A detailed report on the measures taken in the Department of Conservative Dentistry and Periodontology in Munich at the beginning of the COVID-19 outbreak

Clinical Oral Investigations - The corona disease (COVID-19) is developing into one of the greatest challenges for healthcare professionals around the world. In this article, we report the detailed...     

Blockade of epithelial Na+ channels by triamterenes — Underlying mechanisms and molecular basis

The three subunits (α, β, γ) encoding for the rat epithelial Na⁺ channel (rENaC) were expressed in Xenopus oocytes, and the induced Na⁺ conductance was tested for its sensitivity to various triamterene derivatives. Triamterene blocked rENaC in a voltage-dependent manner, and was 100-fold less potent than amiloride at pH 7.5. At −90 mV and −40 mV, the IC₅₀ values were 5 μM and 10 μM, respectively. The blockage by triamterene, which is a weak base with a pK _a of 6.2, was dependent on the extracellular pH. The IC₅₀ was 1 μM at pH 6.5 and only 17 μM at pH 8.5, suggesting that the protonated compound is more potent than the unprotonated one. According to a simple kinetic analysis, the apparent inhibition constants at −90 mV were 0.74 μM for the charged and 100.6 μM for the uncharged triamterene. The main metabolite of triamterene, p-hydroxytriamterene sulfuric acid ester, inhibited rENaC with an approximately twofold lower affinity. Derivatives of triamterene, in which the p-position of the phenylmoiety was substituted by acidic or basic residues, inhibited rENaC with IC₅₀ values in the range of 0.1–20 μM. Acidic and basic triamterenes produced a rENaC blockade with a similar voltage and pH dependence as the parent compound, suggesting that the pteridinemoiety of triamterene is responsible for that characteristic. Expression of the rENaC α-subunit-deletion mutant, Δ278–283, which lacks a putative amiloride-binding site, induced a Na⁺ channel with a greatly reduced affinity for both triamterene and amiloride. In summary, rENaC is a molecular target for triamterene that binds to its binding site within the electrical field, preferably as a positively charged molecule in a voltage- and pH-dependent fashion. We propose that amiloride and triamterene bind to rENaC using very similar mechanisms. Received: 2 January 1996/Accepted: 17 May 1996     

Molecular and functional characterization of the small Ca2+-regulated K+ channel (rSK4) of colonic crypts

Colonic crypt cells possess basolateral Ca(2+)-regulated K+ channels which support Cl- secretion by providing the necessary driving force. The pharmacological characteristics of these channels were examined in Ussing chamber experiments of rat and rabbit colon mucosa by the use of blockers. The chromanol 293B, a blocker of KVLQT1 channels, and clotrimazole (CTZ), a blocker of small Ca(2+)-activated K+ channels, blocked stimulated Cl- secretion completely. Small-conductance Ca(2+)-activated K+ channels (SK) in excised basolateral patches of rat colonic crypts were inhibited concentration dependently by the imidazoles CTZ, NS004 and NS1619 and activated by 1-EBIO. These properties are similar to those of the known human SK channel (hSK4). hSK4-expressing Xenopus laevis oocytes showed ionomycin-activated and CTZ-inhibited K+ currents. When P2Y2 receptors were coexpressed these currents were also activated by ATP. The concentration/response curve was identical to that of rat SK channels. In human colonocytes (T84) exposed to hSK4 antisense probes, but not to sense probes, carbachol-induced K+ currents were attenuated. With RT-PCR an hSK4 could be demonstrated in human colon and in T84 colonocytes. By homology cloning the SK of the rat colon (rSK4) was identified. This protein has a high homology to hSK4 and mouse IK1. These data indicate that the Ca(2+)-activated and imidazole-inhibited basolateral K+ current in the colon is caused by SK4 channels.     

AMPK controls epithelial Na+ channels through Nedd4-2 and causes an epithelial phenotype when mutated

The metabolic sensor adenosine-monophosphate-activated kinase (AMPK) detects the cellular energy status and adjusts metabolic activity according to the cytosolic AMP to ATP ratio. Na⁺ absorption by epithelial Na⁺ channels (ENaC) is a highly energy-consuming process that is inhibited by AMPK. We show that the catalytic subunit α1 of AMPK inhibits ENaC in epithelial tissues from airways, kidney, and colon and that AMPK regulation of ENaC is absent in AMPKα1−/− mice. These mice demonstrate enhanced electrogenic Na⁺ absorption that leads to subtle changes in intestinal and renal function and may also affect Na⁺ absorption and mucociliary clearance in the airways. We demonstrate that AMPK uses the ubiquitin ligase Nedd4-2 to inhibit ENaC by increasing ubiquitination and endocytosis of ENaC. Thus, enhanced expression of epithelial Na⁺ channels was detected in colon, airways, and kidney of AMPKα1−/− mice. Therefore, AMPKα1 is a physiologically important regulator of electrogenic Na⁺ absorption and may provide a novel pharmacological target for controlling epithelial Na⁺ transport. Categories: Membranes and Transport; bioenergetics, anabolic/catabolic processes studied at the molecular level.     

Regulation of Cl− secretion by AMPK in vivo

Previous in vitro studies suggested that Cl(-) currents produced by the cystic fibrosis transmembrane conductance regulator (CFTR; ABCC7) are inhibited by the alpha1 isoform of the adenosine monophosphate (AMP)-stimulated kinase (AMPK). AMPK is a serine/threonine kinase that is activated during metabolic stress. It has been proposed as a potential mediator for transport-metabolism coupling in epithelial tissues. All previous studies have been performed in vitro and thus little is known about the regulation of Cl(-) secretion by AMPK in vivo. Using AMPKalpha1(-/-) mice and wild-type littermates, we demonstrate that phenformin, an activator of AMPK, strongly inhibits cAMP-activated Cl(-) secretion in mouse airways and colon, when examined in ex vivo in Ussing chamber recordings. However, phenformin was equally effective in AMPKalpha1(-/-) and wild-type animals, suggesting additional AMPK-independent action of phenformin. Phenformin inhibited CFTR Cl(-) conductance in basolaterally permeabilized colonic epithelium from AMPKalpha1(+/+) but not AMPKalpha1(-/-) mice. The inhibitor of AMPK compound C enhanced CFTR-mediated Cl(-) secretion in epithelial tissues of AMPKalpha1(-/-) mice, but not in wild-type littermates. There was no effect on Ca(2+)-mediated Cl(-) secretion, activated by adenosine triphosphate or carbachol. Moreover CFTR-dependent Cl(-) secretion was enhanced in the colon of AMPKalpha1(-/-) mice, as indicated in Ussing chamber ex vivo and rectal PD measurements in vivo. Taken together, these data suggest that epithelial Cl(-) secretion mediated by CFTR is controlled by AMPK in vivo.     

Peripheral blood CD4+CD8+ lymphocytes in cynomolgus monkeys are of resting memory T lineage

In this study, we analyzed peripheral blood CD4+CD8+ double-positive (DP) lymphocytes in adult cynomolgus monkeys (Macaca fascicularis). Forty of 55 monkeys had > 5% of the peripheral blood DP subpopulation (9.3 +/- 5.9%; mean +/- SD) in peripheral blood lymphocytes (PBL) in contrast to a low percentage of peripheral blood DP cells in humans and mice. In a cross-sectional study, the peripheral blood DP cells were found to increase in proportion with age. To clarify whether peripheral blood DP lymphocytes were immature precursors released from thymus without prior differentiation, the expressions of CD8 chains and CD1b on peripheral blood DP lymphocytes were compared with those on thymocytes. The peripheral blood DP lymphocytes were CD8 alpha + beta- and CD1b-, while thymic DP lymphocytes were CD8 alpha + beta + and CD1b +, suggesting that the peripheral blood DP cells are extrathymic T lymphocytes. Furthermore, the peripheral blood DP lymphocytes exhibited a resting memory T cell phenotype with CD2hiCD3+CD28-CD29hiCD49dhiCD69- CD80lo. Taken together, adult cynomolgus monkeys possess a unique peripheral blood DP T cell subpopulation which expresses a resting memory T cell phenotype. In addition, similar phenotypic properties of DP lymphocytes were distributed in the spleen and lymph nodes, although the proportion was less in the spleen and much less in lymph nodes than in PBL.      

The cystic fibrosis transmembrane conductance regulator attenuates the endogenous Ca2+ activated Cl– conductance of Xenopus oocytes

 Oocytes from Xenopus laevis activate a Ca²⁺ dependent Cl^– conductance when exposed to the Ca²⁺ ionophore ionomycin. This Ca²⁺ activated Cl^– conductance (CaCC) is strongly outwardly rectifying and has a halide conductivity ratio (G_I– / G_Cl–) of about 4.4. This is in contrast to the cystic fibrosis transmembrane conductance regulator (CFTR)-Cl^– conductance, which produces more linear I/V curves with a G_I– / G_Cl– ratio of about 0.52. Ionomycin enhanced CaCC (ΔG) in water injected and CFTR expressing ooyctes in the absence of 3-isobutyl-1-methylxanthine (IBMX, 1 mmol/l) by (μS) 23 ± 1.9 (n=9) and 23.6 ± 2.3 (n=11). Stimulation by IBMX did not change CaCC in water injected oocytes. CaCC was inhibited in CFTR-expressing ooyctes after stimulation with IBMX or a membrane permeable form of cAMP and was only 5.1 ± 0.48 μS (n=18) and 6.9 ± 0.6 (n=3), respectively. Inhibition of CaCC was correlated to the amount of CFTR-current activated by IBMX. ΔF508-CFTR which demonstrates only a small residual function in activating a cAMP dependent Cl^– channel in oocytes inhibited CaCC to a lesser degree (ΔG=12.1 ± 1.1 μS; n=7). Changes of CFTR and CaCC-Cl^– whole cell conductances were also measured when extracellular Cl^– was replaced by I^–. The results confirmed the reduced activation of CaCC in the presence of activated CFTR. No evidence was found for inhibition of CFTR-currents by increase of intracellular Ca²⁺. Moreover, intracellular cAMP was not changed by ionomycin and stimulation by IBMX did not change the ionomycin induced Ca²⁺ increase in Xenopus oocytes. Taken together, these results suggest that activation of CFTR-Cl^– currents is paralleled by an inhibition of Ca²⁺ activated Cl^– currents in ooyctes of Xenopus laevis. These results provide another example for CFTR-dependent regulation of membrane conductances other than cAMP-dependent Cl^– conductance. They might explain previous findings in epithelial tissues of CF-knockout mice. Received: 17 June 1997 / Received after revision: 4 September 1997 / Accepted: 5 September 1997     

Identification of Candida species in the clinical laboratory: a review of conventional,commercial, and molecular techniques

In healthy individuals, Candida species are considered commensal yeasts of the oral cavity. However, these microorganisms can also act as opportunist pathogens, particularly the so‐called non‐albicans Candida species that are increasingly recognized as important agents of human infection. Several surveys have documented increased rates of C. glabrata, C. tropicalis, C. guilliermondii, C. dubliniensis, C. parapsilosis, and C. krusei in local and systemic fungal infections. Some of these species are resistant to antifungal agents. Consequently, rapid and correct identification of species can play an important role in the management of candidiasis. Conventional methods for identification of Candida species are based on morphological and physiological attributes. However, accurate identification of all isolates from clinical samples is often complex and time‐consuming. Hence, several manual and automated rapid commercial systems for identifying these organisms have been developed, some of which may have significant sensitivity issues. To overcome these limitations, newer molecular typing techniques have been developed that allow accurate and rapid identification of Candida species. This study reviewed the current state of identification methods for yeasts, particularly Candida species.     

The influence of epigenetics in relation to oral health

The immune response is influenced by genetic and epigenetic factors, as well as disease and environmental factors. The term ‘epigenetics’ describes changes in the genome that influence the gene expression without altering the DNA sequence. In contrast to genetic changes in the DNA, epigenetic changes are reversible and are influenced by environmental factors. The aim of this study is to review the literature on epigenetic modifications with respect to oral health and inflammatory conditions in the oral cavity and to discuss the potential use of this new research field for the dental hygienists' and/or dentists' clinical work. Relevant publications were identified using the PubMed database without limits. The searches were conducted during January to March 2012 and resulted in articles published between 1912 and 2012. Key factors such as environment, diet, smoking, bacteria and inflammation were identified to be relevant to oral health. The result of this review article shows that there is a void in the research on epigenetics in relation to oral health. Identification of epigenetic modifications correlating with oral health may not only present a link between the influence of genetics and that of the environment on oral diseases but also provide new treatment models and tools for the dental professionals.