Caractérisation spatiale des syncytia formés par le couplage des astrocytes du noyau sensoriel principal du nerf trijumeau en fonction de la concentration de calcium extracellulaire.

Le mouvement masticatoire est généré et coordonné par un générateur de patron central (GPC) situé au niveau du pont. Plusieurs résultats antérieurs de notre laboratoire soutiennent que le réseau de neurones à l’origine de la rythmogénèse est situé dans le noyau sensoriel principal du nerf trijumeau (NVsnpr). Ces mêmes expériences révèlent que des diminutions de la concentration calcique extracellulaire ([Ca2+]e) tiennent une place importante dans la génération des bouffées de décharges des neurones de cette région. Notre laboratoire tente de vérifier si la contribution des astrocytes à l’homéostasie de la concentration calcique extracellulaire est impliquée dans la genèse du rythme. Cette étude a pour but la caractérisation spatiale du syncytium astrocytaire au sein du NVsnpr dorsal et l’étude de l’effet de la [Ca2+]e sur les propriétés astrocytaires électrophysiologiques et de connectivité. Nous avons utilisés pour ce faire la technique d’enregistrement par patch-clamp sur une préparation en tranche de tronc cérébral de rat. Nous démontrons ici que la diminution de la [Ca2+]e n’affecte pas les propriétés électrophysiologiques astrocytaires, mais induit une augmentation de la taille du syncytium. De plus, nous établissons l’existence au sein du NVsnpr dorsal d’une organisation anatomofonctionnelle du réseau astrocytaire calquée sur l’organisation neuronale.

Regulation of mass and function of pancreatic β-cells: identification of anti-apoptotic peptides and role of GLP-1

Résumé La masse de cellules β sécrétrices d'insuline est un tissu dynamique qui s'adapte aux variations de la demande métabolique pour assurer une normoglycémie. Cette adaptation se fait par un changement de sécrétion d'insuline et de la masse totale des cellules β. Une perte complète ou partielle des cellules β conduit respectivement à un diabète de type 1 et de type 2. Les mécanismes qui régulent la masse de cellules β et maintiennent leur phénotype differencié sont encore peu connus. Leur identification est nécessaire pour comprendre le développement du diabète et développer des stratégies de traitement. La greffe d'îlots est une approche thérapeutique prometteuse pour le diabète de type 1, mais est limitée par une perte précoce des cellules β due à une apoptose induite par des cytokines. Afin d'améliorer la survie des cellules β lors de la greffe d'îlots, le premier but était de trouver des peptides pouvant bloquer l'apoptose induite par FasL et TNF-α. Pour ce faire, deux librairies de phages ont été criblées pour sélectionner des peptides se liant au Fas DD ou au TNFRl DD. Nous avons identifié six peptides différents. Cependant, aucun d'entre eux n'était capable de protéger les cellules de l'apoptose induite par FasL ou TNF-α. Deuxièmement, le GLP-1 est une hormone qui stimule la sécrétion d'insuline, et est impliquée dans la prolifération des cellules β, la différentiation, et inhibe l'apoptose. Nous avons fait l'hypothèse que le GLP-1 joue un rôle crucial dans le contrôle de la masse et de la fonction des cellules β. Afin de l'évaluer, une analyse par puce à ADN a été réalisée en comparant des cellules βTC-Tet traitées avec du GLP-1 à des cellules non-traitées. 376 gènes régulés ont été identifiés, dont RGS2, CREM, ICERI et DUSP14, augmentés significativement par le GLP-1. Nous avons confirmé que le GLP-1 augmente l'expression de ces gènes, aussi bien au niveau des transcripts que des protéines. De plus, nous avons montré que le GLP-1 induit leur expression par activation de la voie cAMP/PKA, et nécessite l'entrée de calcium extracellulaire. D'après leur fonction biologique, nous avons ensuite supposé que ces gènes pourraient agir comme régulateurs négatifs de la signalisation du GLP-l, et donc freiner son effet proliférateur. Pour vérifier notre hypothèse, des siRNAs contre ces gènes ont été développés, et leurs effets sur la prolifération des cellules β seront évalués ultérieurement. Abstract The pancreatic β-cell mass is a dynamic tissue which adapts to variations in metabolic demand in order to ensure normoglycemia. This adaptation occurs through a change in both insulin secretion and the total mass of ,β-cells. An absolute or relative loss of β-cells leads to type 1 and type 2 diabetes, respectively. The mechanisms that regulate the pancreatic β-cell mass and maintain the fully differentiated phenotype of the insulin-secreting β-cells are only poorly defined. Their identification is required to understand the progression of diabetes, but also to design strategies for the treatment of diabetes. Islet transplantation is a promising therapeutic approach for type 1 diabetes, but it is still limited by an early graft loss due to cytokine-induced apoptosis. In order to improve β-cell survival during islet transplantation, our first goal was to find novel blockers of FasL- and TNF-α-mediated cell death in the form of peptides. To that end, we screened two phage display libraries to select Fas DD- or TNFR1 DD-binding peptides. We identified six different small peptides. However, none of these peptides was able to prevent cells from FasL- or TNF-α-mediated apoptosis. Secondly, GLP-1 is a hormone that has been shown to stimulate insulin secretion and to be involved in β-cell proliferation, differentiation and inhibition of apoptosis. We hypothesized that GLP-1 plays a crucial role to control mass and function of β-cells. To evaluate this hypothesis, we performed a cDNA microarray analysis with GLP-1-treated βTC-Tet cells compared to untreated cells. We found 376 regulated genes, among these, RGS2, CREM, ICERI and DUSP14, which were significantly upregulated by GLP-1. We confirmed that both their mRNA and protein levels were strongly and rapidly increased after GLP-1 treatment. Moreover, we found that GLP-1 activates their expression mainly through the activation of the cAMP/PKA signaling pathway, and requires extracellular calcium entry. According to their biological function, we then hypothesized that these genes might act as negative regulators of the GLP-1 signaling. In particular, they might brake the effects of GLP-1 on β-cell proliferation. To verify this hypothesis, siRNAs against these genes were developed. The effect of these siRNAs on GLP-1-induced β-cell proliferation will be evaluated later.

Mode d’action moléculaire de la toxine anti-tumorale : PS1Aa2 du bacille de Thuringe

Les parasporines sont des toxines Cry du bacille de Thuringe actives contre des cellules tumorales. Ce travail montre que la parasporine PS1Aa2 (Cry31Aa2) forme des pores dans des membranes artificielles, comme de nombreuses toxines Cry. Ceux-ci ont plusieurs niveaux de conductance dont les plus fréquents étaient de 11, 16 et 21 pS dans une solution de 150 mM KCl. Nos résultats de microspectrofluorométrie avec la sonde Fura-2 montrent que la présence de la PS1Aa2 peut produire des augmentations du calcium intracellulaire, la plupart du temps sous la forme d’oscillations calciques et parfois des augmentations soutenues. Ces réponses ont été observées en présence et en absence de calcium extracellulaire, dans les lignées tumorales HeLa et HepG2 et dans la lignée non tumorale HEK 293. Bien que quelques études aient montré que le calcium semble intervenir dans leur mode d’action, de telles oscillations calciques n’ont jamais été décrites auparavant pour des toxines Cry. Les expériences ont dû être faites à des concentrations beaucoup plus élevées de toxine que prévues sur la base des résultats publiés de cytotoxicité. Malgré la présence des fragments identifiés auparavant comme actifs, sa faible efficacité semble liée à la présence d’ADN dans les préparations qui entraîne la précipitation de la protéine. Les travaux futurs sur cette toxine seraient donc grandement facilités par une amélioration de sa méthode de préparation.

Microbial Evaluation Of Traumatized Teeth Treated With Triple Antibiotic Paste Or Calcium Hydroxide With 2% Chlorhexidine Gel In Pulp Revascularization.

Revascularization outcome depends on microbial elimination because apical repair will not happen in the presence of infected tissues. This study evaluated the microbial composition of traumatized immature teeth and assessed their reduction during different stages of the revascularization procedures performed with 2 intracanal medicaments. Fifteen patients (7-17 years old) with immature teeth were submitted to the revascularization procedures; they were divided into 2 groups according to the intracanal medicament used: TAP group (n = 7), medicated with a triple antibiotic paste, and CHP group (n = 8), dressed with calcium hydroxide + 2% chlorhexidine gel. Samples were taken before any treatment (S1), after irrigation with 6% NaOCl (S2), after irrigation with 2% chlorhexidine (S3), after intracanal dressing (S4), and after 17% EDTA irrigation (S5). Cultivable bacteria recovered from the 5 stages were counted and identified by means of polymerase chain reaction assay (16S rRNA). Both groups had colony-forming unit counts significantly reduced after S2 (P < .05); however, no significant difference was found between the irrigants (S2 and S3, P = .99). No difference in bacteria counts was found between the intracanal medicaments used (P = .95). The most prevalent bacteria detected were Actinomyces naeslundii (66.67%), followed by Porphyromonas endodontalis, Parvimonas micra, and Fusobacterium nucleatum, which were detected in 33.34% of the root canals. An average of 2.13 species per canal was found, and no statistical correlation was observed between bacterial species and clinical/radiographic features. The microbial profile of infected immature teeth is similar to that of primarily infected permanent teeth. The greatest bacterial reduction was promoted by the irrigation solutions. The revascularization protocols that used the tested intracanal medicaments were efficient in reducing viable bacteria in necrotic immature teeth.

Quantitative Proteomic Analysis Reveals Metabolic Alterations, Calcium Dysregulation, And Increased Expression Of Extracellular Matrix Proteins In Laminin α2 Chain-deficient Muscle.

Congenital muscular dystrophy with laminin α2 chain deficiency (MDC1A) is one of the most severe forms of muscular disease and is characterized by severe muscle weakness and delayed motor milestones. The genetic basis of MDC1A is well known, yet the secondary mechanisms ultimately leading to muscle degeneration and subsequent connective tissue infiltration are not fully understood. In order to obtain new insights into the molecular mechanisms underlying MDC1A, we performed a comparative proteomic analysis of affected muscles (diaphragm and gastrocnemius) from laminin α2 chain-deficient dy(3K)/dy(3K) mice, using multidimensional protein identification technology combined with tandem mass tags. Out of the approximately 700 identified proteins, 113 and 101 proteins, respectively, were differentially expressed in the diseased gastrocnemius and diaphragm muscles compared with normal muscles. A large portion of these proteins are involved in different metabolic processes, bind calcium, or are expressed in the extracellular matrix. Our findings suggest that metabolic alterations and calcium dysregulation could be novel mechanisms that underlie MDC1A and might be targets that should be explored for therapy. Also, detailed knowledge of the composition of fibrotic tissue, rich in extracellular matrix proteins, in laminin α2 chain-deficient muscle might help in the design of future anti-fibrotic treatments. All MS data have been deposited in the ProteomeXchange with identifier PXD000978 (http://proteomecentral.proteomexchange.org/dataset/PXD000978).

Down-regulation Of Slc8a1 As A Putative Apoptosis-evasion Mechanism By Modulation Of Calcium Levels In Penile Carcinoma.

The SLC8A1 gene, which encodes the Na(+)/Ca(2+) exchanger, plays a key role in calcium homeostasis. Our previous gene expression oligoarray data revealed SLC8A1 underexpression in penile carcinoma (PeCa). The aim of this study was to investigate whether the dysregulation of SLC8A1 expression is associated with apoptosis and cell proliferation in PeCa, via modulation of calcium concentration. The underlying mechanisms of SLC8A1 underexpression were also explored, focusing on copy number alteration and microRNA. Transcript levels of SLC8A1 gene and miR-223 were evaluated by quantitative PCR, comparing PeCa samples with normal glans tissues. SLC8A1 copy number was evaluated by microarray-based comparative genomic hybridization (array-CGH). Caspase-3 and Ki-67 immunostaining, as well as calcium distribution by Laser Ablation Imaging Inductively Coupled Plasma Mass Spectrometry [LA(i)-ICP-MS], were investigated in both normal and tumor samples. Confirming our previous data, SLC8A1 underexpression was detected in PeCa samples (P=0.001) and was not associated with gene copy number loss. In contrast, overexpression of miR-223 (P=0.002) was inversely correlated with SLC8A1 (P=0.015, r=-0.426), its putative repressor. In addition, SLC8A1 underexpression was associated with decreased calcium distribution, high Ki-67 and low caspase-3 immunoexpression in PeCa when compared with normal tissues. Down-regulation of the SLC8A1 gene, most likely mediated by its regulator miR-223, can lead to reduced calcium levels in PeCa and, consequently, to suppression of apoptosis and increased tumor cell proliferation. These data suggest that the miR-223-NCX1-calcium-signaling axis may represent a potential therapeutic approach in PeCa.

Effects of a novel calcium aluminate cement on the early events of the progression of osteogenic cell cultures

The present study evaluated the progression of osteogenic cell cultures exposed to a novel calcium aluminate cement (CAC+) in comparison with the gold standard mineral trioxide aggregate (MTA). Cells were enzimatically isolated from newborn rat calvarial bone, plated on glass coverslips containing either CAC+ or a control MTA samples in the center, and grown under standard osteogenic conditions. Over the 10-day culture period, roundening of sample edges was clearly noticed only for MTA group. Although both cements supported osteogenic cell adhesion, spreading, and proliferation, CAC+-exposed cultures showed significantly higher values in terms of total cell number at days 3 and 7, and total protein content and alkaline phosphatase activity at day 10. The present in vitro results indicate that the exposure to CAC+ supports a higher differentiation of osteogenic cells compared with the ones exposed to MTA. Further experimental studies should consider CAC+ as a potential alternative to MTA when the repair of mineralized tissues is one of the desired outcomes in endodontic therapy.

Large apical periodontitis healing following root canal dressing with calcium hydroxide: a case report

PURPOSE: The objective of this paper is to report the clinical case of a patient who presented a chronic apical periodontitis, arising from internal inflammatory resorption followed by pulp necrosis, and a long-term success of a root canal therapy using calcium hydroxide as root canal dressing. CASE DESCRIPTION: A 20-year-old male patient presented for routine dental treatment. By radiographic examination we noted an extensive radioluscent area, laterally to the permanent maxillary right lateral incisor, with possibility of communication with the lateral periodontium, suggestive of a chronic apical periodontitis. Due to external root resorption detection, we used a calcium hydroxide root canal dressing, changed every 15 days, for a period of 2 months. Root canal filling was performed using gutta-percha cones by lateral condensation technique Radiographic follow up held after 19 years of treatment indicated a periodontium in conditions of normality, with the presence of lamina dura. CONCLUSION: Calcium hydroxide is a suitable material to be used as root canal dressing in teeth with apical periodontitis. Long-term evaluation demonstrated the satisfactory clinical outcome following root canal treatment.

Subcutaneous tissue response of isogenic mice to calcium hydroxide-based pastes with chlorhexidine

This study was evaluated the response of subcutaneous connective tissue of isogenic mice to calcium hydroxide-based pastes with chlorhexidine digluconate (CHX). Seventy isogenic male BALB/c mice aged 6-8 weeks and weighing 15-20 g were randomly assigned to 8 groups. The animals received polyethylene tube implants as follows: Groups I, II, and III (n=10) - Calen® paste mixed with 0.4% CHX (experimental paste; Calen/CHX) for 7, 21, and 63 days, respectively; Groups IV, V, and VI (n=10) - UltraCal™ paste mixed with 2% CHX (experimental paste supplied by Ultradent Products Inc.; Ultracal/CHX) for 7, 21, and 63 days, respectively; and Groups VII and VIII (n=5): empty tube for 7 and 21 days, respectively. At the end of the experimental periods, the implants were removed together with the surrounding tissues (skin and subcutaneous connective tissue). The biopsied tissues were subjected to routine processing for histological analysis. Using a descriptive analysis and a four-point (0-3) scoring system, the following criteria were considered for qualitative and quantitative analysis of the tissue around the implanted materials: collagen fiber formation, tissue thickness and inflammatory infiltrate. A quantitative analysis was performed by measuring the thickness (µm), area (µm²) and perimeter (µm) of the reactionary granulomatous tissue formed at the tube ends. Data were analyzed statistically by the Kruskal-Wallis test and Dunn's post-test (α=0.05). Calen/CHX showed biocompatibility with the subcutaneous and reactionary tissues, with areas of discrete fibrosis and normal conjunctive fibrous tissue, though without statistically significant difference (p>0.05) from the control groups. In Groups I to III, there was a predominance of score 1, while in Groups IV to VI scores 2 and 3 predominated for all analyzed parameters. UltraCal/CHX, on the other hand, induced the formation of an inflammatory infiltrate and abundant exudate, suggesting a persistent residual aggression from the material, even 63 days after implant placement. In conclusion, the Calen paste mixed with 0.4% CHX allowed an adequate tissue response, whereas the UltraCal paste mixed with 2% CHX showed unsatisfactory results.

Quantitative radiographic evaluation of periapical bone resorption in dog's teeth contaminated with bacterial endotoxin (LPS) associated or not with calcium hydroxide

The aim of this study was to quantify radiographically the periapical bone resorption in dogs' teeth contaminated with bacterial endotoxin (LPS), associated or not with calcium hydroxide. After pulp tissue removal, 60 premolars were randomly assigned to 4 groups and were either filled with LPS (group 1), filled with LPS plus calcium hydroxide (group 2) or filled with saline (group 3) for a period of 30 days. In group 4, periapical lesion formation was induced with no canal treatment. Standardized radiographs were taken at the beginning of the treatment and after 30 days and the Image J Program was used for measurement of periapical lesion size. Periapical lesions were observed in groups 1 (average of 8.44 mm2) and 4 (average of 3.02 mm2). The lamina dura was intact and there were no areas of periapical bone resorption in groups 2 and 3. It may be concluded that calcium hydroxide was effective in inactivating LPS, as demonstrated by the absence of apical periodontitis in the roots that were filled with bacterial endotoxin plus calcium hydroxide.

Tissue inflammatory response to implantation of calcium hydroxide and iodoform in the back of rats

PURPOSE: This study evaluated the inflammatory reaction caused by the implantation of iodoform and calcium hydroxide in the back of rats. These drugs may be used as intracanal dressings to eliminate residual bacteria of the root canal system. METHODS: Twenty albinic rats (Rattus norvegicus, var Wistar) were divided into four groups: control group 1 (CG1) had normal skin; control group 2 (CG2) had wounded tissue without drugs; in groups 3 and 4, iodoform (IG) and calcium hydroxide (CHG) were inserted into the wounds, respectively. After 3, 5 and 11 days, slices of the implanted areas were macroscopically and microscopically observed regarding to their qualitative and quantitative aspects. RESULTS: In the macroscopical analysis, the CHG showed a large area of necrosis and swelling, which progressively decreased; in the IG the presence of iodoform surrounded by normal tissue was observed. The qualitative and quantitative histological analysis showed that IG promoted a shorter delay in the inflammatory response than the CHG. CONCLUSION: The inflammatory reaction for iodoform had a peak period five days after the drug insertion. By comparison, calcium hydroxide showed a very large area of necrosis that could only be partially eliminated after eleven days.

Evaluation of the diffusion capacity of calcium hydroxide pastes through the dentinal tubules

This study aimed to evaluate the diffusion capacity of calcium hydroxide pastes with different vehicles through dentinal tubules. The study was conducted on 60 extracted single-rooted human teeth whose crowns had been removed. The root canals were instrumented and divided into 4 groups according to the vehicle of the calcium hydroxide paste: Group I - distilled water; Group II - propylene glycol; Group III - 0.2% chlorhexidine; Group IV - 2% chlorhexidine. After placement of the root canal dressings, the teeth were sealed and placed in flasks containing deionized water. After 1, 2, 7, 15, 30, 45 and 60 days, the pH of the water was measured to determine the diffusion of calcium hydroxide through the dentinal tubules. The data were recorded and statistically compared by the Tukey test. The results showed that all pastes presented a similar diffusion capacity through dentin. Group IV did not present difference compared to group I. Group II presented difference compared to the other groups, as did Group III. In conclusion, groups I and IV presented a better diffusion capacity through dentin than groups II and III; 2% chlorhexidine can be used as a vehicle in calcium hydroxide pastes.

Extraction and concentration of biogenic calcium oxalate from plant leaves

The objective of this study was to extract and concentrate calcium oxalate (CaOx) crystals from plant leaves that form the above mentioned crystals. The chemical and physical studies of CaOx from plant to be performed depend on an adequate amount of the crystals. The plant used in this study was croton (Codiaeum variegatum). The leaves were ground in a heavy duty blender and sieved through a 0.20 mm sieve. The suspension obtained was suspended in distilled water. The crystals were concentrated at the bottom of a test tube. The supernatant must be washed until it is free of plant pigments and other organic substances. Biogenic CaOx crystals have well-defined and sharp peaks, indicating very high crystallinity. Moreover, the CaOx crystals were not damaged during the extraction procedure, as can be seen on the scanning electron microscope images. The porposed method can be considered efficient to extract and concentrate biogenic calcium oxalate.

Dietary calcium intake and overweight: an epidemiologic view

OBJECTIVE: We evaluated the relation between overweight and calcium intake in adults living in the municipality of São Paulo, Brazil. METHODS: This was a cross-sectional population-based study on a sample of 1459 adults that was obtained by multistage cluster sampling. Dietary intake was measured by the 24-h recall method. Poisson's and linear regression analyses were performed to evaluate the relation between overweight and quartiles of calcium intake adjusted for energy. RESULTS: The prevalence of overweight was 43.1% and the average adjusted calcium intake was 448.6 mg. In the linear regression analyses, the regression coefficient for adjusted calcium was significant and negative (P = 0.019, beta(1) = -0.0001). Although evaluated by quartiles, the prevalence ratio for overweight in the first quartile of calcium intake was 1.24 (95% confidence interval 1.00-1.54) and that in the second quartile was 1.24 (95% confidence interval 1.03-1.49). CONCLUSION: In the present study, calcium intake showed a significant negative association with body mass index

Relationships of blood lead to calcium, iron, and vitamin C intakes in Brazilian pregnant women

Background & aims. This study aimed to determine the relationship between blood lead concentrations and calcium, iron and vitamin C dietary intakes of pregnant women. Methods. Included in the study were 55 women admitted to a hospital, for delivery, from June to August 2002. A food frequency questionnaire was applied to determine calcium, iron and vitamin C intakes, and a general questionnaire to obtain data on demographic-socioeconomic condition, obstetric history, smoking habit, and alcohol intake. Blood lead and haemoglobin were determined, respectively, by atomic absorption spectrometry and by the haemoglobinometer HemoCue®. Multiple linear regression models were used to determine the relationship between blood lead and calcium, iron and vitamin C intakes, and haemoglobin levels, controlling for confounders. Results. The final model of the regression analysis detected an inverse relationship between blood lead and age of the women (p=0.011), haemoglobin (p=0.001), vitamin C (p=0.012), and calcium intake (p<0.001) (R2=0.952). One hundred percent, 98.2% and 43.6% of the women were below the adequate intake (AI) for calcium, and below the recommended dietary allowances (RDA) for iron, and vitamin C, respectively. Conclusion. Despite the small sample size, the results of this study suggest that maternal age, haemoglobin, vitamin C intake, and calcium intake may interfere with blood concentrations of lead