Measuring principles of frictional coefficients in cartilaginous tissues and its substitutes

The frictional properties of cartilaginous tissues, such as the hydraulic permeability, the electro-osmotic permeability, the diffusion coefficients of various ions and solutes, and the electrical conductance, are vital data to characterise the extracellular environment in which chondrocytes reside. This paper analyses one-dimensional measurement principles of these coefficients. Particular attention is given to the deformation dependence of them and the highly deformable nature of the tissues. A suggested strategy is the combination of a diffusion experiment using radiotracer methods, an electro-osmotic flow experiment and an electro-osmotic pressure experiment at low electric current.     

Specific granules of human eosinophils have lysosomal characteristics: presence of lysosome-associated membrane proteins and acidification upon cellular activation

Eosinophils possess characteristic specific granules. Their content may be important during host defense but it can also cause damage after release at sites of inflammation. We investigated possible lysosomal characteristics of these granules. Lysosome-associated membrane protein (LAMP)-1 and 2, were detected by Western blot, subcellular fractionation, and immunoelectron microscopy (IEM) and were localized to the membrane of specific granules and in vesicles of the cytoplasm, separate from secretory vesicles. No binding of mannose 6-phosphate receptor to proteins of specific granules could be detected, indicating that they are dephosphorylated and mature. Cellular activation by interleukin-5 caused acidification of specific granules, as detected by pH-dependent probes. The acidification was inhibited by concanamycin A (inhibitor of vacuolar H(+)-ATPase). Activation of eosinophils by serum-treated zymosan (STZ) caused degranulation into STZ-containing phagosomes and incorporation of LAMPs to their membranes. In conclusion, specific granules of eosinophils can be regarded as specialized primary lysosomes, a feature that may be important for their function and integrity.     

Chromosomal breakpoint mapping by arrayCGH using flow-sorted chromosomes

Despite the recent completion of the human genome project, the mapping of disease-related chromosomal translocation breakpoints and genes has remained laborious. Here, we describe a novel and rapid procedure to map such translocation breakpoints using flow-sorted chromosomes in combination with array-based comparative genomic hybridization (arrayCGH). To test the feasibility of this approach, we used a t(12;15)(q13;q25)-positive cell line with known breakpoint positions as a model. The derivative 12 chromosomes were flow-sorted, labeled, and hybridized to a genome-wide array containing 3648 well-characterized human genomic clones. The exact locations of the breakpoints on both chromosome 12 and 15 could be determined in a single hybridization experiment. In addition, we have tested the minimal amount of material necessary to perform these experiments and show that it is possible to obtain highly reliable profiles using as little as 10,000 flow-sorted chromosomes.     

Selective enrichment and purification of cultures of Methanosaeta spp

A simple method for the isolation of axenic cultures of members of the obligately acetotrophic methanogenic genus Methanosaeta is described. To overcome the competitive advantage obtained by faster growing acetate-utilizing Methanosarcina spp. in batch enrichment cultures, acetone and isopropanol are used as the growth substrates for the enrichment step. Acetone- and isopropanol-utilizing bacteria slowly ferment these substrates to acetate, which allows Methanosaeta spp. to maintain the acetate concentration at levels below the threshold required for growth of Methanosarcina spp. These enrichments eventually develop dense populations of Methanosaeta spp., which can then be separated from contaminating microorganisms to yield axenic cultures.     

Method development in high-performance liquid chromatography for high-throughput profiling and metabonomic studies of biofluid samples

"Metabonomics" has in the past decade demonstrated enormous potential in furthering the understanding of, for example, disease processes, toxicological mechanisms, and biomarker discovery. The same principles can also provide a systematic and comprehensive approach to the study of food ingredient impact on consumer health. However, "metabonomic" methodology requires the development of rapid, advanced analytical tools to comprehensively profile biofluid metabolites within consumers. Until now, NMR spectroscopy has been used for this purpose almost exclusively. Chromatographic techniques and in particular HPLC, have not been exploited accordingly. The main drawbacks of chromatography are the long analysis time, instabilities in the sample fingerprint and the rigorous sample preparation required. This contribution addresses these problems in the quest to develop generic methods for high-throughput profiling using HPLC. After a careful optimization process, stable fingerprints of biofluid samples can be obtained using standard HPLC equipment. A method using a short monolithic column and a rapid gradient with a high flow-rate has been developed that allowed rapid and detailed profiling of larger numbers of urine samples. The method can be easily translated into a slow, shallow-gradient high-resolution method for identification of interesting peaks by LC-MS/NMR. A similar approach has been applied for cell culture media samples. Due to the much higher protein content of such samples non-porous polymer-based small particle columns yielded the best results. The study clearly shows that HPLC can be used in metabonomic fingerprinting studies.     

Protein-protein interactions as a target for drugs in proteomics

Protein-protein interactions play a central role in numerous processes in the cell and are one of the main fields of functional proteomics. This review highlights the methods of bioinformatics and functional proteomics of protein-protein interaction investigation. The structures and properties of contact surfaces, forces involved in protein-protein interactions, kinetic and thermodynamic parameters of these reactions were considered. The properties of protein contact surfaces depend on their functions. The contact surfaces of permanent complexes resemble domain contacts or the protein core and it is reasonable to consider such complex formation as a continuation of protein folding. Characteristics of contact surfaces of temporary protein complexes share some similarities with active sites of enzymes. The contact surfaces of the temporary protein complexes have unique structure and properties and they are more conservative in comparison with active site of enzymes. So they represent prospective targets for a new generation of drugs. During the last decade, numerous investigations were undertaken to find or design small molecules that block protein dimerization or protein(peptide)-receptor interaction, or, on the contrary, to induce protein dimerization.     

Beyond 100 genomes

By the end of 2002, we witnessed the landmark submission of the 100th complete genome sequence in the databases. An overview of these genomes reveals certain interesting trends and provides valuable insights into possible future developments.     

Phytoseiid predators of whiteflies feed and reproduce on non-prey food sources

Two phytoseiid species, Euseius scutalis (Athias-Henriot) and Typhlodromips swirskii (Athias-Henriot), are able to suppress whitefly populations on single plants and are candidate biological control agents for whiteflies such as Bemisia tabaci (Gennadius). These species can feed on pollen and insect-produced honeydew and these food sources are likely to be available in crops. If the utilization of these food types results in increased reproduction or survival, populations of predators can persist when whitefly prey is scarce or absent. We studied the impact of pollen and whitefly-produced honeydew on the life history of the two phytoseiids. Cattail pollen allowed for survival, development and reproduction of both predators. Whitefly-produced honeydew greatly increased survival of E. scutalis, allowed for development into adulthood and for a sustained low rate of oviposition. The survival of adult T. swirskii was high on cucumber leaf tissue, either with or without pollen or honeydew. Oviposition by adults and juvenile survival of T. swirskii was very low in presence of honeydew. Biological control of whiteflies may benefit from both pollen and honeydew because these non-prey food sources have a positive effect on the life history of the two predator species, especially E. scutalis.     

The expression level determines the surface distribution of the transferrin receptor in Trypanosoma brucei

The transferrin receptor (TfR) of Trypanosoma brucei is a heterodimer attached to the surface membrane by a glycosylphosphatidylinositol (GPI) anchor. The TfR is restricted to the flagellar pocket, a deep invagination of the plasma membrane. The membrane of the flagellar pocket and the rest of the cell surface are continuous, and the mechanism that selectively retains the TfR in the pocket is unknown. Here, we report that the TfR is retained in the flagellar pocket by a specific and saturable mechanism. In bloodstream-form trypanosomes transfected with the TfR genes, TfR molecules escaped flagellar pocket retention and accumulated on the entire surface, even at modest (threefold) overproduction levels. Similar surface accumulation was observed when the TfR levels were physiologically upregulated threefold when trypanosomes were starved for transferrin. These results suggest that the TfR flagellar pocket retention mechanism is easily saturated and that control of the expression level is critical to maintain the restricted surface distribution of the receptor.     

Identification of a novel putative Ran-binding protein and its close homologue

In the process of cloning genes at the breakpoint of t(5;14) (q34;q11), a recurring translocation in acute lymphoblastic leukemia, we isolated and characterized a novel gene at 5q34, and a close human homologue (66% amino acid identity) located at 8p11-12. The presence of an importin-beta N-terminal domain at their N-terminus, their size of approximately 110 kD, their nuclear localization and the identity of the homologue to a gene of a recently submitted RanGTP binding protein (RanBP16), suggest that its protein is a novel member of the importin-beta superfamily of nuclear transport receptors, therefore called RanBP17. Northern blot analysis of human tissues revealed a ubiquitous expression pattern of the RanBP16 gene and a very restricted expression pattern of the RanBP17 gene, showing high expression in testis and pancreas. Both genes are evolutionary conserved and show a high (99 and 94%) amino acid conservation with their murine counterparts and a striking similarity (40%) to a protein product of Caenorhabditis elegans (C35A5.8).