Direct interactions among Ret, GDNF and GFRalpha1 molecules reveal new insights into the assembly of a functional three-protein complex

The glial-cell-line-derived neurotrophic factor (GDNF) ligand activates the Ret receptor through the assembly of a multiprotein complex, including the GDNF family receptor alpha1 (GFRalpha1) molecule. Given the neuroprotective role of GDNF, there is an obvious need to precisely identify the structural regions engaged in direct interactions between the three molecules. Here, we combined a functional approach for Ret activity (in PC12 cells) to cross-linking experiments followed by MS-MALDI to study the interactions among the purified extracellular region of the human Ret, GDNF and GFRalpha1 molecules. This procedure allowed us to identify distinct regions of Ret that are physically engaged in the interaction with GDNF and GFRalpha1. The lack of these regions in a recombinant Ret form results in the failure of both structural and functional binding of Ret to GFRalpha1/GDNF complex. Furthermore, a model for the assembly of a transducing-competent Ret complex is suggested.     

Compartmentalization of hepatitis C virus quasispecies in blood mononuclear cells of patients with mixed cryoglobulinemic syndrome

The aim of this study was to investigate the quasispecies heterogeneity of hepatitis C virus (HCV) in the plasma, cryoprecipitate, and peripheral lymphocytes of chronically infected HCV patients with mixed cryoglobulinemia (MC). We studied 360 clones from 10 HCV-positive patients with MC and 8 age-, gender- and HCV genotype-matched subjects with chronic HCV infection but without MC. A partial nucleotide sequence encompassing the E1/E2 region, including hypervariable region 1 (HVR1), was amplified and cloned from plasma, cryoprecipitates, and peripheral blood mononuclear cells (PBMC), and the genetic diversity and complexity and synonymous and nonsynonymous substitution rates were determined. Heterogeneous selection pressure at codon sites was evaluated. Compartmentalization was estimated by phylogenetic and phenetic (Mantel's test) approaches. The patients with MC had 3.3 times lower nonsynonymous substitution rates (1.7 versus 5.7 substitutions/100 sites). Among the subjects with HCV genotype 1, the MC patients had significantly less complexity than the controls, whereas the diversity and complexity were similar in the genotype 2 patients and controls. Site-specific selection analysis confirmed the low frequency of MC patients showing positive selection. There was a significant correlation between positive selection and the infecting HCV genotype. The quasispecies were less heterogeneous in PBMC than in plasma. Significant compartmentalization of HCV quasispecies was observed in the PBMC of four of nine subjects (three with MC) and seven of nine cryoprecipitates. In one subject with MC, we detected a 5-amino-acid insertion at codons 385 to 389 of HVR1. Our results suggest reduced quasispecies heterogeneity in MC patients that is related to a low selection pressure which is probably due to an impaired immune response, the HCV genotype, and/or the duration of the infection. The frequent HCV quasispecies compartmentalization in patients' PBMC suggests a possible pathogenetic significance.     

Is neutrophil elastase the missing link between emphysema and fibrosis? Evidence from two mouse models

Background

The separation of emphysema from fibrosis is not as clear-cut as it was thought in early studies. These two pathologies may be present at the same time in human lungs and in mice either instilled with elastolytic enzymes or bleomycin or exposed to cigarette-smoke. According to a current view, emphysema originates from a protease/antiprotease imbalance, and a role for antiproteases has also been suggested in the modulation of the fibrotic process. In this study we investigate in experimental animal models of emphysema and fibrosis whether neutrophil elastase may constitute a pathogenic link between these two pathologies.

Methods

This study was done in two animal models in which emphysema and fibrosis were induced either by bleomycin (BLM) or by chronic exposure to cigarette-smoke. In order to assess the protease-dependence of the BLM-induced lesion, a group mice was treated with 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, a serine proteinase inhibitor active toward neutrophil elastase. Lungs from each experimental group were used for the immunohistochemical assessment of transforming growth factor-β (TGF-β) and transforming growth factor-α (TGF-α) and for determination of the mean linear intercept as well as the percent volume densities of fibrosis and of emphysematous changes. Additionally, the lungs were also assessed for desmosine content and for the determination of elastase levels in the pulmonary interstitium by means of immunoelectron microscopy.

Results

We demonstrate that in BLM-treated mice (i) the development of elastolytic emphysema precedes that of fibrosis; (ii) significant amount of elastase in alveolar interstitium is associated with an increased expression of TGF-β and TGF-α; and finally, (iii) emphysematous and fibrotic lesions can be significantly attenuated by using a protease inhibitor active against neutrophil elastase.Also, in a strain of mice that develop both emphysema and fibrosis after chronic cigarette-smoke exposure, the presence of elastase in alveolar structures is associated with a positive immunohistochemical reaction for reaction for both TGF-β and TGF-α.

Conclusion

The results of the present study strongly suggest that neutrophil elastase may represent a common pathogenic link between emphysema and fibrosis. Proteases and in particular neutrophil elastase could act as regulatory factors in the generation of soluble cytokines with mitogenic activity for mesenchymal cells resulting either in emphysema or in fibrosis or both.     

CysMap and CysJoin: database and tools for protein disulphides localisation

We have developed a computer program able to make user-customised databases derived from the public PIR non-redundant reference protein database. When the database of interest has been created, the user will generate the map of all the possible linear peptides containing one and two cysteines for each protein and combine them to calculate the mass of all the possible clusters of linear peptides linked by a disulphide bridge with a cysteine pair. It is also possible to create selected maps corresponding to peptides formed by the action of specific proteases. In this way, mass spectrometric data obtained from the hydrolysis of proteins of unknown sequence can be related to that contained in the database for quick disulphide assignment and protein identification. To confirm signal attribution, the program will also furnish the expected mass of cluster peptides after performing a cycle of Edman degradation. The utility of the program is discussed and examples of application are given.     

Cell therapy of primary myopathies

Mesoangioblasts are multipotent progenitors of mesodermal tissues. In vitro mesoangioblasts differentiate into many mesoderm cell types, such as smooth, cardiac and striated muscle, bone and endothelium. After transplantation mesoangioblasts colonize mostly mesoderm tissues and differentiate into many cell types of the mesoderm. When delivered through the arterial circulation, mesoangioblasts significantly restore skeletal muscle structure and function in a mouse model of muscular dystrophy. Their ability to extensively self-renew in vitro, while retaining multipotency, qualifies mesoangioblasts as a novel class of stem cells. Phenotype, properties and possible origin of mesoangioblasts are addressed in the first part of this paper. In the second part we will focus on the cell therapy approach for the treatment of Muscular Dystrophy and we will describe why mesangioblasts appear to be promising candidates for this strategy.     

Structure of the exopolysaccharide produced by Enterobacter amnigenus

The bacterial species Enterobacter amnigenus was isolated from sugar beets harvested in Finland. It produced an exopolysaccharide rich in l-fucose, which gave viscous water solutions. Its primary structure was determined mainly by NMR spectroscopy and ESIMS of oligosaccharides and a polysaccharide with decreased molecular weight, obtained by Smith degradation of the O-deacetylated native polymer [carbohydrate structure: see text]     

Evidence for a role of mitogen-activated protein kinase 3/mitogen-activated protein kinase in the development of testicular ischemia-reperfusion injury

Mitogen-activated protein kinase (MAPK) 3/MAPK1 (also known as ERK1/ERK2) plays an important role in the signal transduction pathways. To our knowledge, however, its role in the development of testicular ischemia-reperfusion injury has not yet been investigated. Therefore, we studied the pattern of MAPK3/MAPK1 activation in a experimental model of testicular ischemia-reperfusion injury. We also investigated MAPK8 to understand whether an association exists between these two MAPKs. Adult male Sprague-Dawley rats were subjected to 1 h of testicular ischemia followed by 24 h of reperfusion or to a sham testicular ischemia-reperfusion. Animals were randomized to receive PD98059, which is an inhibitor of MAPK3/MAPK1 (10 mg/kg i.p. administered immediately after detorsion), or its vehicle. The time course of MAPK3/MAPK1, MAPK8, and tumor necrosis factor (TNF; also known as TNF alpha) expression and a histological examination in both the ischemic-reperfused testis and the contralateral one were performed. In both testes, MAPK3/MAPK1 and MAPK8 expression appeared following 10 min of reperfusion and reached their highest activation after 30 min. The MAPK levels slowly decreased, and no significant expression of either kinase was observed following 2 h of reperfusion. Expression of TNF was evident after 1 h of reperfusion and reached its maximum increase after 3 h. PD98059 blunted MAPK3/MAPK1 and MAPK8, reduced TNF expression, and improved the testicular damage caused by ischemia-reperfusion injury in both testes. These data emphasize that MAPK3/MAPK1 has a role in testicular damage and that its blockade might have a future therapeutic role for the management of patients with unilateral testicular torsion.     

Inter-residue and solvent-residue interactions in proteins: a statistical study on experimental structures

A large set of protein structures resolved by X-ray or NMR techniques has been extracted from the Protein Data Bank and analyzed using statistical methods. In particular, we investigate the interactions between side chains and the interactions between solvent and side chains, pointing out on the possibility of including the solvent as part of a knowledge-based potential. The solvent-residue contacts are accounted for on the basis of the Voronoi's polyhedron analysis. Our investigation confirms the importance of hydrophobic residues in determining the protein stability. We observe that in general hydrophobic-hydrophobic interactions and, more specifically, aromatic-aromatic contacts tend to be increasingly distally separated in the primary sequence of proteins, thus connecting distinct secondary structure elements. A simple relation expressing the dependence of the protein free energy by the number of residues is proposed. Such a relation includes both the residue-residue and the solvent-residue contributions. The former is dominant for large size proteins, whereas for small sizes (number of residues less than 100) the two terms are comparable. Gapless threading experiments show that the solvent-residue knowledge-based potential yields a significant contribution with respect to discriminating the native structure of proteins. Such contribution is important especially for proteins of small size and is similar to that given by the most favorable residue-residue knowledge-based potential referring to hydrophobic-hydrophobic interactions such as isoleucine-leucine. In general, the inclusion of the solvent-residue interaction produces a relevant increase of the free energy gap between the native structures and decoys.     

Identification of unique transcripts from a mouse full-length, subtracted inner ear cDNA library

A small-scale full-length library construction approach was developed to facilitate production of a mouse full-length cDNA encyclopedia representing approximately 250 enriched, normalized, and/or subtracted cDNA libraries. One library produced using this approach was a subtracted adult mouse inner ear cDNA library (sIEa). The average size of the inserts was approximately 2.5 kb, with the majority ranging from 0.5 to 7.0 kb. From this library 22,574 sequence reads were obtained from 15,958 independent clones. Sequencing and chromosomal localization established 5240 clusters, with 1302 clusters being unique and 359 representing new ESTs. Our sIEa library contributed 56.1% of the 7773 nonredundant Unigene clusters associated with the four mouse inner ear libraries in the NCBI dbEST. Based on homologous chromosomal regions between human and mouse, we identified 1018 UniGene clusters associated with the deafness locus critical regions. Of these, 59 clusters were found only in our sIEa library and represented approximately 50% of the identified critical regions.     

CTAB-urea method purifies RNA from melanin for cDNA microarray analysis

Melanin represents a major problem for the study of melanoma by microarrays since it is retained during RNA extraction and inhibits the enzymatic reactions used for probe preparation. Here we report a new method for cleaning RNA from melanin, based on the use of the cationic detergent cetyl-trimethylammonium bromide (CTAB)-urea for RNA precipitation. This method is easy to perform and has a low cost. Purified RNA is recovered with high quality and good yield. CTAB-urea treated RNA from highly pigmented melanoma cells can be successfully reverse transcribed and labeled to obtain probes which can be subsequently used in cDNA microarray experiments, giving consistent and reproducible results.