Synthesis and biological activity of selective pipecolic acid-based TNF-alpha converting enzyme (TACE) inhibitors

A series of novel, selective TNF-alpha converting enzyme inhibitors based on 4-hydroxy and 5-hydroxy pipecolate hydroxamic acid scaffolds is described. The potency and selectivity of TACE inhibition is dramatically influenced by the nature of the sulfonamide group which interacts with the S1' site of the enzyme. Substituted 4-benzyloxybenzenesulfonamides exhibit excellent TACE potency with >100x selectivity over inhibition of matrix metalloprotease-1 (MMP-1). Alkyl substituents on the ortho position of the benzyl ether moiety give the most potent inhibition of TNF-alpha release in LPS-treated human whole blood.     

Rheological characteristics of microbial suspensions of Pseudomonas aeruginosa and Bacillus cereus

The rheological properties of the bacteria Pseudomonas aeruginosa and Bacillus cereus have been investigated. The apparent viscosity of the bacterial suspensions has been measured at different conditions. The results showed that the bacterial suspensions' apparent viscosity increased with increasing biomass concentration of each of these strains. The P. aeruginosa suspension followed shear thinning behavior while B. cereus suspension followed shear thickening behavior. The shear stress versus shear rate experimental data were best represented by the Herschel-Bulkley model. The apparent viscosity of the P. aeruginosa and B. cereus suspensions decreased with increasing temperature. The relationship between the apparent viscosity and the shearing time highlighted the rheopectic behavior of the suspensions used in this work.     

Tolerance and bioaccumulation of copper in Phragmites australis and Zea mays

The effects of copper on the growth, tolerance indices, mineral composition (N, P, K, Fe, Zn and Mn) and metal uptake of reed (Phragmites australis [Cav. Trin. ex Steudel]) and maize (Zea mays L.) were investigated in hydroponic experiments at copper concentrations ranging from 0.5 to 157 M Cu. A reduction in root length was shown to be a good indicator of copper toxicity, concentrations of 15.7 and 78.7 M Cu inhibiting root growth in maize and reed, respectively. The reed was significantly more tolerant of copper than maize and at 7.85 M Cu (external concentration), reed can be described as a Cu tolerant plant, and maize as a Cu non-tolerant species. As a result of Cu toxicity, the concentrations of macronutrients N, P and K decreased in both shoot and root of maize, while the concentrations were hardly affected in reed tissues. Fe concentration increased in shoots and roots of maize and in roots of reed with increasing Cu treatments, leading to highly significant (p<0.01) linear relationships between tissue Fe and Cu concentrations. The bioconcentration factor (BCF) of Cu was higher in roots than in shoots of both plant species, ranging from 612 to 1592 in reed for the Cu treatments tested. In the roots of maize, BCF of Cu increased from 349 to 1931 when increasing Cu in nutrient solution from 7.85 M to 78.5 M. Therefore, reed could be useful in wastewater treatments for the removal of Cu. However, the use of reed in phytoextraction of Cu from contaminated soils is limited by the low accumulation rate in shoots and although reed can be more efficient than maize for Cu phytoextraction, harvesting the full biomass, including roots, may be required.     

Expression and purification of the recombinant ConBr (Canavalia brasiliensis lectin) produced in Escherichia coli cells

ConBr, a D-glucose/D-mannose-specific lectin from Canavalia brasiliensis seeds, was produced in Escherichia coli from a (c)DNA clone subcloned to pET15b expression vector. The recombinant lectin (rConBr) was purified by one-step immobilized metal-affinity chromatography using an amino-terminal hexahistidine tag. By SDS-PAGE and Western blot, rConBr was highly pure with an apparent molecular mass of 37 kDa. N-terminal sequence analysis revealed a single sequence, confirming the identity of the expressed protein as the pre-pro-ConBr.     

Hedgehog-Gli signalling and the growth of the brain

The development of the vertebrate brain involves the creation of many cell types in precise locations and at precise times, followed by the formation of functional connections. To generate its cells in the correct numbers, the brain has to produce many precursors during a limited period. How this is achieved remains unclear, although several cytokines have been implicated in the proliferation of neural precursors. Understanding this process will provide profound insights, not only into the formation of the mammalian brain during ontogeny, but also into brain evolution. Here we review the role of the Sonic hedgehog-Gli pathway in brain development. Specifically, we discuss the role of this pathway in the cerebellar and cerebral cortices, and address the implications of these findings for morphological plasticity. We also highlight future directions of research that could help to clarify the mechanisms and consequences of Sonic hedgehog signalling in the brain.     

Taking stock of our models: the function and future of stock centres

Stock centres for our animal models are as important as other scientific resources, such as the primary literature or genome databases. But they need forward planning, international cooperation and secure funding to keep pace with the explosion in functional genomics that relies so heavily on them.     

The identification of protein-protein interactions of the nuclear pore complex of Saccharomyces cerevisiae using high throughput matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry

Mass spectrometry has become the technology of choice for detailed identification of proteins in complex mixtures. Although electrophoretic separation, proteolytic digestion, mass spectrometric analysis of unseparated digests, and database searching have become standard methods in widespread use, peptide sequence information obtained by collision-induced dissociation and tandem mass spectrometry is required to establish the most comprehensive and reliable results. Most tandem mass spectrometers in current use employ electrospray ionization. In this work a novel tandem mass spectrometer employing matrix-assisted laser desorption ionization-time-of-flight/time-of-flight operating at 200 Hz has been used to identify proteins interacting with known nucleoporins in the nuclear pore complex of Saccharomyces cerevisiae. Proteins interacting with recombinant proteins as bait were purified from yeast extracts and then separated by one-dimensional SDS-PAGE. Although peptide mass fingerprinting is sometimes sufficient to identify proteins, this study shows the importance of employing tandem mass spectrometry for identifying proteins in mixtures or as covalently modified forms. The rules for incorporating these features into MS-Tag are presented. In addition to providing an evaluation of the sensitivity and overall quality of collision-induced dissociation spectra obtained, standard conditions for ionization and fragmentation have been selected that would allow automatic data collection and analysis, without the need to adjust parameters in a sample-specific fashion. Other considerations essential for successful high throughput protein analysis are discussed.     

Mesothelial cells in suspension expose an enriched integrin repertoire capable of capturing soluble fibronectin and laminin

Pleural cavities are lined by a polarized monolayer of mesothelial cells (MC). During pleuritis, MC are shed into effusions, and pleural obstruction may occur. Integrins are cell surface receptors mediating interactions with extracellular matrix (ECM) proteins. The distribution of beta 1-, beta 3-, beta 4-integrins and fibronectin and laminin in normal and chronically inflamed pleura and in/on MC from pleural effusions was examined by immunomorphology and flow cytometry. Adhesion assays of MC to fibronectin and laminin were performed. In situ, resting MC expressed beta 1-, beta 3-, and beta 4-, and alpha v-subunits. Activated MC were beta 1- and alpha v-positive and also expressed alpha 3 and alpha 6; beta 4 was confined to the basal surface of MC; beta 3 was absent. Floating MC from effusions neoexpressed alpha 5 and reexpressed beta 3. In vitro, MC surface expressed beta 1, beta 3, alpha 3, alpha 5, alpha 6, alpha v, and also alpha 1 and alpha 2. In normal pleura, fibronectin and laminin were components of the basement membrane. In pleuritis, the basement membrane was desintegrated. Instead, newly formed fibronectin/laminin containing fibrils extended into the submesothelial connective tissue. Floating MC freshly isolated from effusions carried fibronectin and laminin on their surface and showed specific binding to these ECM proteins. Binding was blocked by anti-beta 1 or anti-alpha 5 and anti-alpha 6 antibodies, respectively. MC incubated with fibronectin showed a clear shift to the S phase, while laminin had no effect. In conclusion, activated and detached MC progressively enrich their integrin repertoire. By capturing soluble fibronectin and laminin and by matrix-mediated bridging, readhering MC may contribute to pleural obstruction. Further, soluble fibronectin bound to alpha 5 beta 1 might be life-sustaining for floating MC by driving cells into cell cycle.     

Neurobehavioural responses to hypergravity environment in the CD-1 mouse

Behavioural responses of CD-1 mice exposed to 2 g hypergravity (HG; 60 or 120 min) were investigated during adolescence and at adulthood. To characterise motion sickness (MS), pica behaviour, a reliable MS index measured through kaolin consumption, and spontaneous activity were evaluated before, during and after HG exposure. Short- and/or long-lasting effects on emotional responses, exploratory behaviour and spatial learning performances were also investigated and brain levels of nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) assessed. An increased pica behaviour during post rotational days and a reduction in spontaneous activity during rotation indicated a mild sickness associated with HG, and susceptibility to MS was sex and age related. Short- and long-lasting effects of exposure were also observed, rotated mice showing altered emotional/anxiety behavioural profiles and impaired spatial learning performances. Moreover, central NGF levels were markedly increased after rotation, while minor changes were observed in BDNF levels.     

Gaucher's disease

Gaucher disease is an uncommon autosomic recessive disorder. The disease is caused by a deficiency in the activity of the lysosomal enzyme glucocerebrosidase which is responsible for the degradation of glucosylceramide, résulting from the breakdown of red and white cell-membranes. In the absence of enzyme glucosylceramide accumulates in the lysosomes of macrophages. This accumulation leads to hepatomegaly, splenomegaly with subsequent haematologic abnormalities (leucopenia, anemia, thrombopenia) and bone manifestations. Three types of Gaucher disease are described: type 1 is the most common, type 2 and 3 are associated with neurologic symptoms. Macrophages are the likely cellular source of biochemical abnormalities: elevated blood level of ferritin, angiotensin converting enzyme, immunoglobulins and haemostasis abnormalities. Lysosomal perturbations lead to increased blood level of tartrate resistant acid phosphatase and chitotriosidase. Enzyme replacement therapy is available in France since 1991. In 2002, 136 patients are treated. The efficacy is overt on the asthenia, organomegaly and haematological manifestations. Bone pains disappear or decrease in intensity, however bone complications may be irreversible justifying treatment initiations before the appearance of lesions that may lead to serious functional impairment.