The Cbl-interacting protein TULA inhibits dynamin-dependent endocytosis

The Cbl- and ubiquitin-interacting protein T-cell ubiquitin ligand (TULA) has been demonstrated to inhibit endocytosis and downregulation of ligand-activated EGF receptor (EGFR) by impairing Cbl-induced ubiquitination. We presently report that TULA additionally inhibited clathrin-dependent endocytosis in general, as both uptake of transferrin (Tf) and low-density lipoprotein (LDL) was inhibited. Additionally, endocytosis of the raft proteins CD59 and major histocompatibility complex class I (MHC-I), which we demonstrate were mainly endocytosed clathrin-independently, but dynamin-dependently, was blocked in cells overexpressing TULA. By contrast, the uptake of ricin, which is mainly endocytosed clathrin- and dynamin-independently, was not affected by overexpressed TULA. Consistently, TULA and dynamin co-immunoprecipitated and colocalized intracellularly, and upon overexpression of dynamin the TULA-mediated inhibitory effect on endocytosis of Tf, LDL, CD59 and MHC-I was counteracted. Overexpressed dynamin did not restore ubiquitination of the EGFR, and consistently dynamin did not rescue endocytosis of the EGFR in cells overexpressing TULA. We conclude that TULA inhibits both clathrin-dependent and clathrin-independent endocytic pathways by functionally sequestering dynamin via the SH3 domain of TULA binding proline-rich sequences in dynamin.     

Determination of cortisol in human saliva using liquid chromatography-electrospray tandem mass spectrometry

The aim of this work was to develop a method for determination of cortisol in saliva by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Saliva was sampled on Salivette tubes. These were centrifuged, deuterium-labeled cortisol was added as internal standard and the proteins precipitated by acetonitrile. The supernatant was evaporated, dissolved in methanol acidified with acetic acid and analyzed by LC-MS-MS. The with-in run precision, tested by pooling saliva samples from volunteers and then analyzing these in a single run, was found to be 7% at 0.7 microgram l(-1). The between-run precision was tested by analysis of the same samples at different days and found to be 11% at 2.5 microgram l(-1). The limit of quantification was 0.5 microgram l(-1). The method was applied for analysis of saliva samples from three volunteers during their last week before vacation and the first and second week on vacation. In addition, the method was compared to analysis by an immunological method. The values from the immunological method were 2.7 times higher than the LC-MS-MS results.     

Probiotic effect of lactic acid bacteria in the feed on growth and survival of fry of Atlantic cod (Gadus morhua)

A growing concern for the high consumption of antibiotics in aquaculture has initiated a search for alternative methods of disease control. Improved resistance against infectious diseases can be achieved by the use of probiotics. Probiotics are live microorganisms supplemented in food or feed which give beneficial effects on the intestinal microbial balance. In the present study a dry feed containing lactic acid bacteria (Carnobacterium divergens) isolated from Atlantic cod (Gadus morhua)intestines was given to cod fry. After three weeks of feeding the fry was exposed to a virulent strain of Vibrio anguillarum. The death rate was recorded during further three weeks of feeding with lactic acid bacteria supplemented feed. A certain improvement of disease resistance was obtained, and at the end of the experiment lactic acid bacteria dominated the intestinal flora in surviving fish given feed supplemented with lactic acid bacteria. No obvious growth inhibition of V. anguillarum was observed in an in vitro mixed culture of this bacterium and the C. divergens isolated from cod intestines. This revised version was published online in August 2006 with corrections to the Cover Date.     

Formation and activity of covalent conjugates of poliovirus and ligands binding to cell surface structures

Disulfide-linked conjugates of poliovirus with streptavidin or concanavalin A were formed and the binding of the conjugates to mouse L cells that lack natural poliovirus receptors was studied. The conjugate with streptavidin was specifically bound to biotinylated L cells, but not to unmodified L cells. The conjugate with conA was bound to L cells in the absence of, but not in the presence of alpha-methyl mannoside. Incubation of L cells with bound conjugates did not produce virus, although the conjugates were highly infectious in HeLa cells, containing natural poliovirus receptors. This suggests that the artificially bound virus was unable to penetrate the L cells and start replication. The possibility that binding of the virus to the natural receptor is required for efficient infection is discussed.     

Functional analysis of the matricellular protein SPARC with novel monoclonal antibodies.

SPARC (osteonectin, BM-40) is a matricellular glycoprotein that is expressed in many embryogenic and adult tissues undergoing remodeling or repair. SPARC modulates cellular interaction with the extracellular matrix (ECM), inhibits cell adhesion and proliferation, and regulates growth factor activity. To explore further the function and activity of this protein in tissue homeostasis, we have developed several monoclonal antibodies (MAbs) that recognize distinct epitopes on SPARC. The MAbs bind to SPARC with high affinity and identify SPARC by ELISA, Western blotting, immunoprecipitation, immunocytochemistry, and/or immunohistochemistry. The MAbs were also characterized in functional assays for potential alteration of SPARC activity. SPARC binds to collagen I and laminin-1 through an epitope defined by MAb 293; this epitope is not involved in the binding of SPARC to collagen III. The other MAbs did not interfere with the binding of SPARC to collagen I or III or laminin-1. Inhibition of the anti-adhesive effect of SPARC on endothelial cells by MAb 236 was also observed. Functional analysis of SPARC in the presence of these novel MAbs now confirms that the activities ascribed to this matricellular protein can be assigned to discrete subdomains.     

SPARC-like 1 regulates the terminal phase of radial glia-guided migration in the cerebral cortex

Differential adhesion between migrating neurons and transient radial glial fibers enables the deployment of neurons into appropriate layers in the developing cerebral cortex. The identity of radial glial signals that regulate the termination of migration remains unclear. Here, we identified a radial glial surface antigen, SPARC (secreted protein acidic and rich in cysteine)-like 1, distributed predominantly in radial glial fibers passing through the upper strata of the cortical plate (CP) where neurons end their migration. Neuronal migration and adhesion assays indicate that SPARC-like 1 functions to terminate neuronal migration by reducing the adhesivity of neurons at the top of the CP. Cortical neurons fail to achieve appropriate positions in the absence of SPARC-like 1 function in vivo. Together, these data suggest that antiadhesive signaling via SPARC-like 1 on radial glial cell surfaces may enable neurons to recognize the end of migration in the developing cerebral cortex.     

Still rethinking the value of high wood density

? Premise of the study: In a previous paper, we questioned the traditional interpretation of the advantages and disadvantages of high wood density (Functional Ecology 24: 701-705). Niklas and Spatz (American Journal of Botany 97: 1587-1594) challenged the biomechanical relevance of studying properties of dry wood, including dry wood density, and stated that we erred in our claims regarding scaling. ? Methods: We first present the full derivation of our previous claims regarding scaling. We then examine how the fresh modulus of rupture and the elastic modulus scale with dry wood density and compare these scaling relationships with those for dry mechanical properties, using almost exactly the same data set analyzed by Niklas and Spatz. ? Key results: The derivation shows that given our assumptions that the modulus of rupture and elastic modulus are both proportional to wood density, the resistance to bending is inversely proportional to wood density and strength is inversely proportional with the square root of wood density, exactly as we previously claimed. The analyses show that the elastic modulus of fresh wood scales proportionally with wood density (exponent 1.05, 95% CI 0.90-1.11) but that the modulus of rupture of fresh wood does not, scaling instead with the 1.25 power of wood density (CI 1.18-1.31). ? Conclusions: The deviation from proportional scaling for modulus of rupture is so small that our central conclusion remains correct: for a given construction cost, trees with lower wood density have higher strength and higher resistance to bending.