Identification of calcineurin as a predictor of oocyte quality and fertilization competence based on microarray data.

OBJECTIVE: The aim of our study was to detect a biomarker for selection of competent oocytes with acceptable fertilization potential. Calcium ion fluctuation play the most critical role of modulating intercellular signaling pathways in oocyte maturation, egg activation and the egg-to-embryo transition. Since, the stimulatory action of calcium ion is mediated by binding to certain proteins, the calcium/calmodulin-binding genes (CBGs), as the main calcium binding group, was analyzed in detail. METHODS: In this work, bioinformatics analysis was conducted on the CBGs of human cumulus cells (CCs) to elucidate a reliable biomarker for fertile oocyte selection. Calcineurin (CaN) or protein phosphatase 3 (PPP3) was selected which consists of a catalytic subunit A with PPP3CA (Aα), PPP3CB (Aß), and PPP3CC (Aγ) isoforms and a regulatory subunit B. Whereas CaN A regulates calcium ion function, our study gives insights to probable role of related isoforms within human oogenesis process. The presence of CaN A in CCs surrounding growing and mature oocytes was confirmed by western blotting and the expression patterns of related isoforms were examined by reverse transcription-quantitative PCR (RT-qPCR). RESULTS: Our results indicated the increased expression of the catalytic subunit of CaN protein in the CCs of metaphase (M) II oocytes. The expression level of PPP3CB was significantly elevated in CCs of fertile MII compared with those in the germinal vesicle (GV), MI and unfertilized MII oocytes (P ≤ 0.05). CONCLUSION: Elevated level of PPP3CB isoform in the CCs of fertile MII oocyte could be a reliable indication of oocyte fertilization potential. However, further researches are required to introduce CaN Aß as an appropriate biomarker for oocyte selection in assisted reproduction technique (ART) programs.

Biochemical Activity Architectures Visualized-Using Genetically Encoded Fluorescent Biosensors to Map the Spatial Boundaries of Signaling Compartments.

All biological processes arise through the coordinated actions of biochemical pathways. How such functional diversity is achieved by a finite cast of molecular players remains a central mystery in biology. Spatial compartmentation-the idea that biochemical activities are organized around discrete spatial domains within cells-was first proposed nearly 40 years ago and has become firmly rooted in our understanding of how biochemical pathways are regulated to ensure specificity. However, directly interrogating spatial compartmentation and its mechanistic origins has only really become possible in the last 20 or so years, following technological advances such as the development of genetically encoded fluorescent biosensors. These powerful molecular tools permit a direct, real-time visualization of dynamic biochemical processes in native biological contexts, and they are essential for probing the spatial regulation of biochemical activities. In this Account, we review our lab's efforts in developing and using biosensors to map the spatial compartmentation of intracellular signaling pathways and illuminate key mechanisms that establish the boundaries of an intricate biochemical activity architecture. We first discuss the role of regulatory fences, wherein the dynamic activation and deactivation of diffusible messengers produce diverse signaling compartments. For example, we used biosensors for the Ca2+ effector calmodulin and its downstream target calcineurin to reveal a spatial gradient of calmodulin that controls the temporal dynamics of calcineurin signaling. Our studies using cyclic adenosine monophosphate (cAMP) biosensors have similarly elucidated fenced cAMP domains generated by competing production and degradation pathways, ranging in size from cell-spanning gradients to nanoscale hotspots. Second, we describe the role played by intracellular membranes in creating unique signaling platforms with distinctive pathway regulation, as revealed through studies using subcellularly targeted fluorescent biosensors. Using biosensors to visualize subcellular extracellular response kinase (ERK) pathway activity, for example, led us to discover a local signaling circuit that mediates distinct plasma membrane ERK dynamics versus global ERK signaling. Similarly, our work developing biosensors to monitor the subcellular mechanistic target of rapamycin complex 1 (mTORC1) signaling allowed us to not only clarify the presence of mTORC1 activity in the nucleus but also identify a novel mechanism governing the activation of mTORC1 in this location. Finally, we detail how molecular assemblies enable the precise spatial tuning of biochemical activity, through investigations enabled by cutting-edge advances in biosensor design. We recently identified liquid-liquid phase separation as a major factor in cAMP compartmentation aided by a new strategy for targeting biosensors to endogenously expressed proteins via genome editing, for instance, and have also been able to directly visualize nanometer-scale protein kinase signalosomes using an entirely new class of biosensors specifically developed for the dynamic super-resolution imaging of live-cell biochemical activities. Our work provides key insights into the molecular logic of spatially regulated signaling and lays the foundation for a broader exploration of biochemical activity architectures across multiple spatial scales.

[Shendi granules regulates the balance of T cell subsets and down-regulates podocalyxin in rats with mesangial proliferative glomerulonephritis].

Objective To observe the effect of Shendi granules on T cell subsets and podocyte marker protein in rats with mesangial proliferative glomerulonephritis (MsPGN), and study possible mechanism. Methods Totally 40 SD rats were randomly divided into the model group, valsartan group, Shendi granule group and normal group. The Shendi granule group were given 4 g/(kg.d) of Shendi granule by gavage; the valsartan group were given 10.3 mg/(kg.d) of valsartan by gavage; the model group and normal group were given the same amount of saline per day by gavage. The treatments lasted 12 weeks. Routine biochemical method was used to quantify 24-hour urine protein; the numbers of CD4+ and CD8+ T cells were detected by flow cytometry; the serum levels of interleukin 2 (IL-2), IL-4 and IL-17, the levels of urinary podocalyxin (PCX) and B7-1, the renal calcineurin (CaN) content were determined by ELISA. Results Compared with the normal group, the levels of 24-hour urine protein, CD8+ T cells, serum IL-2 and IL-17, urinary PCX and B7-1, CaN in the model group were higher. The above indexes in the valsartan group and control group were lower than those in the model group, they were lower in the Shendi granule group than in the valsartan group. The whole blood CD4+ T cell number and serum IL-4 level in the model group were lower than those in the normal group, they were higher in the valsartan group and control group than in the model group. Compared with the valsartan group, the Shendi granule group had a better improvement. Conclusion Shendi granule could reduce 24-hour urine protein effectively. The mechanism may be related to the regulation of CD4+ T and CD8+ T cell numbers, the down-regulated expressions of serum IL-2, IL-17, the decreased levels of PCX and B7-1 in urine, CaN in kidney tissue, and the up-regulated level of serum IL-4.

The Calcineurin Variant CnAß1 Controls Mouse Embryonic Stem Cell Differentiation by Directing mTORC2 Membrane Localization and Activation.

Embryonic stem cells (ESC) have the potential to generate all the cell lineages that form the body. However, the molecular mechanisms underlying ESC differentiation and especially the role of alternative splicing in this process remain poorly understood. Here, we show that the alternative splicing regulator MBNL1 promotes generation of the atypical calcineurin Aß variant CnAß1 in mouse ESCs (mESC). CnAß1 has a unique C-terminal domain that drives its localization mainly to the Golgi apparatus by interacting with Cog8. CnAß1 regulates the intracellular localization and activation of the mTORC2 complex. CnAß1 knockdown results in delocalization of mTORC2 from the membrane to the cytoplasm, inactivation of the AKT/GSK3ß/ß-catenin signaling pathway, and defective mesoderm specification. In summary, here we unveil the structural basis for the mechanism of action of CnAß1 and its role in the differentiation of mESCs to the mesodermal lineage.

Quality-control method for the determination of biological activity of engineered calcineurin subunit B.

The aim of this study was to establish a quality-control method for calcineurin subunit B (CNB) biological activity determinations. CNB enhances the p-nitrophenylphosphate (pNPP) dephosphorylating activity of calcineurin subunit A Δ316 mutant (CNAΔ316). A series of CNB concentrations were fitted to a four-parameter equation to calculate the corresponding pNPP maximum dephosphorylation rates. Values were calculated based on biological activity references using a parallel line method. The method was then validated for accuracy, precision, linearity, linear range, sensitivity, specificity, and robustness. The recovery results were greater than 98%. Intra-plate precision was 6.7%, with inter-plate precision of 10.8%. The coefficient of determination was greater than 0.98. The linear range was 0.05-50 µg mL(-1), with sensitivity of 50 µg mL(-1). Tested cytokines did not induce CNAΔ316 dephosphorylation of pNPP. The chosen CNAΔ316 concentration range did not affect activity determinations.

Calpain-Calcineurin-Nuclear Factor Signaling and the Development of Atrial Fibrillation in Patients with Valvular Heart Disease and Diabetes.

Calpain, calcineurin (CaN), and nuclear factor of activated T cell (NFAT) play a key role in the development of atrial fibrillation. Patients with valvular heart disease (VHD) are prone to develop atrial fibrillation (AF). Thus, our current study was aimed at investigating whether activation of calpain-CaN-NFAT pathway is associated with the incidence of AF in the patients with VHD and diabetes. The expressions of calpain 2 and alpha- and beta-isoforms of CaN catalytic subunit (CnA) as well as NFAT-c3 and NFAT-c4 were quantified by quantitative reverse transcription-polymerase chain reaction in atrial tissues from 77 hospitalized patients with VHD and diabetes. The relevant protein content was measured by Western blot and calpain 2 in human atrium was localized by immunohistochemistry. We found that the expressions of calpain 2, CnA alpha and CnA beta, and NFAT-c3 but not NFAT-c4 were significantly elevated in the samples from patients with AF compared to those with sinus rhythm (SR). Elevated protein levels of calpain 2 and CnA were observed in patients with AF, and so was the enhanced localization of calpain 2. We thereby concluded that CaN together with its upstream molecule, calpain 2, and its downstream effector, NFAT-c3, might contribute to the development of AF in patients with VHD and diabetes.

[Qibaipingfei Capsule down-regulate the levels of calcineurin and nuclear factor of activated T-cells isoform c3 (NFATc3) in chronic obstructive pulmonary disease].

OBJECTIVE: To investigate the effect of Qibaipingfei Capsule (QPC) on the expressions of calcineurin (CaN) and nuclear factor of activated T-cells isoform c3 (NFATc3) of rat models with phlegm and blood stasis syndrome of chronic obstructive pulmonary disease (COPD), and to explore the possible mechanism underlying the intervention of QPC in pulmonary vascular remodeling of COPD. METHODS: Sixty male Sprague-Dawley (SD) rats were randomly divided into a normal group, a model group, a positive group of nifedipine, a high dose group, a middle dose group and a low dose group of QPC. The rat models with phlegm and blood stasis syndrome of COPD were established by compound methods of forced swimming, smoking and hypoxia. Then the pulmonary function and the pathological alterations of pulmonary vessels were observed. Furthermore, the mRNA and protein levels of CaN and NFATc3 in the lung tissues were determined by real-time quantitative PCR and Western blot analysis. RESULTS: Compared with the normal group, the forced expiratory volume at 0.3 second (FEV0.3), the forced vital capacity (FVC) and FEV0.3/FVC in the model group were significantly reduced, but compared with the model group, the values mentioned above were restored to different extents in the groups of nifedipine and QPC. The lung tissues of the model group showed the thickening of pulmonary vascular wall and the formation of compensating emphysema. The above pathological changes were relieved in all the treatment groups. Compared with the normal group, the expressions of CaN and NFATc3 in the model group were significantly up-regulated in transcription and translation levels. Compared with the model group, these expressions were down-regulated to various degrees in all the treatment groups. CONCLUSION: QPC can decrease the levels of CaN and NFATc3 in the lung tissues of COPD.

Comparison of Microcystis aeruginosa (PCC7820 and PCC7806) growth and intracellular microcystins content determined by liquid chromatography-mass spectrometry, enzyme-linked immunosorbent assay anti-Adda and phosphatase bioassay.

Cyanobacteria are able to produce several metabolites that have toxic effects on humans and animals. Among these cyanotoxins, the hepatotoxic microcystins (MC) occur frequently. The intracellular MC content produced by two strains of Microcystis aeruginosa, PCC7806 and PCC7820, and its production kinetics during the culture time were studied in order to elucidate the conditions that favour the growth and proliferation of these toxic strains. Intracellular MC concentrations measured by liquid chromatography (LC) coupled to electrospray ionization mass spectrometer (MS) were compared with those obtained by enzyme-linked immunosorbent assay (ELISA) anti-Adda and protein phosphatase 2A (PP2A) inhibition assays. It has been demonstrated there are discrepancies in the quantification of MC content when comparing ELISA and LC-MS results. However, a good correlation has been obtained between PP2A inhibition assay and LC-MS. Three MC were identified using LC-MS in the PCC7806 strain: MC-LR, demethylated MC-LR and a new variant detected for the first time in this strain, [L-MeSer(7)] MC-LR. In PCC7820, MC-LR, D-Asp(3)-MCLR, Dglu(OCH3)-MCLR, MC-LY, MC-LW and MC-LF were identificated. The major one was MC-LR in both strains, representing 81 and 79% of total MC, respectively. The total MC content in M. aeruginosa PCC7820 was almost three-fold higher than in PCC7806 extracts.

Nutritional intervention restores muscle but not kidney phenotypes in adult calcineurin Aα null mice.

Mice lacking the α isoform of the catalytic subunit of calcineurin (CnAα) were first reported in 1996 and have been an important model to understand the role of calcineurin in the brain, immune system, bones, muscle, and kidney. Research using the mice has been limited, however, by failure to thrive and early lethality of most null pups. Work in our laboratory led to the rescue of CnAα-/- mice by supplemental feeding to compensate for a defect in salivary enzyme secretion. The data revealed that, without intervention, knockout mice suffer from severe caloric restriction. Since nutritional deprivation is known to significantly alter development, it is imperative that previous conclusions based on CnAα-/- mice are revisited to determine which aspects of the phenotype were attributable to caloric restriction versus a direct role for CnAα. In this study, we find that defects in renal development and function persist in adult CnAα-/- mice including a significant decrease in glomerular filtration rate and an increase in blood urea nitrogen levels. These data indicate that impaired renal development we previously reported was not due to caloric restriction but rather a specific role for CnAα in renal development and function. In contrast, we find that rather than being hypoglycemic, rescued mice are mildly hyperglycemic and insulin resistant. Examination of muscle fiber types shows that previously reported reductions in type I muscle fibers are no longer evident in rescued null mice. Rather, loss of CnAα likely alters insulin response due to a reduction in insulin receptor substrate-2 (IRS2) expression and signaling in muscle. This study illustrates the importance of re-examining the phenotypes of CnAα-/- mice and the advances that are now possible with the use of adult, rescued knockout animals.

Genetic stretching factors in masseter muscle after orthognathic surgery.

Up to 30% of patients relapse after orthognathic operations, and one reason might be incomplete neuromuscular adaptation of the masticatory muscles. Displacement of the mandible in sagittal or vertical directions, or both, leads to stretching or compression of these muscles. The aim of this study was to analyse stretching factors in 35 patients with retrognathism or prognathism of the mandible (Classes II and III). Tissue samples were taken from both sides of the masseter muscle (anterior and posterior) both before and 6 months after operation. Developmental myosin heavy chains MYH3 and MYH8, the fast and slow MYH 1, 2, and 7, and cyclo-oxygenase (COX) 2, forkhead transcription factor (FOX)O3a, calcineurin, and nuclear factor of activated T cells (NFAT)1c (stretching and regeneration-specific), were analysed by real time polymerase chain reaction (PCR). Correlations of Class II and III with sagittal and vertical cephalometric measurements ANB and ML-NL-angle were examined, and the results showed significant differences in amounts of MYH8 (p<0.05), MYH1 (p<0.05), and FOXO3a (p<0.05) between the 2 groups. Regeneration factor COX2 is more dominant in Class II. Surgically, bite opening (ML/NL angle) correlated with stretching indicators FOXO3a, calcineurin, and NFAT1c only in Class II patients. This means that stretching of the masseter muscle caused by lengthening of the mandible and raising of the bite in Class II patients was more likely to lead to relapse (similar to that in patients with open bite) than in Class III patients. In conclusion, deep bite should be reduced more by incisor intrusion than by skeletal opening. The focus in these patients should be directed towards physiotherapeutic strengthening of the muscles of mastication, and more consideration should be given to change in the vertical dimension.

Cell-specific expression of calcineurin immunoreactivity within the rat basolateral amygdala complex and colocalization with the neuropeptide Y Y1 receptor.

Neuropeptide Y (NPY) produces potent anxiolytic effects via activation of NPY Y1 receptors (Y1r) within the basolateral amygdaloid complex (BLA). The role of NPY in the BLA was recently expanded to include the ability to produce stress resilience and long-lasting reductions in anxiety-like behavior. These persistent behavioral effects are dependent upon activity of the protein phosphatase, calcineurin (CaN), which has long been associated with shaping long-term synaptic signaling. Furthermore, NPY-induced reductions in anxiety-like behavior persist months after intra-BLA delivery, which together indicate a form of neuronal plasticity had likely occurred. To define a site of action for NPY-induced CaN signaling within the BLA, we employed multi-label immunohistochemistry to determine which cell types express CaN and if CaN colocalizes with the Y1r. We have previously reported that both major neuronal cell populations in the BLA, pyramidal projection neurons and GABAergic interneurons, express the Y1r. Therefore, this current study evaluated CaN immunoreactivity in these cell types, along with Y1r immunoreactivity. Antibodies against calcium-calmodulin kinase II (CaMKII) and GABA were used to identify pyramidal neurons and GABAergic interneurons, respectively. A large population of CaN immunoreactive cells displayed Y1r immunoreactivity (90%). Nearly all (98%) pyramidal neurons displayed CaN immunoreactivity, while only a small percentage of interneurons (10%) contained CaN immunoreactivity. Overall, these anatomical findings provide a model whereby NPY could directly regulate CaN activity in the BLA via activation of the Y1r on CaN-expressing, pyramidal neurons. Importantly, they support BLA pyramidal neurons as prime targets for neuronal plasticity associated with the long-term reductions in anxiety-like behavior produced by NPY injections into the BLA.

Determinants of sodium removal with tidal automated peritoneal dialysis.

In a comprehensive evaluation of dialysis adequacy, major attention has been recently paid to fluid and Na balance. Removal of Na has been reported to be significantly poorer with automated peritoneal dialysis (APD) than with continuous ambulatory peritoneal dialysis. Only limited data on Na removal with tidal APD have been published. We analyzed peritoneal Na mass balance in 122 separate nightly tidal APD sessions performed by 7 peritonitis-free, clinically stable, patients with negligible residual renal function (< 100 mL urine daily). Correlations with other efficiency measures [ultrafiltration (UF) and small-solute clearances], prescriptive parameters [duration of treatment, initial intraperitoneal fill volume (IPV) and its tidal percentage, and dialysate flux] and peritoneal transport status were tested in univariate and multivariate linear regression models. Removal of Na was 89 +/- 55 mmol per treatment, which correlated with UF (r = 0.29, p = 0.001) and was higher in patients with high-average transport (118 +/- 41 mmol vs. 81 +/- 56 mmol in low-average transporters, p = 0.0004), in whom a significant positive correlation was found with initial IPV and duration of treatment (r = 0.55; 95% confidence interval: 0.21 to 0.77; p = 0.0029; and r = 0.66; 95% confidence interval: 0.38 to 0.83; p = 0.0002 respectively). Removal of Na correlated weakly with UF in tidal APD and showed wide inter-patient variability. It should therefore be measured rather than roughly estimated from UF. Its magnitude exposes the anuric patient on nightly APD with a "dry" day to the risk of Na retention, unless controlled Na intake or dialytic strategies aimed at enhancing Na removal, or both, are implemented.

Molecular diagnostics of calcineurin-related pathologies.

BACKGROUND: The Ca(2+)-dependent protein phosphatase enzyme calcineurin (Cn) (protein phosphatase 3) is best known for its role as director of the adaptive immune response. One of its principal substrates is the nuclear factor of activated T cells (NFAT), which translocates to the nucleus after dephosphorylation to mediate gene transcription. Drugs targeting Cn (the Cn inhibitors tacrolimus and cyclosporin A) have revolutionized posttransplantation therapy in allograft recipients by considerably reducing rejection rates. CONTENT: Owing primarily to intensive study of the side effects of the Cn inhibitors, the unique importance of Cn and Cn/NFAT signaling in the normal physiological processes of many other cell and tissue types is becoming more evident. During the last decade, it has become clear that an extensive and diverse array of clinical conditions can be traced back, at least in part, to a disturbed Cn-signaling axis. Hence, both diagnostics and therapeutic monitoring could benefit from a technique that conveniently reads out Cn/NFAT operative status. SUMMARY: This review outlines the current knowledge on the pathologic conditions that have calcineurin as a common denominator and reports on the progress that has been made toward successfully applying Cn and Cn/NFAT activity markers in molecular diagnostics.

Inmunosupresíón en niños con trasplante hepático / Immunosuppression in children with liver transplantation

La inmunosupresión en niños con trasplante hepático, ha evolucionado con dos momentos clave: la disponibilidad de los inhibidores de calcineurina ciclosporina y tacrolimus. La inmunosupresión primaria se diseña sobre la base de un inhibidor de calcineurina como fármaco principal. Los esteroides se incluyen en la pauta de inmunosupresión primaria en la mayoría de los centros. Las pautas habituales a largo plazo consisten en ciclosporina o tacrolimus, en monoterapia a niveles inferiores a los deseados en el periodo precoz postrasplante, o en combinación con dosis bajas de esteroide. Los inhibidor e s de c a l c ineur ina induc en vasoconstricción arterial aguda y crónica que causa nefrotoxicidad, con disminución del filtrado glomerular y tubulopatía. Los niveles ensangre de ciclosporina o de tacrolimus se determinan para evaluar el estado de inmunosupresión. La edad de adolescente y adulto joven es una etapa de riesgo para el injerto por ser frecuente la omisión accidental de dosis de medicación inmunosupresora, una irregularidad que es difícil de evaluar en su extensión a pesar de una buena relación médicopaciente y frecuentes chequeos. El rechazo tiene una incidencia entre el 30 y 50% de los pacientes, entre los días 5 y 30 postrasplante.

Immunosuppression in children with liver transplantation has evolved with two key moments: the availability of calcineurin inhibitors, cyclosporine and tacrolimus. The primary immunosuppression is designed on the basis of a calcineurin inhibitor as primary drug. Steroids are included in the pr imary immunosuppression regimen in most schools. The long-term normal patterns consist of cyclosporine or tacrolimus as monotherapy to lower than desired levels in the early period aftertransplantation, or in combination with low dose steroid. Calcineurin inhibitors induce arterial vasoconstriction causing acute and chronic nephrotoxicity, with reduced glomerular filtration and tubular. Blood levels of cyclosporine or tacrolimus are determined to assess the state of immunosuppression. The age of adolescence and young adulthood is a time of risk to the graft by the accidental omission to be frequent doses ofimmunosuppressive medication, an irregularitywhich is difficult to assess its extent in spite of a good doctor-patient relationship and frequentcheckups. The rejection has an incidence between 30 and 50% of patients, between 5 and 30 aftertransplantation.

Calcineurin activity is required for the completion of cytokinesis.

Successful completion of cytokinesis requires the spatio-temporal regulation of protein phosphorylation and the coordinated activity of protein kinases and phosphatases. Many mitotic protein kinases are well characterized while mitotic phosphatases are largely unknown. Here, we show that the Ca(2+)- and calmodulin-dependent phosphatase, calcineurin (CaN), is required for cytokinesis in mammalian cells, functioning specifically at the abscission stage. CaN inhibitors induce multinucleation in HeLa cells and prolong the time cells spend connected via an extended intracellular bridge. Upon Ca(2+) influx during cytokinesis, CaN is activated, targeting a set of proteins for dephosphorylation, including dynamin II (dynII). At the intracellular bridge, phospho-dynII and CaN are co-localized to dual flanking midbody rings (FMRs) that reside on either side of the central midbody ring. CaN activity and disassembly of the FMRs coincide with abscission. Thus, CaN activity at the midbody plays a key role in regulating the completion of cytokinesis in mammalian cells.

Expression and activities of N-myristoyltransferase and calcineurin in normal and inflamed lungs.

The role of N-myristoyltransferase and calcineurin is well established in signaling pathways. However, there are no data on their expression and activities in normal and inflamed lungs. The mechanisms of lung inflammation induced following administration of lipopolysaccharides (LPS) or exposure to swine barn air remain unclear. Therefore, we examined expression and activities of N-myristoyltransferase and calcineurin in normal and inflamed lungs of rats. Histopathology showed acute inflammation in the lungs of rats exposed to barn air or LPS but not of control rats. There was no difference in the activities of N-myristoyltransferase and calcineurin among the control, barn-exposed, and LPS-treated rat lungs. Although N-myristoyltransferase and calcineurin were localized in airway epithelium, blood vessel walls, alveolar macrophages, and septa in the lungs of rats from all the groups, the staining intensity was increased in the lungs from rats exposed to intravenous LPS or barn air. Densitometric analyses of Western blots of 55- and 60-kDa polypeptide bands corresponding to N-myristoyltransferase and calcineurin, respectively, in the lung homogenates revealed no differences among the groups. These results show that expression of myristoyltransferase and calcineurin in lung epithelium and endothelium and a cell-specific increase in immunohistochemical expression.

Síndrome de dor óssea induzida por tacrolimo pós-transplante renal: relato de caso e revisão da literatura / Tacrolimus induced bone pain syndrome after kidney transplantation: case report and review of the literature

Objetivo: Relatar um caso de síndrome de dor óssea induzida por inibidores da calcineurina. Relato de caso: Paciente masculino de 54 anos, branco, foi que submetido a transplante renal haploidêntico e, ao fim do terceiro mês pós-transplante, apresentou dor espontânea, de forte intensidade, em joelhos, tornozelos e pés, de maneira simétrica, com incapacidade funcional, induzida por inibidores da calcineurina. O diagnóstico foi comprovado por ressonância magnética e cintilografia óssea. Evolução: Houve remissão espontânea e completa dos sintomas no fim do sexto mês após o transplante. Conclusão: A síndrome de dor osteoarticular induzida por inibidores da calcineurina é uma condição clínica incomum, mas que pode comprometer a qualidade de vida e a boa evolução do paciente transplantado. Seu diagnóstico correto deve ser feito prontamente por meio de estudos de ressonância magnética e de cintilografia óssea.

Objective: To present a case of calcineurin inhibitor-induced bone pain syndrome. Case report: A 54-year-old Caucasian male patient underwent a haploidentical kidney transplant and, at the end of the third postoperative month, developed severe, spontaneous, symmetrical pain in his knees, ankles, and feet, associated with functional impairment, induced by calcineurin inhibitors. The diagnosis was confirmed by MRI and bone scan. Evolution: The patient presented spontaneous remission of symptoms at the end of the sixth postoperative month. Conclusion: Calcineurin inhibitorinduced bone pain syndrome is an uncommon clinical condition, but it can impair the quality of life and good evolution of transplant recipients. Correct and prompt diagnosis with MRI and bone scan is recommended.

Morphological analysis of the mormyrid cerebellum using immunohistochemistry, with emphasis on the unusual neuronal organization of the valvula.

This study used immunohistochemistry, Golgi impregnation, and electron microscopy to examine the circuitry of the cerebellum of mormyrid fish. We used antibodies against the following antigens: the neurotransmitters glutamate and gamma-aminobutyric acid (GABA); the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD); GABA transporter 1; the anchoring protein for GABA and glycine receptors, gephyrin; the calcium binding proteins calbindin and calretinin; the NR1 subunit of the N-methyl-D-aspartate glutamate receptor; the metabotropic glutamate receptors mGluR1alpha and mGluR2/3; the intracellular signaling molecules calcineurin and calcium calmodulin kinase IIalpha (CAMKIIalpha); and the receptor for inositol triphosphate (IP3RIalpha). Purkinje cells are immunoreactive to anti-IP3R1alpha, anticalcineurin, and anti-mGluR1alpha. Cerebellar efferent cells (eurydendroid cells) are anticalretinin and anti-NR1 positive in the valvula but not in the corpus and caudal lobe. In contrast, climbing fibers are anticalretinin and anti-NR1 immunopositive in the corpus and caudal lobe but not in the valvula. Purkinje cells, Golgi cells, and stellate cells are GABA positive, whereas efferent cells are glutamate positive. Unipolar brush cells are immunoreactive to anti-mGluR2/3, anticalretinin, and anticalbindin. We describe a "new" cell type in the mormyrid valvula, the deep stellate cell. These cells are GABA, calretinin, and calbindin positive. They are different from superficial stellate cells in having myelinated axons that terminate massively with GAD- and gephyrin-positive terminals on the cell bodies and proximal dendrites of efferent cells. We discuss how the valvula specializations described here may act in concert with the palisade pattern of Purkinje cell dendrites for analyzing spatiotemporal patterns of parallel fiber activity.

Visualization of phosphatase activity in living cells with a FRET-based calcineurin activity sensor.

Protein kinases and phosphatases are organized into complex intracellular signaling networks designed to coordinate their activities in both space and time. In order to better understand the molecular mechanisms underlying the regulation of signal transduction networks, it is important to define the spatiotemporal dynamics of both protein kinases and phosphatases within their endogenous environment. Herein, we report the development of a genetically-encoded protein biosensor designed to specifically probe the activity of the Ca2+/calmodulin-dependent protein phosphatase, calcineurin. Our reporter design utilizes a phosphatase activity-dependent molecular switch based on the N-terminal regulatory domain of the nuclear factor of activated T-cells as a specific substrate of calcineurin, sandwiched between cyan fluorescent protein and yellow fluorescent protein. Using this reporter, calcineurin activity can be monitored as dephosphorylation-induced increases in fluorescence resonance energy transfer and can be simultaneously imaged with intracellular calcium dynamics. The successful design of a prototype phosphatase activity sensor lays a foundation for studying targeting and compartmentation of phosphatases.

Defective formation of PKA/CnA-dependent annexin 2-S100A10/CFTR complex in DeltaF508 cystic fibrosis cells.

Cystic fibrosis (CF) is characterised by impaired epithelial ion transport and is caused by mutations in the cystic fibrosis conductance regulator protein (CFTR), a cAMP/PKA and ATP-regulated chloride channel. We recently demonstrated a cAMP/PKA/calcineurin (CnA)-driven association between annexin 2 (anx 2), its cognate partner -S100A10 and cell surface CFTR. The complex is required for CFTR and outwardly rectifying chloride channel function in epithelia. Since the cAMP/PKA-induced Cl(-) current is absent in CF epithelia, we hypothesized that the anx 2-S100A10/CFTR complex may be defective in CFBE41o cells expressing the commonest F508del-CFTR (DeltaF-CFTR) mutation. Here, we demonstrate that, despite the presence of cell surface DeltaF-CFTR, cAMP/PKA fails to induce anx 2-S100A10/CFTR complex formation in CFBE41o- cells homozygous for F508del-CFTR. Mechanistically, PKA-dependent serine phosphorylation of CnA, CnA-anx 2 complex formation and CnA-dependent dephosphorylation of anx 2 are all defective in CFBE41o- cells. Immunohistochemical analysis confirms an abnormal cellular distribution of anx 2 in human and CF mouse epithelia. Thus, we demonstrate that cAMP/PKA/CnA signaling pathway is defective in CF cells and suggest that loss of anx 2-S100A10/CFTR complex formation may contribute to defective cAMP/PKA-dependent CFTR channel function.