Domain Swapping and Different Oligomeric States for the Complex Between Calmodulin and the Calmodulin-Binding Domain of Calcineurin A

Background

Calmodulin (CaM) is a ubiquitously expressed calcium sensor that engages in regulatory interactions with a large number of cellular proteins. Previously, a unique mode of CaM target recognition has been observed in the crystal structure of a complex between CaM and the CaM-binding domain of calcineurin A.

Methodology/Principal Findings

We have solved a high-resolution crystal structure of a complex between CaM and the CaM-binding domain of calcineurin A in a novel crystal form, which shows a dimeric assembly of calmodulin, as observed before in the crystal state. We note that the conformation of CaM in this complex is very similar to that of unliganded CaM, and a detailed analysis revels that the CaM-binding motif in calcineurin A is of a novel ‘1-11’ type. However, using small-angle X-ray scattering (SAXS), we show that the complex is fully monomeric in solution, and a structure of a canonically collapsed CaM-peptide complex can easily be fitted into the SAXS data. This result is also supported by size exclusion chromatography, where the addition of the ligand peptide decreases the apparent size of CaM. In addition, we studied the energetics of binding by isothermal titration calorimetry and found them to closely resemble those observed previously for ligand peptides from CaM-dependent kinases.

Conclusions/Significance

Our results implicate that CaM can also form a complex with the CaM-binding domain of calcineurin in a 1∶1 stoichiometry, in addition to the previously observed 2∶2 arrangement in the crystal state. At the structural level, going from 2∶2 association to two 1∶1 complexes will require domain swapping in CaM, accompanied by the characteristic bending of the central linker helix between the two lobes of CaM.     

Gene Expression Profile of Endotoxin-stimulated Leukocytes of the Term New Born: Control of Cytokine Gene Expression by Interleukin-10

Introduction

Increasing evidence now supports the association between the fetal inflammatory response syndrome (FIRS) with the pathogenesis of preterm labor, intraventricular hemorrhage and bronchopulmonary dysplasia. Polymorphonuclear leukocyte (PMNs) and mononuclear cell (MONOs) infiltration of the placenta is associated with these disorders. The aim of this study was to reveal cell-specific differences in gene expression and cytokine release in response to endotoxin that would elucidate inflammatory control mechanisms in the newly born.

Methods

PMNs and MONOs were separately isolated from the same cord blood sample. A genome-wide microarray screened for gene expression and related pathways at 4 h of LPS stimulation (n = 5). RT-qPCR and ELISA were performed for selected cytokines at 4 h and 18 h of LPS stimulation.

Results

Compared to PMNs, MONOs had a greater diversity and more robust gene expression that included pro-inflammatory (PI) cytokines, chemokines and growth factors at 4 h. Only MONOs had genes changing expression (all up regulated including interleukin-10) that were clustered in the JAK/STAT pathway. Pre-incubation with IL-10 antibody, for LPS-stimulated MONOs, led to up regulated PI and IL-10 gene expression and release of PI cytokines after 4 h.

Discussion

The present study suggests a dominant role of MONO gene expression in control of the fetal inflammatory response syndrome at 4 hrs of LPS stimulation. LPS-stimulated MONOs but not PMNs of the newborn have the ability to inhibit PI cytokine gene expression by latent IL-10 release.     

Dependency of Calcium Alternans on Ryanodine Receptor Refractoriness

Background

Rapid pacing rates induce alternations in the cytosolic calcium concentration caused by fluctuations in calcium released from the sarcoplasmic reticulum (SR). However, the relationship between calcium alternans and refractoriness of the SR calcium release channel (RyR2) remains elusive.

Methodology/Principal Findings

To investigate how ryanodine receptor (RyR2) refractoriness modulates calcium handling on a beat-to-beat basis using a numerical rabbit cardiomyocyte model. We used a mathematical rabbit cardiomyocyte model to study the beat-to-beat calcium response as a function of RyR2 activation and inactivation. Bi-dimensional maps were constructed depicting the beat-to-beat response. When alternans was observed, a novel numerical clamping protocol was used to determine whether alternans was caused by oscillations in SR calcium loading or by RyR2 refractoriness. Using this protocol, we identified regions of RyR2 gating parameters where SR calcium loading or RyR2 refractoriness underlie the induction of calcium alternans, and we found that at the onset of alternans both mechanisms contribute. At low inactivation rates of the RyR2, calcium alternans was caused by alternation in SR calcium loading, while at low activation rates it was caused by alternation in the level of available RyR2s.

Conclusions/Significance

We have mapped cardiomyocyte beat-to-beat responses as a function of RyR2 activation and inactivation, identifying domains where SR calcium load or RyR2 refractoriness underlie the induction of calcium alternans. A corollary of this work is that RyR2 refractoriness due to slow recovery from inactivation can be the cause of calcium alternans even when alternation in SR calcium load is present.     

Ryanodine Receptors Selectively Interact with L Type Calcium Channels in Mouse Taste Cells

Introduction

We reported that ryanodine receptors are expressed in two different types of mammalian peripheral taste receptor cells: Type II and Type III cells. Type II cells lack voltage-gated calcium channels (VGCCs) and chemical synapses. In these cells, ryanodine receptors contribute to the taste-evoked calcium signals that are initiated by opening inositol trisphosphate receptors located on internal calcium stores. In Type III cells that do have VGCCs and chemical synapses, ryanodine receptors contribute to the depolarization-dependent calcium influx.

Methodology/Principal Findings

The goal of this study was to establish if there was selectivity in the type of VGCC that is associated with the ryanodine receptor in the Type III taste cells or if the ryanodine receptor opens irrespective of the calcium channels involved. We also wished to determine if the ryanodine receptors and VGCCs require a physical linkage to interact or are simply functionally associated with each other. Using calcium imaging and pharmacological inhibitors, we found that ryanodine receptors are selectively associated with L type VGCCs but likely not through a physical linkage.

Conclusions/Significance

Taste cells are able to undergo calcium induced calcium release through ryanodine receptors to increase the initial calcium influx signal and provide a larger calcium response than would otherwise occur when L type channels are activated in Type III taste cells.     

Local Calcium Elevation and Cell Elongation Initiate Guided Motility in Electrically Stimulated Osteoblast-Like Cells

Background

Investigation of the mechanisms of guided cell migration can contribute to our understanding of many crucial biological processes, such as development and regeneration. Endogenous and exogenous direct current electric fields (dcEF) are known to induce directional cell migration, however the initial cellular responses to electrical stimulation are poorly understood. Ion fluxes, besides regulating intracellular homeostasis, have been implicated in many biological events, including regeneration. Therefore understanding intracellular ion kinetics during EF-directed cell migration can provide useful information for development and regeneration.

Methodology/Principal Findings

We analyzed the initial events during migration of two osteogenic cell types, rat calvarial and human SaOS-2 cells, exposed to strong (10–15 V/cm) and weak (≤5 V/cm) dcEFs. Cell elongation and perpendicular orientation to the EF vector occurred in a time- and voltage-dependent manner. Calvarial osteoblasts migrated to the cathode as they formed new filopodia or lamellipodia and reorganized their cytoskeleton on the cathodal side. SaOS-2 cells showed similar responses except towards the anode. Strong dcEFs triggered a rapid increase in intracellular calcium levels, whereas a steady state level of intracellular calcium was observed in weaker fields. Interestingly, we found that dcEF-induced intracellular calcium elevation was initiated with a local rise on opposite sides in calvarial and SaOS-2 cells, which may explain their preferred directionality. In calcium-free conditions, dcEFs induced neither intracellular calcium elevation nor directed migration, indicating an important role for calcium ions. Blocking studies using cadmium chloride revealed that voltage-gated calcium channels (VGCCs) are involved in dcEF-induced intracellular calcium elevation.

Conclusion/Significance

Taken together, these data form a time scale of the morphological and physiological rearrangements underlying EF-guided migration of osteoblast-like cell types and reveal a requirement for calcium in these reactions. We show for the first time here that dcEFs trigger different patterns of intracellular calcium elevation and positional shifting in osteogenic cell types that migrate in opposite directions.     

Cross-Talk between Signaling Pathways Can Generate Robust Oscillations in Calcium and cAMP

Background

To control and manipulate cellular signaling, we need to understand cellular strategies for information transfer, integration, and decision-making. A key feature of signal transduction is the generation of only a few intracellular messengers by many extracellular stimuli.

Methodology/Principal Findings

Here we model molecular cross-talk between two classic second messengers, cyclic AMP (cAMP) and calcium, and show that the dynamical complexity of the response of both messengers increases substantially through their interaction. In our model of a non-excitable cell, both cAMP and calcium concentrations can oscillate. If mutually inhibitory, cross-talk between the two second messengers can increase the range of agonist concentrations for which oscillations occur. If mutually activating, cross-talk decreases the oscillation range, but can generate ‘bursting’ oscillations of calcium and may enable better filtering of noise.

Conclusion

We postulate that this increased dynamical complexity allows the cell to encode more information, particularly if both second messengers encode signals. In their native environments, it is unlikely that cells are exposed to one stimulus at a time, and cross-talk may help generate sufficiently complex responses to allow the cell to discriminate between different combinations and concentrations of extracellular agonists.     

Calcium Supplementation Increases Blood Creatinine Concentration in a Randomized Controlled Trial

Background

Calcium supplements are widely used among older adults for osteoporosis prevention and treatment. However, their effect on creatinine levels and kidney function has not been well studied.

Methods

We investigated the effect of calcium supplementation on blood creatinine concentration in a randomized controlled trial of colorectal adenoma chemoprevention conducted between 2004–2013 at 11 clinical centers in the United States. Healthy participants (N = 1,675) aged 45–75 with a history of colorectal adenoma were assigned to daily supplementation with calcium (1200 mg, as carbonate), vitamin D₃ (1000 IU), both, or placebo for three or five years. Changes in blood creatinine and total calcium concentration were measured after one year of treatment and multiple linear regression was used to estimate effects on creatinine concentrations.

Results

After one year of treatment, blood creatinine was 0.013±0.006 mg/dL higher on average among participants randomized to calcium compared to placebo after adjustment for other determinants of creatinine (P = 0.03). However, the effect of calcium treatment appeared to be larger among participants who consumed the most alcohol (2–6 drinks/day) or whose estimated glomerular filtration rate (eGFR) was less than 60 ml/min/1.73 m² at baseline. The effect of calcium treatment on creatinine was only partially mediated by a concomitant increase in blood total calcium concentration and was independent of randomized vitamin D treatment. There did not appear to be further increases in creatinine after the first year of calcium treatment.

Conclusions

Among healthy adults participating in a randomized clinical trial, daily supplementation with 1200 mg of elemental calcium caused a small increase in blood creatinine. If confirmed, this finding may have implications for clinical and public health recommendations for calcium supplementation.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00153816","term_id":"NCT00153816"}}NCT00153816     

Calcium Chloride in Neonatal Parenteral Nutrition: Compatibility Studies Using Laser Methodology

Introduction

We have previously reported results of precipitation studies for neonatal parenteral nutrition solutions containing calcium chloride and sodium phosphate using visual methods to determine compatibility. The purpose of this study was to do further testing of compatibility for solutions containing calcium chloride using more sensitive methods.

Methods

Solutions of Trophamine (Braun Medical Inc, Irvine, CA) and Premasol (Baxter Pharmaceuticals, Deerfield, IL) were compounded with calcium chloride and potassium phosphate. Controls contained no calcium or phosphate. After incubation at 37° for 24 hours solutions without visual precipitation were analyzed to determine mean particle size using dynamic light scattering from a laser light source.

Results

Particle sizes were similar for control solutions and those without visual precipitation and a mean particle size <1000 nm. Compatible solutions were defined as those with added calcium and phosphate with no visual evidence of precipitation and mean particle size <1000 nm. In solutions containing 2.5–3% amino acids and 10 mmol/L of calcium chloride the maximum amount of potassium phosphate that was compatible was 7.5 mmol/L.

Conclusion

Maximum amounts of phosphate that could be added to parenteral nutrition solutions containing Trophamine and calcium chloride were about 7.5–10 mmol/L less for a given concentration of calcium based upon laser methodology compared to visual techniques to determine compatibility. There were minor differences in compatibility when adding calcium chloride and potassium phosphate to Premasol versus Trophamine.     

Proteomic characterization of serine hydrolase activity and composition in normal urine

Background

Serine hydrolases constitute a large enzyme family involved in a diversity of proteolytic and metabolic processes which are essential for many aspects of normal physiology. The roles of serine hydrolases in renal function are largely unknown and monitoring their activity may provide important insights into renal physiology. The goal of this study was to profile urinary serine hydrolases with activity-based protein profiling (ABPP) and to perform an in-depth compositional analysis.

Methods

Eighteen healthy individuals provided random, mid-stream urine samples. ABPP was performed by reacting urines (n = 18) with a rhodamine-tagged fluorophosphonate probe and visualizing on SDS-PAGE. Active serine hydrolases were isolated with affinity purification and identified on MS-MS. Enzyme activity was confirmed with substrate specific assays. A complementary 2D LC/MS-MS analysis was performed to evaluate the composition of serine hydrolases in urine.

Results

Enzyme activity was closely, but not exclusively, correlated with protein quantity. Affinity purification and MS/MS identified 13 active serine hydrolases. The epithelial sodium channel (ENaC) and calcium channel (TRPV5) regulators, tissue kallikrein and plasmin were identified in active forms, suggesting a potential role in regulating sodium and calcium reabsorption in a healthy human model. Complement C1r subcomponent-like protein, mannan binding lectin serine protease 2 and myeloblastin (proteinase 3) were also identified in active forms. The in-depth compositional analysis identified 62 serine hydrolases in urine independent of activity state.

Conclusions

This study identified luminal regulators of electrolyte homeostasis in an active state in the urine, which suggests tissue kallikrein and plasmin may be functionally relevant in healthy individuals. Additional serine hydrolases were identified in an active form that may contribute to regulating innate immunity of the urinary tract. Finally, the optimized ABPP technique in urine demonstrates its feasibility, reproducibility and potential applicability to profiling urinary enzyme activity in different renal physiological and pathophysiological conditions.     

Association between CASR Polymorphisms,Calcium Intake,and Colorectal Cancer Risk

Aim

The current study aimed to assess the effect of dietary calcium intake and possible interactions with calcium-sensing receptor (CASR) gene polymorphisms on colorectal cancer risk.

Methods

A total of 420 colorectal cancer cases and 815 controls were included in the analysis. Calcium intake was investigated using a 103 item semi-quantitative food frequency questionnaire, and four single nucleotide polymorphisms (SNPs) within the CASR, rs10934578, rs12485716, rs2270916, and rs4678174, were evaluated.

Results

No SNPs were associated with colorectal cancer risk after adjusting for covariates. Overall, no significant effect modification by CASR polymorphisms on the association between calcium intake and colorectal cancer risk were detected. However, all 4 of the polymorphisms within the CASR showed significantly higher odds ratios for association with colorectal cancer risk in the low-calcium-intake group compared to the high-calcium-intake group. In the case of rs2270916, individuals with the CC genotype and low calcium intake showed an increased colorectal cancer risk compared to their counterparts with the TT genotype and high calcium intake (OR = 2.11, 95% CI = 1.27–3.51).

Conclusions

Subjects with lower calcium intake exhibited a higher colorectal cancer risk compared with subjects with the same genotype who had higher calcium intake. Our results suggest that individuals who have low dietary calcium intake should be aware of their increased colorectal cancer risk and prevention strategies.     

Insulin-Like Growth Factor Binding Proteins Increase Intracellular Calcium Levels in Two Different Cell Lines

Background

Insulin-like growth factor binding proteins (IGFBPs) are six related secreted proteins that share IGF-dependent and -independent functions. If the former functions begin to be well described, the latter are somewhat more difficult to investigate and to characterize. At the cellular level, IGFBPs were shown to modulate numerous processes including cell growth, differentiation and apoptosis. However, the molecular mechanisms implicated remain largely unknown. We previously demonstrated that IGFBP-3, but not IGFBP-1 or IGFBP-5, increase intracellular calcium concentration in MCF-7 cells (Ricort J-M et al. (2002) FEBS lett 527: 293–297).

Methodology/Principal Findings

We perform a global analysis in which we studied, by two different approaches, the binding of each IGFBP isoform (i.e., IGFBP-1 to -6) to the surface of two different cellular models, MCF-7 breast adenocarcinoma cells and C2 myoblast proliferative cells, as well as the IGFBP-induced increase of intracellular calcium concentration. Using both confocal fluorescence microscopy and flow cytometry analysis, we showed that all IGFBPs bind to MCF-7 cell surface. By contrast, only four IGFBPs can bind to C2 cell surface since neither IGFBP-2 nor IGFBP-4 were detected. Among the six IGFBPs tested, only IGFBP-1 did not increased intracellular calcium concentration whatever the cellular model studied. By contrast, IGFBP-2, -3, -4 and -6, in MCF-7 cells, and IGFBP-3, -5 and -6, in C2 proliferative cells, induce a rapid and transient increase in intracellular free calcium concentration. Moreover, IGFBP-2 and -3 (in MCF-7 cells) and IGFBP-5 (in C2 cells) increase intracellular free calcium concentration by a pertussis toxin sensitive signaling pathway.

Conclusions

Our results demonstrate that IGFBPs are able to bind to cell surface and increase intracellular calcium concentration. By characterizing the IGFBPs-induced cell responses and intracellular couplings, we highlight the cellular specificity and complexity of the IGF-independent actions of these IGF binding proteins.     

Sulfate but Not Thiosulfate Reduces Calculated and Measured Urinary Ionized Calcium and Supersaturation: Implications for the Treatment of Calcium Renal Stones

Background

Urinary sulfate (SO₄ ²⁻) and thiosulfate (S₂O₃ ²⁻) can potentially bind with calcium and decrease kidney stone risk. We modeled the effects of these species on the concentration of ionized calcium (iCa) and on supersaturation (SS) of calcium oxalate (CaOx) and calcium phosphate (CaP), and measured their in vitro effects on iCa and the upper limit of stability (ULM) of these salts.

Methods

Urine data from 4 different types of stone patients were obtained from the Mayo Nephrology Clinic (Model 1). A second data set was obtained from healthy controls and hypercalciuric stone formers in the literature who had been treated with sodium thiosulfate (STS) (Model 2). The Joint Expert Speciation System (JESS) was used to calculate iCa and SS. In Model 1, these parameters were calculated as a function of sulfate and thiosulfate concentrations. In Model 2, data from pre- and post STS urines were analyzed. ULM and iCa were determined in human urine as a function of sulfate and thiosulfate concentrations.

Results

Calculated iCa and SS values for all calcium salts decreased with increasing sulfate concentration. Thiosulfate had no effect on these parameters. In Model 2, calculated iCa and CaOx SS increased after STS treatment, but CaP SS decreased, perhaps due to a decrease in pH after STS treatment. In confirmatory in vitro experiments supplemental sulfate, but not thiosulfate, significantly increased the calcium needed to achieve the ULM of CaP and tended to increase the oxalate needed to reach the ULM of CaOx. Sulfate also significantly decreased iCa in human urine, while thiosulfate had no effect.

Conclusion

Increasing urinary sulfate could theoretically reduce CaOx and CaP stone risk. Although STS may reduce CaP stone risk by decreasing urinary pH, it might also paradoxically increase iCa and CaOx SS. As such, STS may not be a viable treatment option for stone disease.     

Photounbinding of calmodulin from a family of CaM binding peptides

Background

Recent studies have shown that fluorescently labeled antibodies can be dissociated from their antigen by illumination with laser light. The mechanism responsible for the photounbinding effect, however, remains elusive. Here, we give important insights into the mechanism of photounbinding and show that the effect is not restricted to antibody/antigen binding.

Methodology/Principal Findings

We present studies of the photounbinding of labeled calmodulin (CaM) from a set of CaM-binding peptides with different affinities to CaM after one- and two-photon excitation. We found that the photounbinding effect becomes stronger with increasing binding affinity. Our observation that photounbinding can be influenced by using free radical scavengers, that it does not occur with either unlabeled protein or non-fluorescent quencher dyes, and that it becomes evident shortly after or with photobleaching suggest that photounbinding and photobleaching are closely linked.

Conclusions/Significance

The experimental results exclude surface effects, or heating by laser irradiation as potential causes of photounbinding. Our data suggest that free radicals formed through photobleaching may cause a conformational change of the CaM which lowers their binding affinity with the peptide or its respective binding partner.     

Calcium Intake and Serum Concentration in Relation to Risk of Cardiovascular Death in NHANES III

Background

Evidence for an association between calcium intake and risk of cardiovascular death remains controversial. By assessing dietary intake, use of supplements, and serum levels of calcium, we aimed to disentangle this link in the third National Health and Nutrition Examination Survey (NHANES III).

Methods

Mortality linkage of NHANES III to death certificate data for those aged 17 years or older (n = 20,024) was used to estimate risk of overall cardiovascular death as well as death from ischemic heart disease (IHD), acute myocardial infarction (AMI), heart failure (HF), and cerebrovascular disease (CD) with multivariate Cox proportional hazards regression analysis.

Results

About 10.0% of the population died of cardiovascular disease and the majority (5.4%) died of IHD. There was increased risk of overall CVD death for those in the bottom 5% of serum calcium compared to those in the mid 90% (HR: 1.51 (95% CI: 1.03–2.22)). For women there was a statistically significant increased risk of IHD death for those with serum calcium levels in the top 5% compared to those in the mid 90% (HR: 1.72 (95%CI: 1.13–2.61)), whereas in men, low serum calcium was related to increased IHD mortality (HR: 2.32 (95% CI 1.14–3.01), P_interaction: 0.306). No clear association with CVD death was observed for dietary or supplemental calcium intake.

Conclusions

Calcium as assessed by serum concentrations is involved in cardiovascular health, though differential effects by sex may exist. No clear evidence was found for an association between dietary or supplementary intake of calcium and cardiovascular death.     

Mesenchymal Stem Cells Augment the Anti-Bacterial Activity of Neutrophil Granulocytes

Background

Mesenchymal stem cells (MSCs) participate in the regulation of inflammation and innate immunity, for example by responding to pathogen-derived signals and by regulating the function of innate immune cells. MSCs from the bone-marrow and peripheral tissues share common basic cell-biological functions. However, it is unknown whether these MSCs exhibit different responses to microbial challenge and whether this response subsequently modulates the regulation of inflammatory cells by MSCs.

Methodology/Principal Findings

We isolated MSCs from human bone-marrow (bmMSCs) and human salivary gland (pgMSCs). Expression levels of TLR4 and LPS-responsive molecules were determined by flow cytometry and quantitative PCR. Cytokine release was determined by ELISA. The effect of supernatants from unstimulated and LPS-stimulated MSCs on recruitment, cytokine secretion, bacterial clearance and oxidative burst of polymorphonuclear neutrophil granulocytes (PMN) was tested in vitro. Despite minor quantitative differences, bmMSCs and pgMSCs showed a similar cell biological response to bacterial endotoxin. Both types of MSCs augmented anti-microbial functions of PMNs LPS stimulation, particularly of bmMSCs, further augmented MSC-mediated activation of PMN.

Conclusions/Significance

This study suggests that MSCs may contribute to the resolution of infection and inflammation by promoting the anti-microbial activity of PMNs. This property is exerted by MSCs derived from both the bone-marrow and peripheral glandular tissue.     

Fractalkine Depresses Cardiomyocyte Contractility

Background

Our laboratory reported that male mice with cardiomyocyte-selective knockout of the prostaglandin E₂ EP4 receptor sub-type (EP4 KO) exhibit reduced cardiac function. Gene array on left ventricles (LV) showed increased fractalkine, a chemokine implicated in heart failure. We therefore hypothesized that fractalkine is regulated by PGE₂ and contributes to depressed contractility via alterations in intracellular calcium.

Methods

Fractalkine was measured in LV of 28–32 week old male EP4 KO and wild type controls (WT) by ELISA and the effect of PGE₂ on fractalkine secretion was measured in cultured neonatal cardiomyocytes and fibroblasts. The effect of fractalkine on contractility and intracellular calcium was determined in Fura-2 AM-loaded, electrical field-paced cardiomyocytes. Cardiomyocytes (AVM) from male C57Bl/6 mice were treated with fractalkine and responses measured under basal conditions and after isoproterenol (Iso) stimulation.

Results

LV fractalkine was increased in EP4 KO mice but surprisingly, PGE₂ regulated fractalkine secretion only in fibroblasts. Fractalkine treatment of AVM decreased both the speed of contraction and relaxation under basal conditions and after Iso stimulation. Despite reducing contractility after Iso stimulation, fractalkine increased the Ca²⁺ transient amplitude but decreased phosphorylation of cardiac troponin I, suggesting direct effects on the contractile machinery.

Conclusions

Fractalkine depresses myocyte contractility by mechanisms downstream of intracellular calcium.     

Effects of Intracellular Calcium and Actin Cytoskeleton on TCR Mobility Measured by Fluorescence Recovery

Background

The activation of T lymphocytes by specific antigen is accompanied by the formation of a specialized signaling region termed the immunological synapse, characterized by the clustering and segregation of surface molecules and, in particular, by T cell receptor (TCR) clustering.

Methodology/Principal Findings

To better understand TCR motion during cellular activation, we used confocal microscopy and photo-bleaching recovery techniques to investigate the lateral mobility of TCR on the surface of human T lymphocytes under various pharmacological treatments. Using drugs that cause an increase in intracellular calcium, we observed a decrease in TCR mobility that was dependent on a functional actin cytoskeleton. In parallel experiments measurement of filamentous actin by FACS analysis showed that raising intracellular calcium also causes increased polymerization of the actin cytoskeleton. These in vitro results were analyzed using a mathematical model that revealed effective binding parameters between TCR and the actin cytoskeleton.

Conclusion/Significance

We propose, based on our results, that increase in intracellular calcium levels leads to actin polymerization and increases TCR/cytoskeleton interactions that reduce the overall mobility of the TCR. In a physiological setting, this may contribute to TCR re-positioning at the immunological synapse.     

Utility of Intraoperative Frozen Section in the Diagnosis of Periprosthetic Joint Infection

Purpose

Intraoperative frozen section (FS) is an effective diagnostic test for periprosthetic joint infection (PJI). We evaluated the diagnostic characteristics of single- and multiplex-site intraoperative FS, and evaluated the results of single-site FS combined with those of C-reactive protein (CRP) level and erythrocyte sedimentation rate (ESR) for assessing PJI.

Methods

We studied 156 painful joint arthroplasties in 152 consecutive patients presenting for revision total joint arthroplasty due to PJI. Receiver operating characteristic analysis was used to determine the optimal cutoff values for CRP level, ESR, and intraoperative FS histopathology. Sensitivity, specificity, positive and negative predictive values, and accuracy of the diagnostic tests were assessed using a 2×2 table.

Results

We investigated the diagnostic utility of polymorphonuclear leukocyte number (PMN) per high-power field (HPF) on FS. Our data showed that 5 PMNs per HPF is a suitable diagnostic threshold, with a high accuracy in single- and multiplex-site FS. Five PMNs in any 1 of 5 sites had the highest sensitivity of 0.86 and a specificity of 0.96. Five PMNs in every 1 of 5 sites had greater diagnostic utility, with a specificity of 1; however, the sensitivity of this measure fell to 0.62. Five PMNs in single-site FS had a sensitivity of 0.70 and a specificity of 0.94. Five PMNs in single-site FS or CRP level ≥15 mg/L increased the sensitivity to 0.92; however, the specificity decreased to 0.79.

Conclusion

Compared with single-site FS, any 1 positive site on multiplex-site FS may improve sensitivity, while every 1 positive site on multiplex-site FS may improve specificity. Five PMNs in any 1 of 5 sites on FS has excellent utility for the diagnosis of PJI. Additional systematic large-scale studies are needed to verify this result.     

Peeping into Human Renal Calcium Oxalate Stone Matrix: Characterization of Novel Proteins Involved in the Intricate Mechanism of Urolithiasis

Background

The increasing number of patients suffering from urolithiasis represents one of the major challenges which nephrologists face worldwide today. For enhancing therapeutic outcomes of this disease, the pathogenic basis for the formation of renal stones is the need of hour. Proteins are found as major component in human renal stone matrix and are considered to have a potential role in crystal–membrane interaction, crystal growth and stone formation but their role in urolithiasis still remains obscure.

Methods

Proteins were isolated from the matrix of human CaOx containing kidney stones. Proteins having MW>3 kDa were subjected to anion exchange chromatography followed by molecular-sieve chromatography. The effect of these purified proteins was tested against CaOx nucleation and growth and on oxalate injured Madin–Darby Canine Kidney (MDCK) renal epithelial cells for their activity. Proteins were identified by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF MS) followed by database search with MASCOT server. In silico molecular interaction studies with CaOx crystals were also investigated.

Results

Five proteins were identified from the matrix of calcium oxalate kidney stones by MALDI-TOF MS followed by database search with MASCOT server with the competence to control the stone formation process. Out of which two proteins were promoters, two were inhibitors and one protein had a dual activity of both inhibition and promotion towards CaOx nucleation and growth. Further molecular modelling calculations revealed the mode of interaction of these proteins with CaOx at the molecular level.

Conclusions

We identified and characterized Ethanolamine-phosphate cytidylyltransferase, Ras GTPase-activating-like protein, UDP-glucose:glycoprotein glucosyltransferase 2, RIMS-binding protein 3A, Macrophage-capping protein as novel proteins from the matrix of human calcium oxalate stone which play a critical role in kidney stone formation. Thus, these proteins having potential to modulate calcium oxalate crystallization will throw light on understanding and controlling urolithiasis in humans.