Plants constantly adjust their repertoire of plasma membrane proteins that mediates transduction of environmental and developmental signals as well as transport of ions, nutrients, and hormones. The importance of regulated secretory and endocytic trafficking is becoming increasingly clear; however, our knowledge of the compartments and molecular machinery involved is still fragmentary. We used immunogold electron microscopy and confocal laser scanning microscopy to trace the route of cargo molecules, including the BRASSINOSTEROID INSENSITIVE1 receptor and the REQUIRES HIGH BORON1 boron exporter, throughout the plant endomembrane system. Our results provide evidence that both endocytic and secretory cargo pass through the trans-Golgi network/early endosome (TGN/EE) and demonstrate that cargo in late endosomes/multivesicular bodies is destined for vacuolar degradation. Moreover, using spinning disc microscopy, we show that TGN/EEs move independently and are only transiently associated with an individual Golgi stack.

Stomatal formation is regulated by multiple developmental and environmental signals, but how these signals are integrated to control this process is not fully understood. In Arabidopsis thaliana, the basic helix-loop-helix transcription factor SPEECHLESS (SPCH) regulates the entry, amplifying and spacing divisions that occur during stomatal lineage development. SPCH activity is negatively regulated by mitogen-activated protein kinase (MAPK)-mediated phosphorylation. Here, we show that in addition to MAPKs, SPCH activity is also modulated by brassinosteroid (BR) signalling. The GSK3/SHAGGY-like kinase BIN2 (BR INSENSITIVE2) phosphorylates residues overlapping those targeted by the MAPKs, as well as four residues in the amino-terminal region of the protein outside the MAPK target domain. These phosphorylation events antagonize SPCH activity and limit epidermal cell proliferation. Conversely, inhibition of BIN2 activity in vivo stabilizes SPCH and triggers excessive stomatal and non-stomatal cell formation. We demonstrate that through phosphorylation inputs from both MAPKs and BIN2, SPCH serves as an integration node for stomata and BR signalling pathways to control stomatal development in Arabidopsis.

A novel hydrogenase has recently been found in methanogenic archaea. It catalyzes the reversible dehydrogenation of methylenetetrahydromethanopterin (CH2=H4MPT) to methenyltetrahydromethanopterin (CHEH4MPT') and H2 and was therefore named H2-forming methylenetetrahydromethanopterin dehydrogenase. The hydrogenasc, which is composed of only one polypeptide with an apparent molecular mass of 43 kDa, does not mediate the reduction of viologen dyes with either Hz or CH2=H4MPT. We report here that the purified enzyme from Methanobacterium thernioautotrophicum exhibits the following other unique properties : (a) the colorless protein with a specific activity or 2000 Uimg (V,,,,,) did not contain iron-sulfur clusters, nickel, or flavins; (b) the activity was not inhibited by carbon monoxide, acetylene. nitrite, cyanide, or azide; (cj the enzyme did not catalyze an isotopic exchange between 'H, and 'H+ ; (d) the enzyme catalyzed the reduction of CHFH4MPT+ with 'H, generating [n2ethylenc-'HH]CH,=H4MPT; and (e) the primary structure contained at most four conserved cysteines as revealed by a comparison of the DNA-deduced amino acid sequence of [he proteins from M . theumuautotrophicum and Methanopyrus kundleri. None of the four cysteines were closely spaced as would be indicative for a (NiFe) hydrogenase or a ferredoxintype iron-sulfur protein.Properties of the H,-forming methylenetetrahydromethanopterin dehydrogenase from Methmobacterium i v d f e i are also described indicating that thc enzyme from this methanogenic archaeon is very similar to the enzyme from M . thermoautotrophicum with respect both to molecular and catalytic properties.

For the first time estrogen DNA-adducts were identified in DNA human breast tumor tissue using nano-LC coupled to nano-Electrospray Tandem Mass Spectrometry. Normal breast tissue was analyzed analogously. The data obtained in the five breast tumor and five adjacent normal tissue samples were compared qualitatively, but no straightforward difference was observed. Prior to LC-MS analysis the DNA was enzymatically hydrolyzed to a nucleoside pool. The DNA-hydrolysates were directly injected onto a column switching system developed for on-line sample clean-up and subsequent analysis of the DNA-adducts. In four patients using Premarin, DNA-adducts of 4-hydroxy-equilenin (4OHEN) were detected. All except three samples contained DNA-adducts from 4-hydroxy-estradiol or 4-hydroxy-estrone. Also DNA isolated from eight alcohol fixed and paraffin embedded breast tumor tissue showed the presence of different estrogen DNA-adducts. Worthwhile mentioning is the presence of adducts responding to m/z 570 Ͼ m/z 454 transition. This is a well-known SRM-transition indicative for the presence of the 2'-deoxyguanosine (dGuo) adduct of Benzo H ormone treatment is widespread for women of all ages. In the United States, for instance, 30% of post-menopausal women use hormone eeplacement therapy (HRT) [1], e.g., Premarin. Studies in which the development of breast and endometrial cancer was associated with estrogen therapy [2][3][4][5][6] were supported by a more recent follow-up study in which it was demonstrated that post-menopausal women have an increased risk of breast cancer when using estrogens, especially in combination with progestin [7]. In animals, too, a relationship between the administration of estrogens and the development of cancer was shown [8].The carcinogenic properties of estrogens are explained by direct stimulation of cell proliferation via estrogen receptor mediated mechanisms [9,10] and by mechanisms based on metabolic activation [11][12][13][14], leading to DNA damage such as oxidative damage [15][16][17][18] and the formation of DNA-adducts [19 -28], which can cause mutations and induce cancer [29].The latter pathways are the result of metabolic activation of estrogens by the cytochrome P-450 system leading to 2-and 4-hydroxy derivatives. The 2-hydroxy estrogens are excreted in the urine as a result of their fast transformation to water-soluble compounds [13,14]. The 4-hydroxy form, however, has a longer half-life

This is the first study to report on concentrations of perfluorinated organochemicals (FOCs) in marine mammals stranded along the southern North Sea coast in relation to stable nitrogen and carbon isotope ratios (delta15N and delta13C). The presence of FOCs in top predators such as marine mammals would indicate a potential biomagnification of these compounds and their widespread occurrence. Liver and kidney tissues of nine marine mammal species have been sampled. Among all the measured FOCs compounds, PFOS (perfluorooctane sulfonate) was predominant in terms of concentration. The highest PFOS concentrations were found in the liver of harbor seal compared to white-beaked dolphin, harbor porpoise, gray seal, sperm whale, white-sided dolphin, striped dolphin, fin whale, and hooded seal. PFOS concentrations differed significantly between sexes and age classes in harbor porpoises. Stable isotope measurements (delta13C and delta15N) were used in this study to describe the behavior of contaminants in food webs. We found a significant (p < 0.05) linear relationship between PFOS concentrations in livers of harbor porpoises and both muscle delta13C and delta15N measurements. Harbor and gray seals and white-beaked dolphin, which displayed the highest trophic position, contained the highest PFOS levels, while offshore feeders such as sperm whales, fin whales, striped dolphin, and white-sided dolphin showed lower PFOS concentrations than inshore species.

No abstract

Owing to the low abundance of signaling proteins and transcription factors, their protein complexes are not easily identified by classical proteomics. The isolation of these protein complexes from endogenous plant tissues (rather than plant cell cultures) is therefore an important technical challenge. Here, we describe a sensitive, quantitative proteomics-based procedure to determine the composition of plant protein complexes. The method makes use of fluorophore-tagged protein immunoprecipitation (IP) and label-free mass spectrometry (MS)-based quantification to correct for nonspecifically precipitated proteins. We provide procedures for the isolation of membrane-bound receptor complexes and transcriptional regulators from nuclei. The protocol consists of an IP step (~6 h) and sample preparation for liquid chromatography-tandem MS (LC-MS/MS; 2 d). We also provide a guide for data analysis. Our single-step affinity purification protocol is a good alternative to two-step tandem affinity purification (TAP), as it is shorter and relatively easy to perform. The data analysis by label-free quantification (LFQ) requires a cheaper and less challenging experimental setup compared with known labeling techniques in plants.

A liquid chromatography/mass spectrometry (LC/MS) method for the analysis of complex mixtures of nucleoside mono-, di- and triphosphates has been developed. A short capillary column (35mm x 0.3mm i.d.) was operated under ion-pair high-performance liquid chromatography conditions and hyphenated to (negative) electrospray (tandem) mass spectrometry. As such, the separation of 12 nucleotides was performed by a binary gradient elution using CH(3)OH/H(2)O and N,N-dimethylhexylamine (N,N-DMHA) as ion-pairing agent. The influence of different N,N-DMHA concentrations on the chromatographic and mass spectrometric performance was evaluated to achieve optimal LC/MS conditions. In addition it was demonstrated that a controlled admission of ammonium dihydrogen phosphate (NH(4)H(2)PO(4)) improved both chromatographic performance and mass spectrometric detection. Because the system was hyphenated to an orthogonal designed electrospray interface (Z-spraytrade mark), long acquisition times were possible without loss of sensitivity.