Specific processing of tenascin-C by the metalloprotease meprin beta neutralizes its inhibition of cell spreading

The metalloprotease meprin has been implicated in tissue remodelling due to its capability to degrade extracellular matrix components. Here, we investigated the susceptibility of tenascin-C to cleavage by meprin beta and the functional properties of its proteolytic fragments. A set of monoclonal antibodies against chicken and human tenascin-C allowed the mapping of proteolytic fragments generated by meprin beta. In chicken tenascin-C, meprin beta processed all three major splicing variants by removal of 10 kDa N-terminal and 38 kDa C-terminal peptides, leaving a large central part of subunits intact. IN similar cleavage pattern was found for large human tenascin-C variant where two N-terminal peptides (10 or 15 kDa) and two C-terminal fragments (40 and 55 kDa) were removed from the intact subunit. N-terminal sequencing revealed the exact amino acid positions of cleavage sites. In both chicken and human tenascin-C N-terminal cleavages occurred just before and/or after the heptad repeats involved in subunit oligomerization. In the human protein, an additional cleavage site was identified in the alternative fibronectin type III repeat D. Whereas all these sites are known to be attacked by several other proteases, a unique cleavage by meprin beta was located to the 7th constant fibronectin type III repeat in both chicken and human tenascin-C, thereby removing the C-terminal domain involved in its anti-adhesive activity. In cell adhesion assays meprin beta-digested human tenascin-C was not able to interfere with fibronectin-mediated cell spreading, confirming cleavage in the anti-adhesive domain. Whereas the expression of meprin beta and tenascin-C does not overlap in normal colon tissue, inflamed lesions of the mucosa from patients with Crohn's disease exhibited many meprin beta-positive leukocytes in regions where tenascin-C was strongly induced. Our data indicate that, at least under pathological conditions, meprin beta might attack specific functional sites in tenascin-C that are important for its oligomerization and anti-adhesive activity. (C) 2009 Elsevier B.V. All rights reserved.

Proteomic analysis of mononuclear cells of patients with minimal-change nephrotic syndrome of childhood.

Background/Aims. Recently, peripheral blood mononuclear cell transcriptome analysis has identified genes that are upregulated in relapsing minimal-change nephrotic syndrome (MCNS). In order to investigate protein expression in peripheral blood mononuclear cells (PBMC) from relapsing MCNS patients, we performed proteomic comparisons of PBMC from patients with MCNS in relapse and controls. METHODS: PBMC from a total of 20 patients were analysed. PBMC were taken from five patients with relapsing MCNS, four in remission, five patients with other glomerular diseases and six controls. Two dimensional electrophoresis was performed and proteome patterns were compared. RESULTS: Automatic heuristic clustering analysis allowed us to pool correctly the gels from the MCNS patients in the relapse and in the control groups. Using hierarchical population matching, nine spots were found to be increased in PBMC from MCNS patients in relapse. Four spots were identified by mass spectrometry. Three of the four proteins identified (L-plastin, alpha-tropomyosin and annexin III) were cytoskeletal-associated proteins. Using western blot and immunochemistry, L-plastin and alpha-tropomyosin 3 concentrations were found to be enhanced in PBMC from MCNS patients in relapse. Conclusions. These data indicate that a specific proteomic profile characterizes PBMC from MCNS patients in relapse. Proteins involved in PBMC cytoskeletal rearrangement are increased in relapsing MCNS. We hypothesize that T-cell cytoskeletal rearrangement may play a role in the pathogenesis of MCNS by altering the expression of cell surface receptors and by modifying the interaction of these cells with glomerular cells.

Experimental and computational approaches to quantitative proteomics: status quo and outlook.

Proteomics has come a long way from the initial qualitative analysis of proteins present in a given sample at a given time ("cataloguing") to large-scale characterization of proteomes, their interactions and dynamic behavior. Originally enabled by breakthroughs in protein separation and visualization (by two-dimensional gels) and protein identification (by mass spectrometry), the discipline now encompasses a large body of protein and peptide separation, labeling, detection and sequencing tools supported by computational data processing. The decisive mass spectrometric developments and most recent instrumentation news are briefly mentioned accompanied by a short review of gel and chromatographic techniques for protein/peptide separation, depletion and enrichment. Special emphasis is placed on quantification techniques: gel-based, and label-free techniques are briefly discussed whereas stable-isotope coding and internal peptide standards are extensively reviewed. Another special chapter is dedicated to software and computing tools for proteomic data processing and validation. A short assessment of the status quo and recommendations for future developments round up this journey through quantitative proteomics.

Determination of oseltamivir and oseltamivir carboxylate in human plasma by liquid chromatography-tandem mass spectrometry

Introduction: Oseltamivir phosphate (OP), the prodrug of oseltamivir carboxylate (OC; active metabolite), is marketed since 10 years for the treatment of seasonal influenza flu. It has recently received renewed attention because of the threat of avian flu H5N1 in 2006-7 and the 2009-10 A/H1N1 pandemic. However, relatively few studies have been published on OP and OC clinical pharmacokinetics. The disposition of OC and the dosage adaptation of OP in specific populations, such as young children or patients undergoing extrarenal epuration, have also received poor attention. An analytical method was thus developed to assess OP and OC plasma concentrations in patients receiving OP and presenting with comorbidities or requiring intensive care. Methods: A high performance liquid chromatography coupled to tandem mass spectrometry method (HPLC-MS/MS) requiring 100-µL aliquot of plasma for quantification within 6 min of OP and OC was developed. A combination of protein precipitation with acetonitrile, followed by dilution of supernant in suitable buffered solvent was used as an extraction procedure. After reverse phase chromatographic separation, quantification was performed by electro-spray ionization-triple quadrupole mass spectrometry. Deuterated isotopic compounds of OP and OC were used as internal standards. Results: The method is sensitive (lower limit of quantification: 5 ng/mL for OP and OC), accurate (intra-/inter-assay bias for OP and OC: 8.5%/5.5% and 3.7/0.7%, respectively) and precise (intra-/inter-assay CV%: 5.2%/6.5% and 6.3%/9.2%, respectively) over the clinically relevant concentration range (upper limits of quantification 5000 ng/mL). Of importance, OP, as in other previous reports, was found not to be stable ex vivo in plasma on standard anticoagulants (i.e. EDTA, heparin or citrate). This poor stability of OP has been prevented by collecting blood samples on commercial fluoride/oxalate tubes. Conclusions: This new simple, rapid and robust HPLC-MS/MS assay for quantification of OP and OC plasma concentrations offers an efficient tool for concentration monitoring of OC. Its exposure can probably be controlled with sufficient accuracy by thorough dosage adjustment according to patient characteristics (e.g. renal clearance). The usefulness of systematic therapeutic drug monitoring in patients appears therefore questionable. However, pharmacokinetic studies are still needed to extend knowledge to particular subgroups of patients or dosage regimens.

The effect of treatment with calcitonin on vertebral fracture rate in osteoporosis.

The efficacy of treatments for osteoporosis does not become evident when evaluated by fracture incidence (FI). Vertebral FI decreased in all controlled studies on calcitonin, but not significantly. Small sample sizes and short periods of treatment may have masked a possible therapeutic benefit, but longer, controlled studies with sodium fluoride or etidronate in larger groups of patients also failed to show a decrease in FI. The present analysis of nine published, therapeutic studies which indicate the FI per year and the initial prevalence of vertebral fractures, examines the question of whether the initial prevalence of fractures has an effect on the subsequent incidence of new fractures and whether the therapeutic effects have to be evaluated as a function of the initial prevalence of fractures. Bearing in mind the differences in roentgenological evaluation and in the size and quality of the various studies, the analysis revealed (1) that in the control groups there was a higher FI in patients with more than three vertebral fractures at baseline (estimated odds ratio (OR) = 49, p = 0.011); (2) that a similar trend, although not statistically significant, was observed in treated patients; (3) that the groups of control patients treated for more than 1 year showed in general an increase in FI beyond the first year and that the reverse was true in treated patients. In conclusion, failure to allow for the initial prevalence of vertebral fractures at the individual level in therapeutic trials of calcitonin to treat osteoporosis and prevent new fractures might have contributed to the absence of a demonstrable benefit of the treatment in those studies.

Amyloid and non-amyloid forms of 5q31-linked corneal dystrophy resulting from kerato-epithelin mutations at Arg-124 are associated with abnormal turnover of the protein.

Mutations in kerato-epithelin are responsible for a group of hereditary cornea-specific deposition diseases, 5q31-linked corneal dystrophies. These conditions are characterized by progressive accumulation of protein deposits of different ultrastructure. Herein, we studied the corneas with mutations at kerato-epithelin residue Arg-124 resulting in amyloid (R124C), non-amyloid (R124L), and a mixed pattern of deposition (R124H). We found that aggregated kerato-epithelin comprised all types of pathological deposits. Each mutation was associated with characteristic changes of protein turnover in corneal tissue. Amyloidogenesis in R124C corneas was accompanied by the accumulation of N-terminal kerato-epithelin fragments, whereby species of 44 kDa were the major constituents of amyloid fibrils. R124H corneas with prevailing non-amyloid inclusions showed accumulation of a new 66-kDa species altogether with the full-size 68-kDa form. Finally, in R124L cornea with non amyloid deposits, we found only the accumulation of the 68-kDa form. Two-dimensional gels revealed mutation-specific changes in the processing of the full-size protein in all affected corneas. It appears that substitutions at the same residue (Arg-124) result in cornea-specific deposition of kerato-epithelin via distinct aggregation pathways each involving altered turnover of the protein in corneal tissue.

cAMP-dependent chloride secretion mediates tubule enlargement and cyst formation by cultured mammalian collecting duct cells.

Polycystic kidney diseases result from disruption of the genetically defined program that controls the size and geometry of renal tubules. Cysts which frequently arise from the collecting duct (CD) result from cell proliferation and fluid secretion. From mCCD(cl1) cells, a differentiated mouse CD cell line, we isolated a clonal subpopulation (mCCD-N21) that retains morphogenetic capacity. When grown in three-dimensional gels, mCCD-N21 cells formed highly organized tubular structures consisting of a palisade of polarized epithelial cells surrounding a cylindrical lumen. Subsequent addition of cAMP-elevating agents (forskolin or cholera toxin) or of membrane-permeable cAMP analogs (CPT-cAMP) resulted in rapid and progressive dilatation of existing tubules, leading to the formation of cystlike structures. When grown on filters, mCCD-N21 cells exhibited a high transepithelial resistance as well as aldosterone- and/or vasopressin-induced amiloride-sensitive and -insensitive current. The latter was in part inhibited by Na(+)-K(+)-2Cl(-) cotransporter (bumetanide) and chloride channel (NPPB) inhibitors. Real-time PCR analysis confirmed the expression of NKCC1, the ubiquitous Na(+)-K(+)-2Cl(-) cotransporter and cystic fibrosis transmembrane regulator (CFTR) in mCCD-N21 cells. Tubule enlargement and cyst formation were prevented by inhibitors of Na(+)-K(+)-2Cl(-) cotransporters (bumetanide or ethacrynic acid) or CFTR (NPPB or CFTR inhibitor-172). These results further support the notion that cAMP signaling plays a key role in renal cyst formation, at least in part by promoting chloride-driven fluid secretion. This new in vitro model of tubule-to-cyst conversion affords a unique opportunity for investigating the molecular mechanisms that govern the architecture of epithelial tubes, as well as for dissecting the pathophysiological processes underlying cystic kidney diseases.

A collagen-poly(lactic acid-co-ɛ-caprolactone) hybrid scaffold for bladder tissue regeneration.

Scaffold materials should favor cell attachment and proliferation, and provide designable 3D structures with appropriate mechanical strength. Collagen matrices have proven to be beneficial scaffolds for tissue regeneration. However, apart from small intestinal submucosa, they offer a limited mechanical strength even if crosslinking can enhance their mechanical properties. A more cell-friendly way to increase material strength is to combine synthetic polymer meshes with plastic compressed collagen gels. This work describes the potential of plastic compressed collagen-poly(lactic acid-co-ɛ-caprolactone) (PLAC) hybrids as scaffolds for bladder tissue regeneration. Human bladder smooth muscle and urothelial cells were cultured on and inside collagen-PLAC hybrids in vitro. Scaffolds were analyzed by electron microscopy, histology, immunohistochemistry, and AlamarBlue assay. Both cell types proliferated in and on the hybrid, forming dense cell layers on top after two weeks. Furthermore, hybrids were implanted subcutaneously in the backs of nude mice. Host cell infiltration, scaffold degradation, and the presence of the seeded bladder cells were analyzed. Hybrids showed a lower inflammatory reaction in vivo than PLAC meshes alone, and first signs of polymer degradation were visible at six months. Collagen-PLAC hybrids have potential for bladder tissue regeneration, as they show efficient cell seeding, proliferation, and good mechanical properties.

Leishmania major: differential regulation of the surface metalloprotease in amastigote and promastigote stages.

During its life cycle, the protozoan parasite Leishmania major alternates from an intracellular amastigote form in the mammalian host to a flagellated promastigote form in the insect vector. The expression of the surface metalloprotease (PSP) during differentiation in vitro was investigated by Western and Northern blots, by immunoprecipitation of cells metabolically labeled with [35S]methionine or labeled at the surface with radioactive iodine, and by quantification of the proteolytic activity in substrate-containing polyacrylamide gels. We report that the surface metalloprotease is down-regulated at both the mRNA and the protein level in amastigotes, where it represents less than 1% of the equivalent proteolytic activity detected in promastigotes. A significant amount of mRNA is detected 4 hr after the onset of differentiation. The expression of the protease begins at that time and reaches steady state 8 hr later. The synthesis of PSP precedes the complete morphological differentiation to the promastigote stage and the appearance of the lipophosphoglycan, another major promastigote surface component. In contrast to PSP, a family of mercaptoethanol-activated proteases present in the amastigote exists only at a reduced level in the promastigote. The confinement of the surface metalloprotease to the insect stage of the parasite suggests that it has no physiological function in the parasitism maintenance of mammalian host macrophages.

New formulation of vasoactive intestinal peptide using liposomes in hyaluronic acid gel for uveitis.

We evaluated the benefits of a novel formulation of vasoactive intestinal peptide (VIP) based on the incorporation of VIP-loaded rhodamine-conjugated liposomes (VIP-Rh-Lip) within hyaluronic acid (HA) gel (Gel-VIP-Rh-Lip) for the treatment of endotoxin-induced uveitis (EIU) in comparison with VIP-Rh-Lip alone. In vitro release study and rheological analysis showed that interactions between HA chains and liposomes resulted in increased viscosity and reinforced elasticity of the gel. In vivo a single intravitreal injection of Gel-VIP-Rh-Lip was performed in rats 7 days prior to uveitis induction by subcutaneous lipopolysaccharide injection. The maximal ocular inflammation occurs within 16-24 h in controls (VIP-Rh-Lip, unloaded-Rh-Lip). Whereas intraocular injection of VIP-Rh-Lip had no effect on EIU severity compared with controls, Gel-VIP-Rh-Lip reduced significantly the clinical score and number of inflammatory cells infiltrating the eye. The fate of liposomes, VIP and HA in the eyes, regional and inguinal lymph nodes and spleen was analyzed by immunostaining and fluorescence microscopy. Retention of liposomes by HA gel was observed in vitro and in vivo. Inflammation severity seemed to impact on system stability resulting in the delayed release of VIP. Thus, HA gel containing VIP-Rh-Lip is an efficient strategy to obtain a sustained delivery of VIP in ocular and lymph node tissues.

Multivitamins/multiminerals in Switzerland: not as good as it seems.

BACKGROUND: Multivitamin/multimineral (MVM) supplements are commonly consumed by the general population, but little is known regarding their composition and compliance with local regulations. We assessed the composition and compliance with regulations [no indication in the label of vitamin/minerals amounting <15% of the acceptable daily intake (ADI)] of MVM available in Switzerland. METHODS: The composition of vitamin/minerals supplements was obtained from the Swiss drug compendium, the Internet, pharmacies, parapharmacies and supermarkets. MVM was defined as the presence of at least 5 vitamins and/or minerals. RESULTS: Of the 254 vitamin/mineral supplements collected, 95 (37%) were considered as MVM. The most frequent vitamins were B₆ (73.7%), C (71.6%), B₂ (69.5%) and B₁ (67.4%); the least frequent were K (17.9%), biotin (51.6%), pantothene (55.8%) and E (56.8%). Approximately half of MVMs provided >150% of the ADI for vitamins. The most frequent minerals were zinc (66.3%), calcium (55.8%), magnesium (54.7%) and copper (48.4%), and the least frequent were fluoride (3.2%), phosphorous (17.9%), chrome (22.1%) and iodine (25%). More than two thirds of MVMs provided between 50 and 150% of the ADI for minerals, and few MVMs provided >150% of the ADI. While few MVMs provided <15% of the ADI for vitamins, a considerable fraction did so for minerals (32.7% for magnesium, 26.1% for copper and 22.6% for calcium). CONCLUSION: There is a great variability regarding the composition of MVMs available in Switzerland. Several MVM do not comply with Swiss regulations, which calls for monitoring and corrective measures.

Plasticity of protein expression during culture of fetal skin cells.

In order to gain insight into the biology of fetal skin during culture, cellular proteins were studied during four culture passages (P00, P01, P04 as well as P10) using high-resolution two-dimensional (2-D) gel electrophoresis and mass spectrometry (MS). Bioinformatic analyses were focused on a region of each gel corresponding to pI between 4 and 8 and M(r) from 8000 to 35 000. In this area, 373 +/- 42 spots were detected (N = 18). Twenty-six spots presented an integrated intensity that increased in the higher passages, whereas five spots showed a progressively lower intensity in subsequent passaging. MS analysis was performed on spots that were unambiguously identified on preparative 2-D gels. Among the 26 spots showing an increased size between P00 and P10, 9 were identified, and corresponded to 3 proteins: (i) peptidyl-prolyl cis-trans isomerase A (P05092; cyclophilin A or cyclosporin A-binding protein), (ii) triosephosphate isomerase (P00938), and (iii) enoyl-CoA hydratase (P30084). Among these nine identified spots, three were absent at P00, but were present at P10. They corresponded to isoforms of peptidyl-prolyl cis-trans isomerase and triosephosphate isomerase, respectively. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of the acidic isoforms of triosephosphate isomerase showed modifications of cysteine residues to cysteic acid. All these isoforms were clearly present in the skin cells of a 4-year-old child, as well as in skin cells from a 80-year-old man, at P00. These observations probably reflect either an oxidative stress related to cell culture, or, alternatively, maturation, differentiation and the aging of the cells.

Optimization of preservation conditions of As (III) bioreporter bacteria.

Long-term preservation of bioreporter bacteria is essential for the functioning of cell-based detection devices, particularly when field application, e.g., in developing countries, is intended. We varied the culture conditions (i.e., the NaCl content of the medium), storage protection media, and preservation methods (vacuum drying vs. encapsulation gels remaining hydrated) in order to achieve optimal preservation of the activity of As (III) bioreporter bacteria during up to 12 weeks of storage at 4 degrees C. The presence of 2% sodium chloride during the cultivation improved the response intensity of some bioreporters upon reconstitution, particularly of those that had been dried and stored in the presence of sucrose or trehalose and 10% gelatin. The most satisfying, stable response to arsenite after 12 weeks storage was obtained with cells that had been dried in the presence of 34% trehalose and 1.5% polyvinylpyrrolidone. Amendments of peptone, meat extract, sodium ascorbate, and sodium glutamate preserved the bioreporter activity only for the first 2 weeks, but not during long-term storage. Only short-term stability was also achieved when bioreporter bacteria were encapsulated in gels remaining hydrated during storage.

Atorvastatin-loaded hydrogel affects the smooth muscle cells of human veins

L'hyperplasie intimale est la cause majeure de sténoses de pontages veineux. Différents médicaments tels que les statines permettent de prévenir les sténoses mais leur administration systémique n'a que peu d'effet. Nous avons développé une matrice d'hydrogel d'acide hyaluronique qui permet d'avoir un relargage contrôlé d'atorvastatine sur un site désiré. L'enjeu de ce projet de recherche est de démontrer que l'atorvastatine relarguée par l'hydrogel a un effet similaire sur les cellules musculaires lisses de veines saphènes humaines comparé à l'atorvastatine directement diluée dans le milieu de culture. La recherche a été conduite conjointement par le laboratoire de médecine expérimentale du département de chirurgie thoracique et vasculaire du Centre Hospitalier Universitaire Vaudois et de l'Ecole de sciences pharmaceutiques des Universités de Lausanne et de Genève. On a incorporé de l'atorvastatine calcium (Chemos GmbH, Regenstauf Allemagne) dans des gels d'acide hyaluronique (Fortelis extra) à des concentrations déterminées afin de pouvoir analyser le relargage de l'Atovastatine dans le milieu de culture cellulaire par rapport aux concentrations d'atorvastatine directement ajoutées dans le milieu. Des cellules musculaires lisses primaires ont été cultivées à partir d'expiants de veines saphènes humaines. Elles ont été identifiées grâce à l'immunohistochimie par des anticorps contre la desmine et l'alpha-smooth muscle actine. La prolifération et la viabilité de ces cellules ont été analysées à l'aide du test MTT, leur transmigration avec le test de la chambre de Boyden et leur migration avec le principe de cicatrisation de plaies (wound healing assey). L'expression de gènes connus pour participer au développement de l'hyperplasie intimale, tels que la gap junction protein Connexin43 (Cx43), l'inhibiteur du plasminogène PAI-1, Thème oxygénase HO-1, la métalloproteinase-9 et l'inhibiteur de l'activateur du plasminogène tissulaire tPA, a été déterminée par niveau de mRNA exprimé en PCR. Leur expression en protéines a été analysée en utilisant la méthode par Western blots ainsi que l'immunohistochimie. Les expériences ont été effectuées à triple reprise en duplicats en parallèles avec de l'atorvastatine calcium directement ajoutée dans le milieu de culture et avec l'atorvastatine relarguée par l'hydrogel d'acide hyaluronique. Conclusions L'atorvastatine est relarguée par l'hydrogel de façon contrôlée. L'hydrogel contenant l'atorvastatine diminue la viabilité et la transmigration des cellules musculaires lisses de veines saphènes humaines de façon similaire à l'atorvastatine directement introduite dans le milieu de culture. L'hydrogel contenant l'atorvastatine module de façon sélective l'expression de marqueurs de la différentiation cellulaire de cellules musculaires lisses de veines saphènes humaines avec un retard de 24 heures comparé avec les effets de l'atorvastatine directement ajoutée au milieu de culture, sans néanmoins changer la distribution intra-cellulaire des protéines Cx43, HO-1 et PAI-1. Perspectives Il s'agit d'un projet d'importance clinique majeure permettant de réaliser des améliorations du traitement des artériopathies occlusives, ainsi que de relevance pharmacologique permettant de réaliser des dépôts de molécules avec un relargage stable et contrôlé à un site spécifique.

Sedimentation and electrophoretic migration of DNA knots and catenanes.

Various site-specific recombination enzymes produce different types of knots or catenanes while acting on circular DNA in vitro and in vivo. By analysing the types of knots or links produced, it is possible to reconstruct the order of events during the reaction and to deduce the molecular "architecture" of the complexes that different enzymes form with DNA. Until recently it was necessary to use laborious electron microscopy methods to identify the types of knots or catenanes that migrate in different bands on the agarose gels used to analyse the products of the reaction. We reported recently that electrophoretic migration of different knots and catenanes formed on the same size DNA molecules is simply related to the average crossing number of the ideal representations of the corresponding knots and catenanes. Here we explain this relation by demonstrating that the expected sedimentation coefficient of randomly fluctuating knotted or catenated DNA molecules in solution shows approximately linear correlation with the average crossing number of ideal configurations of the corresponding knots or catenanes.