The tautomerase active site of macrophage migration inhibitory factor is a potential target for discovery of novel anti-inflammatory agents

Macrophage migration inhibitory factor (MIF) is an immunoregulatory protein that is a potential therapeutic target for a number of inflammatory diseases. Evidence exists that an unexpected catalytic active site of MIF may have a biological function. To gain further insight into the role of the catalytic active site, a series of mutational, structural, and biological activity studies were performed. The insertion of an alanine between Pro-1 and Met-2 (PAM) abolishes a non-physiological catalytic activity, and this mutant is defective in the in vitro glucocorticoid counter-regulatory activity of MIF. The crystal structure of MIF complexed to (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), an inhibitor of MIF d-dopachrome tautomerase activity, reveals that ISO-1 binds to the same position of the active site as p-hydroxyphenylpyruvic acid, a substrate of MIF. ISO-1 inhibits several MIF biological activities, further establishing a role for the catalytic active site of MIF.     

IL-13-induced chemokine responses in the lung: role of CCR2 in the pathogenesis of IL-13-induced inflammation and remodeling

IL-13 stimulates inflammatory and remodeling responses and contributes to the pathogenesis of human airways disorders. To further understand the cellular and molecular events that mediate these responses, we characterized the effects of IL-13 on monocyte chemotactic proteins (MCPs) and compared the tissue effects of transgenic IL-13 in mice with wild-type (+/+) and null (-/-) CCR2 loci. Transgenic IL-13 was a potent stimulator of MCP-1, -2, -3, and -5. This stimulation was not specific for MCPs because macrophage-inflammatory protein (MIP)-1alpha, MIP-1beta, MIP-2, MIP-3alpha, thymus- and activation-regulated chemokine, thymus-expressed chemokine, eotaxin, eotaxin 2, macrophage-derived chemokines, and C10 were also induced. The ability of IL-13 to increase lung size, alveolar size, and lung compliance, to stimulate pulmonary inflammation, hyaluronic acid accumulation, and tissue fibrosis, and to cause respiratory failure and death were markedly decreased, whereas mucus metaplasia was not altered in CCR2(-/-) mice. CCR2 deficiency did not decrease the basal or IL-13-stimulated expression of target matrix metalloproteinases or cathepsins but did increase the levels of mRNA encoding alpha1-antitrypsin, tissue inhibitor of metalloproteinase-1, -2, and -4, and secretory leukocyte proteinase inhibitor. In addition, the levels of bioactive and total TGF-beta(1) were decreased in lavage fluids from IL-13 transgenic mice with -/- CCR2 loci. These studies demonstrate that IL-13 is a potent stimulator of MCPs and other CC chemokines and document the importance of MCP-CCR2 signaling in the pathogenesis of the IL-13-induced pulmonary phenotype.     

A new isoelectric focusing gel for two-dimensional electrophoresis constructed in microporous hollow fiber membranes

We describe the preparation of IEF tube gels inside a nonwetting microporous plastic tubing. The gel in the tube need not be extruded after the first dimension separation. Instead, the porous structure of the tubes is made wettable, and the proteins are electrophoresed "through-the-wall" into the second dimension PAGE gel. Commercial ampholytes and reagents are suitable for the procedure. A useful p/ range of 4.5-9.5 can be obtained when p/ 3-10 ampholyte mixtures are used. Because of the high surface area of the porous material, precautions must be exercised to reduce oxygen inhibition during polymerization and dehydration of the gel during storage and use. A sheath device is described that satisfies these requirements. The plastic tubes can be disposed of by incineration and pose no biohazard.     

Pulmonary type II cell hypertrophy and pulmonary lipoproteinosis are features of chronic IL-13 exposure

Interleukin (IL)-13, a key mediator of Th2-mediated immunity, contributes to the pathogenesis of asthma and other pulmonary diseases via its ability to generate fibrosis, mucus metaplasia, eosinophilic inflammation, and airway hyperresponsiveness. In these studies, we compared surfactant accumulation in wild-type mice and mice in which IL-13 was overexpressed in the lung. When compared with littermate controls, transgenic animals showed alveolar type II cell hypertrophy under light and electron microscopy. Over time, their alveoli also filled with surfactant in a pulmonary alveolar proteinosis pattern. At the same time, prominent interstitial fibrosis occurs. Bronchoalveolar lavage fluid from these mice had a three- to sixfold increase in surfactant phospholipids. Surfactant proteins (SP)-A, -B, and -C showed two- to threefold increases, whereas SP-D increased 70-fold. These results indicate that IL-13 is a potent stimulator of surfactant phospholipid and surfactant accumulation in the lung. IL-13 may therefore play a central role in the broad range of chronic pulmonary conditions in which fibrosis, type II cell hypertrophy, and surfactant accumulation occur.     

Nuclear transport kinetics in microarrays of nuclear envelope patches

Optical Single Transporter Recording (OSTR) is a technique for analyzing membrane transport kinetics at high sensitivity, selectivity, and spatial resolution. Cellular membranes are firmly attached to microarrays of small test compartments (TCs) with diameters between approximately 0.1 and 100 microm and depths between approximately 10 and 100 microm. This permits to generate either "small" membrane patches containing few transporters or "large" patches containing many transporters. Transport of substrates across membrane patches is recorded by confocal microscopy. The present article reviews recent applications of OSTR to the nuclear pore complex (NPC). The results show that the transport functions of the NPC, previously studied almost exclusively in intact and permeabilized cells, are conserved in isolated nuclei and can be fully reconstituted in purified nuclear envelopes by addition of recombinant transport factors. This opens new avenues to the analysis of nuclear transport including the export of nucleic-acid-protein and ribosomal particles.     

Cloning, chromosomal organization and expression analysis of Neurl, the mouse homolog of Drosophila melanogaster neuralized gene

The Drosophila neuralized (neur) gene belongs to the neurogenic group of genes involved in regulating cell-cell interactions required for neural precursor development. neur mutant phenotypes include strong overcommitment to neural fates at the expense of epidermal fates. The human neuralized homolog (NEURL) has been recently determined and found to map to chromosome 10q25.1 within the region frequently deleted in malignant astrocytomas. Because of its potential importance in developmental processes, we analyzed the structure of the mouse homolog, Neurl, and its expression pattern in embryonic tissues. Neurl activity is detected from early developmental stages in several tissues and organs including neural tissues, limbs, the skeletal system, sense organs and internal organs undergoing epithelial-mesenchymal interactions. Neurl encodes a polypeptide associated with the plasma membrane but also detected in the cytoplasm. Similarly to the Drosophila gene, mammalian neuralized may code for an important regulatory factor.     

Mitochondrial RNase P RNAs in ascomycete fungi: lineage-specific variations in RNA secondary structure

The RNA subunit of mitochondrial RNase P (mtP-RNA) is encoded by a mitochondrial gene (rnpB) in several ascomycete fungi and in the protists Reclinomonas americana and Nephroselmis olivacea. By searching for universally conserved structural elements, we have identified previously unknown rnpB genes in the mitochondrial DNAs (mtDNAs) of two fission yeasts, Schizosaccharomyces pombe and Schizosaccharomyces octosporus; in the budding yeast Pichia canadensis; and in the archiascomycete Taphrina deformans. The expression of mtP-RNAs of the predicted size was experimentally confirmed in the two fission yeasts, and their precise 5' and 3' ends were determined by sequencing of cDNAs generated from circularized mtP-RNAs. Comparative RNA secondary structure modeling shows that in contrast to mtP-RNAs of the two protists R. americana and N. olivacea, those of ascomycete fungi all have highly reduced secondary structures. In certain budding yeasts, such as Saccharomycopsis fibuligera, we find only the two most conserved pairings, P1 and P4. A P18 pairing is conserved in Saccharomyces cerevisiae and its close relatives, whereas nearly half of the minimum bacterial consensus structure is retained in the RNAs of fission yeasts, Aspergillus nidulans and Taphrina deformans. The evolutionary implications of the reduction of mtP-RNA structures in ascomycetes will be discussed.     

Long-term release and improved intracellular penetration of oligonucleotide-polyethylenimine complexes entrapped in biodegradable microspheres

The aim of this work was to design a biodegradable delivery system for oligonucleotides providing both a sustained release and an improved intracellular penetration. To this purpose oligonucleotide/polyethylenimine (ON/PEI) complexes at nitrogen to phosphate (N/P) molar ratios of about 15 or 40 were encapsulated into poly(lactide-co-glycolide) microspheres by the multiple emulsion-solvent evaporation technique. ON/PEI complexes were efficiently entrapped inside microspheres. The introduction of salts within the external aqueous phase allowed an improvement of microsphere characteristics. In particular, the use of sodium chloride led to a reduced microsphere porosity and a more homogeneous ON distribution inside the polymeric matrix. These effects were attributed to the reduced flux of water from the external aqueous phase toward the internal aqueous droplets, due to the osmotic effect of sodium chloride. Both, the reduced porosity and the improved ON distribution inside the matrix, were considered responsible for the lower burst effect and the slower ON release rate from microsphere prepared with sodium chloride. ON/PEI complexes encapsulated inside microspheres were also protected toward enzymatic degradation in fetal calf serum. Interestingly, ON/PEI complexes slowly released from microspheres efficiently penetrated inside HeLa cells and oligonucleotides were preferentially located in the nucleus.