Expression of biologically active mouse ciliary neutrophic factor (CNTF) and soluble CNTFRalpha in Escherichia coli and characterization of their functional specificities

Ciliary neurotrophic factor (CNTF) is a neuroprotective cytokine initially identified in chick embryo. It has been evaluated for the treatment of neurodegenerative diseases. CNTF also acts on non-neuronal cells such as oligodendrocytes, astrocytes, adipocytes and skeletal muscles cells. CNTF has regulatory effects on body weight and is currently in clinical trial for the treatment of diabetes and obesity. CNTF mediates its function by activating a tripartite receptor comprising the CNTF receptor alpha chain (CNTFRalpha), the leukemia inhibitory factor receptor beta chain (LIFRbeta) and gp130. Human, rat and chicken CNTF have been expressed as recombinant proteins, and most preclinical studies in murine models have been performed using rat recombinant protein. Rat and human CNTF differ in their fine specificities: in addition to CNTFR, rat CNTF has been shown to activate the LIFR (a heterodimer of LIFRbeta and gp130), whereas human CNTF can bind and activate a tripartite receptor comprising the IL-6 receptor alpha chain (IL-6Ralpha) and LIFR. To generate tools designed for mouse models of human diseases; we cloned and expressed in E. coli both mouse CNTF and the CNTFRalpha chain. Recombinant mouse CNTF was active and showed a high level of specificity for mouse CNTFR. It shares the arginine residue with rat CNTF which prevents binding to IL-6Ralpha. It did not activate the LIFR at all concentrations tested. Recombinant mouse CNTF is therefore specific for CNTFR and as such represents a useful tool with which to study CNTF in mouse models. It appears well suited for the comparative evaluation of CNTF and the two additional recently discovered CNTFR ligands, cardiotrophin-like cytokine\cytokine-like factor-1 and neuropoietin.     

Preparation and in vivo toxicity study of solid lipid microparticles as carrier for pulmonary administration

The purpose of this research was to investigate the effects of processing conditions on the characteristics of solid lipid microparticles (SLM) with a potential application as carriers for pulmonary administration. Compritol (5.0% wt/wt) SLM dispersions were prepared by rotor-stator homogenization, at different surfactant concentrations and emulsification times. The SLM were characterized, in terms of morphology and size, after lyophilization and sterilization by autoclaving process. In vivo assessment was carried out in rats by intratracheal instillation of either placebo or SLM dispersion, and by bronchoalveolar lavage for cytological analysis. Mean particle size of 4 to 5 μm was achieved using 0.3% and 0.4% (wt/wt) of emulsifier (Poloxamer 188) and emulsification times of 2 and 5 minutes. The particles showed spherical shape and smooth surface. The morphology of microparticles, the size, and the size distribution were not substantially modified after lyophilization and sterilization. Total cell counts showed no significant differences between placebo and SLM 0.5% or 2.5% groups. Regarding cytology, percentage of polymorphonuclear neutrophils and macrophages did not significantly differ between groups. These results suggest that a single intratracheal administration of the SLMs does not induce a significant inflammatory airway response in rats and that the SLMs might be a potential carrier for encapsulated drug via the pulmonary route.     

Bioactive recombinant neuregulin-1, -2, and -3 expressed in Escherichia coli

The neuregulins (NRGs) are a family of signaling proteins that are ligands for receptor tyrosine kinase of the ErbB family (namely ErbB3 and ErbB4). To date, four different neuregulin genes have been identified (neuregulin1-4). While NRG1 isoforms have been extensively studied, little is yet known about the other genes of the family. We report the expression of recombinant NRG1beta1, NRG2alpha, NRG2beta, and NRG3 as recombinant fusion proteins in Escherichia coli. The cDNA encoding for the EGF-like domain of each protein was cloned from the mouse olfactory bulb and inserted into the pET-19b vector allowing for bacterial expression of the protein fused to an N-terminal His tag. The recombinant NRGs expressed in the inclusion bodies were solubilized under denaturing conditions, purified by affinity chromatography, and refolded via dialysis in the presence of reducing agents. Purified recombinant NRGs were active as they bound to their receptors and induced their phosphorylation. In particular, and in agreement with data on the native proteins, all the molecules were able to bind and activate ErbB4 while only the rNRG1 and the two rNRG2 (but not rNRG3) bound ErbB3.     

The Proteome Analysis database: a tool for the in silico analysis of whole proteomes

The Proteome Analysis database (http://www.ebi.ac.uk/proteome/) has been developed by the Sequence Database Group at EBI utilizing existing resources and providing comparative analysis of the predicted protein coding sequences of the complete genomes of bacteria, archeae and eukaryotes. Three main projects are used, InterPro, CluSTr and GO Slim, to give an overview on families, domains, sites, and functions of the proteins from each of the complete genomes. Complete proteome analysis is available for a total of 89 proteome sets. A specifically designed application enables InterPro proteome comparisons for any one proteome against any other one or more of the proteomes in the database.     

Identification of stop codon readthrough genes in Saccharomyces cerevisiae

We specifically sought genes within the yeast genome controlled by a non-conventional translation mechanism involving the stop codon. For this reason, we designed a computer program using the yeast database genomic regions, and seeking two adjacent open reading frames separated only by a unique stop codon (called SORFs). Among the 58 SORFs identified, eight displayed a stop codon bypass level ranging from 3 to 25%. For each of the eight sequences, we demonstrated the presence of a poly(A) mRNA. Using isogenic [PSI⁺] and [psi^–] yeast strains, we showed that for two of the sequences the mechanism used is a bona fide readthrough. However, the six remaining sequences were not sensitive to the PSI state, indicating either a translation termination process independent of eRF3 or a new stop codon bypass mechanism. Our results demonstrate that the presence of a stop codon in a large ORF may not always correspond to a sequencing error, or a pseudogene, but can be a recoding signal in a functional gene. This emphasizes that genome annotation should take into account the fact that recoding signals could be more frequently used than previously expected.     

Increased albumin plasma efflux contributes to hypoalbuminemia only during early phase of sepsis in rats

The mechanisms leading to hypoalbuminemia in sepsis were explored by measuring plasma volume, albumin distribution, plasma albumin transcapillary escape rate (TER), and efflux (TER x albumin intravascular pool). These parameters were quantified in infected rats, injected intravenously with live Escherichia coli, and pair-fed and well-fed rats using an injection of (35)S-albumin and measuring plasma and whole body albumin concentrations. Animals were studied on days 1, 6, and 10 after infection. In pair-fed rats, neither albumin distribution nor exchange rate between the intra- and extravascular compartments was modified. The increase of plasma volume after infection partly explained hypoalbuminemia. Infection resulted in a reduction of the total albumin pool of the body all along the experimental period, indicating a net loss of the protein. Albumin TER (%/day) was significantly increased 1 and 6 days after infection, but the absolute efflux was increased only on day 1. Normal values were observed on day 10. Therefore, an accelerated plasma efflux contributes to hypoalbuminemia only during the early period of sepsis. During this phase, the protein was retained in the extravascular space where it was probably catabolized. Later on, other factors are probably involved.     

Alpha 1-adrenergic and cholinergic agonists activate MAPK by separate mechanisms to inhibit secretion in lacrimal gland

Thepurpose of this study was to determine the role of p42/p44mitogen-activated protein kinase (MAPK) in₁-adrenergically and cholinergically stimulated proteinsecretion in rat lacrimal gland acinar cells and the pathways used bythese agonists to activate MAPK. Acini were isolated by collagenasedigestion and incubated with the ₁-adrenergic agonistphenylephrine or the cholinergic agonist carbachol, and activation ofMAPK and protein secretion were then measured. Phenylephrine andcarbachol activated MAPK in a time- and concentration-dependent manner.Inhibition of MAPK significantly increased phenylephrine- andcarbachol-induced protein secretion. Inhibition of EGF receptor (EGFR)with AG1478, an inhibitor of the EGFR tyrosine kinase activity,significantly increased phenylephrine- but not carbachol-inducedprotein secretion. Whereas phenylephrine-induced activation of MAPK wascompletely inhibited by AG1478, activation of MAPK by carbachol wasnot. Phenylephrine stimulated tyrosine phosphorylation of the EGFR, whereas carbachol stimulated p60^Src, and possibly Pyk2, toactivate MAPK. We conclude that, in the lacrimal gland, activation ofMAPK plays an inhibitory role in ₁-adrenergically andcholinergically stimulated protein secretion and that these agonistsuse different signaling mechanisms to activate MAPK.

     

Determinants of S. cerevisiae dynein localization and activation: implications for the mechanism of spindle positioning

BACKGROUND: During anaphase in budding yeast, dynein inserts the mitotic spindle across the neck between mother and daughter cells. The mechanism of dynein-dependent spindle positioning is thought to involve recruitment of dynein to the cell cortex followed by capture of astral microtubules (aMTs). RESULTS: We report the native-level localization of the dynein heavy chain and characterize the effects of mutations in dynein regulators on its intracellular distribution. Budding yeast dynein displays discontinuous localization along aMTs, with enrichment at the spindle pole body and aMT plus ends. Loss of Bik1p (CLIP-170), the cargo binding domain of Bik1p, or Pac1p (LIS1) resulted in diminished targeting of dynein to aMTs. By contrast, loss of dynactin or a mutation in the second P loop domain of dynein resulted in an accumulation of dynein on the plus ends of aMTs. Unexpectedly, loss of Num1p, a proposed dynein cortical anchor, also resulted in selective accumulation of dynein on the plus ends of anaphase aMTs. CONCLUSIONS: We propose that, rather than first being recruited to the cell cortex, dynein is delivered to the cortex on the plus ends of polymerizing aMTs. Dynein may then undergo Num1p-dependent activation and transfer to the region of cortical contact. Based on the similar effects of loss of Num1p and loss of dynactin on dynein localization, we suggest that Num1p might also enhance dynein motor activity or processivity, perhaps by clustering dynein motors.