Reduction of alpha-Gal expression by relocalizing alpha-galactosidase to the trans-Golgi network and cell surface

Historically, the most effective means of modifying cell surface carbohydrates has required the intracellular overexpression of glycosyltransferases or glycosidases and is dependent on the enzymes occupying a cellular localization close to the carbohydrate structures they modify. We report on relocalizing the lysosomal resident glycosidase human alpha-galactosidase to other regions of the cell, Golgi and cell surface, where it is in closer proximity for cleaving the carbohydrate structure Galalpha(1,3)Gal. Relocalization of alpha-galactosidase was achieved by using the transmembrane and cytoplasmic domains from the human protein furin, which is known to localize in the trans-Golgi network (TGN) and cell surface. Two chimeric forms of alpha-galactosidase were generated, one directing it to the TGN of the cell and the other to the cell surface, as shown by confocal microscopy. The relocalized enzymes have the ability to cleave terminal alpha-galactose as detected by expression on the cell surface. Furthermore, when expressed as a transgene in mice, the TGN form of alpha-galactosidase was more effective at decreasing cell surface terminal alpha-galactose than was the native lysosomal form. When expressed in conjunction with the alpha1,2fucosyltransferase that also decreases Galalpha(1,3)Gal, the reduction was additive. The ability to relocalize enzymes that modify cell surface carbohydrate structures has far-reaching implications in biology and may be useful in such fields as xenotransplantation and treatment of glycosidase disorders.     

Pollen recognition during the self-incompatibility response in plants

Recent work has identified the elusive male (pollen) determinant that underlies self-incompatibility in Brassica (cabbage). The key pollen factor, recognized by the stigma of an incompatible plant, is a small cysteine-rich protein that interacts directly with the receptor domain of a stigma receptor serine-threonine kinase to initiate haplotype-specific pollen recognition and rejection.     

Tissue engineering: chondrocytes and cartilage

Tissue engineering offers new strategies for developing treatments for the repair and regeneration of damaged and diseased tissues. These treatments, using living cells, will exploit new developments in understanding the principles in cell biology that control and direct cell function. Arthritic diseases that affect so many people and have a major impact on the quality of life provide an important target for tissue engineering. Initial approaches are in cartilage repair; in our own programme we are elucidating the signals required by chondrocytes to promote new matrix assembly. These principles will extend to other tissues of the musculoskeletal system, including the repair of bone, ligament and tendon.     

Gammadelta T cells: functional plasticity and heterogeneity

Gammadelta T cells remain an enigma. They are capable of generating more unique antigen receptors than alphabeta T cells and B cells combined, yet their repertoire of antigen receptors is dominated by specific subsets that recognize a limited number of antigens. A variety of sometimes conflicting effector functions have been ascribed to them, yet their biological function(s) remains unclear. On the basis of studies of gammadelta T cells in infectious and autoimmune diseases, we argue that gammadelta T cells perform different functions according to their tissue distribution, antigen-receptor structure and local microenvironment; we also discuss how and at what stage of the immune response they become activated.     

Hyphenation of sedimentation field flow fractionation with flow cytometry

Interest in the development of field flow fractionation (FFF) systems for cell sorting recently increased with the possibility of collecting and characterizing viable cellular materials. There are various tools for the analysis of cell characteristics, but the reference is small- and large-angle light scattering often coupled with fluorimetric measurements. The well-known flow cytometry (FC) cell analysis techniques can be associated with FFF leading to the possibility of collecting information provided by a remarkable separation technique for micron-sized particles (cells) operating in the steric-hyperlayer elution mode with multiparametric detection provided by flow cytometry. Moreover FFF derived cell characteristics can be correlated with FC characteristics to describe in a unique way the nature of the eluted materials. Experimental demonstrations are described herein using nucleated cells (HL-60 cell lineage) and human red blood cells (HRBC).     

Improved chip design for integrated solid-phase microextraction in on-line proteomic sample preparation

A recently introduced silicon microextraction chip (SMEC), used for on-line proteomic sample preparation, has proved to facilitate the process of protein identification by sample clean up and enrichment of peptides. It is demonstrated that a novel grid-SMEC design improves the operating characteristics for solid-phase microextraction, by reducing dispersion effects and thereby improving the sample preparation conditions. The structures investigated in this paper are treated both numerically and experimentally. The numerical approach is based on finite element analysis of the microfluidic flow in the microchip. The analysis is accomplished by use of the computational fluid dynamics-module FLOTRAN in the ANSYS software package. The modeling and analysis of the previously reported weir-SMEC design indicates some severe drawbacks, that can be reduced by changing the microextraction chip geometry to the grid-SMEC design. The overall analytical performance was thereby improved and also verified by experimental work. Matrix-assisted laser desorption/ionization mass spectra of model peptides extracted from both the weir-SMEC and the new grid-SMEC support the numerical analysis results. Further use of numerical modeling and analysis of the SMEC structures is also discussed and suggested in this work.     

Global Positioning System (GPS) location accuracy improvement due to selective availability removal

Global Positioning System (GPS) is an important new technology for spatio-temporal behaviour studies of animals. Differential correction improves location accuracy. Previously, it mostly removed partially the influence of Selective Availability (SA). SA was deactivated in May 2000. The aim of this study was to quantify the influence of SA cancellation on location accuracy of various GPS receivers. We tested the accuracy of locations obtained from non-differential and differential GPS animal collars before and after SA removal. We found a significant improvement in accuracy for both types of GPS collars. However, differential GPS still provides more accurate locations.