Metalloprotease Meprinβ in Rat Kidney: Glomerular Localization and Differential Expression in Glomerulonephritis

Meprin (EC 3.4.24.18) is an oligomeric metalloendopeptidase found in microvillar membranes of kidney proximal tubular epithelial cells. Here, we present the first report on the expression of meprinβ in rat glomerular epithelial cells and suggest a potential involvement in experimental glomerular disease. We detected meprinβ in glomeruli of immunostained rat kidney sections on the protein level and by quantitative RT-PCR of laser-capture microdissected glomeruli on the mRNA level. Using immuno-gold staining we identified the membrane of podocyte foot processes as the main site of meprinβ expression. The glomerular meprinβ expression pattern was altered in anti-Thy 1.1 and passive Heymann nephritis (PHN). In addition, the meprinβ staining pattern in the latter was reminiscent of immunostaining with the sheep anti-Fx1A antiserum, commonly used in PHN induction. Using Western blot and immunoprecipitation assays we demonstrated that meprinβ is recognized by Fx1A antiserum and may therefore represent an auto-antigen in PHN. In anti-Thy 1.1 glomerulonephritis we observed a striking redistribution of meprinβ in tubular epithelial cells from the apical to the basolateral side and the cytosol. This might point to an involvement of meprinβ in this form of glomerulonephritis

The Metalloprotease Meprinβ Processes E-Cadherin and Weakens Intercellular Adhesion

BACKGROUND: Meprin (EC 3.4.24.18), an astacin-like metalloprotease, is expressed in the epithelium of the intestine and kidney tubules and has been related to cancer, but the mechanistic links are unknown. METHODOLOGY/PRINCIPAL FINDINGS: We used MDCK and Caco-2 cells stably transfected with meprin alpha and or meprin beta to establish models of renal and intestinal epithelial cells expressing this protease at physiological levels. In both models E-cadherin was cleaved, producing a cell-associated 97-kDa E-cadherin fragment, which was enhanced upon activation of the meprin zymogen and reduced in the presence of a meprin inhibitor. The cleavage site was localized in the extracellular domain adjacent to the plasma membrane. In vitro assays with purified components showed that the 97-kDa fragment was specifically generated by meprin beta, but not by ADAM-10 or MMP-7. Concomitantly with E-cadherin cleavage and degradation of the E-cadherin cytoplasmic tail, the plaque proteins beta-catenin and plakoglobin were processed by an intracellular protease, whereas alpha-catenin, which does not bind directly to E-cadherin, remained intact. Using confocal microscopy, we observed a partial colocalization of meprin beta and E-cadherin at lateral membranes of incompletely polarized cells at preconfluent or early confluent stages. Meprin beta-expressing cells displayed a reduced strength of cell-cell contacts and a significantly lower tendency to form multicellular aggregates. CONCLUSIONS/SIGNIFICANCE: By identifying E-cadherin as a substrate for meprin beta in a cellular context, this study reveals a novel biological role of this protease in epithelial cells. Our results suggest a crucial role for meprin beta in the control of adhesiveness via cleavage of E-cadherin with potential implications in a wide range of biological processes including epithelial barrier function and cancer progression

Relationship between PPI and baseline startle response

Prepulse inhibition (PPI) of the startle response to a sudden noise is the reduction in startle observed when the noise is preceded shortly by a mild sensory event, which is often a tone. A part of the literature is based on the assumption that PPI is independent of the baseline startle. A simple model is presented and experimental validation provided. The model is based on the commonly accepted observation that the neuronal circuit of PPI differs from that of startle. But, by using a common output, the measures of both phenomena become linked to each other. But, how can we interpret the numerous experimental data showing PPI to be independent of the startle level? It is suggested that in a number of such cases the baseline startle would have been stabilized by a ceiling effect in the startle/PPI neuronal networks. Reducing the startle level, for example in a PPI evaluation procedure, may disclose properties of startle masked by this ceiling effect. Disclosure of habituation to the startle eliciting noise produced an increase of PPI along its initial measurements. Taken together, even if the neuronal process that sustains startle and PPI are distinct, separating them experimentally requires careful parametric methods and caution in the interpretation of the corresponding observations

Isolated Hypoxic Hepatic Perfusion with Retrograde Outflow in Patients with Irresectable Liver Metastases; A New Simplified Technique in Isolated Hepatic Perfusion

Background: Isolated hepatic perfusion with high-dose chemotherapy is a treatment option for patients with irresectable metastases confined to the liver. Prolonged local control and impact on survival have been claimed. Major drawbacks are magnitude and costs of the procedure. We developed an isolated hypoxic h

Brown Spider (Loxosceles genus) Venom Toxins: Tools for Biological Purposes

Venomous animals use their venoms as tools for defense or predation. These venoms are complex mixtures, mainly enriched of proteic toxins or peptides with several, and different, biological activities. In general, spider venom is rich in biologically active molecules that are useful in experimental protocols for pharmacology, biochemistry, cell biology and immunology, as well as putative tools for biotechnology and industries. Spider venoms have recently garnered much attention from several research groups worldwide. Brown spider (Loxosceles genus) venom is enriched in low molecular mass proteins (5–40 kDa). Although their venom is produced in minute volumes (a few microliters), and contain only tens of micrograms of protein, the use of techniques based on molecular biology and proteomic analysis has afforded rational projects in the area and permitted the discovery and identification of a great number of novel toxins. The brown spider phospholipase-D family is undoubtedly the most investigated and characterized, although other important toxins, such as low molecular mass insecticidal peptides, metalloproteases and hyaluronidases have also been identified and featured in literature. The molecular pathways of the action of these toxins have been reported and brought new insights in the field of biotechnology. Herein, we shall see how recent reports describing discoveries in the area of brown spider venom have expanded biotechnological uses of molecules identified in these venoms, with special emphasis on the construction of a cDNA library for venom glands, transcriptome analysis, proteomic projects, recombinant expression of different proteic toxins, and finally structural descriptions based on crystallography of toxins