Characterization of Arsenate Uptake by Cultured Primary Rat Astrocytes

Arsenate is known to be accumulated by cultured astrocytes and to stimulate astrocytic glutathione export, but the arsenate uptake into astrocytes has not been characterized so far. To address this topic, we have exposed primary rat astrocyte cultures to arsenate and determined the cellular arsenic content by atomic absorption spectroscopy. Viable astrocytes accumulated arsenate in a time- and concentration-dependent manner. Their cellular arsenic content increased almost proportional with time for up to 60 min after application of arsenate. Analysis of the concentration-dependent increase in the specific arsenic content of the cells after 30 min of arsenate exposure revealed that cultured astrocytes take up arsenate with saturable kinetics by a transport process that has apparent K_M- and V_max-values of 1.7 ± 0.2 mM and 28 ± 4 nmol/(mg protein × 30 min), respectively. Arsenate uptake in viable astrocytes was strongly inhibited by the presence of phosphate or by lowering the incubation temperature to 4 °C and was completely abolished in a sodium ion-free medium. These results strongly suggest that the saturable temperature-dependent arsenate uptake into astrocytes is mediated by a sodium-dependent phosphate cotransporter.     

Emotion recognition in children and adolescents with attention-deficit/hyperactivity disorder (ADHD)

Children with attention-deficit/hyperactivity disorder (ADHD) are impaired in social adaptation and display deficits in social competence. Deficient emotion recognition has been discussed to underlie these social problems. However, comorbid conduct problems have not been considered in the majority of studies conducted so far, and the influence of medication on emotion recognition has rarely been studied. Here, emotion recognition performance was assessed in children with ADHD without medication compared with children with ADHD under stimulant medication and a matched control group. In order to rule out confounding by externalizing symptoms, children with comorbid conduct problems were excluded. Video clips with neutral faces developing a basic emotion (happiness, sadness, disgust, fear and anger) were presented in order to assess emotion recognition. Results indicated between-group differences neither concerning the number of correctly identified emotions nor concerning reaction times and their standard deviations. Thus, we suggest that ADHD per se is not associated with deficits in emotion recognition.     

Organization of Ca2+ release units in excitable smooth muscle of the guinea-pig urinary bladder

Ca(2+) release from internal stores (sarcoplasmic reticulum or SR) in smooth muscles is initiated either via pharmaco-mechanical coupling due to the action of an agonist and involving IP3 receptors, or via excitation-contraction coupling, mostly involving L-type calcium channels in the plasmalemma (DHPRs), and ryanodine receptors (RyRs), or Ca(2+) release channels of the SR. This work focuses attention on the structural basis for the coupling between DHPRs and RyRs in phasic smooth muscle cells of the guinea-pig urinary bladder. Immunolabeling shows that two proteins of the SR: calsequestrin and the RyR, and one protein the plasmalemma, the L-type channel or DHPR, are colocalized with each other within numerous, peripherally located sites located within the caveolar domains. Electron microscopy images from thin sections and freeze-fracture replicas identify feet in small peripherally located SR vesicles containing calsequestrin and distinctive large particles clustered within small membrane areas. Both feet and particle clusters are located within caveolar domains. Correspondence between the location of feet and particle clusters and of RyR- and DHPR-positive foci allows the conclusion that calsequestrin, RyRs, and L-type Ca(2+) channels are associated with peripheral couplings, or Ca(2+) release units, constituting the key machinery involved in excitation-contraction coupling. Structural analogies between smooth and cardiac muscle excitation-contraction coupling complexes suggest a common basic mechanism of action.     

Do mitochondria make nitric oxide? no?

Several papers have claimed that mitochondria contain nitric oxide synthase (NOS) and make nitric oxide (NO*) in amounts sufficient to affect mitochondrial respiration. However, we found that the addition of L-arginine or the NOS inhibitor L-NMMA to intact rat liver mitochondria did not have any effect on the respiratory rate in both State 3 and State 4. We did not detect mitochondrial NO* production by the oxymyoglobin oxidation assay, or electrochemically using an NO* electrode. An apparent NO* production detected by the Griess assay was identified as an artifact. NO* generated by eNOS added to the mitochondria could easily be detected, although succinate-supplemented mitochondria appeared to consume NO*. Our data show that NO* production by normal rat liver mitochondria cannot be detected in our laboratory, even though the levels of production claimed in the literature should easily have been measured by the techniques used. The implications for the putative mitochondrial NOS are discussed.     

Species-specific variation in the importance of the spectral quality gradient in canopies as a signal for photosynthetic resource partitioning

BACKGROUND AND AIMS: Plants adjust the distribution of photosynthetic capacity and chlorophyll to canopy density. The importance of the gradient in the red : far-red ratio (R : FR) relative to the irradiance gradient was studied for its perception with respect to this partitioning of photosynthetic resources. Whether the relative importance of these two signals varied between six species of different growth habit (Phaseolus vulgaris, Lysimachia vulgaris, Hedera helix, Ficus benjamina, Carex acutiformis and Brachypodium pinnatum) was investigated further. METHODS: Single leaves of plants were shaded in daylight by a spectrally neutral filter or a leaf. In another experiment, leaves were treated with supplemental FR. In most cases, treatment effects were evaluated after 2 weeks. KEY RESULTS: Nitrogen and photosynthetic capacity (Amax) per leaf area, parameters pertaining to between-leaf resource partitioning, were strongly reduced in neutral shade but not additionally by spectral leaf shade. Supplemental FR reduced these parameters also, except in Carex. Acceleration of induction of senescence was observed in spectral leaf shade in primary bean leaves. Amax per unit chlorophyll, a parameter pertaining to within-leaf resource partitioning, was reduced in neutral shade, but not in spectral leaf shade or supplemental FR. CONCLUSIONS: Signalling mechanisms associated with perception of the R : FR gradient in canopies were less important than those associated with the irradiance gradient for between-leaf and within-leaf partitioning of photosynthetic resources. The relative importance of the signals differed between species because Carex was the only species for which no indications were found for an involvement of the spectral gradient in perception of canopy density.     

Disease-associated polyglutamine stretches in monomeric huntingtin adopt a compact structure

Abnormal polyglutamine (polyQ) tracts are the only common feature in nine proteins that each cause a dominant neurodegenerative disorder. In Huntington's disease, tracts longer than 36 glutamines in the protein huntingtin (htt) cause degeneration. In situ, monoclonal antibody 3B5H10 binds to different htt fragments in neurons in proportion to their toxicity. Here, we determined the structure of 3B5H10 Fab to 1.9?? resolution by X-ray crystallography. Modeling demonstrates that the paratope forms a groove suitable for binding two β-rich polyQ strands. Using small-angle X-ray scattering, we confirmed that the polyQ epitope recognized by 3B5H10 is a compact two-stranded hairpin within monomeric htt and is abundant in htt fragments unbound to antibody. Thus, disease-associated polyQ stretches preferentially adopt compact conformations. Since 3B5H10 binding predicts degeneration, this compact polyQ structure may be neurotoxic.     

Investigation of Dry Powder Inhaler (DPI) Resistance and Aerosol Dispersion Timing on Emitted Aerosol Aerodynamic Particle Sizing by Multistage Cascade Impactor when Sampled Volume Is Reduced from Compendial Value of 4 L

Compendial methods determining dry powder inhaler (DPI)-emitted aerosol aerodynamic particle size distribution (APSD) collect a 4-L air sample containing the aerosol bolus, where the flow, which propagates through the cascade impactor (CI) measurement system from the vacuum source, is used to actuate the inhaler. A previous article described outcomes with two CIs (Andersen eight-stage cascade impactor (ACI) and Next-Generation Pharmaceutical Impactor (NGI)) when the air sample volume was ≤4 L with moderate-resistance DPIs. This article extends that work, examining the hypothesis that DPI flow resistance may be a factor in determining outcomes. APSD measurements were made using the same CI systems with inhalers representing low and high flow resistance extremes (Cyclohaler® and HandiHaler® DPIs, respectively). The ratio of sample volume to internal dead space (normalized volume (V*)) was varied from 0.25 to 1.98 (NGI) and from 0.43 to 3.46 (ACI). Inhaler resistance was a contributing factor to the rate of bolus transfer; the higher resistance DPI completing bolus relocation to the NGI pre-separator via the inlet when V* was as small as 0.25, whereas only ca. 50% of the bolus mass was collected at this condition with the Cyclohaler® DPI. Size fractionation of the bolus from either DPI was completed within the ACI at smaller values of V* than within the NGI. Bolus transfer from the Cyclohaler® capsule and from the HandiHaler® to the ACI system were unaffected by the different flow rise time observed in the two different flow controller systems, and the effects the ACI-based on APSD measurements were marginal.     

cAMP-dependent Protein Kinase and c-Jun N-terminal Kinase Mediate Stathmin Phosphorylation for the Maintenance of Interphase Microtubules during Osmotic Stress

Dynamic microtubule changes after a cell stress challenge are required for cell survival and adaptation. Stathmin (STMN), a cytoplasmic microtubule-destabilizing phosphoprotein, regulates interphase microtubules during cell stress, but the signaling mechanisms involved are poorly defined. In this study ectopic expression of single alanine-substituted phospho-resistant mutants demonstrated that STMN Ser-38 and Ser-63 phosphorylation were specifically required to maintain interphase microtubules during hyperosmotic stress. STMN was phosphorylated on Ser-38 and Ser-63 in response to hyperosmolarity, heat shock, and arsenite treatment but rapidly dephosphorylated after oxidative stress treatment. Two-dimensional PAGE and Phos-tag gel analysis of stress-stimulated STMN phospho-isoforms revealed rapid STMN Ser-38 phosphorylation followed by subsequent Ser-25 and Ser-63 phosphorylation. Previously, we delineated stress-stimulated JNK targeting of STMN. Here, we identified cAMP-dependent protein kinase (PKA) signaling as responsible for stress-induced STMN Ser-63 phosphorylation. Increased cAMP levels induced by cholera toxin triggered potent STMN Ser-63 phosphorylation. Osmotic stress stimulated an increase in PKA activity and elevated STMN Ser-63 and CREB (cAMP-response element-binding protein) Ser-133 phosphorylation that was substantially attenuated by pretreatment with H-89, a PKA inhibitor. Interestingly, PKA activity and subsequent phosphorylation of STMN were augmented in the absence of JNK activation, indicating JNK and PKA pathway cross-talk during stress regulation of STMN. Taken together our study indicates that JNK- and PKA-mediated STMN Ser-38 and Ser-63 phosphorylation are required to preserve interphase microtubules in response to hyperosmotic stress.