The NKG2D receptor and its ligands-recognition beyond the "missing self"?

NKG2D is a surface receptor that activates natural killer (NK) cells and delivers a co-stimulatory signal to CD8-positive T cells. The ligands of NKG2D are induced by cellular stress and are specifically expressed by some tumor cells. This sparked the idea of an alternative regulation of NK cells by expression of "induced self" ligands on target cells which can overcome the inhibition imparted by MHC class I-specific inhibitory receptors.     

Clonality analysis after retroviral-mediated gene transfer to CD34+ cells from the cord blood of ADA-deficient SCID neonates

A clinical trial of retroviral-mediated transfer of the adenosine deaminase (ADA) gene into umbilical cord blood CD34(+) cells was started in 1993. ADA-containing peripheral blood mononuclear cells (PBMCs) have persisted in patients from this trial, with T lymphocytes showing the highest prevalence of gene marking. To gain a greater understanding of the nature and number of the transduced cells that were engrafted, we used linear amplification-mediated PCR (LAM-PCR) to identify clonal vector proviral integrants. In one patient, a single vector integrant was predominant in T lymphocytes at a stable level over most of the eight-year time span analyzed and was also detected in some myeloid samples. T-cell clones with the predominant integrant, isolated after eight years, showed multiple patterns of T-cell receptor (TCR) gene rearrangement, indicating that a single pre-thymic stem or progenitor cell served as the source of the majority of the gene-marked cells over an extended period of time. It is important to distinguish the stable pattern of monoclonal gene marking that we observed here from the progressive increase of a T-cell clone with monoclonal gene marking that results from leukemic transformation, as observed in two subjects in a clinical trial of gene therapy for X-linked severe combined immunodeficiency (SCID).     

Production of supercoiled multimeric plasmid DNA for biopharmaceutical application

Production of nucleic acids as an active pharmaceutical ingredient (API) in gene therapy and genetic vaccination is gaining more and more importance. Non-viral vectors like plasmid DNA are currently investigated in various clinical trials. Supercoiled multimeric plasmids are of particular interest for pharmaceutical purpose because they contain multiple copies of a therapeutic gene and can therefore be more efficient vectors. A process for the preparation of Escherichia coli strains replicating dimers, trimers, and tetramers of a 4.6 kb plasmid is presented. Cultivation of these clones on semi-defined glycerol medium in a 7 l bioreactor shows structural stability of dimers and trimers during the whole cultivation process. Plasmid concentrations and selectivities are compared to the corresponding cultivation with the plasmid monomer. Cultivation of the tetramer replicating strain shows a disintegration of the plasmid multimer and reconstitution of the monomer and smaller multimers.     

Electrostimulation: a future treatment option for patients with neurogenic urodynamic disorders?

This paper provides an overview of electrical stimulation of the nervous system as a treatment option for urodynamic dysfunction and of some of the recent results in this field. The set-up used in our studies for improved bladder filling in spinal cord injured patients by conditional stimulation of the dorsal penile/clitoral nerve is a highly efficient way to limit neurogenic detrusor overactivity and increase bladder capacity. Ongoing studies suggest that recording of bladder nerve activity is stable over time and may be a technique for chronic monitoring of bladder activity. Bladder emptying exploiting an anodal blocking technique permits bladder emptying without simultaneous urethral-perineal contraction, thus enabling a physiological voiding pattern in one continuous sequence. In patients with supraspinal lesions, deep brain electrical stimulation is established only as treatment for a subgroup of patients suffering from Parkinson's disease. Yet, with improved electrode designs and increased clinical experience and experimental results, probably other groups of patients may be candidates for deep brain stimulation. In our study in pigs there was a trend towards increased bladder capacity and compliance in response to stimulation, which is encouraging as several neurological diseases are accompanied by overactive bladder with reduced capacity.     

Taxol impairs anterograde axonal transport of microinjected horseradish peroxidase in dorsal root ganglia neurons in vitro

We have investigated the effects of taxol on the axonal transport of horseradish peroxidase (HRP) in dorsal root ganglia (DRG) cells and their neuronal cytoskeleton. The former were analysed by microinjection of HRP into single DRG cells and the latter was studied by means of immunohistochemistry and cryo-electron microscopy. In cultured and untreated DRG cells, microinjected HRP was typically transported anterogradely several hundred micrometres along their neurites. Different exposure periods (1, 2 and 3 days) to taxol were analysed. The axonal transport of HRP in DRG cells was time-dependently impeded by taxol. After the drug had been washed out, a recovery of the axonal transport of HRP was observed and confirmed by quantitative analysis. Cryo-electron microscopy revealed an abnormal aggregation of axonal and cytoplasmic microtubules, associated with a decreased amount of cross-linking structures, in taxol-treated DRG cell cultures. After 3 days of taxol exposure, microtubule-associated proteins and Tau-protein were restricted to the cellular somata but the neurofilament network and tubulin-proteins seemed to be unaffected. Our results demonstrate, for the first time, an inhibition of anterograde axonal transport of HRP in single neurons by taxol. This effect is reversible and seems not to be caused by cellular damage, but is rather a consequence of an altered organisation of microtubules and/or microtubule-associated proteins.     

Cloning and heterologous expression of a rape cDNA encoding UDP-glucose:sinapate glucosyltransferase

A cDNA encoding a UDP-glucose:sinapate glucosyltransferase (SGT) that catalyzes the formation of 1-O-sinapoylglucose, was isolated from cDNA libraries constructed from immature seeds and young seedlings of rape (Brassica napus L.). The open reading frame encoded a protein of 497 amino acids with a calculated molecular mass of 55,970 Da and an isoelectric point of 6.36. The enzyme, functionally expressed in Escherichia coli, exhibited broad substrate specificity, glucosylating sinapate, cinnamate, ferulate, 4-coumarate and caffeate. Indole-3-acetate, 4-hydroxybenzoate and salicylate were not conjugated. The amino acid sequence of the SGT exhibited a distinct sequence identity to putative indole-3-acetate glucosyltransferases from Arabidopsis thaliana and a limonoid glucosyltransferase from Citrus unshiu, indicating that SGT belongs to a distinct subgroup of glucosyltransferases that catalyze the formation of 1-O-acylglucosides (β-acetal esters). Received: 14 July 2000 / Accepted: 8 August 2000