Volume Contents

Volume Contents

Volume Contents     

Transcriptional regulation of cytosol and membrane alanyl-aminopeptidase in human T cell subsets

Aminopeptidase inhibitors strongly affect the proliferation and function of immune cells in man and animals and are promising agents for the pharmacological treatment of inflammatory or autoimmune diseases. Membrane alanyl-aminopeptidase (mAAP) has been considered as the major target of these anti-inflammatory aminopeptidase inhibitors. Recent evidence also points to a role of the cytosol alanyl-aminopeptidase (cAAP) in the immune response. In this study we used quantitative RT-PCR to determine the mRNA expression of both cAAP and mAAP in resting and activated peripheral T cells and also in CD4+, CD8+, Th1, Th2 and Treg (CD4+ CD25+) subpopulations. Both mAAP and cAAP mRNAs were expressed in all cell types investigated, and in response to activation their expression appeared to be upregulated in CD8+ cells, but downregulated in Treg cells. In CD4+ cells, mAAP and cAAP mRNAs were affected in opposite ways in response to activation. The cAAP-specific inhibitor, PAQ-22, did not affect either cAAP or mAAP expression in activated CD4+ or CD8+ cells, whereas in activated Treg cells it markedly upregulated the mRNA levels of both aminopeptidases. The non-discriminatory inhibitor, phebestin, significantly increased the amount of mAAP and cAAP mRNA in CD4+ and that of cAAP in Treg cells.     

Neural organization of the circadian system of the cockroach Leucophaea maderae

The cockroach Leucophaea maderae was the first animal in which lesion experiments localized an endogenous circadian clock to a particular brain area, the optic lobe. The neural organization of the circadian system, however, including entrainment pathways, coupling elements of the bilaterally distributed internal clock, and output pathways controlling circadian locomotor rhythms are only recently beginning to be elucidated. As in flies and other insect species, pigment-dispersing hormone (PDH)-immunoreac- tive neurons of the accessory medulla of the cockroach are crucial elements of the circadian system. Lesions and transplantation experiments showed that the endogeneous circadian clock of the brain resides in neurons associated with the accessory medulla. The accessory medulla is organized into a nodular core receiving photic input, and into internodular and peripheral neuropil involved in efferent output and coupling input. Photic entrainment of the clock through compound eye photoreceptors appears to occur via parallel, indirect pathways through the medulla. Light-like phase shifts in circadian locomotor activity after injections of γ-aminobutyric acid (GABA)- or Mas-allatotropin into the vicinity of the accessory medulla suggest that both substances are involved in photic entrainment. Extraocular, cryptochrome-based photoreceptors appear to be present in the optic lobe, but their role in photic entrainment has not been examined. Pigment-dispersing hormone-immunoreactive neurons provide efferent output from the accessory medulla to several brain areas and to the peripheral visual system. Pigment-dispersing hormone-immunoreactive neurons, and additional heterolateral neurons are, furthermore, involved in bilateral coupling of the two pacemakers. The neuronal organization, as well as the prominent involvement of GABA and neuropeptides, shows striking similarities to the organization of the suprachiasmatic nucleus, the circadian clock of the mammalian brain.     

Identification of a hydroxy substituted calamenene--a sesquiterpene associated with wound reactions in non-infected xylem of Tilia spp

Xylem of lime trees (Tilia spp.) with wound reactions was structurally investigated by scanning (SEM) and transmission electron microscopy (TEM) as well as chemically analyzed by direct thermal desorption-gas chromatography-mass spectrometry (DTD-GC-MS). Wound reactions in the outer xylem lead to distinct discolorations around the wound. Within a 4-week response no fungal infection occurred in discoloured xylem. At the fine structural level, wound reactions become primarily visible as the secretion of dark-staining substances from parenchyma cells into lumens of vessels and fibres. With increasing reaction time vessels aggregate large amounts of secretion products, whereas in fibres wall-associated linings are formed and the inner secondary wall appears incrusted. After 2-3 months a narrow, greenish-brown boundary developed at the transition between the discoloured outer and the unchanged inner xylem. This green-brown boundary layer remained non-infected also in older wounds. DTD-GC-MS analyses revealed that the sesquiterpene Hydroxycalamenene represents a key substance of wound reactions in non-infected lime trees. Other substances such as fatty acids or their esters and coniferyl aldehydes or their derivatives were also found. TEM investigations of the samples after DTD-GC-MS showed less pronounced cell wall-attached linings in fibres as well as reduced incrustation of inner secondary walls. The massive deposits in the vessel lumens remained unchanged. The role of these wound reaction products and their ways of synthesis are discussed.     

Role of mass spectrometry in the purification of peptides and proteins

Experiments were carried out to evaluate the fractionation of proteins and peptides according to mass. Model mixtures were separated by either reversed-phase or ion-exchange chromatography with mass spectrometry-compatible mobile phase additives. Fraction collection was triggered by the mass/charge ratio of each one of the components of the mixture. Chromatography was additionally monitored with a UV-Vis detector in order to compare the new technique with generally accepted in separations. The results indicated that adequate purification is achieved by this new technique. Fraction collection triggered by changes in the mass/charge ratio reduces sample handling and analysis time. This study demonstrates the utility of mass-directed fractionation of peptides and proteins when mass spectrometry-compatible mobile phase additives are used.     

AtDUR3 encodes a new type of high-affinity urea/H+ symporter in Arabidopsis

Urea is the major nitrogen form supplied as fertilizer in agricultural plant production but also an important nitrogen metabolite in plants. We report the cloning and functional characterization of AtDUR3, a high-affinity urea transporter in plants. AtDUR3 contains 14 putative transmembrane-spanning domains and represents an individual member in Arabidopsis that belongs to a superfamily of sodium-solute symporters. Heterologous expression in urea uptake-defective yeast as well as two-electrode voltage clamp and uptake studies using (14)C-labeled urea in AtDUR3-expressing oocytes demonstrated that AtDUR3 mediates urea transport. In both heterologous systems, urea transport was stimulated at low pH. In oocytes, inward currents indicated that urea is cotransported with protons. By contrast, a supply of Na(+) ions could not stimulate urea transport. Transport of (14)C-labeled urea by AtDUR3 in oocytes exhibited saturation kinetics with a K(m) of approximately 3 micro M. AtDUR3 was expressed in shoots and roots and upregulated during early germination and under nitrogen deficiency in roots. We propose a role of AtDUR3 in urea uptake by plant cells at low external urea concentrations.     

Hybrid-cell vaccines for cancer immune therapy

Hybrid cells generated by fusing allogenic-presenting cells, such as dendritic cells, with tumor cells are a new tool in cancer immunotherapy which are designed to enhance the immunogenicity of antigenic tumors by presenting the whole spectrum of tumor-associated antigens, by providing the co-stimulatory molecules required for T-cell activation, and by the expression of allogenic MHC molecules for recruitment and activation of T-cell help. This approach has been successfully tested in animal models as well as in clinical phaseI/II trials with various tumors. Besides clinical repsonses, induction of tumor-specific cytolytic T-cells were observed. The electrofusion protocol described here has the advantage of high fusion efficiency, high hybrid-cell viability, as well as high reproducibility, and can be used for various tumor cell types after minor adjustments are made to the instrument settings in order to process large numbers of dendritic cells with consistent efficiencies.