A metabolite sensor subunit of the Atg1/ULK complex regulates selective autophagy.

Cells convert complex metabolic information into stress-adapted autophagy responses. Canonically, multilayered protein kinase networks converge on the conserved Atg1/ULK kinase complex (AKC) to induce non-selective and selective forms of autophagy in response to metabolic changes. Here we show that, upon phosphate starvation, the metabolite sensor Pho81 interacts with the adaptor subunit Atg11 at the AKC via an Atg11/FIP200 interaction motif to modulate pexophagy by virtue of its conserved phospho-metabolite sensing SPX domain. Notably, core AKC components Atg13 and Atg17 are dispensable for phosphate starvation-induced autophagy revealing significant compositional and functional plasticity of the AKC. Our data indicate that, instead of functioning as a selective autophagy receptor, Pho81 compensates for partially inactive Atg13 by promoting Atg11 phosphorylation by Atg1 critical for pexophagy during phosphate starvation. Our work shows Atg11/FIP200 adaptor subunits bind not only selective autophagy receptors but also modulator subunits that convey metabolic information directly to the AKC for autophagy regulation.

Immunocytochemical determination of the subcellular distribution of ascorbate in plants.

Ascorbate is an important antioxidant in plants and fulfills many functions related to plant defense, redox signaling and modulation of gene expression. We have analyzed the subcellular distribution of reduced and oxidized ascorbate in leaf cells of Arabidopsis thaliana and Nicotiana tabacum by high-resolution immuno electron microscopy. The accuracy and specificity of the applied method is supported by several observations. First, preadsorption of the ascorbate antisera with ascorbic acid or dehydroascorbic acid resulted in the reduction of the labeling to background levels. Second, the overall labeling density was reduced between 50 and 61% in the ascorbate-deficient Arabidopsis mutants vtc1-2 and vtc2-1, which correlated well with biochemical measurements. The highest ascorbate-specific labeling was detected in nuclei and the cytosol whereas the lowest levels were found in vacuoles. Intermediate labeling was observed in chloroplasts, mitochondria and peroxisomes. This method was used to determine the subcellular ascorbate distribution in leaf cells of plants exposed to high light intensity, a stress factor that is well known to cause an increase in cellular ascorbate concentration. High light intensities resulted in a strong increase in overall labeling density. Interestingly, the strongest compartment-specific increase was found in vacuoles (fourfold) and in plastids (twofold). Ascorbate-specific labeling was restricted to the matrix of mitochondria and to the stroma of chloroplasts in control plants but was also detected in the lumen of thylakoids after high light exposure. In summary, this study reveals an improved insight into the subcellular distribution of ascorbate in plants and the method can now be applied to determine compartment-specific changes in ascorbate in response to various stress situations.

High-performance liquid chromatography determination of alpha1-acid glycoprotein in small volumes of plasma from neonates.

In order to investigate how the alpha1-acid glycoprotein (AGP) concentrations of neonates change in response to surgical stress, a simple high-performance liquid chromatography (HPLC)-assay for the measurement of alpha1-acid glycoprotein levels was developed. A fraction containing alpha1-acid glycoprotein was isolated from the bulk of plasma protein by addition of 0.6M perchloric acid and was then analysed directly on a short PLRP-S 4000A reversed phase column column. The method was validated by analysis of pooled plasma from healthy adults both in comparison with a calibration curve and by standard additions. The procedure was able to isolate alpha1-acid glycoprotein rapidly (<30 min) and required only 50 microl of plasma. The mean extraction recovery was 79.1% (CV 6.4%). The within-run precision for the analysis of three replicates of quality control sample ranged from +/-1.2 to +/-3.8% and the between-run precision was +/-6.1%. The method was linear (r(2)=0.988) over a concentration range from 6 to 100.0 mg/100 ml. The AGP levels in neonatal samples ranged from 25 to 93 mg/100 ml.

A pathogen-inducible divinyl ether synthase (CYP74D) from elicitor-treated potato suspension cells.

In elicitor-treated potato cells, 9-lipoxygenase-derived oxylipins accumulate with the divinyl ether colneleic acid as the major metabolite. Here, the identification of a potato cDNA is described, whose predicted amino acid sequence corresponds to divinyl ether synthases, belonging to the recently identified new P450 subfamily CYP74D. The recombinant protein was expressed in Escherichia coli and shown to metabolize 9-hydroperoxy linoleic acid to colneleic acid at pH 6.5. This fatty acid derivative has been implicated in functioning as a plant antimicrobial compound. RNA blot analyses revealed accumulation of divinyl ether synthase transcripts both upon infiltration of potato leaves with Pseudomonas syringae and after infection with Phytophthora infestans.

Determination of free concentrations of ropivacaine and bupivacaine in plasma from neonates using small-scale equilibrium-dialysis followed by liquid chromatography-mass spectrometry.

Attempts to determine a safe plasma concentration of ropivacaine and bupivacaine in neonates have not been consistent. This might be due to an underestimation of free drug in small plasma samples by currently used techniques, e.g., ultrafiltration. We describe a simple microscale equilibrium-dialysis technique for the separation of free and bound ropivacaine and bupivacaine. The free drug in the dialysate was determined using solid-phase extraction and liquid chromatography with mass spectrometry. Pentycaine was used as an internal standard and added to the dialysates prior to extraction. The method is very selective and sensitive, as no compounds other than the analyte and internal standard were observed in the resulting chromatograms at low ng/ml levels. The limit of quantitation was 2.5 ng/ml. The calibration curve was linear in the range of 2 to 1000 ng/ml. The precision of the whole procedure was 8.1% (n=10) and 6.5% (n=7) for ropivacaine and bupivacaine, respectively. The method was tested in the analysis of plasma samples taken from neonates who had received epidural injections.