Caries Resistance of Lased Human Root Surface with 10.6 mu m CO(2) Laser-Thermal, Morphological, and Microhardness Analysis

Although the cariostatic effects of CO(2) laser on enamel have been shown, its effects on root surface demineralization remains uncertain. The objectives of this in vitro research was to establish safe parameters for a pulsed 10.6 mu m CO(2) laser and to evaluate its effect on morphological features of the root surface, as well as on the reduction of root demineralization. Ninety-five human root surfaces were randomly divided into five groups: G1-No treatment (control); G2-2.5 J/cm(2); G3-4.0 J/cm(2); G4-5.0 J/cm(2); and G5-6.0 J/cm(2). Intrapulpal temperature was evaluated during root surface irradiation by a thermocouple and morphological changes were evaluated by SEM. After the surface treatment, the specimens were submitted to a 7-day pH-cycling model. Subsequently, the cross-sectional Knoop microhardness values were measured. For all irradiated groups, intrapulpal temperature changes were less than 1.5 degrees C. Scanning electron microscopy images indicated that fluences as low as 4.0 J/cm(2) were sufficient to induce morphological changes in the root surface. Additionally, for fluences reaching or exceeding 4.0 J/cm(2), laser-induced inhibitory effects on root surface demineralization were observed. It was concluded that laser energy density in the range of 4.0 to 6.0 J/cm(2) could be applied to a dental root to reduce demineralization of this surface without compromising pulp vitality.

The Potential Role of Suppressors of Cytokine Signaling in the Attenuation of Inflammatory Reaction and Alveolar Bone Loss Associated with Apical Periodontitis

Inflammatory cytokines contribute to periapical tissue destruction. Their activity is potentially regulated by suppressors of cytokine signaling (SOCS), which down-regulate signal transduction as part of an inhibitory feedback loop. We investigated the expression of the cytokines tumor necrosis factor alpha (TNF-alpha); interleukin (IL)-10 and RANKL; and SOCS-1, -2, and -3 by real-time polymerase chain reaction in 57 periapical granulomas and 38 healthy periapical tissues. Periapical granulomas exhibited significantly higher SOCS-1, -2, and -3, TNF-alpha, IL-10, and RANKL messenger RNA levels when compared with healthy controls. Significant positive correlations were found between SOCS1 and IL-10 and between SOCS3 and IL-10. Significant inverse correlations were observed between SOCS1 and TNF-alpha, SOCS1 and RANKL, and SOCS3 and TNF-alpha. Increased SOCS-1, -2, and -3 messenger RNA levels in periapical granulomas may be related to the downregulation of inflammatory cytokines in these lesions; therefore, SOCS molecules may play a role in the dynamics of periapical granulomas development. (J Endod 2008;34:1480-1484)

Regulation of Trypanosoma cruzi-Induced Myocarditis by Programmed Death Cell Receptor

Trypanosoma cruzi infection causes intense myocarditis, leading to cardiomyopathy and severe cardiac dysfunction. Protective adaptive immunity depends on balanced signaling through a T cell receptor and coreceptors expressed on the T cell surface. Such coreceptors can trigger stimulatory or inhibitory signals after binding to their ligands in antigen-presenting cells (APC). T. cruzi modulates the expression of coreceptors in lymphocytes after infection. Deregulated inflammation may be due to unbalanced expression of these molecules. Programmed death cell receptor 1 (PD-1) is a negative T cell coreceptor that has been associated with T cell anergy or exhaustion and persistent intracellular infections. We aimed to study the role of PD-1 during T. cruzi-induced acute myocarditis in mice. Cytometry assays showed that PD-1 and its ligands are strongly upregulated in lymphocytes and APC in response to T. cruzi infection in vivo and in vitro. Lymphocytes infiltrating the myocardium exhibited high levels of expression of these molecules. An increased cardiac inflammatory response was found in mice treated with blocking antibodies against PD-1, PD-L1, and to a lesser extent, PD-L2, compared to that found in mice treated with rat IgG. Similar results in PD-1(-/-) mice were obtained. Moreover, the PD-1 blockade/deficiency led to reduced parasitemia and tissue parasitism but increased mortality. These results suggest the participation of a PD-1 signaling pathway in the control of acute myocarditis induced by T. cruzi and provide additional insight into the regulatory mechanisms in the pathogenesis of Chagas` disease.

The anti-MMP activity of benzalkonium chloride

Objective: This study evaluated the ability of benzalkonium chloride (BAC) to bind to dentine and to inhibit soluble recombinant MMPs and bound dentine matrix metalloproteinases (MMPs). Methods: Dentine powder was prepared from extracted human molars. Half was left mineralized; the other half was completely demineralized. The binding of BAG to dentine powder was followed by measuring changes in the supernatant concentration using UV spectrometry. The inhibitory effects of BAC on rhMMP-2, -8 and -9 were followed using a commercially available in vitro proteolytic assay. Matrix-bound endogenous MMP-activity was evaluated in completely demineralized beams. Each beam was either dipped into BAG and then dropped into 1 mL of a complete medium (CM) or they were placed in 1 mL of CM containing BAG for 30 days. After 30 days, changes in the dry mass of the beams or in the hydroxyproline (HYP) content of hydrolysates of the media were quantitated as indirect measures of matrix collagen hydrolysis by MMPs. Results: Demineralized dentine powder took up 10-times more BAG than did mineralized powder. Water rinsing removed about 50% of the bound BAC, whilst rinsing with 0.5 M NaCl removed more than 90% of the bound BAG. BAG concentrations 0.5 wt% produced 100% inhibition of soluble recombinant MMP-2, -8 or -9, and inhibited matrix-bound MMPs between 55 and 66% when measured as mass loss or 76-81% when measured as solubilization of collagen peptide fragments. Conclusions: BAC is effective at inhibiting both soluble recombinant MMPs and matrix-bound dentine MMPs in the absence of resins. (C) 2010 Elsevier Ltd. All rights reserved.

Ionotropic glutamatergic receptors in the rostral medullary raphe modulate hypoxia and hypercapnia-induced hyperpnea

It has been suggested that the medullary raphe (MR) plays a key role in the physiological responses to hypoxia and hypercapnia. We assessed the role of ionotropic glutamate receptors in the rostral MR (rMR) in the respiratory responses to hypoxia and hypercapnia by measuring pulmonary ventilation (V(E)) and body temperature (Tb) of male Wistar rats before and after microinjecting Kynurenic acid (KY, an ionotropic glutamate receptors antagonist, 0.1 mM) into the rMR followed by 60 min of hypoxia (7% O(2)) or hypercapnia exposure (7% CO(2)). Compared to the control group, the ventilatory response to hypoxia was attenuated in animals treated with KY intra-rMR, however the ventilatory response to hypercapnia increased significantly. No differences in Tb among groups were observed during hypoxia or hypercapnia. These data suggest that the glutamate acting on ionotropic receptors in the rMR exerts an excitatory modulation on hyperventilation induced by hypoxia but an inhibitory modulation on the hypercapnia-induced hyperpnea. (C) 2010 Elsevier B.V. All rights reserved.

Developmental changes of gene expression after spinal cord injury in neonatal opossums

Changes in gene expression have been measured 24 h after injury to mammalian spinal cords that can and cannot regenerate In opossums there is a critical period of development when regeneration stops being possible at 9 days postnatal cervical spinal cords regenerate, at 12 days they do not By the use of marsupial cDNA microarrays we detected 158 genes that respond differentially to injury at the two ages critical for regeneration For selected candidates additional measurements were made by real time PCR and sites of their expression were shown by immunostaining Candidate genes have been classified so as to select those that promote or prevent regeneration Up regulated by injury at 8 days and/or down regulated by injury at 13 days were genes known to promote growth, such as Mitogen activated protein kinase kinase 1 or transcripton factor TCF7L2 By contrast, at 13 days up regulation occurred of Inhibitory molecules including annexins ephrins and genes related to apoptosis and neurodegeneranve diseases Certain genes such as calmodulin 1 and NOGO changed expression similarly in animals that could and could not regenerate without any additional changes in response to injury These findings confirmed and extended changes of gene expression found in earlier screens on 9 and 12 day preparations without lesions and provide a comprehensive list of genes that serve as a basis for testing how identified molecules singly or in combination, promote and prevent central nervous system regeneration (C) 2010 Elsevier B V All rights reserved

Reduced stress fever is accompanied by increased glucocorticoids and reduced PGE(2) in adult rats exposed to endotoxin as neonates

Immune challenges during neonatal period may permanently program immune responses later in life, including endotoxin fever. We tested the hypothesis that neonatal endotoxin exposure affects stress fever in adult rats. In control rats (treated with saline as neonates; nSal) body temperature peaked similar to 1.5 degrees C during open-field stress, whereas in rats exposed to endotoxin (lipopolysaccharide, LPS) as neonates (nLPS) stress fever was significantly attenuated. Following stress, plasma corticosterone levels significantly increased from 74.29 +/- 7.05 ng ml(-1) to 226.29 +/- 9.87 ng ml(-1) in nSal rats, and from 83.43 +/- 10.31 ng ml(-1) to 324.7 +/- 36.87 ng ml(-1) in nLPS rats. Animals treated with LPS as neonates and adrenalectomized one week before experimentation no longer displayed the attenuated febrile response to stress. This attenuated stress fever caused by an increased corticosterone secretion is likely to be linked to an inhibitory effect of glucocorticoids on cyclooxygenase activity/PGE(2) production in preoptic/anteroventral third ventricular region (AV3V) since stress failed to cause a significant increase in PGE(2) in nLPS rats, and this effect was reverted by adrenalectomy. Altogether, the present results indicate that endogenous glucocorticoids are key modulators of the attenuated stress fever in adult rats treated with LPS as neonates, and they act downregulating PGE(2) production in AV3V. Moreover, our findings also support the notion that neonatal immune stimulus affects programming of stress responses during adulthood, despite the fact that inflammation and stress are two distinct processes mediated largely by different neurobiological mechanisms. (C) 2010 Elsevier B.V. All rights reserved.

Serotonergic mechanisms on breathing modulation in the rat locus coeruleus

The locus coeruleus (LC) is a noradrenergic nucleus that plays an important role in the ventilatory response to hypercapnia. This nucleus is densely innervated by serotonergic fibers and contains high density of serotonin (5-HT) receptors, including 5-HT(1A) and 5-HT(2). We assessed the possible modulation of respiratory response to hypercapnia by 5-HT, through 5-HT(1A) and 5-HT(2) receptors, in the LC. To this end, we determined the concentrations of 5-HT and its metabolite 5-hydroxyindole-3-acetic acid (5-HIAA) in the LC after hypercapnic exposure. Pulmonary ventilation (V(E), plethysmograph) was measured before and after unilateral microinjection (100 nL) of WAY-100635 (5-HT(1A) antagonist, 5.6 and 56 mM), 8-OHDPAT (5-HT(1A/7) agonist, 7 and 15 mM), Ketanserin (5-HT(2A) antagonist, 3.7 and 37 mM), or (+/-)-2,5-dimethoxy-4-iodoamphetaminehydrochloride (DOI; 5-HT(2A) agonist, 6.7 and 67 mM) into the LC, followed by a 60-min period of 7% CO(2) exposure. Hypercapnia increased 5-HTIAA levels and 5-HIAA/5-HT ratio within the LC. WAY-100635 and 8-OHDPAT intra-LC decreased the hypercapnic ventilatory response due to a lower tidal volume. Ketanserin increased CO(2) drive to breathing and DOI caused the opposite response, both acting on tidal volume. The current results provide evidence of increased 5-HT release during hypercapnia in the LC and that 5-HT presents an inhibitory modulation of the stimulatory role of LC on hypercapnic ventilatory response, acting through postsynaptic 5-HT(2A) receptors in this nucleus. In addition, hypercapnic responses seem to be also regulated by presynaptic 5-HT(1A) receptors in the LC.

GABAergic antagonist blocks the reduction of tonic immobility behavior induced by activation of 5-HT(2) receptors in the basolateral nucleus of the amygdala in guinea pigs

Tonic immobility (TI) is a temporary state of profound motor inhibition induced by situations that generate intense fear, with the objective of protecting an animal from attacks by predators. A preliminary study by our group demonstrated that microinjection into the basolateral nucleus of the amygdala (BLA) of an agonist to 5-HT(1A) and 5-HT(2) receptors promoted a decrease in TI duration. In the current study, the effects of GABAergic stimulation of the BLA and the possible interaction between GABA(A) and 5-HT(2) receptors on TI modulation were investigated. Observation revealed that GABAergic agonist muscimol (0.26 nmol) reduced the duration of TI episodes, while microinjection of the GABAergic antagonist bicuculline (1 nmol) increased TI duration. Additionally, microinjection of 5-HT(2) agonist receptors (alpha-methyl-5-HT, 0.32 nmol) into the BLA decreased TI duration, an effect reversed by pretreatment with bicuculline (at the dose that had no effect per se, 0.2 nmol). Moreover, the activation of GABA(A) and 5-HT(2) receptors in the BLA did not alter the spontaneous motor activity in the open field test. These experiments demonstrated that the activation of GABA(A) and 5-HT(2) receptors of the BLA possibly produce a reduction in unconditioned fear that decreases the TI duration in guinea pigs, but this is not due to increased spontaneous motor activity, which could affect a TI episode nonspecifically. Furthermore, these results suggest an interaction between GABAergic and serotoninergic mechanisms mediated by GABA(A) and 5-HT(2) receptors. In addition, the GABAergic circuit of the BLA presents a tonic inhibitory influence on TI duration. (C) 2009 Elsevier Inc. All rights reserved.

Tissue-specific induction of SOCS gene expression by PRL

The mechanisms whereby tissue sensitivity to PRL is controlled are not well understood. Here we report that expression of mRNA and protein for members of the SOCS/CIS/JAB family of cytokine signaling inhibitors is increased by PRL administration in ovary and adrenal gland of the lactating rat deprived of circulating PRL and pups for 24 h but not in mammary gland. Moreover, suckling increases SOCS mRNA in the ovary but not in the mammary gland of pup-deprived rats. Deprivation of PRL and pups for 48 h allows the mammary gland to induce SOCS genes in response to PRL administration, and this is associated with a decrease in basal SOCS-3 mRNA and protein expression to the level seen in other tissues, suggesting that SOCS-3 induced refractoriness related to filling of the gland. In reporter assays, SOCS-1, SOCS-3, and CIS, but not SOCS-2, are able to inhibit transactivation of the STAT 5-responsive beta -lactoglobulin promoter in transient transfection assays. Moreover, suckling results in loss of ovarian and adrenal responsiveness to PRL administered 2 h after commencement of suckling, as determined by STAT 5 gel shift assay. Immunohistochemistry was used to localize the cellular sites of SOCS-3 and CIS protein expression in the ovary and adrenal gland. We propose that induced SOCS-1, SOCS-3, and CIS are actively involved in the cellular inhibitory feedback response to physiological PRL surges in the corpus luteum and adrenal cortex during lactation, but after pup withdrawal, the mammary gland is rendered unresponsive to PRL by increased levels of SOCS-3.

Cervical mobilisation: concurrent effects on pain, sympathetic nervous system activity and motor activity

Recent findings that spinal manual therapy (SMT) produces concurrent hypoalgesic and sympathoexcitatory effects have led to the proposal that SMT may exert its initial effects by activating descending inhibitory pathways from the dorsal periaqueductal gray area of the midbrain (dPAG). In addition to hypoalgesic and sympathoexcitatory effects, stimulation of the dPAG in animals has been shown to hal e a facilitatory effect on motor activity. This study sought to further investigate the proposal regarding SMT and the FAG by including a test of motor function in addition to the variables previously investigated, Using a condition randomised, placebo-controlled, double blind, repeated measures design, 30 subjects with mid to lon er cervical spine pain of insidious onset participated in the study. The results indicated that the cervical mobilisation technique produced a hypoalgesic effect as revealed by increased pressure pain thresholds on the side of treatment (P = 0.0001) and decreased resting visual analogue scale scores (P = 0.049). The treatment technique also produced a sympathoexcitatory effect with an increase in skin conductance (P < 0.002) and a decrease in skin temperature (P = < 0.02). There was a decrease in superficial neck flexor muscle activity (P < 0.0002) at the lower levels of a staged cranio-cervical flexion test. This could imply facilitation of the deep neck flexor muscles with a decreased need for co-activation of the superficial neck flexors, The combination of all findings,would support the proposal that SMT may, at least initially, exert part of its influence via activation of the PAG, (C) 2000 Harcourt Publishers Ltd.

Control of the cystic fibrosis transmembrane conductance regulator by alpha G(i) and RGS proteins

The cystic fibrosis transmembrane conductance regulator (CFTR) has been shown previously to be regulated by inhibitory G proteins. In the present study, we demonstrate inhibition of CFTR by alphaG(i2) and alphaG(i1), but not alphaG(0), in Xenopus oocytes. We further examined whether regulators of G protein signaling (RGS) proteins interfere with alphaG(i)-dependent inhibition of CFTR. Activation of CFTR by IBMX and forskolin was attenuated in the presence of alphaG(i2), indicating inhibition of CFTR by alphaG(i2) in Xenopus oocytes. Coexpression of the proteins RGS3 and RGS7 together with CFTR and alphaG(i2) partially recovered activation by IBMX/forskolin. 14-3-3, a protein that is known to interfere with RGS proteins, counteracted the effects of RGS3. These data demonstrate the regulation of CFTR by alphaG(i) in Xenopus oocytes. Because RGS proteins interfere with the G protein-dependent regulation of CFTR, this may offer new potential pathways for pharmacological intervention in cystic fibrosis. (C) 2001 Academic Press.

Interpretation of the reversible inhibition of adenosine deaminase by small cosolutes in terms of molecular crowding

Published results on the inhibitory effects of small cosolutes on adenosine deamination by adenosine deaminase [Kurz. L. C.. Weitkamp, E., and Frieden, C. (1987) Biochemistry 26, 3027-3032; Dzingeleski, G., and Wolfenden, R. (1993) Biochemistry 32, 9143 -9147] have been reexamined. Results for sucrose, dioxane, methanol, and ethanol are shown to be qualitatively consistent with thermodynamic interpretation in terms of molecular crowding effects arising from the occurrence of a minor increase in enzyme volume and/or asymmetry during the kinetic reaction-a conformational transition that could be either preexisting or ligand induced. Direct evidence for the existence of the putative isomeric transition is provided by active enzyme gel chromatography on Sephadex G-100, which demonstrates a negative dependence of enzyme elution volume upon substrate concentration and is therefore consistent with substrate-mediated conformational changes that favor a larger (or more asymmetric) isomeric state of the enzyme. There are thus experimental grounds for adopting the present description of the inhibitory effects of unrelated cosolutes on the kinetics of adenosine deamination by adenosine deaminase in terms of thermodynamic nonideality.

Ultracentrifugal studies of the effect of molecular crowding by trimethylamine N-oxide on the self-association of muscle glycogen phosphorylase b

The suitability of sedimentation equilibrium for characterizing the self-association of muscle glycogen phosphorylase b has been reappraised. Whereas sedimentation equilibrium distributions for phosphorylase b in 40 mM Hepes buffer (pH 6.8) supplemented with 1 mM AMP signify a lack of chemical equilibrium attainment, those in buffer supplemented additionally with potassium sulfate conform with the requirements of a dimerizing system in chemical as we:ll as sedimentation equilibrium. Because the rate of attainment of chemical equilibrium under the former conditions is sufficiently slow to allow resolution of the dimeric and tetrameric enzyme species by sedimentation velocity, this procedure has been used to examine the effects of thermodynamic nonideality arising from molecular crowding try trimethylamine N-oxide on the self-association behaviour of phosphorylase b. In those terms the marginally enhanced extent of phosphorylase b self-association observed in the presence of high concentrations of the cosolute is taken to imply that the effects of thermodynamic nonideality on the dimer-tetramer equilibrium are being countered by those displacing the T reversible arrow R isomerization equilibrium for dimer towards the smaller, nonassociating T state. Because the R state is the enzymically active form, an inhibitory effect is the predicted consequence of molecular crowding by high concentrations of unrelated solutes. Thermodynamic nonideality thus provides an alternative explanation for the inhibitory effects of high concentrations of glycerol, sucrose and ethylene glycol on phosphorylase b activity, phenomena that have been attributed to extremely weak interaction of these cryoprotectants with the T state of the enzyme.

Analysis of the TGF beta functional pathway in epithelial ovarian carcinoma

Epithelial ovarian carcinoma is often diagnosed at an advanced stage of disease and is the leading cause of death from gynaecological neoplasia. The genetic changes that occur during the development of this carcinoma are poorly understood. It has been proposed that IGFIIR, TGF beta1 and TGF beta RII act as a functional unit in the TGF beta growth inhibitory pathway, and that somatic loss-of-function mutations in any one of these genes could lead to disruption of the pathway and subsequent loss of cell cycle control. We have examined these 3 genes in 25 epithelial ovarian carcinomas using single-stranded conformational polymorphism analysis and DNA sequence analysis. A total of 3 somatic missense mutations were found in the TGF beta RII gene, but none in IGFRII or TGF beta1. An association was found between TGF beta RII mutations and histology, with 2 out of 3 clear cell carcinomas having TGF beta RII mutations. This data supports other evidence from mutational analysis of the PTEN and beta -catenin genes that there are distinct developmental pathways responsible for the progression of different epithelial ovarian cancer histologic subtypes. (C) 2001 Cancer Research Campaign.