability of PCB19 to inhibit the SOCE current and thapsigargin-induced Mn2+ influx. These results imply that one of the molecular mechanism by which NDL-PCBs cause neurobehavioral disturbances involves NDL-PCB-mediated inhibition of SOCE, thereby interfering with GPCR-mediated Ca2+ signaling.Polychlorinated biphenyls (PCBs) are ubiquitous pollutants which accumulate in the food chain. Recently, several molecular mechanisms by which non-dioxin-like (NDL) PCBs mediate neurodevelopmental and neurobehavioral toxicity have been elucidated. However, although the G-protein coupled receptor (GPCR) is a significant target for neurobehavioral disturbance, our understanding of the effects of PCBs on GPCR signaling remains unclear. In this study, we investigated the effects of NDL-PCBs on GPCR-mediated Ca Polychlorinated biphenyls (PCBs) are some of the most ubiquitous environmental contaminants presently originating from industry. Their characteristics (e.g. inflammability and electric conductivity) have brought them to a range of applications such as coolants, flame retardants, and hydraulic fluids , 2. Furtmeta or para position of the phenyl ring show coplanar or dihedral molecular shape. On the other hand, NDL-PCBs are molecules containing chlorine substitutions at the ortho position of the phenyl ring and exhibiting a toxicological profile distinct from that of DL-PCBs i increases in PC12 cells, and confirmed the previous finding that 50 μM PCB19 induces a sustained increase in intracellular Ca2+ levels  = 3.653)  nor TCDD (AhR-activating dioxin) stimulated any Ca2+ increase by themselves, and both were also less effective than PCB19 to inhibit subsequent bradykinin-induced Ca2+ increase  . These dincrease .2+]i through a mechanism involving inositol 1,4,5-trisphosphate (InsP3)-dependent Ca2+ release from internal stores and subsequent SOCE from the extracellular space i because of the interplay between Ca2+ release and SOCE. In this condition, PCB19 decreased thapsigargin-induced sustained Ca2+ levels  [+/Ca2+ exchangers (NCX) [2+ influx. To test the possibility of an indirect SOCE inhibition via a fast recovery of [Ca2+]i levels, we used bepridil (Na+ site specific NCX inhibitor), and caloxin (isoform-nonselective PMCA inhibitor). Interestingly PCB19-mediated SOCE inhibition is not affected by the presence of bepridil or caloxin, implying that PCB19’s effect is not contributed by the activation of PMCA or NCX , its fluorescence is not affected by cytosolic Ca2+, whether originating from the intracellular pool or the extracellular space. Therefore, if Mn2+ is added in the extracellular solution, any changes in fluorescence result from binding of fura-2 to Mn2+, the latter of which is only able to enter cells from extracellular space. We found that PCB19 decreased the rate of thapsigargin-induced fluorescence quenching, indicating that thapsigargin-induced SOCE is specifically inhibited by PCB19 like as a SOCE inhibitor 1-{-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenyl}-1H- imidazole hydrochloride (SK&F96365)  Inositol 1,4,5-trisphosphate (InsP3) was purchased from GE Healthcare Life Sciences . Bovine calf serum and horse serum were obtained from HyClone . RPMI 1640 and penicillin/streptomycin were purchased from Gibco .PCB4, PCB19, PCB36, PCB50, and PCB100 were obtained from Chem Service . ATP, bradykinin, GTP, sodium methane sulfonate, glucose, N-hydroxyethylethylenediamine triacetic acid, 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’- tetraacetic acid (BAPTA), HEPES, and sulfinpyrazone were purchased from Sigma . SK&F96365 and 2-aminoethoxyphenyl borate (2-APB) were obtained from Tocris . Thapsigargin was purchased from Alomone Labs . Fura-2/acetoxymethylester (Fura-2/AM) was obtained from Molecular Probes . i according to previously reported methods InsP3 (100 nCi/ml). The resultant mixture was incubated for 15 min on ice and then centrifuged at 2,000 x g for 10 min. 100 μl of water and 1 ml of liquid scintillation cocktail were added to the pellet to measure its radioactivity. The InsP3 concentrations of the samples were determined by comparison to a standard curve and expressed as pmol/mg of protein. Total cellular protein concentrations were determined with the Bradford method after sonication of cells.InsPeviously ,50. Brie2+ quenching assay was performed as previously described i are expressed as % of controls (thapsigargin-induced Ca2+ levels without PCB19 treatment). Number of experiments are depicted in bar graph and each point represents mean ± SEM.(TIF)Click here for additional data file.S3 FigA, Fura-2-loaded PC12 cells were treated with 1 μM thapsigargin (TG), then sequentially challenged with indicated concentration of 2APB , and then treated with 50 μM PCB19. B, The [Ca2+]i level at point a, b, and c were quantitatively analyzed using calcium traces. Number of experiments are depicted in bar graph and each point represents mean ± SEM.(TIF)Click here for additional data file.


Endothelial dysfunction, including increased endothelial permeability, is considered an early marker for atherosclerosis. High-mobility group box 1 protein (HMGB1) and extracellular Ca2+ entry, primarily mediated through store-operated Ca2+ entry (SOCE), are known to be involved in increasing endothelial permeability. The aim of this study was to clarify how HMGB1 could lead to endothelia hyperpermeability.2+ sensor mediating SOCE, inhibited the HMGB1-induced influx of Ca2+ and Src activation followed by significant suppression of endothelial permeability. Moreover, knockdown of Orai1, an essential pore-subunit of SOCE channels, decreased HMGB1-induced endothelial hyperpermeability.We have shown that human vascular endothelial cell permeability is increased, while transendothelial electrical resistance and VE-cadherin expression were reduced by HMGB1 treatment. Two SOCE inhibitors and knockdown of stromal interaction molecule 1 (STIM1), a Ca2+ entry in the modulation of endothelial cell permeability. STIM1 may thus represent a possible new therapeutic target against atherosclerosis.These data suggest that SOCE, acting via STIM1, might be the predominant mechanism of Ca Atherosclerosis remains one of the most important and common causes of death and disability in developed countries , 2. It h2+ plays a critical role in endothelial permeabilityi) were estimated as ΔF/F0, where ΔF was defined as the fluorescence intensity after subtracting the basal intensity, and F0 was derived from the average intensity of the first 10–20 frames minus the background in the cell-free region.Intracellular Ca5’-GGCUCUGGAUACAGUGCUCTT-3’ and STIM1 siRNA-2: 5’-GAAGCUGCGCGAUGAGAUCTT-3’) and Two siRNAs against Orai1 (Orai1 siRNA-1: 5’-CGAGCACTCCATGCAGGCG-3’ and Orai1 siRNA-2: 5’-GAAGCUGCGCGAUGAGAUCTT-3’) were designed and obtained from Shanghai GenePharma Co, Ltd.  and were transfected into EA.hy926 cells using a Lipo2000 Transfection Kit . A scrambled, non-targeting siRNA (5’-UUCUCCGAACGUGUCACGUTT-3’ and 3’-ACGUGACACGUUCGGAGAATT-5’) was used as a control. Oligofectamine was diluted and mixed in OptiMEM I (Invitrogen) to allow the formation of siRNA-liposome complexes. After 20 min incubation at room temperature, the mixture was overlayed onto EA.hy926 cells cultured in medium without antibiotics. The transfected cells were analyzed 48 h after transfection.Two siRNAs against STIM1 -regulated influx, and may be a common component of SOCs . We ther release . Thapsig release . We furt release . Src act release .Since STIM1 may promote endothelial permeability independently of Ca2+ entry, Orai1, 2+ influx, endothelial cell hyperpermeability and Src activation,. Taken together, these data suggest that SOCE, acting through STIM1, plays an important role in the regulation of HMGB1-stimulated endothelial cell hyperpermeability through Src activation.The present study indicates the existence of a novel mechanism responsible for modulating vascular permeability. Our results demonstrated the following: (1) HMGB1 increased endothelial cell permeability in time- and concentration- dependent manners; (2) VE-cadherin protein expression was decreased and detectable intercellular gaps were induced in endothelial cells treated with HMGB1; (3) Inhibition of SOCE or STIM1 knockdown significantly attenuated HMGB1-induced Ca-/- transgenic mice[HMGB-1 is a chromatin-associated nuclear protein that stabilizes nucleosome formation and facilitates transcription-factor binding by bending DNA . HMGB1 henic mice. In the enic mice. VE-cadhenic mice. Previouenic mice. The expenic mice. Our datenic mice. Further2+ through transient receptor potential channel 1, the essential component of SOC, has been established as the initial pivotal signal that precedes endothelial cell cytoskeletal reorganization and the disassembly of VE-cadherin [2+ influx in endothelial cells, without affecting intracellular Ca2+ store release. These results reveal that SKF96365 and 2-APB reduced endothelial hyperpermeability by inhibiting Ca2+ influx via SOCE. However, although SKF96365 and 2-APB have been widely used to inhibit SOCE, they are both non-specific and can also suppress voltage-operated and receptor-operated Ca2+ channels [2+ concentration in the lumen of the ER, which plays a vital role in mediating SOCE [2+-bound STIM1 molecules are located predominantly in the ER but also appear in the plasma membrane. Upon Ca2+ depletion of the ER as a result of cell activation, STIM1 in the ER becomes aggregated into puncta, relocated in distinct regions of the ER close to the plasma membrane, and associated and reorganized in the plasma membrane, which is sufficient to activate SOCs and mediate Ca2+ influx. Recent studies indicate that STIM1 is required for endothelial SOCE [2+ influx without affecting intracellular Ca2+ store release. In addition, STIM1 was recently reported to promote endothelial permeability independently of Ca2+ entry [2+ influx.Endothelial permeability is a complex process that is regulated by SOC in chronic pulmonary hypertension . The riscadherin , 40. In channels , 42. STIing SOCE , 44. Wheial SOCE , 46. In 2+ entry . Orai1 c2+ entry , 47, wasRecent evidence has shown that Src family of kinases is associated with the regulation of microvascular barrier function and various endothelial responses, including hyperpermeability, to different proinflammatory mediators , 49. Oth2+ entry via SOCE, which in turn activates the Src pathways. In addition to indicating the relevance of SOCE to endothelial permeability, this study provides the first evidence for STIM1 involvement in the signaling pathways regulating HMGB1-induced endothelial hyperpermeability.The results of this study indicate the existence of a novel mechanism for modulating vascular permeability: HMGB1 activates Ca


The [Ca2+]i rise evoked by BK also involves the influx of extracellular Ca2+via canonical transient receptor potential (TRPC) channels. Vasoinhibins likely interfere with TRPC-mediated Ca2+ entry since La3+, which is an enhancer of TRPC4 and TRPC5 channel activity, prevented vasoinhibins from blocking the stimulation by BK of endothelial cell NO production and proliferation, and vasoinhibins reduced the BK-induced increase of TRPC5 mRNA expression. Finally, vasoinhibins prevented the BK-induced phosphorylation of eNOS at Ser1179, a post-translational modification that facilitates Ca2+-calmodulin activation of eNOS. Together, our data show that vasoinhibins, by lowering NO production through the inhibition of both [Ca2+]i mobilization and eNOS phosphorylation, prevent the BK-induced stimulation of endothelial cell proliferation. Thus, vasoinhibins help to regulate BK effects on angiogenesis and vascular homeostasis.Vasoinhibins, a family of antiangiogenic peptides derived from prolactin proteolysis, inhibit the vascular effects of several proangiogenic factors, including bradykinin (BK). Here, we report that vasoinhibins block the BK-induced proliferation of bovine umbilical vein endothelial cells. This effect is mediated by the inactivation of endothelial nitric oxide synthase (eNOS), as the NO donor DETA-NONOate reverted vasoinhibin action. It is an experimentally proven fact that the elevation of intracellular Ca Vasoinh2+/calmodulin, and its activation depends on the elevation of intracellular Ca2+ levels. The stimulation of B2 receptors by BK initiates the classical G(q) protein-coupled receptor activation of phospholipase C-β (PLC-β) that catalyzes the formation of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol, leading to a biphasic increase in intracellular free Ca2+ concentration ([Ca2+]i). The initial transient component reflects an IP3-induced release of Ca2+ from endoplasmic reticulum, whereas the longer-lasting elevation reflects Ca2+ influx from the extracellular space diazen-1-ium-1,2-diolate or DETA-NONOate was purchased from Alexis Corporation . Recombinant human vasoinhibins (corresponding to both 16 and 18 kDa fragments of prolactin) were produced by site-directed mutagenesis using a baculovirus expression system as described thymidine incorporation , and once validated, we used the reduction of 3--2,5-diphenyltetrazolium bromide (MTT) as an index of proliferation to avoid radioisotopes. The 3H-thymidine incorporation assay consisted in seeding cells in 48-well plates (Nunc) at an initial density of 5000 cells/well and subjecting them to a series of treatments  that included a 48 h-incubation with BK (10 μM) combined or not with increasing doses of vasoinhibins , and in the presence or the absence of DETA-NONOate (10 μM), as indicated. When required, treatments were performed in Ca2+-free medium obtained by adding 10 mM EDTA to the standard F12K medium, which normally contains 0.4 mM Ca2+. Twenty-four h before ending the treatment, 1 μL of 3H-thymidine per well was added. After the 48-h treatment, wells were washed thrice in 5% trichloroacetic acid, with the last wash for 20 min at 4 °C. Next, 250 μL boiling 0.25 N NaOH was added, and samples were transferred to scintillation vials for quantification. For the MTT assay, cells were seeded in 96-well plates (Nunc) at an initial density of 2500 cells/well and treated for 48 h with 10% FBS-culture medium supplemented or not with BK (10 μM), vasoinhibins (60 nM), and the nonselective TRP channel blockers SKF-96365 (10 μM)  radioreceptor assay procedure , a competitive ligand binding assay.The accumulation of IPs et al. . Briefly2.6.2+ was measured using an Aminco-Bowman Series-2 luminescence spectrometer with a 150-W xenon source . BUVEC were mechanically dispersed after a 2-min incubation with 0.01% trypsin-EDTA solution, centrifuged, and resuspended at a final density of 106 cells/mL in HBSS  containing (in mM): 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 0.3 Na2HPO4, 0.4 KH2PO4, 4 NaHCO3, 5 glucose, and 10 HEPES (adjusted to pH 7.4 with NaOH). Cells were loaded with Fura-2 by exposure to 2 μM Fura-2 AM  at 37 °C for 1 h in HBSS. After incubation, cells were washed three times in HBSS. Cell suspensions were then incubated under constant perfusion with Ca2+-free or 2 mM Ca2+-containing HBSS containing BK (10 μM) or thapsigargin , as indicated. Ca2+-free medium was prepared by omitting CaCl2 and adding 10 mM EGTA. Prior to BK perfusion, BUVEC were incubated for 2 min with vasoinhibins (60 nM), heat-inactivated vasoinhibins, or prolactin (10 nM). Similarly, prior to Tg perfusion, BUVEC were incubated for 2 min with vasoinhibins (60 nM). Fura-2 fluorescence, at an emission wavelength of 510 nm, was recorded by exciting the probe alternately at 340 and 380 nm. Cytosolic [Ca2+] was derived from the 340 nm/380 nm signal ratio using the Grynkiewicz equation l-arginine and 50 μg of BUVEC protein in a final volume of 100 μL. After 1 h, ice-cold stop medium  was added for 10 min. BUVEC extracts were then applied onto 1-mL columns of Dowex AG50WX8. [3H]L-citrulline was eluted with 1 mL water and quantified by liquid scintillation counting.eNOS activity was determined by L-citrulline assay in BUVEC grown in 12-well plates until 80% confluence that were then pretreated with okadaic acid (50 nM) for 10 min, then vasoinhibins (60 nM) for 1 h and followed or not by BK (10 μM) for 10 min. Protein concentration was evaluated using the Bradford assay (Bio-Rad). The reaction was carried out in reaction buffer  prior to reverse transcription using Moloney murine leukemia virus reverse transcriptase . cDNA was amplified using TRPC5 , TRPC6 , TRPM6 , TRPM7 , actin , and L19 , and subsequently analyzed by agarose gel electrophoresis. Because identical amounts of cDNA were added to the PCR reaction, we estimated the changes in transcript expression levels relative to those of housekeeping genes.2.10.1179-eNOS antibody  overnight, and the antigen-antibody complex was detected using HRP-coupled secondary antibodies  and enhanced chemiluminescence . Membranes were then stripped and probed with monoclonal anti-eNOS  and the alkaline phosphatase secondary antibody kit .BUVEC were pretreated with vasoinhibins (60 nM) for 1 h followed by incubation with BK (10 μM) for 1, 5, or 10 min. Protein samples were denatured by incubation in Laemmli buffer, and then subjected to SDS-PAGE. Immunoblots were incubated with polyclonal anti-phospho-Ser2.11.Values are expressed as mean ± SEM. Statistical significance between groups was determined by analysis of variance (ANOVA) followed by unpaired Student's T-test. Differences in means with P < 0.05 were considered statistically significant. The statistical analysis was performed using the Sigma Stat 7.0 software .3.3.1.Compared to untreated control cultures, BK induced a 50% increase in BUVEC proliferation that was significantly diminished by vasoinhibins at a dose of 60 nM . The NO 2+ mobilization was postulated to mediate vasoinhibin blockage of eNOS activation [2+ from intracellular stores by generating IP3 [3 production  dephosphorylates eNOS at Ser1179 , and vas1179 , we used1179 , to inve4.in vitro.The vascular effects of BK have been largely described and associated with disorders that include diabetic retinopathy -11. The 2+, an event necessary for eNOS binding to calmodulin. As previously hypothesized [3-mediated release of intracellular Ca2+ stores (2+. In addition, our data identified a new target for vasoinhibins to regulate intracellular Ca2+ levels, the TRPC channels. Despite growing evidence that these channels participate in the regulation of angiogenesis [2+ entry, which would enable the increase in intracellular Ca2+ levels necessary for eNOS activation. The eNOS-derived NO could then act as a positive feed-back messenger in the BK signaling cascade as it is able to directly activate the TRPC5 channel [2+ influx, but vasoinhibin action on TRPC5 channels probably represents an additional mechanism that potently regulates BK actions on vascular homeostasis. In addition, vasoinhibins promote dephosphorylation of eNOS at Ser1179, a modification that reduces eNOS sensitivity to Ca2+-calmodulin.The deleterious consequences of uncontrolled NO production likely contributed to the evolution of complex regulatory mechanisms to control eNOS activity. Strikingly, our current and previous work shows that vasoinhibins target several of these mechanisms to reduce NO levels. First of all, vasoinhibins block pathways participating in the increment of intracellular Cathesized , we demo+ stores  and 3 anogenesis , how the channel . FurtherThis study provides new insights into the signal transduction pathway and ion channels mediating vasoinhibin angiostatic activity that could help define the nature of the vasoinhibin receptor, an issue that warrants further investigation.


Typical carcinoid syndrome is characterized by flushing, abdominal pain and diarrhea and occurs in <10 % of carcinoid tumor patients. Very rarely, initial signs include skin manifestations. Our purpose is to highlight cutaneous manifestations in the diagnosis and assessment of a patient with atypical manifestation of type I gastric carcinoid tumor.A 50-year-old woman presented with anemia, chronic urticaria and angioedema. Urticaria was triggered principally by seafood and appeared in the first hour after. Urticaria Activity Score 7 was 24, and quality of life (CU-Q2oL) was 3.61. P. Laboratory findings showed anemia, diminished iron, ferritin, and vitamin B12, with increased gastrin and anti-parietal cell antibody levels. 15 gastric carcinoids 5 mm in diameter were observed in the greater curvature of the stomach during gastric endoscopy and confirmed by biopsy, suggesting that this patient had type I gastric carcinoids. Four additional tumors were found in the small intestine upon examination via video capsule. Endoscopic argon plasma therapy was performed. The patient experienced definitive improvement in quality of life and urticaria activity score.This patient, whose principal symptoms were anemia, urticaria and angioedema, was found to have atypical carcinoid syndrome, with tumors located in the stomach. Allergists, immunologists, internists and primary care physicians should consider the possibility of neuroendocrine malignancies, specifically type I carcinoid tumors, when evaluating patients with urticaria, and consider screening patients with chronic urticaria for elevated anti-parietal cell antibody levels. Carcinoid tumors are rare, slow-growing neuroendocrine tumors arising from the enterochromaffin cells. They are most commonly found in the gastrointestinal tract 64 %) , althoug % , althGastric carcinoid tumors are classified as those associated with chronic gastritis type A , those that concur with the Zollinger-Ellison syndrome (type II), and sporadic gastric tumors (type III). Type I gastric carcinoids account for 70 to 80 % of neuroendocrine gastric malignancies. They are associated with autoimmune atrophic gastritis, hypergastrinemia, and pernicious anemia . Type I More than half of patients with CAG-A–associated carcinoids also have pernicious anemia. Pernicious anemia is associated with increased the risk of gastric carcinoid tumors, presumably due to prolonged achlorhydria. It results in parietal cell loss, compensatory hypergastrinemia, and argyrophilic cell hyperplasia .Persistent achlorhydria produces G cell hyperplasia and more gastrin secretion, leading to hypergastrinemia . GastrinType II carcinoids present with hypergastrinemia and are associated with Zollinger-Ellison syndrome. In contrast to type I gastric carcinoids, the hypergastrinemia associated with type II gastric carcinoids is secondary to gastrin secreting G cell neoplasia rather than parietal cell loss. Type II gastric carcinoids are very uncommon; the majority have good prognosis. The last type of gastric carcinoid, type III, may be associated with atypical carcinoid syndrome , 13. AtyHere we report urticaria, angioedema and anemia as symptoms in an atypical presentation of type I gastric carcinoid. Our purpose is to highlight the relevance of cutaneous manifestations in the diagnosis and assessment of a patient with an underlying neuroendocrine malignancy. We also remark on the effectiveness of endoscopic ablation. And we encourage all medical practitioners to be aware that skin changes may provide an early clue for clinical management of gastric carcinoids.We report a case of a 50-year-old woman with atypical type I gastric carcinoid tumor. The patient presented with anemia, chronic urticaria and angioedema. The urticaria began one year prior to her evaluation by us, and was triggered by seafood. Other triggers included vitamin B12, desserts, and maize, as well as friction from shoes while running, and doing handicrafts. Lesions usually began in the first hour after the stimuli. Three months prior to developing urticaria, the patient was diagnosed with anemia. In the year since her urticaria began, the patient had lost 20 pounds without effort. She reported that her symptoms improved after taking the proton pump inhibitor (PPI) pantoprazole, but she only used the PPI sporadically. The reason for this response to PPI is not clear.Initial symptoms of the urticaria were nausea accompanied by erythema, edema, pruritus and wheals. Wheals were larger than 5 mm, and were accompanied by erythema. They were associated with pain, burning and morning-time livedo reticularis. Wheals lasted more than 12 h, and were localized in palm, soles, armpits, feet, hands, buttocks, shoulders, trunk, legs and back. The patient also experienced generalized angioedema, which was triggered by seafood, corn, some desserts and ibuprofen, as well as blurred vision and sore throat. Urticaria Activity Score 7 (UAS 7) was 24, and the mean score on the CU-Q2oL quality of life measure was 3.61. Fecal exam, immunological tests (complement levels and immunoglobulins) and skin prick allergy tests for shrimp, crab, dust mite, and dog hair were all normal. There were no signs of vasculitis in the skin biopsy.Laboratory tests showed diminished iron, ferritin, and vitamin B12 . The gastrin level was 286 pg/ml  and parietal cell antibody was 106.53 U/ml  . RDW . Three in the antrum were extirpated (biopsy) and sent to pathology; the rest were subjected to plasma argon ablation Fig. -b. Also,This patient had four additional tumors in the small intestine, which were found upon examination via video capsule. The patient underwent endoscopic argon plasma therapy, which proved to be effective in resolution of urticaria. Quality of life greatly improved, with a mean CU-Q2oL score of 0 after treatment. Urticaria was successfully treated, with the UAS 7 score dropping to 0.While endoscopic ablation has been associated with a high rate of recurrence, it is considered a safe procedure with 100 % survival, and has been demonstrated to be effective. We recommend further endoscopic follow-up to screen any possible recurrence.Guidelines on follow up management of patients who have had endoscopic ablation of type I gastric carcinoid tumors have not yet been established. However, Yarzu et al. reported using omalizumab in postoperative care of a male patient with food allergy with pulmonary carcinoid tumor. In the post-surgery period the patient had recurrent laryngeal edema and urticaria attacks. Omalizumab treatment was prescribed because the patient was resistant to anti-histamines and steroids and was used for eight months in symptomatic therapy of recurrent laryngeal edema and urticaria attacks During the four years of follow up, no carcinoid tumor recurrence was noted .There are only a few reported cases of urticaria or angioedema associated with carcinoid tumors , 18. TheThis patient presented with urticaria and angioedema as symptoms. Remarkably, urticaria was the reason for medical consultation. The patient had no diarrhea, and no signs or symptoms of bronchoconstriction or heart failure. This, then, would not fit the definition of a classical carcinoid syndrome . The patWe strongly encourage all physicians who are looking for the etiology of any chronic urticaria, be they allergists, dermatologists, internists or primary care physicians, to consider the possibility of neuroendocrine malignancies and to screen these patients for elevated anti-parietal cell antibodies.Written informed consent was obtained from the patient for publication of this Case Report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


The cause of airway smooth muscle (ASM) hypercontractility in asthma is not fully understood. The relationship of spontaneous intracellular calcium oscillation frequency in ASM to asthma severity was investigated. Oscillations were increased in subjects with impaired lung function abolished by extracellular calcium removal, attenuated by caffeine and unaffected by verapamil or nitrendipine. Whether modulation of increased spontaneous intracellular calcium oscillations in ASM from patients with impaired lung function represents a therapeutic target warrants further investigation. Asthma affects over 300 million people worldwide. It is characterized by variable airflow obstruction (AFO) and airway hyperresponsiveness as a consequence of increased airway smooth muscle (ASM) contractility.52+]i) oscillations are reported in foetal ASM and are implicated in ASM contraction. Agonist-induced [Ca2+]i oscillations are also reported in adult ASM and are implicated in augmenting ASM contraction and Ca2+-dependent transcriptional regulation.2+ oscillations have not hitherto been reported in ASM from adults. We hypothesized that spontaneous [Ca2+]i oscillations are maintained in ASM derived from subjects with asthma and are related to disease severity and disordered airway physiology.Spontaneous ASM contraction is considered of fundamental importance in foetal lung development.2+]i, subconfluent ASM cells were loaded with 2 μmol/L Fura-2AM in the presence of 2.5 mmol/L probenecid and 0.04% w/v pluronic F-127, and visualized on an inverted epifluorescence microscope . Fura-2 fluorescence (F) emission intensity at 510 nm was measured following excitation at wavelengths of 340 and 380 nm, and reported as a ratio, R = F340/F380, such that R is directly proportional to [Ca2+]i. [Ca2+]i oscillatory behaviour was analysed using two methods. First, the [Ca2+]i oscillation frequency was measured by a single-blinded observer counting the number of oscillations reaching a 340/380 ratio >10% greater than the baseline derived for each donor for 10 min. Second, the [Ca2+]i oscillation dominant frequency (and its amplitude) were determined by fast Fourier transform (FFT) spectral analysis using software previously developed for MATLAB.s = 0.58; P = 0.0002). The FFT-derived Ca2+ oscillation dominant frequency data are reported here. The effect of removal of extracellular Ca2+, IP3 receptor inhibition  and voltage-gated L-type Ca2+ channel inhibition (1 μmol/L verapamil and 1 μmol/L nitrendipine) on [Ca2+]i oscillations in highly oscillating cells were investigated.Primary ASM was derived from bronchial biopsies obtained from well-characterized volunteers. All subjects gave written informed consent. The Leicestershire, Northamptonshire and Rutland Ethics committee approved the study. ASM cells were cultured and characterized as previously describedt-tests, and across groups Kruskal–Wallis test and post-hoc pairwise comparisons using Dunn's test. Spearman rank correlation coefficients were used to assess the correlations. Previously, Ressmeyer et al.P value less than 0.05 was considered statistically significant.Statistical analyses were performed using Prism version 6  and IBM SPSS version 20 . Data are presented as median (interquartile ranges) or maximum dominant frequency for the cells for an individual donor. Comparisons before and after pharmacological interventions used paired Student's P = 0.71). The spirometry measurements for all subjects  I-III and severe asthma GINA IV and V respectively (mean (standard error of the mean)) were: forced expiratory volume in 1 s (FEV1)% predicted: 97 (3), 91 (8) and 77 (6) (P < 0.05); FEV1/forced vital capacity (FVC)%: 79 (2), 70 (3) and 64 (3) (P < 0.05). The dominant [Ca2+]i oscillation frequency was determined for each cell (minimum 5 cells per donor). Spontaneous ASM [Ca2+]i oscillations were observed in both health and disease. Example traces are shown . The amplitude of spontaneous [Ca2+]i oscillations were attenuated by caffeine, being restored following its washout  and with FEV1/FVC%  compared with those without persistent AFO the median [Ca2+]i dominant oscillation frequency was significantly increased (5.17 (4.5–7.4) vs 2.4 (2.0–3.7) mHz), P = 0.001; Fig. 1/FVC% and oscillation frequency r = −0.47, P = 0.005; other data not shown).We recruited 34 volunteers , 9 with mild-moderate asthma  and 11 severe asthmatics ). Subjects were age-matched: 60 (5) years healthy, 46 (5) years mild/moderate asthma and 48 (5) years severe asthma (2+]i oscillations are observed in primary ASM cells in asthma and health. The maximum dominant frequency of these spontaneous [Ca2+]i oscillations was increased in severe asthma. The median dominant frequency was related to impaired lung function and was increased in asthmatics with persistent AFO. Agonist-induced [Ca2+]i oscillations have been consistently observed, and previous reports have primarily implicated the intracellular Ca2+ stores.2+ stores and influx pathways.2+ homeostasis has previously been reported as a consequence of altered mitochondrial biogenesis2+-handling in ASM in asthma might be altered via a number of interrelated mechanisms. The consequences of spontaneous [Ca2+]i oscillations in asthma is not fully understood but have been implicated in increased ASM contraction and Ca2+-transcriptional coupling.2+]i oscillation frequency and percentage airway contraction in human airway slices.2+]i oscillations we describe here, predominantly in patients with severe asthma, might be sufficient to induce contraction and may also enhance the response to agonists. In addition to hypercontractility, primary ASM from asthmatics have increased capacity to release several important pro-inflammatory chemokines and matrix proteins compared with ASM derived from healthy volunteers.2+]i oscillations in these mechanisms warrants further study, the associations observed here with asthma severity and disordered airway physiology suggests that these observations might be clinically important. The presence of spontaneous [Ca2+]i oscillations in foetal ASMWe report here for the first time that spontaneous [Ca2+]i oscillations in ASM cells from subjects with impaired lung function. The mechanisms of this aberrant spontaneous [Ca2+]i oscillatory behaviour in asthma need to be fully elucidated and might identify new therapeutic targets.In conclusion, we have observed increased frequency of spontaneous [Ca


In this paper we show that Caldendrin is abundantly present in brain while the shorter splice isoforms L- and S-CaBP1 are not detectable at the protein level. Caldendrin binds both Ca2+ and Mg2+ with a global Kd in the low µM range. Interestingly, the Mg2+-binding affinity is clearly higher than in S-CaBP1, suggesting that the extended N-terminus might influence Mg2+-binding of the first EF-hand. Further evidence for intra- and intermolecular interactions of Caldendrin came from gel-filtration, surface plasmon resonance, dynamic light scattering and FRET assays. Surprisingly, Caldendrin exhibits very little change in surface hydrophobicity and secondary as well as tertiary structure upon Ca2+-binding to Mg2+-saturated protein. Complex inter- and intramolecular interactions that are regulated by Ca2+-binding, high Mg2+- and low Ca2+-binding affinity, a rigid first EF-hand domain and little conformational change upon titration with Ca2+ of Mg2+-liganted protein suggest different modes of binding to target interactions as compared to classical neuronal Ca2+-sensors.Caldendrin, L- and S-CaBP1 are CaM-like Ca It lacks a N-myristoylation sequence, which is present in many NCS proteins and in the shorter splice isoforms L- and S-CaBP1 Caldendrin is the first identified member of the nCaBP family 2+, whereas EF-hand 3 and 4 of the C-terminal lobe are high affinity sites for Ca2+ binding 2+ or Ca2+2+-binding in S-CaBP1 In contrast to S-CaBP1, very little is known about the molecular characteristics, structure and ion binding properties of Caldendrin. The four common EF-hands in L−/S-CaBP1 and Caldendrin are organized into an N- and C-terminal lobe 2+-binding are currently unknown. Studies on the interaction of S-CaBP1 and Caldendrin with the L-type voltage-gated calcium channel (VGCC), Cav1.2, have shown that S-CaBP1 and Caldendrin interact with the channel using different molecular determinants and that both proteins have different effects on channel activity The basic N-terminal half of Caldendrin is unique to the protein and the consequences for the structure, possible interaction with the acidic C-terminal region and the impact on CaIn the present experiments, animal care and procedures were approved and conducted under established standards of the German federal state of Sachsen-Anhalt , Germany in accordance with the European Communities Council Directive (86/609/EEC).All basic cloning steps were performed according to standard protocols of molecular biology and molecular cloning. Caldendrin, Caldendrin-N-terminus , Caldendrin-C-terminus , L- and S-CaBP1 proteins were cloned into the pMXB10 vector  replacing MBP. Newly generated constructs were verified by sequencing analysis. A trptophane (Trp) residue was introduced in place of a phenylalanine (Phe) in each of the EF1W and EF3W mutants. Mutations were introduced by Pfu PCR using mutagenic oligonucleotides following the QuikChange II site-directed mutagenesis kit protocol .Sprague Dawley rats from the animal facilities of the Leibniz-Institute were anesthetized with isofluorane and then killed by decapitation. Freshly dissected adult rat tissue was snap frozen in liquid nitrogen and stored at –80°C. Homogenization was done in TBS containing protease inhibitor cocktail  using a homogenizer (Potter Elvehjem tissue grinder) in a ratio of 10 ml/g wet weight. Tissue homogenates were then mixed with 4x SDS sample buffer  SDS, 40% (v/v) glycerol, 5% (v/v) β-mercaptoethanol, 0.01% (v/v) bromphenol blue) in a ratio of 2∶1, boiled for 10 min and centrifuged at 17000×g for 5 min. Total protein concentration was determined by a amidoblack assay and tissue extracts were then subjected to SDS-PAGE according to standard protocols. Rabbit Caldendrin antisera directed against a full-length fusion protein were generated by EXBIO Diagnostics (Czech Republic). Standard protein purification procedures were used to purify anti-Caldendrin specific antibodies E.coli BL21 DE3) were inoculated (1% v/v) into secondary cultures and induced at an OD600 of 0.6 with 1 mM IPTG. L- and S-CaBP1 were co-expressed with N-myristoyltransferase according to published procedures Caldendrin full length, Caldendrin-Nt, Caldendrin-Ct, L- and S-CaBP1 proteins were purified via Intein system  according to the manufacturer’s protocol. Briefly, overnight grown primary cultures (2+ (1 mM), Ca2+ (50 µM) and Mg2+ (1 mM)+Ca2+ (50 µM) (saturation was observed with the given ion concentrations). The respective blank spectra were subtracted from individual spectra.8-Anilinonaphthalene-1-sulfonate (ANS) fluorescence was used to measure the surface hydrophobicity of Caldendrin and its shorter splice isoforms. The ANS solution (10 mM) was prepared in 100% methanol. 10 µl of this solution was added to the protein sample and incubated for 10 min before recording the spectrum. ANS fluorescence was recorded on a Hitachi F-7000 fluorescence spectrophotometer. Excitation was done at 370 nm and spectra were recorded at wavelengths between 400–600 nm. All spectra were recorded at room temperature in corrected spectra mode using an excitation and emission band pass of 5 nm and 10 nm respectively. The response time was set to 2 sec with a scan speed 100 nm/min to 240 nm/min. Changes in fluorescence spectra were monitored with titration of Mg2+- 5 mM (near-UV), 1 mM (far UV), Ca2+- 5 mM (near UV), 100 µM (far UV).Circular dichroism (CD) spectroscopy was performed on a Jasco-715 spectropolarimeter. Near-UV CD spectra were recorded at room temperature between 250–340 nm using a quartz cuvette of 0.5 cm path length with a chelex-treated protein sample at a concentration of 10–11 mg/ml. Far-UV CD spectra were recorded at room temperature between 195–250 nm using quartz sandwich cuvettes of 0.1 cm path length with a protein sample at a concentration of 0.1–0.2 mg/ml. The common C-terminus of Caldendrin/CaBP1 was dissolved in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, whereas Caldendrin was kept in 20 mM Tris-HCl pH 8.5, 500 mM NaCl and 5 mM TCEP, to ease solubility of the full length protein at the high concentrations used in the near-UV CD experiments. Each spectrum was obtained from 4 accumulations. 0.5 nm data pitch, 50 nm/min scan speed and 0.5 s response time were selected for the recordings. The working concentrations of ligands used were as follows: Mg2+1 mM EGTA , 5 mM MgCl2+1 mM CaCl2 , 1 mM CaCl2  or nil . Ellipticity at 220 nm for each of the 35 samples in each set was plotted against GdmCl. The plots were fit using the ‘two state model of unfolding’ described by the equation:N = ellipticity of the native state; YU = ellipticity of the unfolded state; ΔGU° = standard free energy change of unfolding; [D] = concentration of the denaturant; m = slope of the linear relationship between ΔGU° and [D]). The ΔGU° and D½ ([D] at which 50% of protein was unfolded) obtained from the fit was compared between different groups and conclusions were drawn.Chemical equilibrium unfolding of full length Caldendrin under ligand-free (apo) and various ligand-bound conditions was monitored by far-UV CD spectroscopy. For each set, 35 samples were made, each containing the protein at 0.75 mg/ml concentration in 20 mM Tris-HCl pH 8.5, 500 mM NaCl and 5 mM TCEP and an increasing concentration of guanidinium chloride (GdmCl), ranging from 0–6 M with an average increment of 0.17 M/sample. Each set differed in its ligand condition, their working concentration being 5 mM MgCl2+ and Ca2+ binding affinities for Caldendrin and the common Caldendrin/CaBP1 C-terminus were measured using an isothermal titration calorimeter  as decribed previously 2+ titration of apo-Caldendrin, 46 µM of Ca2+- free Caldendrin in chelex treated buffer containing 50 mM Tris-HCl pH 7.5 and 100 mM KCl was loaded into the cell and 10 mM of MgCl2 prepared in the same buffer was injected. For Ca2+ titration of Mg2+-bound Caldendrin, 43 µM of Ca2+-free Caldendrin in chelex treated buffer containing 50 mM Tris-HCl pH 7.5, 100 mM KCl and 5 mM MgCl2 was loaded into the sample cell and 5 mM of CaCl2 prepared in the same buffer was injected. For Mg2+ titration of the common apo-Caldendrin/CaBP1 C-terminus 173 µM of Ca2+- free protein in chelex treated buffer containing 50 mM Tris-HCl pH 7.5 and 100 mM KCl was loaded and 15 mM of MgCl2 prepared in the same buffer was injected. Ca2+ titration of Mg2+-saturated protein was done with 120 µM of the common C-terminus in chelex treated buffer containing 50 mM Tris-HCl pH 7.5, 100 mM KCl and 5 mM MgCl2 and 5 mM of CaCl2 prepared in the same buffer was injected. Isotherms were fitted using different models of non-linear curve fitting like one set of sites, two sets of independent sites or sequentially binding sites using the Origin software (version 7) supplied by Microcal. The values for the number of sites (n), association constants (Ka), enthalpy change (ΔH) and entropy change (ΔS) for each set of sites obtained from the best fit were accepted. Each experiment was repeated at least three times and the average values are shown in Macroscopic Mg2+ and/or Mg2+-concentrations. Each analytic run was performed at 10 µl/min flow rate under the following conditions: 1 min equilibration of the chip with the indicated analysis buffer. Afterwards the analyte was injected in a 3-minute pulse (association time) followed by a 3-minute pulse with analysis buffer alone (dissociation time). Sequential sensorgrams were recorded at a flow rate of 10 µl/min. Controls were done with coupling of an unrelated GST-protein to the chip surface which resulted in no specific binding.Binding studies for Caldendrin dimerization were carried out using the Biacore X-100 instrument and sensor chip CM5  at 25°C as described previously 2+ -buffer , Ca2+-free buffer  and Mg2+-buffer . Eluted protein chromatograms were recorded at 214 nm. Eluted protein was processed for immunoblotting and the identity of the two peaks with Caldendrin was confirmed with a Caldendrin antibody.Homodimerization properties of Caldendrin were investigated by size exclusion chromatography performed on a ÄKTA FPLC system . Untagged full length Caldendrin was applied on a superdex 75 column in Ca2+500 µM). The refractive index (1.337) and viscosity (0.8791) of 1x Tris-KCl was calculated and measurements were done with different protein concentrations with suitable attenuation power. The experiment was run in Ca2+ buffer  and Ca2+ free buffer . Experiments were repeated several times to ensure accuracy.Dynamic light scattering (DLS) experiments were performed on filtered and ultracentrifuged samples of Caldendrin at 25°C using a Zetasizer Nano S instrument . Untagged Caldendrin was buffer exchanged with 1x Tris-KCl buffer  fluorescence spectroscopy. We saw that the overall changes in the spectra due to Mggligible +B. Howev S-CaBP1 .2+ sensors like Toponin-C and CaM undergo a close to open conformational change upon Ca2+ binding that exposes a hydrophobic surface which then can act as an interface for target recognition. Accordingly a close to open conformational change upon Ca2+ binding was observed in the NMR and crystal structures of the C-terminal EF-hand domain of S-CaBP1 2+  resulted in no specific binding (data not shown). Thus, if a second low-affinity binding site exists it might be either located in the second EF-hand domain of Caldendrin or is based on the coordination of Mg2+ by amino acids from the N and C terminus.Taken together the CD data suggest that Mg-binding  as S-CaB-binding . This af process  [19]. Caing site . The err2+-coordination at least of EF-hand 1 2+- and Ca2+-binding affinities to those reported for S-CaBP1 .Taken together the data suggest that the presence of the N-terminus appears to influence the mechanism of Mg2+-binding. To prove whether the N-terminus can directly interact with the C-terminus we immobilized the common C-terminus (residues 137–298) on the surface of a sensor chip via amide coupling for subsequent surface plasmon resonance (SPR) analysis  displayed two peaks at the size of a dimer and monomer  experiments that allow an estimatimation of the hydrodynamic radius and oligomerization state of the protein in solution were performed. The theoretical mass of the bacterially expressed Caldendrin full length protein is 33.08 kDa. The DLS size histograms showed estimated molecular weights of 69.8 and 80.6 kDa with poly-dispersities of 17 and 3.4% for Mg2+- and Ca2+-bound Caldendrin, respectively, suggesting a lower level heterogeneous population of protein species  form they associate with their targets, whereas in their apo-form (OFF), they dissociate from them; the exposure of the interfacial hydrophobic surface controls this switch. An enhancement of surface hydrophobicity upon Ca2+-binding was observed in the current and previous studies for S-CaBP1 2+ for target interactions of Caldendrin as opposed to the classical switch-like role. Several interactions of the common C-terminus are Ca2+-independent and conceivable is therefore a Ca2+-independent pre-association with a functional outcome that is then modulated by Ca2+. In conjunction with low Ca2+-binding affinity of Caldendrin and the high abundance of CaM, a binding mode that cannot be competed by other Ca2+-sensors is likely and it is an interesting question whether a bound target might alter the Ca2+-binding dynamics of Caldendrin with respect to affinity as well as on/off rates for Ca2+.This is of particular relevance in light of the absence of a major change in surface hydrophobicity of Caldendrin upon Ca2+. Mg2+-binding causes a conformational change in Caldendrin and surprisingly Ca2+- titration of the Mg2+-bound protein had no further impact on the structure. In near-UV CD experiments, we found that Mg2+-binding alters the structure of Caldendrin and equilibrium chemical unfolding experiments revealed that Mg2+-binding increases the structural stability of Caldendrin, indicating a structural role of Mg2+ for protein folding. It is possible that folding back of the N-terminal domain of Caldendrin impacts on Mg2+-binding, and that Caldendrin might therefore behave different from S-CaBP1 and neuronal Ca2+-sensors in this regard. The first EF-hand domain will always be Mg2+-bound, which excludes interdomain cooperativity in Ca2+-binding as known for other CaM-like four EF-hand Ca2+-sensor proteins. The rigid first EF-hand domain and folding back of the N-terminus might enable unique Ca2+-dependent target association. Of interest in this regard is the fact that Caldendrin contains several PxxP motifs in the N-terminal domain. It is conceivable that SH3 domain binding could influence the folding back of the N-terminus and thereby Mg2+-binding affinitiy and at the same time stabilizes a Ca2+-independent interaction of the C-terminus that is then subsequently modulated by Ca2+-binding of the second EF-hand domain. Alternatively, folding back of the N-terminus appears to be modulated by Ca2+ and the PxxP motifs might be more accessible in Ca2+-bound Caldendrin.A surprising finding was the high affinity of Caldendrin for Mg2+ in biochemical as well as biophysical experiments. Binding of Ca2+ has a modulatory influence on self-association and folding back of the N-terminus. Collectively the data suggest that Caldendrin in a Ca2+ free state might be a dimer via self-association of the N-terminus, whereas binding of Ca2+ facilitates dimerization of the EF-hand domain containing part and loosens the association of the N- with the C-terminus. Intra- and intermolecular interactions of the N-terminus might happen in parallel. Thus both, folding back and dimerization appear not to be mutually exclusive and several intermediate states are conceivable. Along these lines it is tempting to speculate that dimerization, folding back and Ca2+-binding might affect the interface for protein-protein interactions.Besides this intramolecular interaction, we also found evidence for intermolecular interactions of the N- and C-terminus. Structural studies carried out on S-CaBP1 have yielded variable results with respect to its dimer status 2+- and low Ca2+-binding affinity and little conformational change upon titration with Ca2+, indicating a different mode of target binding than other neuronal Ca2+-sensors.Caldendrin exhibits complex self-association, high MgFigure S1Scheme of Caldendrin/CaBP1 protein organization, production and purification. (A) Purification of Caldendrin, L- and S-CaBP1 by the Impact Intein purification system. Coomassie staining shows Caldendrin full-length at different steps of purification starting from soluble protein in the supernatant (SN), flow through (FT), matrix bound (MB), eluted to dialyzed and concentrated protein. Myristoylated protein purification by the Impact purification system (right panel). Molecular masses are indicated.(TIF)Click here for additional data file.Figure S2Caldendrin adopts a back-folded conformation: in vitro FRET analysis. (A) Fluorescence spectra showing increased FRET efficiency in the positive control (CFP-YFP) and no FRET is observed in the negative control (CFP+YFP), where CFP and YFP were expressed by different vectors. (B) A clear FRET signal was detected in the YFP channel when YFP was fused to the N- and CFP to the C-terminus of Caldendrin (YFP-CDD-CFP). (C) Co-transfection of Caldendrin fused with YFP- to the N-terminus and Caldendrin fused with CFP to the C-terminus resulted in no FRET signal in the YFP-channel.(TIF)Click here for additional data file.Figure S3Size exclusion chromatography. (A) Overlaid gel filtration chromatograms of Caldendrin injected in the presence of Mg2+  or Mg2++Ca2+ . Chromatograms of Caldendrin were compared with low molecular weight calibration kit proteins . The first peak (10–11 elution volume in ml) eluted at the size of the dimer and the second peak (11.5–12 ml) eluted at the size of the monomer. Absorption of all chromatograms was done at the wavelength 214 and 280 nm and with the 500 µl fractions size. (B) The chromatogram shows that recombinant Caldendrin elutes at the size of a monomer (11–11.5 ml) in the presence of 0.02% sodium dodecyl sulfate (SDS/black). A Caldendrin dimer was prominent when we incubated the protein with 0.01% Glutaraldehyde (GA) (red). Chromatograms of Caldendrin were compared with low molecular weight calibration kit proteins .(TIF)Click here for additional data file.