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Drug induced phospholipidosis (PLD) may be observed in the preclinical phase of drug development and pose strategic questions. As lysosomes have a central role in pathogenesis of PLD, assessment of lysosomal concentrations is important for understanding the pharmacokinetic basis of PLD manifestation and forecast of potential clinical appearance. Herein we present a systematic approach to provide insight into tissue-specific PLD by evaluation of unbound intracellular and lysosomal (reflecting acidic organelles) concentrations of two structurally related diprotic amines, GRT1 and GRT2. Their intratissue distribution was assessed using brain and lung slice assays. GRT1 induced PLD both in vitroand in vivo. GRT1 showed a high intracellular accumulation that was more pronounced in the lung, but did not cause cerebral PLD due to its effective efflux at the blood–brain barrier. Compared to GRT1, GRT2 revealed higher interstitial fluid concentrations in lung and brain, but more than 30-fold lower lysosomal trapping capacity. No signs of PLD were seen with GRT2. The different profile of GRT2 relative to GRT1 is due to a structural change resulting in a reduced basicity of one amino group. Hence, by distinct chemical modifications, undesired lysosomal trapping can be separated from desired drug delivery into different organs. In summary, assessment of intracellular unbound concentrations was instrumental in delineating the intercompound and intertissue differences in PLD induction in vivoand could be applied for identification of potential lysosomotropic compounds in drug development. more...

Truncated trkB.T1 (T1) neurotrophin receptor inhibits full-length trkB.TK+ (TK+) signaling. At least two possible mechanisms have been proposed for this action: T1 could trap the ligand or function as a dominant negative receptor. To differentiate between these possibilities we have studied survival of serum-deprived PC12-trkB cells stably expressing TK+. PC12-trkB cells were observed to display constitutive trkB kinase activity which leads to survival of a cell subpopulation in the absence of added brain-derived neurotrophic factor (BDNF) and serum. Exogenous BDNF significantly increased cell survival, and this increase was inhibited by BDNF neutralizing antibody. The antibody treatment had no effect on the constitutive TK+ activity. Transfected T1 completely inhibited survival by BDNF or constitutive trkB kinase activity in PC12-trkB cells similarly to tyrosine kinase inhibitor K252a. In addition, T1 coimmunoprecipitated with TK+ and inhibited its autophosphorylation by BDNF. These data suggest that truncated T1 inhibits TK+ signaling by dominant negative action. more...

This study examines the mechanisms by which the tyrosine kinase receptor TrkB is down-regulated following binding of brain-derived neurotrophic factor (BDNF). In primary cultures of cerebellar granule neurons, BDNF-induced reduction of TrkB receptors was largely prevented by the addition of specific proteasome inhibitors. HN10 cells, a neuronal cell line that can be readily transfected, also showed a marked down-regulation of cell surface TrkB following BDNF exposure. In addition, we observed that prolonged exposure to nerve growth factor of TrkA-transfected cells did not lead to the down-regulation seen with BDNF and TrkB. TrkA and TrkB chimeric molecules were therefore expressed in HN10 cells and tested for ligand-induced regulation. These experiments led to the conclusion that the motives responsible for down-regulation are contained in the cytoplasmic domain of TrkB, and a short sequence in the juxtamembrane domain of TrkB was identified that confers nerve growth factor-induced down-regulation when inserted into TrkA. more...

Brain-derived neurotrophic factor (BDNF) has potent effects on neuronal survival and plasticity during development and after injury. In the nervous system, neurons are considered the major cellular source of BDNF. We demonstrate here that in addition, activated human T cells, B cells, and monocytes secrete bioactive BDNF in vitro. Notably, in T helper (Th)1- and Th2-type CD4+ T cell lines specific for myelin autoantigens such as myelin basic protein or myelin oligodendrocyte glycoprotein, BDNF production is increased upon antigen stimulation. The BDNF secreted by immune cells is bioactive, as it supports neuronal survival in vitro. Using anti-BDNF monoclonal antibody and polyclonal antiserum, BDNF immunoreactivity is demonstrable in inflammatory infiltrates in the brain of patients with acute disseminated encephalitis and multiple sclerosis. The results raise the possibility that in the nervous system, inflammatory infiltrates have a neuroprotective effect, which may limit the success of nonselective immunotherapies. more...

Studies on the pH dependence of ligand binding to A1 adenosine receptors revealed that protonation of a histidine residue in the binding pocket is accompanied by high affinity agonist binding.

RNA-protein cross-links were introduced into E. coli 50S ribosomal subunits by treatment with 2-iminothiolane followed by mild ultraviolet irradiation. After partial digestion of the RNA, the cross-linked RNA-protein complexes were separated by our recently published three-step procedure. In cases where this separation was inadequate, a further purification step was introduced, involving affinity chromatography with antibodies to the ribosomal 50S proteins. Analysis of the isolated complexes enabled four new cross-link sites on the 23S RNA to be identified, as well as re-confirming several previously established sites. The new sites are as follows: Protein L2 is cross-linked within an oligonucleotide at positions 1818–1823 in the 23S RNA, protein L4 within positions 320–325, protein L24 within positions 99–107, and protein L27 within positions 2320–2323. more...