The 49 K subunit of NADH: ubiquinone reductase (complex I) from Neurospora crassa mitochondria: primary structure of the gene and the protein

Summary The primary structure of the 49 K subunit of the respiratory chain NADH:ubiquinone reductase (complex I) from Neurospora crassa was determined by sequencing cDNA, genomic DNA and the N-terminus of the mature protein. The sequence lengths correlate to a molecular mass of 54002 daltons for the preprotein and 49239 daltons for the mature protein. The presequence consists of 42 amino acids of typical composition for sequences which target nuclear-encoded proteins into mitochondria. The mature protein consists of 436 amino acids and shows 64% similarity to a 49 K subunit of bovine heart NADH:ubiquinone reductase and 33% to a predicted translation product of an open reading frame in the chloroplast DNAs of Marchantia polymorpha and Nicotiana tabacum. Evidence for an iron-sulfur cluster in the subunit is discussed.     

Anti-high endothelial venule monoclonal antibody HECA-452 recognizes plasmacytoid T cells and delineates an ”extranodular“ compartment in the reactive lymph node

So-called plasmacytoid T cells represent a subset of monocyte related cells, which share with endothelium the CD36⁺CD11b⁻ (OKM5⁺OKM1⁻) phenotype. The reactivity of plasmacytoid T cells with rat monoclonal antibody HECA-452, highly specific for high endothelial venules, was analyzed in reactive lymph nodes. In all cases, HECA-452 not only labelled the endothelium of high endothelial venules, but also strongly reacted with singular and clustered plasmacytoid T cells. The HECA-452 positivity for high endothelial venules and plasmacytoid T cells visualized a lymph node compartment extending from the subcapsular sinus to the corticomedullary junction. This compartment surrounded the composite nodule and was designated the ”extranodular“ compartment. The cooccurrence of plasmacytoid T cells and high endothelial venules in this extranodular compartment, together with their immunophenotypical similarities, may be indicative of functional co-operations.     

Adenoviral vectors expressing siRNAs for discovery and validation of gene function

RNA interference is a powerful tool for studying gene function and for drug target discovery in diverse organisms and cell types. In mammalian systems, small interfering RNAs (siRNAs), or DNA plasmids expressing these siRNAs, have been used to down-modulate gene expression. However, inefficient transfection protocols, in particular, for primary cell types, have hampered the use of these tools in disease-relevant cellular assays. To be able to use this technology for genome-wide function screening, a more robust transduction protocol, resulting in a longer duration of the knock-down effect, is required. Here, we describe the validation of adenoviral vectors that express hairpin RNAs that are further processed to siRNAs. Infection of cell lines, or primary human cells, with these viruses leads to an efficient, sequence-specific, and prolonged reduction of the corresponding target mRNA, resulting in a reduction of the encoded protein level in the cell. For knock-down of one of the targets, GalphaS, we have measured inhibition of ligand-dependent, G-protein-coupled signaling. It is expected that this technology will prove to be of great value in target validation and target discovery efforts.     

Anatomical evidence for direct connections between the shell and core subregions of the rat nucleus accumbens

The nucleus accumbens is thought to subserve different aspects of adaptive and emotional behaviors. The anatomical substrates for such actions are multiple, parallel ventral striatopallidal output circuits originating in the nucleus accumbens shell and core subregions. Several indirect ways of interaction between the two subregions and their associated circuitry have been proposed, in particular through striato-pallido-thalamic and dopaminergic pathways. In this study, using anterograde neuroanatomical tracing with Phaseolus vulgaris-leucoagglutinin and biotinylated dextran amine as well as single-cell juxtacellular filling with neurobiotin, we investigated the intra-accumbens distribution of local axon collaterals for the identification of possible direct connections between the shell and core subregions. Our results show widespread intra-accumbens projection patterns, including reciprocal projections between specific parts of the shell and core. However, fibers originating in the core reach more distant areas of the shell, including the rostral pole (i.e. the calbindin-poor part of the shell anterior to the core) and striatal parts of the olfactory tubercle, than those arising in the shell and projecting to the core. The latter projections are more restricted to the border region between the shell and core. The density of the fiber labeling within both the shell and core was very similar. Moreover, specific intrinsic projections within shell and core were identified, including a relatively strong projection from the rostral pole to the rostral shell, reciprocal projections between the rostral and caudal shell, as well as projections within the core that have a caudal-to-rostral predominance. The results of the juxtacellular filling experiments show that medium-sized spiny projection neurons and medium-sized aspiny neurons (most likely fast-spiking) contribute to these intra-accumbens projections. While such neurons are GABAergic, the intrastriatal projection patterns indicate the existence of lateral inhibitory interactions within, as well as between, shell and core subregions of the nucleus accumbens.     

Premature senescence involving p53 and p16 is activated in response to constitutive MEK/MAPK mitogenic signaling

Oncogenic Ras transforms immortal rodent cells to a tumorigenic state, in part, by constitutively transmitting mitogenic signals through the mitogen-activated protein kinase (MAPK) cascade. In primary cells, Ras is initially mitogenic but eventually induces premature senescence involving the p53 and p16^INK4a tumor suppressors. Constitutive activation of MEK (a component of the MAPK cascade) induces both p53 and p16, and is required for Ras-induced senescence of normal human fibroblasts. Furthermore, activated MEK permanently arrests primary murine fibroblasts but forces uncontrolled mitogenesis and transformation in cells lacking either p53 or INK4a. The precisely opposite response of normal and immortalized cells to constitutive activation of the MAPK cascade implies that premature senescence acts as a fail-safe mechanism to limit the transforming potential of excessive Ras mitogenic signaling. Consequently, constitutive MAPK signaling activates p53 and p16 as tumor suppressors.     

Anaphylactoid shock – a common cause of death in heroin addicts?

We measured mast-cell tryptase in postmortem blood from 22 heroin addicts dying suddenly after injection. In 32%, the concentration of tryptase was elevated (≥10 μg/1), and the mean value of tryptase was significantly different from a control group dying from known, nonimmunologic causes ( P <0.05). The increased tryptase concentrations indicate that death was preceded by systemic mast-cell degranulation. All victims of drug deaths had morphine in blood, most below 0.2 μg/ml. In 71% of the victims of drug-related deaths with tryptase values ≥10 μg/1, the intermediate degradation product, 6–monoacetyl-morphine, was not found in blood, whereas this was the case in only two victims with values below that cutoff point. This indicates that those with high tryptase concentrations survived longer than those with lower values. No correlation was found between the IgE levels and tryptase in either group, supporting the hypothesis that tryptase release was not mediated by an allergic reaction. The well-known property of opiates to stimulate unspecifically the liberation of histamine and other constituents of mast-cell granules offers one explanation of our observations. The results suggest that many heroin fatalities are caused by an anaphylactoid reaction.     

High-performance liquid chromatography of the neuroactive steroids alphaxalone and pregnanolone in plasma using dansyl hydrazine as fluorescent label: application to a pharmacokinetic-pharmacodynamic study in rats

This report describes a rapid and sensitive analytical method for the quantification of the neuroactive steroids alphaxalone and pregnanolone in rat plasma using derivatization with dansyl hydrazine as fluorescent label. The method involves protein precipitation, alkaline derivatization and extraction of the compounds and internal standard pregnenolone with dichloromethane, followed by isocratic reversed-phase high-performance liquid chromatography on a 3-microm Microsphere C18 column with fluorescence detection at wavelengths 332 nm and 516 nm for excitation and emission, respectively. The mobile phase consists of a mixture of 25 mM acetate buffer (pH 3.7)-acetonitrile (45:55, v/v for alphaxalone and 40:60, v/v for pregnanolone) with a flow-rate of 1 ml/min. The total run time was approximately 35 min. In the concentration range of 0.010-10 microg ml(-1), the intra- and inter-assay coefficients of variation were less than 17% for both methods. In 50 microl plasma samples the corresponding limits of detection were 10 ng ml(-1) (signal-to-noise ratio=3). The utility of the analytical method was established by analyzing plasma samples from rats, which had received an intravenous administration of 5 mg kg(-1) alphaxalone or pregnanolone. Values for clearance, volume of distribution at steady state and terminal half life were 71.9 ml min(-1) kg(-1), 814 mg kg(-1) and 13.5 min for alphaxalone and 69.2 ml min(-1) kg(-1), 1,638 ml kg(-1) and 27.8 min for pregnanolone, respectively. Due to its simplicity and sensitivity this method can be used on a routine basis for pharmacokinetic analysis of neuroactive steroids.     

Model systems to study the parameters determining the success of phage antibody selections on complex antigens

Phage antibody display technology offers a powerful tool for the isolation of specific antibodies to defined target antigens. Most selection strategies described to date have relied on the availability of purified and often recombinant antigen, providing the possibility to perform selections on a well-defined antigen source. However, when the target antigen cannot be purified (e.g., an integral membrane protein), or if the antigen is unknown (e.g., when searching for novel markers on cells or tissues), panning of phage antibody libraries has to be performed on complex antigen sources such as cell surfaces or tissue sections, or even by in vivo selection methods. This provides a series of technical and experimental challenges. One focus of our research is to select antibodies directed to novel cancer-induced antigens expressed by tumours and by the tumour vasculature. To understand the parameters governing selection on complex antigen sources and to assess the efficiency of these phage library selections, we have set up two model selection systems in which both tumour cells and vascular endothelial cells serve as target "antigen". We describe a model based on phage antibodies directed to the tumour antigen epithelial glycoprotein-2, to compare phage antibody selections on a range of different antigen sources including purified and recombinant antigen, whole live cells, tissue cryosections and in vivo grown solid tumours. Secondly, we describe a model based on a phage antibody directed against the endothelial cell inducible adhesion molecule E-selectin. We compare selections on cultured cell monolayers with selections on cell suspensions immobilised on columns, to determine which selection approach is most suitable for the identification of novel tumour endothelial cell markers. Our data provide insight into the efficiency and thus potency of different selection strategies and show that there are very large differences in the recovery and enrichment of binding phage between the different methods tested. Our results further demonstrate the feasibility of phage antibody selections on whole, intact cells and show that these may sometimes compare favourably to selections on purified antigen. Selections on endothelial cells immobilised on columns compare favourably with selections on cell-monolayers; the most favourable conditions for both selection procedures are described. The implications of our data for phage antibody selections on these different complex antigen sources using either non-immune or immune phage antibody repertoires are discussed. The use of model systems such as the ones described here will help to determine optimal experimental conditions for phage library selections on complex antigens and aid in developing more powerful selection procedures for target discovery.     

The New Millennium Palliative Care Project (2000-2003): the impact of specialised GP advisors.

This study describes a novel type of support for GPs caring for patients dying at home: the establishment and evaluation of a telephone advisory service for GPs, run by GPs with a special interest in palliative care (GPwSIs) in the Netherlands 2000-2003. A growing number of GPs called for advice, 10% during out of hours. Prognosis of the patients was generally short (days to weeks in 70% of cases). Most advice sought by GPs concerned symptom management and on evaluation, 85% of the GPs followed the advice.