CNS-specific regulatory elements in brain-derived HIV-1 strains affect responses to latency-reversing agents with implications for cure strategies.

Latency-reversing agents (LRAs), including histone deacetylase inhibitors (HDACi), are being investigated as a strategy to eliminate latency in HIV-infected patients on suppressive antiretroviral therapy. The effectiveness of LRAs in activating latent infection in HIV strains derived from the central nervous system (CNS) is unknown. Here we show that CNS-derived HIV-1 strains possess polymorphisms within and surrounding the Sp transcription factor motifs in the long terminal repeat (LTR). These polymorphisms result in decreased ability of the transcription factor specificity protein 1 to bind CNS-derived LTRs, reducing the transcriptional activity of CNS-derived viruses. These mutations result in CNS-derived viruses being less responsive to activation by the HDACi panobinostat and romidepsin compared with lymphoid-derived viruses from the same subjects. Our findings suggest that HIV-1 strains residing in the CNS have unique transcriptional regulatory mechanisms, which impact the regulation of latency, the consideration of which is essential for the development of HIV-1 eradication strategies.

NF-κB Enhances Androgen Receptor Expression through 5'-UTR Binding in Gingival Cells.

Dihydropyridine-induced gingival overgrowth (DIGO) is a side effect observed in patients treated for hypertension. The disease is aggravated by inflammation. Nifedipine (Nif), a dihydropyridine, causes gingival overgrowth by increasing the expression of the androgen receptor (AR). Furthermore, the proinflammatory cytokine interleukin 1ß (IL-1ß) induces collagen α1(I) expression through the AR in DIGO fibroblasts. These observations prompted us to investigate whether and how nuclear factor kappa B (NF-κB) affects AR expression in DIGO. Therefore, gingival fibroblasts obtained from the tissues of patients with DIGO and healthy subjects were stimulated with IL-1ß, Nif, or both. mRNA and protein expression was detected with real-time polymerase chain reaction and Western blotting. High correlation coefficients were observed for the mRNA expression of the AR, connective tissue growth factor, and collagen α1(I) induced by both drugs. Western blot analysis showed that IL-1ß and Nif increased and activated NF-κB more in DIGO cells than in healthy cells. An electrophoretic mobility shift assay demonstrated that the promoter and 5'-untranslated regions (5'-UTRs) of the AR gene contains 3 binding sites for the NF-κB p65 subunit. A chromatin immunoprecipitation assay revealed that the NF-κB p65 subunit was associated with AR 5'-UTRs in gingival fibroblasts. A site-directed mutagenesis study indicated that a mutation of NF-κB binding sites reduced Nif- and IL-1ß-induced AR promoter activities. Collectively, these data indicate that NF-κB is an essential transcriptional regulator of AR gene expression and thus plays a crucial role in collagen overproduction in DIGO fibroblasts.

Comparison of treatment effects of guided tissue regeneration on infrabony lesions between animal and human studies: a systematic review and meta-analysis.

BACKGROUND AND OBJECTIVE: For ethical reasons it is becoming increasingly more difficult to obtain, from clinical studies, histological data on infrabony defects treated with guided tissue regeneration (GTR) techniques. The aim of this systematic review was to find the value of extrapolating animal data on treatment of periodontal infrabony lesions, using GTR only or GTR + bone grafts, to human clinical results. MATERIAL AND METHODS: Searches of the PubMed and Cochrane databases were combined with hand searching of articles published from 1 January 1969 to 1 August 2012. The search included any type of barrier membrane, with or without grafted materials, used to treat periodontal infrabony lesions. All studies with histological or re-entry methodology outcome parameters that evaluated bone-filling and/or new-cementum-formation ratios from a defect depth were collected. When comparing animal and human outcomes, a meta-analysis was used to evaluate the bone-filling ratio, but only a descriptive analysis of the histological studies was performed. RESULTS: In total, 22 studies were selected for the meta-analysis. In the GTR + bone graft groups the weighted-average bone-filling ratios were 52% (95% CI: 18-85%) in animals and 57% (95% CI: 30-83%) in humans, which were not statistically significantly different (p = 0.825). Similar results were found in the GTR-only groups, in which the weighted-average bone-filling ratios were 54% (95% CI: 37-72%) in animals and 59% (95% CI: 42-77%) in humans (p = 0.703). New-cementum formation of GTR only and GTR + bone grafts showed comparable ratio outcomes, and both were superior to the control group in animals only (p = 0.042). CONCLUSION: Although quality assessments differed between animal and human studies, our analysis indicated that animal models and human results showed similar bone-filling ratios in infrabony defects treated with GTR only or with GTR + bone grafting.

Nitric oxide inhibits androgen receptor-mediated collagen production in human gingival fibroblasts.

UNLABELLED: Lin S-J, Lu H-K, Lee H-W, Chen Y-C, Li C-L, Wang L-F. Nitric oxide inhibits androgen receptor-mediated collagen production in human gingival fibroblasts. J Periodont Res 2012; 47: 701-710. © 2012 John Wiley & Sons A/S Background and Objective: In our previous study, we found that flutamide [an androgen receptor (AR) antagonist] inhibited the up-regulation of collagen induced by interleukin (IL)-1ß and/or nifedipine in gingival fibroblasts. The present study attempted to verify the role of nitric oxide (NO) in the IL-1ß/nifedipine-AR pathway in gingival overgrowth. MATERIAL AND METHODS: Confluent gingival fibroblasts derived from healthy individuals (n = 4) and those with dihydropyridine-induced gingival overgrowth (DIGO) (n = 6) were stimulated for 48 h with IL-1ß (10 ng/mL), nifedipine (0.34 µm) or IL-1ß + nifedipine. Gene and protein expression were analyzed with real-time RT-PCR and western blot analyses, respectively. Meanwhile, Sircol dye-binding and the Griess reagent were, respectively, used to detect the concentrations of total soluble collagen and nitrite in the medium. RESULTS: IL-1ß and nifedipine simultaneously up-regulated the expression of the AR and type-I collagen α1 [Colα1(I)] genes and the total collagen concentration in DIGO cells (p < 0.05). IL-1ß strongly increased the expression of inducible nitric oxide synthase (iNOS) mRNA and the nitrite concentration in both healthy and DIGO cells (p < 0.05). However, co-administration of IL-1ß and nifedipine largely abrogated the expression of iNOS mRNA and the nitrite concentration with the same treatment. Spearman's correlation coefficients revealed a positive correlation between the AR and total collagen (p < 0.001), but they both showed a negative correlation with iNOS expression and the NO concentration (p < 0.001). The iNOS inhibitor, 1400W, enhanced IL-1ß-induced AR expression; furthermore, the NO donor, NONOate, diminished the expression of the AR to a similar extent in gingival fibroblasts derived from both healthy patients and DIGO patients (p < 0.05). CONCLUSION: IL-1ß-induced NO attenuated AR-mediated collagen production in human gingival fibroblasts. The iNOS/NO system down-regulated the axis of AR/Colα1(I) mRNA expression and the production of AR/total collagen proteins by DIGO cells.

Flutamide inhibits nifedipine- and interleukin-1 beta-induced collagen overproduction in gingival fibroblasts.

BACKGROUND AND OBJECTIVE: To understand the role of the androgen receptor in gingival overgrowth, the effects of flutamide on interleukin-1 beta- and nifedipine-induced gene expression of connective tissue growth factor (CTGF/CCN2) and collagen production in gingival fibroblasts were examined. MATERIAL AND METHODS: Gingival fibroblasts from healthy subjects and patients with dihydropyridine-induced gingival overgrowth (DIGO) were used. Confluent cells were treated with nifedipine, interleukin-1 beta or both. The mRNA expression was examined using real-time polymerase chain reaction, and the concentration of total soluble collagen in conditioned media was analysed by Sircol Collagen Assay. In addition, the protein expressions of androgen receptor, CTGF/CCN2 and type I collagen in gingival tissue were determined by western blot. RESULTS: Interleukin-1 beta was more potent than nifedipine in stimulating CTGF/CCN2 and procollagen alpha1(I) mRNA expression, and there was an additive effect of the two drugs. Healthy cells exhibited an equal or stronger response of procollagen alpha1(I) than those with DIGO, but DIGO cells displayed a stronger response in the secretion of soluble collagen in the same conditions. Flutamide, an androgen receptor antagonist, inhibited stimulation by nifedipine or interleukin-1 beta. Additionally, the protein expressions of androgen receptor and type I collagen were higher in DIGO gingival tissue than those in healthy gingival tissue. CONCLUSION: The data suggest that both nifedipine and interleukin-1 beta play an important role in DIGO via androgen receptor upregulation and that gingival overgrowth is mainly due to collagen accumulation. Flutamide decreases the gene expression and protein production of collagen from dihydropyridine-induced overgrowth cells.

An acute injection of Porphyromonas gingivalis lipopolysaccharide modulates the OPG/RANKL system and interleukin-6 in an ovariectomized mouse model.

BACKGROUND/AIMS: In the present study, we attempted to develop a simulated model to explore the causal effects of periodontal pathogens on skeletal homeostasis in postmenopausal osteoporosis. METHODS: Fifty-three female adult ICR mice were randomly assigned to an experimental group (ovariectomized) or a control group. A single injection of Porphyromonas gingivalis lipopolysaccharide (P. gingivalis-LPS, ATCC 33277) or Escherichia coli lipopolysaccharide (E. coli-LPS) was administered intraperitoneally 4 weeks after an ovariectomy. Concentrations of interleukin-6 (IL-6), osteoprotegerin (OPG), and the receptor activator of nuclear factor-kappaB ligand (RANKL) in serum were subsequently analyzed using an enzyme-linked immunosorbent assay (ELISA). RESULTS: Under stimulation with P. gingivalis-LPS or E. coli-LPS, the concentration of OPG rose in both groups. The serum level of RANKL showed a decreasing trend 24 h after the injection in both groups. After injection of P. gingivalis-LPS in both the experimental and control animals, the OPG : RANKL ratio increased 24 h after the booster (22.26-620.99, P < 0.05). The serum level of IL-6 in the experimental group significantly increased 1-6 h after administration of E. coli-LPS and 1-3 h after administration of P. gingivalis-LPS (P < 0.05). CONCLUSIONS: A single booster injection of P. gingivalis-LPS induced short-term changes in OPG, RANKL, and IL-6 serum levels in this ovariectomized mouse model.

Stimulation of cells derived from nifedipine-induced gingival overgrowth with Porphyromonas gingivalis, lipopolysaccharide, and interleukin-1beta.

The purpose of this study was to clarify the main contributory factor of nifedipine-induced gingival overgrowth either by Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) or interleukin-1beta (IL-1beta). Human gingival fibroblasts from healthy tissues and nifedipine-induced gingival overgrowth tissues were stimulated with nifedipine, IL-1beta, Escherichia coli lipopolysaccharide (Ec-LPS), and Pg-LPS, and the gene expressions were analyzed by RT-PCR. Analysis of the data showed no strong evidence of a synergistic effect of nifedipine and Pg-LPS on IL-6, connective tissue growth factor (CTGF), and type 1 collagen gene expression of either healthy cells or nifedipine-induced gingival overgrowth cells. Among the three stimulants--IL-1beta, Pg-LPS, and Ec-LPS--androgen receptor and IL-6 gene expressions in both the healthy and nifedipine-induced gingival overgrowth groups were strongly up-regulated by the presence of IL-1beta only. Furthermore, the responses to IL-1beta in the nifedipine-induced gingival overgrowth group were stronger than those of the healthy group. It can be concluded that IL-1beta is an important mediator responsible for the higher IL-6 and androgen receptor expression of nifedipine-induced gingival overgrowth cells.

Is coronally positioned flap procedure adjunct with enamel matrix derivative or root conditioning a relevant predictor for achieving root coverage? A systemic review.

BACKGROUND AND OBJECTIVE: This study is a systemic review of coronally positioned flap, coronally positioned flap+chemical root surface conditioning, or coronally positioned flap+enamel matrix derivative (EMD) for the treatment of Miller class I and II gingival recession. MATERIAL AND METHODS: All studies available through the Medline database by the end of October 2005 were used. Each study provided mean clinical attachment level, keratinized tissue, probing pocket depth, gingival recession depth and root coverage percentage before and after treatment with coronally positioned flap alone, coronally positioned flap+chemical root surface conditioning, or coronally positioned flap+EMD. Effectiveness was evaluated by comparing the weighted mean average in gingival recession depth, probing pocket depth, clinical attachment level, keratinized tissue and root coverage percentage achieved with the three treatments. RESULTS: Seven studies for the coronally positioned flap+EMD group, four studies for the coronally positioned flap+chemical root surface conditioning group, and seven studies for the coronally positioned flap group were retrieved for this weighted mean analysis. The results of clinical attachment level, gingival recession depth, and root coverage percentage in the coronally positioned flap+EMD group were statistically significantly better than the changes in the coronally positioned flap and coronally positioned flap+chemical root surface conditioning group at 6 and 12 mo (p<0.001). There was no significant difference at the 6-mo comparison among clinical attachment level, keratinized tissue, probing pocket depth, and gingival recession depth, except in the root coverage percentage for coronally positioned flap and coronally positioned flap+chemical root surface conditioning groups. CONCLUSION: The results suggest that root coverage by the coronally positioned flap and coronally positioned flap+chemical root surface conditioning procedures were unpredictable but became more predictable when the coronally positioned flap procedure was improved by the modification of adding EMD.

Effect of freeze-thaw cycles on serum measurements of AFP, CEA, CA125 and CA19-9.

AFP, CEA, CA125 and CA19-9 are commonly used serum tumour markers (TMs) in clinical practice, although their quantification by immunoassay may be influenced by pre-analytical sample handling. Though the effect of repetitive freeze-thaw cycles is generally recognized, it is not clear in detail. The present study measured (CLIA) these TMs in serum samples freshly separated and after each of five freeze-thaw cycles, in which the samples were frozen at -40 degrees C for 10 months at cycle 4 and 2 h at other cycles. Statistical analysis with the General Linear Model for Repeated Measures revealed significant decreases in the measurements of the four TMs, with the least decrease of 6.8 % for CA125 and the most decrease of 18.2 % for CA19-9 after the last cycle, and an overwhelming single cycle decrease of mean 7.7 % at cycle 4 for AFP, CEA and CA125, of 7.5 % and 9.3 % at cycles 4 and 5 for CA19-9. So it seems that measurements of AFP, CEA and CA125 are more readily affected by long-term frozen storage compared with frequent freezing-thawing, while CA19-9 is relatively unstable under both conditions.

Identification of the osteoprotegerin/receptor activator of nuclear factor-kappa B ligand system in gingival crevicular fluid and tissue of patients with chronic periodontitis.

BACKGROUND AND OBJECTIVE: Recent findings have suggested that osteoclastogenesis is directly regulated by receptor activator of nuclear factor-kappa B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG). However, no studies have described interactions of OPG/RANKL and the gp130 cytokine family in periodontal disease. This study aimed to identify and quantify OPG/RANKL in the gingival crevicular fluid (GCF) and connective tissue of patients with periodontitis, and to clarify possible correlations with disease severity and interleukin-6 (IL-6) cytokines. MATERIAL AND METHODS: Ninety-five sites in 20 patients with generalized chronic periodontitis were divided into four groups by site based on probing depth (PD) and bleeding on probing (BOP). In periodontitis patients, GCF was obtained using sterile paper strips from clinically healthy sites (PD 6 mm with BOP, n = 27). Fourteen clinically healthy sites from four periodontally healthy individuals were used as the control group. The levels of OPG, RANKL and two gp130 cytokines - IL-6 and oncostatin M (OSM) - in the GCF were determined by an enzyme-linked immunosorbent assay (ELISA) and are expressed as total amounts (pg/site). Immunohistochemical localization of OPG- and RANKL-positive cells was also performed on gingival connective tissues harvested from patients with periodontitis (inflammatory group, n = 8 biopsies) and from non-diseased individuals (healthy group, n = 8 biopsies). RESULTS: GCF RANKL, but not OPG, was elevated in diseased sites of patients with periodontitis. However, the expressions of OPG and RANKL showed no correlation with disease severity (r = 0.174 and 0.056, respectively), but the content of RANKL in the GCF was significantly positively correlated with those of IL-6 (r = 0.207) and OSM (r = 0.231) (p < 0.01). Immunohistochemical staining showed that RANKL-positive cells were significantly distributed in the inflammatory connective tissue zone of diseased gingiva, compared with those of samples from non-diseased persons (p < 0.01). However, few OPG-positive cells were found in connective tissue zones of either the diseased gingiva or healthy biopsies. CONCLUSION: These findings imply that in this cross-sectional study of GCF, RANKL, IL-6 and OSM were all prominent in periodontitis sites, whereas OPG was inconsistently found in a few samples of diseased sites but was undetectable in any of the control sites. The results also imply that the expression of RANKL was positively correlated with IL-6 and OSM in the GCF.

Immunohistochemical analysis of Th1/Th2 cytokine profiles and androgen receptor expression in the pathogenesis of nifedipine-induced gingival overgrowth.

BACKGROUND: Numerous studies have demonstrated that gingival overgrowth may be associated with androgen and cytokine expression in tissues. OBJECTIVES: The aim of this study was to compare the expression of androgen receptor-presenting cells (AR+ cells) and Th1/Th2 cytokine [Th1: interleukin (IL)-2, interferon-gamma (IFN-gamma); Th2: IL-4, IL-10, IL-13] expression cells in tissue sections of patients with gingival overgrowth. MATERIALS AND METHODS: Tissue samples were collected from patients with healthy periodontium (H group), adult periodontitis (P group), surgically extracted teeth (S group), and nifedipine-induced gingival overgrowth (NIGO group). The clinical periodontal parameters of pocket depth (PD), bleeding on probing (BOP), and plaque control record (PCR) were measured around selected sample teeth. Gingival biopsies were further processed by immunohistochemical staining method. The expressions of cells positive for AR, IL-2, IFN-gamma, IL-4, IL-10, and IL-13 were counted by predetermined semiquantitative methods. RESULTS: Our results indicated that AR, IL-2, IFN-gamma, IL-4, IL-10, and IL-13 were intensively expressed in the nuclei of inflammatory cells and fibroblasts of gingival connective tissue. Stronger expressions of AR, IL-2, and IFN-gamma were found in the NIGO group. The AR+ cells/0.01 mm2 in gingival fibroblasts were significantly higher in the NIGO group (80.2 +/- 10.7) than those of the periodontitis group (52.5 +/- 11.8) and control group (37.4 +/- 11.3) (P < 0.05). The cytokine expression of the NIGO group showed a trend towards Th1-type expression (IL-2; P = 0.0001). In the surgically extracted tooth group, a stronger expression of Th2-type cytokine (IL-4, Il-10, IL-13; P < 0.05) was found in inflammatory cells. In a comparison of the IL-2/IL-4-labeled cell ratio of the four groups, a descending sequence was discovered as NIGO group (0.92 +/- 0.97) > H group (0.81 +/- 0.61) > P group (0.77 +/- 0.82) > S group (0.58 +/- 1.77). CONCLUSIONS: Our data support the following: (i) taking nifedipine may elevate the expression of AR in susceptible oral tissue, e.g. gingiva; (ii) the cytokine profile of T-cells in NIGO tissue indicates a trend preferentially towards Th1 activity; and (iii) elevation of AR expression cells and prominent Th1 cytokine-labeled cells are two significant factors in the pathogenesis of NIGO.

Quantitative classification of life-style types in predaceous phytoseiid mites.

Classification of species into different functional groups based on biological criteria has been a difficult problem in ecology. The difficulty mainly arises because natural classification patterns are not necessarily mutually exclusive. The more group characteristics overlap, the more difficult it is to identify the membership of a species in the overlapping portions of any two groups. In this paper, we present an application of discriminant analysis by creating classification models from life history and morphological data for two specialist and two generalist life-styles type of predaceous phytoseiid mites. Two stages can be distinguished in our method: life-style group membership assignment and trait variable evaluation. We use a Bayesian framework to create a classifier system to locate or assign species within a mixture of trait distributions. The method assumes that a mixture of trait distributions can represent the multiple dimensions of biological data. The mixture is most evident near the boundaries between groups. Because of the complexity of analytical solution, an iterative method is used to estimate the unknown means, variances, and mixing proportion between groups. We also developed a criterion based on information theory to evaluate model performance with different combinations of input variables and different hypotheses. We present a working example of our proposed methods. We apply these methods to the problem of selecting key species for inoculative release and for classical introductions of biological pest control agents.

Stimulation of unitary T-type Ca(2+) channel currents by calmodulin-dependent protein kinase II.

The effect of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) stimulation on unitary low voltage-activated (LVA) T-type Ca(2+) channel currents in isolated bovine adrenal glomerulosa (AG) cells was measured using the patch-clamp technique. In cell-attached and inside-out patches, LVA channel activity was identified by voltage-dependent inactivation and a single-channel conductance of approximately 9 pS in 110 mM BaCl(2) or CaCl(2). In the cell-attached patch, elevation of bath Ca(2+) from 150 nM to 1 microM raised intracellular Ca(2+) in K(+)-depolarized (140 mM) cells and evoked an increase in the LVA Ca(2+) channel probability of opening (NP(o)) by two- to sixfold. This augmentation was associated with an increase in the number of nonblank sweeps, a rise in the frequency of channel opening in nonblank sweeps, and a 30% reduction in first latency. No apparent changes in the single-channel open-time distribution, burst lengths, or openings/burst were apparent. Preincubation of AG cells with lipophilic or peptide inhibitors of CaMKII in the cell-attached or excised (inside-out) configurations prevented the rise in NP(o) elicited by elevated Ca(2+) concentration. Furthermore, administration of a mutant recombinant CaMKIIalpha exhibiting cofactor-independent activity in the absence of elevated Ca(2+) produced a threefold elevation in LVA channel NP(o). These data indicate that CaMKII activity is both necessary and sufficient for LVA channel activation by Ca(2+).

Elastic modulus, permeation time and swelling ratio of a new porcine dermal collagen membrane.

The goal of a single step therapy has been an important consideration in the development of guided tissue regeneration devices. It would spare the patient from the need for repeated surgery and eliminate many problems associated with a non-resorbable barrier. Animal studies of a collagen membrane extracted from porcine dermis (PDCM), as conditioned by different concentrations of glutaraldehyde (GA), have shown it to be biocompatible and biodegradable (up to 9 wk). This in vitro study further investigated the physical properties of this membrane. A PDCM modified and cross-linked with various concentrations (0.01%, 0.05% and 3.00%) of GA was used. A similar control series was not conditioned. At least 4 specimens for each experimental condition were prepared. The elastic modulus (EM) was measured by a universal testing machine. In the permeability test, Al2O3 particles of different sizes (5-23 microns) were mixed with normal saline to make 5 v/v% suspension and the time needed for collecting 7.5 ml of the filtered suspension from 10 ml suspension was recorded. Swelling ratio (gamma) was also measured according to gamma = 1/Vf (volume fraction). Data were analysed using ANOVA and Tukey's LSD test. The EM (40.8 +/- 3.8 gf/mm2) for the GA conditioned membranes showed no significant difference but was greater (p < 0.05) than that of the control. There was a significant increase (100-300%) in the permeation time with GA concentration (control 0.168 vs. 3% GA 0.100). The results suggest that the physical properties of the GA conditioned PDCM (especially in 3%) may fit the clinical requirements of membrane materials used in guided tissue regeneration techniques.

Angiotensin II stimulates T-type Ca2+ channel currents via activation of a G protein, Gi.

Angiotensin II (ANG II) is the most potent and the most physiologically important stimulator of aldosterone synthesis and secretion from the adrenal zona glomerulosa. Because steroidogenesis by adrenal glomerulosa (AG) cells is mediated in part by Ca2+ influx through T- and L-type Ca2+ channels, we evaluated whether T-type Ca2+ channels are regulated by ANG II. We observe that ANG II enhances T-type Ca2+ current by shifting the voltage dependence of channel activation to more negative potentials. This shift is transduced by the ANG II type 1 receptor. The effect of the hormone is not mediated by Ca2+/calmodulin-dependent protein kinase II (CaMKII) as it is not prevented by CaMKII(281-302), a peptide inhibitor of the catalytic region of the kinase. Rather, this shift is mediated by the activation of a G protein, Gi, because it is abolished by cell pretreatment with pertussis toxin and by cell dialysis with a monoclonal antibody generated against recombinant Gi alpha. This effect of ANG II on T-type Ca2+ channels should increase Ca2+ entry in AG cells at physiologically relevant voltages and result in a sustained increase in aldosterone secretion.

Ca2+/calmodulin-dependent protein kinase II activation and regulation of adrenal glomerulosa Ca2+ signaling.

We recently reported that elevations in the intracellular Ca2+ concentration ([Ca2+]i) enhance low-voltage-activated, T-type, Ca2+ channel activity via Ca2+/calmodulin-dependent protein kinase II (CaMKII). Here, we document CaMKII activity in bovine adrenal glomerulosa (AG) cells and assess the importance of CaMKII in depolarization-induced Ca2+ signaling. AG cell extracts exhibited kinase activity toward a CaMKII-selective peptide substrate that was dependent on both Ca2+ [half-maximal concentration for Ca2+ activation (K0.5) = 1.5 microM] and calmodulin (K0.5 = 46 nM) and was sensitive to a calmodulin antagonist and a CaMKII peptide inhibitor. On cell treatment with elevated extracellular potassium (10-60 mM) or angiotensin II, Ca(2+)-independent CaMKII activity increased to 133-205% of basal activity. Ca(2+)-independent kinase activity in agonist-stimulated extracts was inhibited by the CaMKII inhibitor peptide, 1(-)[N,O-bis(1,5- isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), a cell-permeable inhibitor of CaMKII, reduced the agonist-induced stimulation of Ca(2+)-independent CaMKII activity. KN-62 also diminished depolarization-induced increases in [Ca2+]i without affecting the membrane potential. These observations suggest that CaMKII is activated in situ by aldosterone secretagogues and augments Ca2+ signaling through voltage-gated Ca2+ channels.

Translocation of ferritin and biomineralization of goethite in the radula of the limpet Cellana toreuma reeve.

The radula of the limpet, Cellana toreuma, consists of a continuous series of teeth in various stages of iron biomineralization. The major iron-binding protein of the limpet's iron-containing granule (siderosome) has been purified and identified as ferritin. Limpet ferritin has a M(r) of 575 kDa and can be resolved into two bands by SDS-PAGE analysis, with respective M(r)s of 26 and 21 kDa. The partial N-terminal amino acid sequences of these two subunits were confirmed, and antisera against them were respectively generated. The specifity of these two antisera shows no difference between them. By using transmission electron microscopy and immunogold staining techniques the following two events were revealed: (1) in the superior epithelial cell of the radula, ferritin was disassembled through autophagy or heterophagy before exocytosis; (2) of the limpet ferritin, at least the 26-kDa subunit was found to pass through the microvilli, resulting in the accumulation of iron in the extracellular tooth chamber and the formation of goethite needles. Intracellular ferritin being translocated to the extracellular environment is discussed in the text.

Ca(2+)-dependent activation of T-type Ca2+ channels by calmodulin-dependent protein kinase II.

The T-type Ca2+ channel is unique among voltage-dependent Ca2+ channels in its low threshold for opening and its slow kinetics of deactivation. Here, we evaluate the importance of intracellular Ca2+ (Cai2+) in promoting low-threshold gating of T-type channels in adrenal glomerulosa cells. We observe that 390 nM to 1.27 microM Cai2+ enhances T-type current by shifting the voltage dependence of channel activation to more negative potentials. This Ca(2+)-induced shift is mediated by calmodulin-dependent protein kinase II (CaMKII), because it is abolished by inhibitors of CaMKII but not of protein kinase C and is subsequently restored by exogenous calmodulin. This Ca(2+)-induced reduction in gating threshold would render T-type Ca2+ channels uniquely suited to transduce depolarizing stimuli of low amplitude into a Ca2+ signal sufficient to support a physiological response.

Treatment of an osseous lesion associated with a severe palato-radicular groove: a case report.

This is a case report that describes the management of a severe periodontal defect associated with a palato-radicular (palato-gingival) groove affecting the maxillary right lateral incisor of a 50 year-old male. The patient presented with pain, gingival swelling, and a 10 mm periodontal pocket on the distopalatal aspect of the right maxillary lateral incisor. The defect was initially treated by scaling and root planing. Several days later a flap was elevated, the osseous defect was debrided, and odontoplasty was performed to eliminate the groove. The root surface was treated with citric acid for 3 minutes, the osseous defect was filled with non-porous hydroxyapatite, a periodontal membrane was placed, and the flap was readapted to the tooth. Postoperative care included systemic (minocycline) and local (chlorhexidine) antimicrobial therapy. The membrane was removed 6 weeks postoperatively and 14 months postoperatively the gingiva appeared healthy; radiographs suggested substantial resolution of the osseous defect and about 7 mm of probing attachment gain was recorded. Further studies are necessary to determine which of the several modes of therapy used to treat this lesion are necessary for success.

Topographical characteristics of root trunk length related to guided tissue regeneration.

Thirty-seven molars with 94 furcations were selected for a topographical study of root trunks to clarify the possible factors which may affect the clinical application of guided tissue regeneration technique. A pre-determined plane was marked on the root trunk of each tooth 1 or 2 mm below the cemento-enamel junction (CEJ). The plane followed the presumed position of the occlusal border of the Teflon membrane. Cross sectioning of the root was then performed following the plane, and the width of the gap between the membrane and root surface was measured with the aid of a stereomicroscope. The results revealed, within the limited samples of this study, 94% of the furcations possessed variant depth of developmental concavities on the root trunks. These superficial irregularities at the entrances of furcations may prevent complete adaptation of the coronal microstructure of the Teflon membrane along their root surfaces. The width of the gaps between root surfaces and membranes ranged from 0.000 mm to 2.250 mm for all tooth samples. The study implied that the subgingival application of guided tissue membranes 1 to 2 mm below CEJ cannot ensure complete adaptation of furcation defects with their coronal microstructures in the majority of molars.