NOMA-GAP/ARHGAP33 regulates synapse development and autistic-like behavior in the mouse.

Neuropsychiatric developmental disorders, such as autism spectrum disorders (ASDs) and schizophrenia, are typically characterized by alterations in social behavior and have been linked to aberrant dendritic spine and synapse development. Here we show, using genetically engineered mice, that the Cdc42 GTPase-activating multiadaptor protein, NOMA-GAP, regulates autism-like social behavior in the mouse, as well as dendritic spine and synapse development. Surprisingly, we were unable to restore spine morphology or autism-associated social behavior in NOMA-GAP-deficient animals by Cre-mediated deletion of Cdc42 alone. Spine morphology can be restored in vivo by re-expression of wild-type NOMA-GAP or a mutant of NOMA-GAP that lacks the RhoGAP domain, suggesting that other signaling functions are involved. Indeed, we show that NOMA-GAP directly interacts with several MAGUK (membrane-associated guanylate kinase) proteins, and that this modulates NOMA-GAP activity toward Cdc42. Moreover, we demonstrate that NOMA-GAP is a major regulator of PSD-95 in the neocortex. Loss of NOMA-GAP leads to strong upregulation of serine 295 phosphorylation of PSD-95 and moreover to its subcellular mislocalization. This is associated with marked loss of surface α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor and defective synaptic transmission, thereby providing a molecular basis for autism-like social behavior in the absence of NOMA-GAP.

Sorafenib and dacarbazine as first-line therapy for advanced melanoma: phase I and open-label phase II studies.

METHOD: The safety of oral sorafenib up to a maximum protocol-specified dose combined with dacarbazine in patients with metastatic, histologically confirmed melanoma was investigated in a phase I dose-escalation study and the activity of the combination was explored in an open-label phase II study. RESULTS: In the phase I study, three patients were treated with sorafenib 200 mg twice daily (b.i.d.) plus 1000 mg m(-2) dacarbazine on day 1 of a 21-day cycle and 15 patients had the sorafenib dose escalated to 400 mg b.i.d. without reaching the maximum tolerated dose of the combination. In the phase II study (n=83), the overall response rate was 12% (95% CI: 6, 21): one complete and nine partial, with median response duration of 46.7 weeks. Stable disease was the best response in 37%; median duration was 13.3 weeks. Median overall survival (OS) was 37.0 weeks (95% CI: 33.9, 46.0). CONCLUSION: Oral sorafenib combined with dacarbazine had acceptable toxicity and some antineoplastic activity against metastatic melanoma.

Final results of the European Advanced Renal Cell Carcinoma Sorafenib (EU-ARCCS) expanded-access study: a large open-label study in diverse community settings.

BACKGROUND: The European Advanced Renal Cell Carcinoma Sorafenib (EU-ARCCS) expanded-access study provided sorafenib to advanced renal cell carcinoma (RCC) patients in whom previous systemic therapy had failed. The study assessed the safety and use of sorafenib for the treatment of advanced RCC in a large community-based patient population across 11 countries in Europe. PATIENTS AND METHODS: EU-ARCCS was a single-arm, open-label trial of sorafenib in advanced RCC patients. End points included safety, time to progression, progression-free survival (PFS), and disease control rate (DCR). Subgroup analyses included age, Eastern Cooperative Oncology Group performance status, histology, prior therapy, and number and sites of metastases. RESULTS: About 1159 advanced RCC patients were enrolled. Most patients (94%) experienced drug-related adverse events (AEs) of any grade, with the most common grade ≥3 AEs including hand-foot skin reaction (13%), diarrhea (7%), fatigue (7%), hypertension (6%), and rash/desquamation (5%). The incidence of AEs in the subgroups was similar to that in the overall population. Median PFS was 6.6 months; DCR at ≥8 and ≥12 weeks was 85% and 78%, respectively. CONCLUSIONS: The sorafenib safety profile in European community-based practice settings was similar to that reported in clinical trials. The heterogeneous advanced RCC patient population in EU-ARCCS permitted assessment of sorafenib in important subpopulations of advanced RCC patients.

Thermodynamics of hydrophobic polyacids.

The thermodynamics of deprotonating hydrolyzed 1-1 copolymers of maleic anhydride with pentyl, hexyl and octyl vinyl ether was investigated by calorimetric and potentiometric methods. These polyacids undergo a transition from a compact to a random coil conformation upon ionization in aqueous media. The results were compared with those obtained previously for similar copolymers with smaller alkyl side-chains. The contributions of the enthalpy and entropy to the free energy were analyzed. The major effects appeared to be related to the charging of the compact form of the polyacids, the electrostriction of water by the completely ionized dicarboxylate groups and the reorganization of water around newly exposed alkyl side-chains arising from the conformational transition.

Simultaneous direct high-performance liquid chromatographic enantioseparation of 2-methylglycidol-1-benzyl ether and 2-methylglycerol-1-benzyl ether using a solvent-switching technique.

Simultaneous HPLC separation of the enantiomers of 3-benzyloxy-2-methyl-1,2-propanediol and the corresponding 3-benzyloxy-2-methyl-1,2-propene oxide could be accomplished on amylose derived Chiralpak AD switching between 10% 2-propanol and 3% 1,2-dimethoxyethane as polar modifier in n-heptane.

Suitability of different cytotoxicity assays for screening combination effects of environmental chemicals in human fibroblasts.

Cytotoxicity screening assays measuring survival, growth, colony forming ability, DNA and protein synthesis in human fibroblasts were tested for their suitability to determine combination effects. Thereby, the dose-response curves of a hydrophilic substance A alone and after pretreatment with a membrane damaging substance B were compared. Substances B were applied at concentrations which did not induce toxic effects in the assays (noec). Synergistic combination effects were demonstrated by reduction of the EC20 value of substances A in the combination in comparison to substances A alone. The following substance pairs (substance B/substance A = membrane damaging/hydrophilic) were tested: n-dodecylbenzenesulfonic acid/2,4-dichlorophenoxyacetic acid, 2,4,6-trichlorophenol/2,4-dichlorophenoxyacetic acid, 1,1,2,2-tetrachloroethane/4-chloroaniline, pentachlorophenol/CrCl3. While survival, growth, and DNA synthesis assays were suitable methods for detecting synergistic combination effects, the growth assay was the most sensitive. Here, all four substance pairs showed synergistic combination effects.

Synergistic DNA damage by oxidative stress (induced by H2O2) and nongenotoxic environmental chemicals in human fibroblasts.

Genotoxic combination effects of oxidative stress (induced by H2O2) and eight nongenotoxic environmental chemicals (4-chloroaniline, 2,3,4,6-tetrachlorophenol, lindane, 2,4-dichloroacetic acid (2,4-D), m-xylene, glyphosate, nitrilotriacetic acid and n-hexanol) were determined in human fibroblasts. Genotoxicity was measured quantitatively by the single cell gel electrophoresis assay. The nongenotoxic chemicals were used in non cytotoxic concentrations. H2O2 was used in concentrations producing low (50 microM) and no cytotoxicity (40 microM). All environmental chemicals acted in a synergistic way with H2O2 except DMSO which effectively inhibited H2O(2)-induced DNA damage. The most effective enhancers were 4-chloroaniline, 2,3,4,6-tetrachlorophenol, m-xylene, and n-hexanol. Synergistic effects of hexanol/H2O2 were still evident at a concentration of 0.09 noec (no observed effect concentration). In contrast to synergistic DNA damage in the cell antagonism was found measuring DNA breakage in isolated PM2 DNA. From the results we concluded that synergisms between H2O2 and nongenotoxic chemicals may be a general phenomenon which is not observed on the level of isolated DNA.

Synergistic DNA damaging effects of 4-nitroquinoline-1-oxide and non-effective concentrations of methyl methanesulfonate in human fibroblasts.

DNA damage and DNA repair in human fibroblasts induced by the combination mixture of the genotoxic agents methyl methanesulfonate (MMS) and 4-nitroquinoline-1-oxide (4-NQO) were studied using the comet assay and the unscheduled DNA synthesis (UDS), respectively. Cells were simultaneously treated for 1h with the no observed effect concentration (noec) of MMS and increasing concentrations of 4-NQO or vice versa. Different results were obtained with the two types of mixtures. When the noec of 4-NQO was combined with increasing concentrations of MMS, no combination effects were observed. However, in experiments with increasing concentrations of 4-NQO and the noec of MMS, an increase in DNA damage and repair (and an enhancement of cytotoxicity) was demonstrated. Quantitative analysis of the effects by the isobologram method confirmed synergistic responses in both tests. We are proposing interactive actions between 4-NQO and MMS, whereby 4-NQO facilitates the attack of MMS on the DNA bases.

Volume effects of starch-water interactions.

Volume changes arising from the sorption of water by three starches have been determined by dilatometry. By a recently described method, parameters of the G.A.B. equation have been used to resolve the volume changes into contributions from strongly sorbed water molecules in contact with the surface and from weakly sorbed water molecules in the remaining water layers. The latter produced only small volume effects. In contrast, the contact sorption caused very large volume decreases. Changes in these decreases with changing water content indicated two types of sorption sites, possibly crystalline and amorphous. In the case of waxy maize starch, for which literature data concerning its degree of crystallinity were available, the contact sorption could be quantitatively resolved into contributions from the presumably crystalline and amorphous portions, with the former exhibiting both higher affinity and larger volume changes for water molecules than the latter.

Correlation of synergistic cytotoxic effects of environmental chemicals in human fibroblasts with their lipophilicity.

The cytotoxic combination effects of 2,4-D with 12 xenobiotics having different lipophilicity were investigated in human fibroblasts at their no effect concentrations (NOEC). Each of the chemicals tested in binary combinations enhanced the toxicity of 2,4-D. These synergistic combination effects were independent of the chemical structure of the test compounds. However, the NOEC's of the xenobiotics used in the combinations varied by a factor of 10,000. For strongly lipophilic compounds the lowest NOEC's were needed to induce synergistic cytotoxicity. A linear regression analysis of the concentrations (NOEC's) of the 12 combined xenobiotics against their lipophilicity revealed a correlation with r = 0.96 for 11 agents. This close correlation may be explained by the membrane damaging properties of lipophilic compounds which enhance the uptake of hydrophilic agents.

K+ currents of encephalitogenic memory T cells decrease with encephalitogenicity while interleukin-2 (IL-2) receptor expression remains stable during IL-2 dependent cell expansion.

Previous studies revealed a linkage between increased K+ current and lymphocyte activation upon non-specific stimulation with mitogenic lectins and antibodies. So far no information is available about the behaviour of K+ currents in specifically autoantigen-stimulated lymphocytes. Therefore, we have investigated K+ currents in encephalitogenic T line cells, specifically stimulated with myelin basic protein, using the whole-cell patch-clamp technique. In parallel, the T cell activation marker interleukin-2 (IL-2) receptor was measured quantitatively by flow cytometry. Outward currents were observed in response to depolarizing voltage steps from a holding potential of -80mV. The peak current density increased with more positive membrane potentials, where the current threshold was about -40mV and the maximum conductance was 1.22nS/pF. This current was characterized by a fast activation and a fast inactivation with half maximal inactivation at -67mV. The sensitivity of the peak current to K+ channel blocking agents was as follows: 4-aminopyridine (4-AP) had a half blocking concentration of 0.4mM and a maximal block of 83.7% at 10mM 4-AP, tetraethyl-ammonium caused a block of 6% at 0.1mM, 15% at 1mM and 40% at 10mM, charybdotoxin blocked 90% at 100nM, whereas iberiotoxin had no effect (all values at a clamped membrane potential of +30mV). The encephalitogenic T cells used in our study reach their highest encephalitogenic potency on day 3 to 4 after the onset of restimulation. Furthermore, K+ currents were measured during the whole course of an in vitro restimulation cycle. The peak currents normalized to cell capacitance reached their maximum on day 2 (326+/- 52.8pA/pF, n = 4) and decreased thereafter as follows: day 3: 139.7 +/- 7.87pA/pF (n = 27), day 4: 85.4 +/- 8.95pA/pF (n = 28) and day 5: 40.9 +/- 7.45pA/pF (n = 17). The activation and inactivation characteristics of the current and its responses to selective blockers were similar at all days after restimulation. In contrast to the K+ current, IL-2 receptor expression was maintained on > 95% of cells until day 6 after restimulation. In conclusion, the K+ currents measured in rat encephalitogenic T cells resemble n-type voltage-gated K+ currents described in mice and man. The comparison of K+ current, IL-2 receptor expression and encephalitogenic potency let us suppose that the observed K+ current represents an early event of specific T cell activation and can serve as a parameter of high functional activity of T cells corresponding to their encephalitogenicity.

Whole-cell patch-clamp: true perforated or spontaneous conventional recordings?

Perforated whole-cell patch-clamp recordings obtained with nystatin are frequently used to preserve intracellular integrity. However, the perforated-patch configuration may sometimes undergo a spontaneous change into the conventional whole-cell configuration, especially when lymphocytes are investigated. The electrophysiological criteria-- previously described--for establishing the existence of the perforated whole-cell configuration have been shown to be insufficient. Thus, the dye eosin, applied to the pipette solution, was tested as a tool for discriminating between the perforated and the conventional whole-cell configurations on rat T-lymphocytes. The dye never entered the cell from the pipette during the entire measurement in the perforated whole-cell configuration. In contrast, all cells in the conventional whole-cell configuration became red immediately after membrane rupture. Eosin barely changed the currents studied. The results suggest that eosin is a dye of choice for verifying a true perforated-patch configuration.

Cicutoxin from Cicuta virosa--a new and potent potassium channel blocker in T lymphocytes.

The effect of cicutoxin, the poisonous principle of the genus Cicuta, on K+ currents of activated T lymphocytes was investigated using the patch clamp technique. Cicutoxin produced a dose-dependent [5 x 10(-6) to 7 x 10(-5) mol/l] and completely reversible block of K+ currents with an EC50 of 1.8 x 10(-5) mol/l. A maximum block of 71% was achieved with cicutoxin at a concentration of 7 x 10(-5) mol/l. Since previous studies have shown that T lymphocyte proliferation is associated with K+ currents, the effect of cicutoxin on T lymphocyte proliferation was studied by means of 3H-thymidine uptake assays. At noncytotoxic concentrations [10(-7) to 5 x 10(-5) mol/l] cicutoxin reduced the 3H-thymidine incorporation dose-dependently. In conclusion, cicutoxin is a potent K+ current blocker which inhibits K+ channel-dependent proliferation of naive and memory T lymphocytes.

K(+) channel-blocking alkoxypsoralens inhibit the immune response of encephalitogenic T line cells and lymphocytes from Lewis rats challenged for experimental autoimmune encephalomyelitis.

Alkoxypsoralens, known as DNA photomodifying agents, have been shown to block voltage-dependent K(+) channels (Kv) as well as to alleviate functional deficits in certain multiple sclerosis (MS) patients in a manner similar to 4-aminopyridine. Since Kv channel blockers are known to inhibit T cell-mediated immune responses both in vitro and in vivo, we investigated the effects of three alkoxypsoralens, 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP), and 5,8-diethoxypsoralen (H37), on the following parameters: (1) whole-cell K(+) currents of encephalitogenic, myelin basic protein-specific memory T cell line cells (MBP-TCLC) derived from Lewis rats as measured by patch-clamp technique, (2) proliferation of MBP-TCLC and lymph node cells (LNC) from Lewis rats challenged for experimental autoimmune encephalomyelitis (EAE) by immunisation with spinal cord homogenate as measured by 3H-thymidine incorporation, (3) interferon-gamma (IFN-gamma) secretion of MBP-TCLC as measured by ELISA, and (4) IFN-gamma gene expression of LNC as measured by quantitative reverse transcription polymerase chain reaction (RT-PCR) with ELISA-detection. The examined alkoxypsoralens exhibited suppressive effects on the measured parameters with the same sequence of efficacy: H37>5-MOP>8-MOP. We, therefore, conclude that Kv channel-blocking alkoxypsoralens interfere with voltage-controlled signal transduction in lymphocytes and might thereby suppress immune responses in autoimmune diseases of the central nervous system and most likely also in other autoimmune disorders. Thus, alkoxypsoralens, especially the non-phototoxic substance H37, are new candidates for further studies on K(+) channel blocking immunosuppressive drugs. The agents may exert a dual beneficial effect on demyelinating diseases like MS, because they could attenuate the inflammatory process and improve axonal conductivity.

All-optical spatial light modulator with megahertz modulation rates.

We demonstrate a novel all-optical spatial light modulator capable of megahertz modulation rates. It is based on the quantum-confined Stark effect, but the modulating electric field is entirely photogenerated by strongly asymmetric photocarrier transfer in GaAs/AlAs layers. In a nonoptimized sample, cw optical excitation of approximately 50 W/cm(2) created a 30-kV/cm electric field, inducing a 9-meV exciton red shift at room temperature. Under pulsed excitation the photogenerated electric field can be switched on in a few tens of picoseconds and relaxes in a few hundred nanoseconds, permitting megahertz modulation rates.