Proteomic analysis of highly purified prolamellar bodies reveals their significance in chloroplast development

The prolamellar body (PLB) proteome of dark-grown wheat leaves was characterized. PLBs are formed not only in etioplasts but also in chloroplasts in young developing leaves during the night, yet their function is not fully understood. Highly purified PLBs were prepared from 7-day-old dark-grown leaves and identified by their spectral properties as revealed by low-temperature fluorescence spectroscopy. The PLB preparation had no contamination of extra-plastidal proteins, and only two envelope proteins were found. The PLB proteome was analysed by a combination of 1-D SDS-PAGE and nano-LC FTICR MS. The identification of chlorophyll synthase in the PLB fraction is the first time this enzyme protein was found in extracts of dark-grown plants. This finding is in agreement with its previous localization to PLBs using activity studies. NADPH:protochlorophyllide oxidoreductase A (PORA), which catalyses the reduction of protochlorophyllide to chlorophyllide, dominates the proteome of PLBs. Besides the identification of the PORA protein, the PORB protein was identified for the first time in dark-grown wheat. Altogether 64 unique proteins, representing pigment biosynthesis, photosynthetic light reaction, Calvin cycle proteins, chaperones and protein synthesis, were identified. The in number of proteins’ largest group was the one involved in photosynthetic light reactions. This fact strengthens the assumption that the PLB membranes are precursors to the thylakoids and used for the formation of the photosynthetic membranes during greening. The present work is important to enhance our understanding of the significance of PLBs in chloroplast development.     

Tubificidae (oligochaeta) from the Ross Sea (Antarctica), with descriptions of one new genus and two new species

Three species,Torodrilus gelidus sp. nov. (subfamily Rhyacodrilinae),Rossidrilus terraenovae gen. et sp. nov. (Limnodriloidinae), and a second unnamed species of Limnodriloidinae, are reported from marine sediments in Terra Nova Bay, Ross Sea. Torodrilus gelidus is distinguished fromT. lowryi Cook, 1970 by its setal pattern (with few exceptions, both anterior and posterior setae are single-pointed in sexually mature specimens ofT. gelidus) and the morphology of its male protuberances (the latter folded over a midventral bursa in segment XI).Rossidrilus terraenovae is characterized by large diverticula attached to the oesophagus in the posterior part of segment IX, unpaired male and spermathecal pores, heavily muscular and entally ciliated atrial ampullae, elongate prostatic pads, and a deep, unpaired and muscular, copulatory sac. It is the first species of its subfamily to be described from Antarctic waters.     

A Phosphothreonine Residue at the C-Terminal End of the Plasma Membrane H+-ATPase Is Protected by Fusicoccin-Induced 14–3–3 Binding

We have isolated the plasma membrane H⁺−ATPase in a phosphorylated form from spinach (Spinacia oleracea L.) leaf tissue incubated with fusicoccin, a fungal toxin that induces irreversible binding of 14–3–3 protein to the C terminus of the H⁺-ATPase, thus activating H⁺ pumping. We have identified threonine-948, the second residue from the C-terminal end of the H⁺-ATPase, as the phosphorylated amino acid. Turnover of the phosphate group of phosphothreonine-948 was inhibited by 14–3–3 binding, suggesting that this residue may form part of a binding motif for 14–3–3. This is the first identification to our knowledge of an in vivo phosphorylation site in the plant plasma membrane H⁺-ATPase.     

The Redox State of Glutathione,Cysteine and Homocysteine in the Extracellular Fluid in the Skin

Glutathione, the most abundant low-molecular weight thiol in the skin, has been shown to protect the skin from both photobiological and chemical injury. The thiols, glutathione in particular, have also been shown to be crucially involved in defence against contact allergens. Since the levels of extracellular thiol concentrations are important determinants of intracellular thiol status, we have compared the normal concentrations and the redox status of the main low-molecular weight thiol components in the extracellular fluid at the dermo-epidermal junction with the corresponding plasma levels. In their sulfhydryl form, all three thiols, i.e. glutathione, cysteine and homocysteine, were more abundant in experimental skin blister fluid than in plasma, as were the free disulfides of glutathione and homocysteine, whereas the free disulfides of cysteine were about the same in blister fluid and in plasma. Protein mixed disulfide levels were higher in plasma than in blister fluid. The present results provide information concerning the extracellular defence in the skin.     

A selection strategy in plant transformation based on antisense oligodeoxynucleotide inhibition

Antisense oligodeoxynucleotide (asODN) inhibition was developed in the 1970s, and since then has been widely used in animal research. However, in plant biology, the method has had limited application because plant cell walls significantly block efficient uptake of asODN to plant cells. Recently, we have found that asODN uptake is enhanced in a sugar solution. The method has promise for many applications, such as a rapid alternative to time‐consuming transgenic studies, and high potential for studying gene functionality in intact plants and multiple plant species, with particular advantages in evaluating the roles of multiple gene family members. Generation of transgenic plants relies on the ability to select transformed cells. This screening process is based on co‐introduction of marker genes into the plant cell together with a gene of interest. Currently, the most common marker genes are those that confer antibiotic or herbicide resistance. The possibility that traits introduced by selectable marker genes in transgenic field crops may be transferred horizontally is of major public concern. Marker genes that increase use of antibiotics and herbicides may increase development of antibiotic‐resistant bacterial strains or contribute to weed resistance. Here, we describe a method for selection of transformed plant cells based on asODN inhibition. The method enables selective and high‐throughput screening for transformed cells without conferring new traits or functions to the transgenic plants. Due to their high binding specificity, asODNs may also find applications as plant‐specific DNA herbicides.     

EPR studies on the photosystem II donor side in salt-washed and reconstituted inside-out thylakoids

EPR measurements on inside-out thylakoids revealed that salt-washing, known to inhibit oxygen evolution and release a 23 and a 16 kDa protein, induced a Signal II_f and decreased the EPR signal from state S₂. Readdition of the released 23 kDa protein restored the oxygen evolution and decreased the Signal II_f, but did not relieve the decrease in the state S₂ signal. It is suggested that salt-washing inhibits the electron transfer from the oxygen-evolving site to Z, the physiological donor to P680. In inhibited photosystem II units lacking Signal II_f, Z⁺ is rapidly reduced, possibly by a modified S-cycle unable to evolve oxygen.     

Spatio-temporal processing of surface EMG signals from the sternocleidomastoideus muscle to assess effects of radiotherapy on motor unit conduction velocity and firing rate—A pilot study

Radiation therapy causes both muscle and nerve tissue damage. However, the evolution and mechanisms of these damages are not fully understood. Information on the state of active muscle fibres and motoneurons can be obtained by measuring sEMG signals and calculating the conduction velocity (CV) and firing rate of individual motor units, respectively. The aim of this pilot study was to evaluate if the multi-channel surface EMG (sEMG) technique could be applied to the sternocleidomastoideus muscle (SCM) of radiotherapy patients, and to assess if the CV and firing rate are altered as a consequence of the radiation.

Surface EMG signals were recorded from the radiated and healthy SCM muscles of 10 subjects, while subjects performed isometric rotation of the head. CV and firing rate were calculated using two recently proposed methods based on spatio-temporal processing of the sEMG signals. The multi-channel sEMG technique was successfully applied to the SCM muscle and CV and firing rates were obtained. The measurements were fast and simple and comfortable for the patients. Sufficient data quality was obtained from both sides of seven and four subjects for the CV and firing rate analysis, respectively. No differences in CV or firing rate were found between the radiated and non-radiated sides (p = 0.13 and p = 0.20, respectively). Firing rate and CV were also obtained from a myokymic discharge pattern. It was found that the CV decreased significantly (p = 0.01) during the bursts.     

Lipoproteomics I: mapping of proteins in low-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry

The molecular mechanisms underlying the relationship between low-density lipoprotein (LDL) and the risk of atherosclerosis are not clear. Therefore, detailed information about the protein composition of LDL may contribute to reveal its role in atherogenesis and the mechanisms that lead to coronary disease in humans. Here, we sought to map the proteins in human LDL by a proteomic approach. LDL was isolated by two-step discontinuous density-gradient ultracentrifugation and the proteins were separated with two-dimensional gel electrophoresis and identified with peptide mass fingerprinting, using matrix assisted laser desorption/ionization-time of flight-mass spectrometry and with amino acid sequencing using electrospray ionization tandem mass spectrometry. These procedures identified apo B-100, apo C-II, apo C-III (three isoforms), apo E (four isoforms), apo A-I (two isoforms), apo A-IV, apo J and apo M (three isoforms not previously described). In addition, three proteins that have not previously been identified in LDL were found: serum amyloid A-IV (two isoforms), calgranulin A, and lysozyme C. The identities of apo M, calgranulin A, and lysozyme C were confirmed by sequence information obtained after collision-induced dissociation fragmentation of peptides characteristic for these proteins. Moreover, the presence of lysozyme C was further corroborated by demonstrating enriched hydrolytic activity in LDL against Micrococcus lysodeikticus. These results indicate that in addition to the dominating apo B-100, LDL contains a number of other apolipoproteins, many of which occur in different isoforms. The demonstration, for the first time, that LDL contains calgranulin A and lysozyme C raises the possibility that LDL proteins may play hitherto unknown role(s) in immune and inflammatory reactions of the arterial wall.     

Cerebral gene expression and neurobehavioural development after perinatal exposure to an environmentally relevant polybrominated diphenylether (BDE47)

Nutrients in seafood are known to be beneficial for brain development. Effects of maternal exposure to 2,2′,4,4′ tetrabromo diphenylether (BDE47) was investigated, alongside the potential ameliorating impact of seafood nutrients, through assessment of neurobehaviour and gene expression in brain and liver. Developing mice were exposed during gestation and lactation via dams dosed through casein- or salmon-based feed, spiked with BDE47. Two concentrations were used: a low level (6 μg/kg feed) representing an environmentally realistic concentration and a high level (1,900 μg/kg feed) representing a BDE47 intake much higher than expected from frequents consumption of contaminated seafood. Experimental groups were similar with respect to reproductive success, growth and physical development. Minor, transient changes in neurobehavioural metrics were observed in groups given the highest dose of BDE47. No significant differences in behaviour or development were seen on postnatal day 18 among maternally exposed offspring. Cerebral gene expression investigated by microarray analyses and validated by RT-qPCR showed low fold changes for all genes, despite dose-dependent accumulation of BDE47 in brain tissue. The gene for glutamate ammonia ligase was upregulated compared to control in the casein-based high BDE47diet, suggesting potential impacts on downstream synaptic transmission. The study supported a previously observed regulation of Igfbp2 in brain with BDE47 exposure. Genes for hepatic metabolic enzymes were not influenced by BDE47. Potential neurotoxic effects and neurobehavioural aberrations after perinatal exposure to high levels of BDE47 were not readily observed in mice pups with the present experimental exposure regimes and methods of analysis.