Assessment of some tools for the characterization of the human osteoarthritic cartilage proteome.

Since the proteome of osteoarthritic articular cartilage has been poorly investigated as yet, we adapted proteomic technologies to the study of the proteins secreted or released by fresh human osteoarthritic cartilage in culture. Fresh cartilage explants were obtained from three donors undergoing surgery for knee joint replacement. The explants were dissected out, minced, and incubated in serum-free culture medium. After 48 h, proteins in the medium were identified by two-dimensional or off-gel electrophoresis coupled to tandem mass spectrometry, or by using an antibody-based protein microarray designed to detect angiogenic factors, growth factors, chemokines, and cytokines. We identified a series of 43 proteins. Some of these proteins were already described as secretion products of chondrocytes, such as YKL-39 or osteoprotegerin, while several other were known proteins but have never been reported previously in cartilage, such as the serum amyloid P-component, the vitamin D binding protein, the pigment epithelium derived factor, the pulmonary and activation-regulated chemokine, lyl-1, thrombopoietin, fibrinogen, angiogenin, gelsolin, and osteoglycin/mimecan. While this study enabled the identification of novel proteins secreted or released by human osteoarthritic cartilage, the goal of the present work was essentially to describe the technical approach necessary for a systematic study of osteoarthritic cartilages from a large population of donors, in order to be able to select the good markers and/or targets for this poorly explored disease.     

Low temperature induces the delivery of mature and immature CFTR to the plasma membrane

Deletion of phenylalanine 508 (ΔF508) is the most prevalent disease-causing mutation resulting in retention of the immature CFTR in the endoplasmic reticulum. The most common strategy to induce the delivery of ΔF508-CFTR to the surface of cells is by reducing the incubation temperature (≈28 °C). Cell surface biotinylation of HEK293T cells grown at 37 °C for 48 h, confirmed the presence of mature wild-type CFTR, but not ΔF508-CFTR at the cell surface. On the other hand, cells incubated at 28 °C for 16 h showed both mature and immature ΔF508-CFTR at their surface. The trafficking of immature ΔF508-CFTR, but not mature ΔF508-CFTR, to the cell surface occurred at low temperature even upon addition of BFA, suggesting the involvement of a Golgi-independent pathway. These results suggest that low temperature induces the appearance of a mix population of mature and immature CFTR molecules at the plasma membrane through distinct pathways.     

Identification and functional expression of a second human beta-galactoside alpha2,6-sialyltransferase, ST6Gal II.

BLAST analysis of the human and mouse genome sequence databases using the sequence of the human CMP-sialic acid:beta-galactoside alpha-2,6-sialyltransferase cDNA (hST6Gal I, EC2.4.99.1) as a probe allowed us to identify a putative sialyltransferase gene on chromosome 2. The sequence of the corresponding cDNA was also found as an expressed sequence tag of human brain. This gene contained a 1590 bp open reading frame divided in five exons and the deduced amino-acid sequence didn't correspond to any sialyltransferase already known in other species. Multiple sequence alignment and subsequent phylogenic analysis showed that this new enzyme belonged to the ST6Gal subfamily and shared 48% identity with hST6Gal-I. Consequently, we named this new sialyltransferase ST6Gal II. A construction in pFlag vector transfected in COS-7 cells gave raise to a soluble active form of ST6Gal II. Enzymatic assays indicate that the best acceptor substrate of ST6Gal II was the free disaccharide Galbeta1-4GlcNAc structure whereas ST6Gal I preferred Galbeta1-4GlcNAc-R disaccharide sequence linked to a protein. The alpha2,6-linkage was confirmed by the increase of Sambucus nigra agglutinin-lectin binding to the cell surface of CHO transfected with the cDNA encoding ST6Gal II and by specific sialidases treatment. In addition, the ST6Gal II gene showed a very tissue specific pattern of expression because it was found essentially in brain whereas ST6Gal I gene is ubiquitously expressed.     

Release of ferulic acid from agroindustrial by-products by the cell wall-degrading enzymes produced by Aspergillus niger I-1472

Aspergillus niger I-1472 was grown on sugar beet pulp to produce cell wall polysaccharide-degrading enzymes, including feruloyl esterases. Compared to enzymatic activities measured in commercially available mixtures previously used for the release of ferulic acid, the A. niger enzymes were more various. These enzymes were tested to release ferulic acid from sugar beet pulp, maize bran, or autoclaved maize bran. They were as efficient as the commercial mixture to release ferulic acid from sugar beet pulp. On the other hand, they were much more efficient to release ferulic acid from maize bran after autoclaving pretreatment, as 95% of ferulic acid ester were solubilized. Thus, A. niger enzymes exhibited a high interest in the release of ferulic acid from various agro-industrial by-products.     

MiR-101 and miR-144 Regulate the Expression of the CFTR Chloride Channel in the Lung

The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a chloride channel that plays a critical role in the lung by maintaining fluid homeostasis. Absence or malfunction of CFTR leads to Cystic Fibrosis, a disease characterized by chronic infection and inflammation. We recently reported that air pollutants such as cigarette smoke and cadmium negatively regulate the expression of CFTR by affecting several steps in the biogenesis of CFTR protein. MicroRNAs (miRNAs) have recently received a great deal of attention as both biomarkers and therapeutics due to their ability to regulate multiple genes. Here, we show that cigarette smoke and cadmium up-regulate the expression of two miRNAs (miR-101 and miR-144) that are predicted to target CFTR in human bronchial epithelial cells. When premature miR-101 and miR-144 were transfected in human airway epithelial cells, they directly targeted the CFTR 3′UTR and suppressed the expression of the CFTR protein. Since miR-101 was highly up-regulated by cigarette smoke in vitro, we investigated whether such increase also occurred in vivo. Mice exposed to cigarette smoke for 4 weeks demonstrated an up-regulation of miR-101 and suppression of CFTR protein in their lungs. Finally, we show that miR-101 is highly expressed in lung samples from patients with severe chronic obstructive pulmonary disease (COPD) when compared to control patients. Taken together, these results suggest that chronic cigarette smoking up-regulates miR-101 and that this miRNA could contribute to suppression of CFTR in the lungs of COPD patients.     

Characterization of G3BPs: Tissue specific expression,chromosomal localisation and rasGAP120 binding studies

The G3BP (ras‐GTPase‐Activating Protein SH3‐Domain‐Binding Protein) family of proteins has been implicated in both signal transduction and RNA‐metabolism. We have previously identified human G3BP‐1, G3BP‐2, and mouse G3BP‐2. Here, we report the cloning of mouse G3BP‐1, the discovery of two alternatively spliced isoforms of mouse, and human G3BP‐2 (G3BP‐2a and G3BP‐2b), and the chromosomal localisation of human G3BP‐1 and G3BP‐2, which map to 5q14.2‐5q33.3 and 4q12‐4q24 respectively. We mapped the rasGAP¹²⁰ interactive region of the G3BP‐2 isoforms and show that both G3BP‐2a and G3BP‐2b use an N‐terminal NTF2‐like domain for rasGAP¹²⁰ binding rather than several available proline‐rich (PxxP) motifs found in members of the G3BPs. Furthermore, we have characterized the protein expression of both G3BP‐1 and G3BP‐2a/b in adult mouse tissues, and show them to be both tissue and isoform specific. J. Cell. Biochem. 84: 173–187, 2002. © 2001 Wiley‐Liss, Inc.     

The plant hormone auxin: insight in sight.

Physiological experiments conducted over the last 60 years indicate that the plant hormone auxin regulates a diverse set of developmental processes via changes in cell division, cell elongation and cell differentiation. Recent studies using transgenic plants with altered auxin levels support these conclusions and promise to provide more detailed information on the role of auxin during plant development. Although it is possible that all auxin responses are mediated by the same primary biochemical events, the studies described in this review are more consistent with multiple modes of auxin action. The development of molecular and genetic approaches to the study of hormone action should resolve this issue. The accelerated rate of progress in this field suggests that real insight into the mechanism of auxin action may be forthcoming.     

A multidimensional <Superscript>1</Superscript>H NMR lipidomics workflow to address chemical food safety issues

Introduction

Although it is still at a very early stage compared to its mass spectrometry (MS) counterpart, proton nuclear magnetic resonance (NMR) lipidomics is worth being investigated as an original and complementary solution for lipidomics. Dedicated sample preparation protocols and adapted data acquisition methods have to be developed to set up an NMR lipidomics workflow; in particular, the considerable overlap observed for lipid signals on 1D spectra may hamper its applicability.

Objectives

The study describes the development of a complete proton NMR lipidomics workflow for application to serum fingerprinting. It includes the assessment of fast 2D NMR strategies, which, besides reducing signal overlap by spreading the signals along a second dimension, offer compatibility with the high-throughput requirements of food quality characterization.

Method

The robustness of the developed sample preparation protocol is assessed in terms of repeatability and ability to provide informative fingerprints; further, different NMR acquisition schemes—including classical 1D, fast 2D based on non-uniform sampling or ultrafast schemes—are evaluated and compared. Finally, as a proof of concept, the developed workflow is applied to characterize lipid profiles disruption in serum from β-agonists diet fed pigs.

Results

Our results show the ability of the workflow to discriminate efficiently sample groups based on their lipidic profile, while using fast 2D NMR methods in an automated acquisition framework.

Conclusion

This work demonstrates the potential of fast multidimensional ¹H NMR—suited with an appropriate sample preparation—for lipidomics fingerprinting as well as its applicability to address chemical food safety issues.