The Presence of Essential and Non-Essential Stratum Corneum Proteases: The Vital Need for Protease Inhibitors

Dry skin is one of the most important concerns of consumers worldwide. Despite huge efforts over several decades, the personal care industry still does not offer complete solutions that satisfy the unmet needs of consumers for moisturizing treatments. The paucity of data for the underlying biochemical problems in and the effects of moisturizers on facial skin biology and physiology may partly explain this. Our recent color mapping studies based on bio-instrumental evaluations of skin capacitance and transepidermal water loss have revealed the complexity of facial skin. However, the biomolecular reasons for these subtle differences in the different zones of the face are unknown so far. As the maturation of the stratum corneum is vital for skin moisturization and optimal barrier function, we believe that the protease / proteaseinhibitor balance particularly of the plasminogen system may be key in these processes. Thus, our aim was to develop a specific dual plasmin and urokinase inhibitor for topical application to barrier-impaired skin and demonstrate its efficac

Competitive Reporter Monitored Amplification (CMA) - Quantification of Molecular Targets by Real Time Monitoring of Competitive Reporter Hybridization

Background: State of the art molecular diagnostic tests are based on the sensitive detection and quantification of nucleic acids. However, currently established diagnostic tests are characterized by elaborate and expensive technical solutions hindering the development of simple, affordable and compact point-of-care molecular tests. Methodology and Principal Findings: The described competitive reporter monitored amplification allows the simultaneous amplification and quantification of multiple nucleic acid targets by polymerase chain reaction. Target quantification is accomplished by real-time detection of amplified nucleic acids utilizing a capture probe array and specific reporter probes. The reporter probes are fluorescently labeled oligonucleotides that are complementary to the respective capture probes on the array and to the respective sites of the target nucleic acids in solution. Capture probes and amplified target compete for reporter probes. Increasing amplicon concentration leads to decreased fluorescence signal at the respective capture probe position on the array which is measured after each cycle of amplification. In order to observe reporter probe hybridization in real-time without any additional washing steps, we have developed a mechanical fluorescence background displacement technique. Conclusions and Significance: The system presented in this paper enables simultaneous detection and quantification of multiple targets. Moreover, the presented fluorescence background displacement technique provides a generic solution fo

Die Nutzung hepatischer Funktionen fuer In-vitro-Verfahren zur Pruefung von Stoffen mit dem Ziel der Einsparung von Tierversuchen. Teilprojekt 11: Nutzung von Praezisionsleberschnitten Schlussbericht

It was the aim of the project to extensively characterize functions in precision-cut rat liver slices in order to use this in vitro model for pharmacological and toxicological objectives instead of in vivo experiments, e.g. investigations of xenobiotic metabolism and its induction, and thus to reduce the number of animal experiments. After the optimization of experimental conditions rat liver slices were of high viability until 48 h of incubation, which was indicated by no decrease in glutathione and potassium concentration and little LDH release. Cytochrome P450-dependent biotransformation activities were stable during this time or decreased a little. The latter is also true of albumin synthesis. Precision-out liver slices are well suitable for the detection of in vitro induction. Only 6 h after exposure of the slices to #beta#-naphthoflavone, an at least 1000-fold increase in CYP1A1-mRNA was detected by means of RT-PCR. An induction was also possible with cryopreserved liver slices. The use of human liver slices will be of great importance in the future. (orig.)SIGLEAvailable from TIB Hannover: DtF QN1(68,31) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekBundesministerium fuer Bildung, Wissenschaft, Forschung und Technologie, Bonn (Germany)DEGerman

Inhibition of Matriptase Activity Results in Decreased Intestinal Epithelial Monolayer Integrity In Vitro

<div><p>Barrier dysfunction in inflammatory bowel diseases implies enhanced paracellular flux and lowered transepithelial electrical resistance (TER) causing effective invasion of enteropathogens or altered intestinal absorption of toxins and drug compounds. To elucidate the role of matriptase-driven cell surface proteolysis in the maintenance of intestinal barrier function, the 3-amidinophenylalanine-derived matriptase inhibitor, MI-432 was used on porcine IPEC-J2 cell monolayer. Studies with two fluorescent probes revealed that short (2 h) treatment with MI-432 caused an altered distribution of oxidative species between intracellular and extracellular spaces in IPEC-J2 cells. This perturbation was partially compensated when administration of inhibitor continued for up to 48 h. Significant decrease in TER between apical and basolateral compartments of MI-432-treated IPEC-J2 cell monolayers proved that matriptase is one of the key effectors in the maintenance of barrier integrity. Changes in staining pattern of matriptase and in localization of the junctional protein occludin were observed suggesting that inhibition of matriptase by MI-432 can also exert an effect on paracellular gate opening via modulation of tight junctional protein assembly. This study confirms that non-tumorigenic IPEC-J2 cells can be used as an appropriate small intestinal model for the <i>in vitro</i> characterization of matriptase-related effects on intestinal epithelium. These findings demonstrate indirectly that matriptase plays a pivotal role in the development of barrier integrity; thus matriptase dysfunction can facilitate the occurence of leaky gut syndrome observed in intestinal inflammatory diseases.</p></div

Summary of <i>in vitro</i> and <i>ex vivo</i> findings [31] after externally induced changes in matriptase-activities in the maintenance of intestinal epithelial integrity.

<p>TER stands for transepithelial electrical resistance, ST-14 means suppression of tumorigenicity-14.–indicates inhibition and + is for activation of matriptase-involved proteolytic pathways. Recent findings confirm that physiological barrier function of intestinal epithelium including transepithelial electrical resistance and paracellular efflux regulated by TJ protein assembly requires matriptase activity.</p

Relative TER (actual TER values divided by initial TERs) of both the control and the inhibited cell cultures (at 10, 25 and 50 μM of inhibitor MI-432).

<p>At 2 hours (*p < 0.05) and 24 hours (**p < 0.01) significant decreases in TER can be seen only with the 50 μM concentration while at 48 hours, for all concentrations of MI-432 (***p < 0.001) there are significant decreases in the TERs when compared to control cells.</p

Quantitative analyses of ROS levels in IPEC-J2 cells incubated for 2 hours (2A, 2B), 24 hours (2C, 2D) and 48 hours (2E, 2F) with inhibitor MI-432 at different concentrations (10, 25, 50 μM).

<p>Fig 2 A, C, E represent intracellular ROS levels determined by relative fluorescence intensities ± SDs using DCF. Fig 2 B, D, F represent extracellular H₂O₂ levels measured by relative fluorescence intensities ± SDs using Amplex Red. * Indicates significant differences between the fluorescence values of the treated and control cells (*p < 0.05, **p< 0.01, ***p< 0.001, n = 3).</p