Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRzeta /CD28 receptor.

Artificial receptors provide a promising approach to target T lymphocytes to tumor antigens. However, the receptors described thus far produce either an activation or a co-stimulatory signal alone, thus limiting the spectrum of functions accomplished by the genetically modified cells. Here we show that human primary T lymphocytes expressing fusion receptors directed to prostate-specific membrane antigen (PSMA) and containing combined T-cell receptor-zeta (TCRzeta), and CD28 signaling elements, effectively lyse tumor cells expressing PSMA. When stimulated by cell-surface PSMA, retrovirally transduced lymphocytes undergo robust proliferation, expanding by more than 2 logs in three weeks, and produce large amounts of interleukin-2 (IL-2). Importantly, the amplified cell populations retain their antigen-specific cytolytic activity. These data demonstrate that fusion receptors containing both TCR and CD28 signaling moieties are potent molecules able to redirect and amplify human T-cell responses. These findings have important implications for adoptive immunotherapy of cancer, especially in the context of tumor cells that fail to express major histocompatibility complex antigens and co-stimulatory molecules.     

Releases of Norepinephrine and Dopamine in Ventriculocisternal Perfusions in Hepatectomized and Laparotomized Rats

Abstract: The completely hepatectomized rat has frequently been used as a model to study changes in the economy of norepinephrine (NE) and dopamine (DA) in hepatic coma. Hypothermia characteristically develops in hepatectomized rats and also occurs in patients in hepatic coma and is associated with improved survival in both. The aims of the present study were to measure both release and uptake of NE and release of DA in brain in warm (37°C) and cool (30–32°C) rats at 3–5 h after laparotomy or hepatectomy. Ventriculocisternal perfusions of the brain were performed on rats under basal conditions and during releases evoked by 40 m M K⁺. Basal releases of NE and DA and evoked release of DA were greater in the warm hepatectomized rats than in all other groups. In some studies, 10⁻⁵ M amitriptyline was added to the perfusates to assess whether neuronal uptake was changed after hepatectomy. Uptake of released NE was equally robust in cool hepatectomized as in cool laparotomized rats but could not be measured in warm hepatectomized rats because of amitriptyline toxicity in these rats. Decreases in NE and increases in DA content were found in most areas of the brain after perfusion. Increased releases of NE and DA may contribute to the pathogenesis of hepatic encephalopathy.     

D2 Dopamine Receptors Stimulate Mitogenesis Through Pertussis Toxin-Sensitive G Proteins and Ras-Involved ERK and SAP/JNK Pathways in Rat C6-D2L Glioma Cells

Abstract: Dopamine D2 receptors are members of the G protein-coupled receptor superfamily and are expressed on both neurons and astrocytes. Using rat C6 glioma cells stably expressing the rat D2L receptor, we show here that dopamine (DA) can activate both the extracellular signal-regulated kinase (ERK) and c-Jun NH₂-terminal kinase (JNK) pathways through a mechanism involving D2 receptor-G protein complexes and the Ras GTP-binding protein. Agonist binding to D2 receptors rapidly activated both kinases within 5 min, reached a maximum between 10 and 15 min, and then gradually decreased by 60 min. Maximal activation of both kinases occurred with 100 nM DA, which produced a ninefold enhancement of ERK activity and a threefold enhancement of JNK activity. DA-induced kinase activation was prevented by either (+)-butaclamol, a selective D2 receptor antagonist, or pertussis toxin, an uncoupler of G proteins from receptors, but not by (?)-butaclamol, the inactive isomer of (+)-butaclamol. Cotransfection of RasN17, a dominant negative Ras mutant, prevented DA-induced activation of both ERK and JNK. PD098059, a specific MEK1 inhibitor, also blocked ERK activation by DA. Transfection of SEK1(K → R) vector, a dominant negative SEK1 mutant, specifically prevented DA-induced JNK activation and subsequent c-Jun phosphorylation without effect on ERK activation. Furthermore, stimulation of D2 receptors promoted [³H]thymidine incorporation with a pattern similar to that for kinase activation. DA mitogenesis was tightly linked to Ras-dependent mitogen-activated protein kinase (MAPK) and JNK pathways. Transfection with RasN17 and application of PD098059 blocked DA-induced DNA synthesis. Transfection with FlagΔ169, a dominant negative c-Jun mutant, also prevented stimulation of [³H]thymidine incorporation by DA. The demonstration of D2 receptor-stimulated MAPK pathways may help to understand dopaminergic physiological functions in the CNS.     

Über Schollen- und Kristallaggregatbildung im extraplasmatischen Raum vitalgefärbter Zellen

Ohne Zusammenfassung     

Tree growth and competition in a post-logging Quercus mongolica forest on Mt. Sobaek, South Korea

Secondary woodlands in South Korea cover most mountains from low to middle elevations. While general patterns of forest succession are well understood, little is known about mechanisms of stand recovery after disturbance. We examined the spatio-temporal variations in establishment, growth, size inequality, and mode of competition among trees in a 50-year-old post-logging Quercus mongolica-dominated stand. We further compared the growth and stem allometry of single trees, presumably of seed origin, with multi-stemmed trees resprouting from stumps. Q. mongolica formed the upper canopy 16–22 m tall, 88.3% of total stand basal area, and 36.2% of total stem density, with most trees established during the first post-logging decade (51.2% were resprouts). During the first three decades, the Q. mongolica recruits grew exponentially, and disproportionately more in diameter than few older reserved trees left after the last cutting. This substantially decreased size inequality. The reverse trend was observed from 1994 to 2004: larger trees grow more, indicating an increasing asymmetry of competition for light. Neighborhood analysis revealed that when target trees had more or larger neighbors, their exponential phase of growth was reduced and maximum size was decreased. After the 50 years of stand development, more than 70% of Q. mongolica showed growth decline as a result of competitive stress, and mortality was about 30%, concentrated in smaller size classes. Compared to single stems, resprouts within clones do not seem to compete less asymmetric as might be expected based on studies of clonal herbaceous plants and physiological integration within genets. As Q. mongolica was also negatively affected by competition from woody species currently prevailing in the lower tree stratum (Tilia amurensis, Acer mono, Fraxinus rhynchophylla, Acer pseudosieboldianum), we predict the stand will become increasingly dominated by these more shade-tolerant trees.     

Removal of pattern-breaking sequences in microtubule binding repeats produces instantaneous tau aggregation and toxicity

Aggregated and highly phosphorylated tau protein is a pathological hallmark of Alzheimer's disease (AD) and other tauopathies. We identified motifs of alternating polar and apolar amino acids within the microtubule-binding repeats of tau which were interrupted by small breaking stretches. Minimal mutation of these breaking sequences yielded a unique instantly aggregating tau mutant containing longer stretches of polar/apolar amino acids without losing its microtubule-binding capacity. These modifications produced rapid aggregation and cytotoxicity with accompanying occurrence of pathologic tau phosphoepitopes (AT8, AT180, AT270, AT100, Ser(422), and PHF-1) and conformational epitopes (MC-1 and Alz50) in cells. Similar to pathological tau in the pretangle state, toxicity appeared to occur early without the requirement for extensive fibril formation. Thus, our mutant protein provides a novel platform for the investigation of the molecular mechanisms for toxicity and cellular behavior of pathologically aggregated tau proteins and the identification of its interaction partners.