Iron stress responses in the cyanobacterium Synechococcus sp. PCC7942

In the present study, we describe the sequential events by which the cyanobacterium Synechococcus sp. PCC 7942 adapts to iron deficiency. In doing so, we have tried to elucidate both short and long-term acclimation to low iron stress in order to understand how the photosynthetic apparatus adjusts to low iron conditions. Our results show that after an initial step, where CP43' is induced and where ferredoxin is partly replaced by flavodoxin, the photosynthetic unit starts to undergo major rearrangements. All measured components of Photosystem I (PSI), PSII and cytochrome (Cyt) ƒ decrease relative to chlorophyll (Chl) a . The photochemical efficiencies of the two photosystems also decline during this phase of acclimation. The well-known drop in phycobilisome content measured as phycocyanin (PC)/Chl was not due to an increased degradation, but rather to a decreased rate of synthesis. The largest effects of iron deficiency were observed on PSI, the most iron-rich structure of the photosynthetic apparatus. In the light of the recent discovery of an iron deficiency induced CP43' ring around PSI a possible dual function of this protein as both an antenna and a quencher is discussed. We also describe the time course of a blue shift in the low temperature Chl emission peak around 715 nm, which originates in PSI. The shift might reflect the disassembly and/or degradation of PSI during iron deficiency and, as a consequence, PSI might under these conditions be found predominantly in a monomeric form. We suggest that the observed functional and compositional alterations represent cellular acclimation enabling growth and development under iron deficiency, and that growth ceases when the acclimation capacity is exhausted. However, the cells remain viable even after growth has ceased, since they resumed growth once iron was added back to the culture.     

Interaction of transcriptional intermediary factor 2 nuclear receptor box peptides with the coactivator binding site of estrogen receptor alpha

The activation function 2/ligand-dependent interaction between nuclear receptors and their coregulators is mediated by a short consensus motif, the so-called nuclear receptor (NR) box. Nuclear receptors exhibit distinct preferences for such motifs depending both on the bound ligand and on the NR box sequence. To better understand the structural basis of motif recognition, we characterized the interaction between estrogen receptor alpha and the NR box regions of the p160 coactivator TIF2. We have determined the crystal structures of complexes between the ligand-binding domain of estrogen receptor alpha and 12-mer peptides from the Box B2 and Box B3 regions of TIF2. Surprisingly, the Box B3 module displays an unexpected binding mode that is distinct from the canonical LXXLL interaction observed in other ligand-binding domain/NR box crystal structures. The peptide is shifted along the coactivator binding site in such a way that the interaction motif becomes LXXYL rather than the classical LXXLL. However, analysis of the binding properties of wild type NR box peptides, as well as mutant peptides designed to probe the Box B3 orientation, suggests that the Box B3 peptide primarily adopts the "classical" LXXLL orientation in solution. These results highlight the potential difficulties in interpretation of protein-protein interactions based on co-crystal structures using short peptide motifs.     

Genotype/age interactions on aggressive behavior in gonadally intact estrogen receptor beta knockout (betaERKO) male mice

Estrogen, as an aromatized metabolite of testosterone, has a facilitatory effect on male aggressive behavior in mice. Two subtypes of estrogen receptors, alpha (ER-alpha) and beta (ER-beta), in the brain are known to bind estrogen. Previous studies revealed that the lack of ER-alpha gene severely reduced the induction of male aggressive behavior. In contrast, mice that lacked the ER-beta gene tended to be more aggressive than wild type (WT) control mice, although the behavioral effects of ER-beta gene disruption were dependent on their social experience. These findings lead us to hypothesize that estrogen may facilitate aggression via ER-alpha whereas it may inhibit aggression via ER-beta. In the present study, we further investigated the role of ER-beta in the regulation of aggressive behavior by examining developmental changes starting at the time of first onset, around the age of puberty. Aggressive behaviors of ER-beta gene knockout (betaERKO) mice were examined in three different age groups, puberty, young-adult, and adult. Each mouse was tested every other day for three times in a resident-intruder paradigm against olfactory bulbectomized intruder mice and their trunk blood was collected for measurements of serum testosterone after the completion of the study. Overall, betaERKO mice were significantly more aggressive than WT. These genotype differences were more pronounced in puberty and young adult age groups, but not apparent in the adult age group, in which betaERKO mice were less aggressive than those in two younger age groups. Serum testosterone levels of betaERKO mice were significantly higher than those of WT mice only in the pubertal age group, but not in young adult (when betaERKO mice were still significantly more aggressive than WT mice) and adult (when no genotype differences in aggression were found) age groups. These results suggest that ER-beta mediated actions of gonadal steroids may more profoundly be involved in the inhibitory regulation of aggressive behavior in pubertal and young adult mice.     

Uptake and intracellular transportation of a bacterial surface protein in lymphoid cells

Some strains of the human pathogen Streptococcus pyogenes express a surface protein called protein H, which is released from the streptococcal surface by a cysteine proteinase produced by the bacteria. Here, we find that soluble protein H binds to the surface of lymphocytes and granulocytes, and that the molecule is taken up by lymphocytes and transported to the perinuclear region. The translocation over the cell membrane is rapid, and the uptake and intracellular transportation is not dependent on actin polymerization. Protein H could be immunoprecipitated from cell extracts and nuclear preparations of lymphocytes, and analysis of molecular interactions between protein H and proteins of different cellular compartments demonstrated a binding to nucleophosmin/ B23, a protein known to shuttle between the cytoplasm and the nucleus, and to the nuclear proteins SET and hnRNP A2/B1. Nucleophosmin/B23 was co-immunoprecipitated with protein H from cell and nuclear extracts, and binding experiments, including kinetic analyses, suggest that protein H dissociating from nucleophosmin/B23 complexes in the perinuclear region or in the nucleus binds to proteins SET and hnRNP A2/B1. Finally, the uptake and intracellular transportation of protein H was found to result in a cytostatic effect on B and T lymphocytes.     

Improvement of the composting time for household waste during an initial low pH phase by mesophilic temperature control

Earlier studies indicated that the activity in the initial phase of composting may be reduced when the temperature rises too fast under low pH conditions. A compost reactor experiment on household waste was designed to test whether the degradation time could be reduced by actively preventing the temperature from rising until the pH had reached a certain value. This experiment was performed by monitoring pH in the condensate from the cooled compost gas. The results from 3 + 3 runs with and without temperature control confirmed our hypothesis and a considerable reduction in composting time was achieved. One possible explanation for the results is that the microbes active in the low pH phase are hampered by high temperature. The abrupt rise in pH when the fatty acids are consumed seems to be a good marker of the point when temperature control can be discontinued.     

Prediction of food protein allergenicity: a bioinformatic learning systems approach

Food hypersensitivity is constantly increasing in Western societies with a prevalence of about 1-2% in Europe and in the USA. Among children, the incidence is even higher. Because of the introduction of foods derived from genetically modified crops on the marketplace, the scientific community, regulatory bodies and international associations have intensified discussions on risk assessment procedures to identify potential food allergenicity of the newly introduced proteins. In this work, we present a novel biocomputational methodology for the classification of amino acid sequences with regard to food allergenicity and non-allergenicity. This method relies on a computerised learning system trained using selected excerpts of amino acid sequences. One example of such a successful learning system is presented which consists of feature extraction from sequence alignments performed with the FASTA3 algorithm (employing the BLOSUM50 substitution matrix) combined with the k-Nearest-Neighbour (kNN) classification algorithm. Briefly, the two features extracted are the alignment score and the alignment length and the kNN algorithm assigns the pair of extracted features from an unknown sequence to the prevalent class among its k nearest neighbours in the training (prototype) set available. 91 food allergens from several specialised public repositories of food allergy and the SWALL database were identified, pre-processed, and stored, yielding one of the most extensively characterised repositories of allergenic sequences known today. All allergenic sequences were classified using a standard one-leave-out cross validation procedure yielding about 81% correctly classified allergens and the classification of 367 non-allergens in an independent test set resulted in about 98% correct classifications. The biocomputational approach presented should be regarded as a significant extension and refinement of earlier attempts suggested for in silico food safety assessment. Our results show that the framework described here is powerful enough to become useful as part of a multiple-procedure test scheme that also depicts other evaluation approaches such as solid phase immunoassay and tests for stability to digestions.     

Preparation of partially 2H/13C-labelled RNA for NMR studies. Stereo-specific deuteration of the H5" in nucleotides

An effective in vitro enzymatic synthesis is described for the production of nucleoside triphosphates (NTPs) which are stereo-specifically deuterated on the H5" position with high selectivity (>98%), and which can have a variety of different labels (13C, 15N, 2H) in other positions. The NTPs can subsequently be employed in the enzymatic synthesis of RNAs using T7 polymerase from a DNA template. The stereo-specific deuteration of the H5" immediately provides the stereo-specific assignment of H5' resonances in NMR spectra, giving access to important structural parameters. Stereo-chemical H-exchange was used to convert commercially available 1,2,3,4,5,6,6-2H-1,2,3,4,5,6-13C-D-glucose (d7-13C6-D-glucose) into [1,2,3,4,5,6(R)-2H-1,2,3,4,5,6-13C]-D-glucose (d6-13C6-D-glucose). [1',3',4',5"-2H-1',2',3',4',5'-13C]GTP (d4-13C5-GTP) was then produced from d6-13C6-D-glucose and guanine base via in vitro enzymatic synthesis employing enzymes from the pentose-phosphate, nucleotide biosynthesis and salvage pathways. The overall yield was approximately 60 mg NTP per 1 g glucose, comparable with the yield of NTPs isolated from Escherichia coli grown on enriched media. The d4-13C5-GTP, together with in vitro synthesised d5-UTP, d5-CTP and non-labelled ATP, were used in the synthesis of a 31 nt RNA derived from the primer binding site of hepatitis B virus genomic RNA. (13C,1H) hetero-nuclear multiple-quantum spectra of the specifically deuterated sample and of a non-deuterated uniformly 13C/15N-labelled sample demonstrates the reduced spectral crowding and line width narrowing compared with 13C-labelled non-deuterated RNA.     

Specific regulation of lipocalin-type prostaglandin D synthase in mouse heart by estrogen receptor beta

Estrogens have important physiological roles in the cardiovascular system. We use DNA microarray technology to study the molecular mechanism of estrogen action in the heart and to identify novel estrogen-regulated genes. In this investigation we identify genes that are regulated by chronic estrogen treatment of mouse heart. We present our detailed characterization of one of these genes, lipocalin-type prostaglandin D synthase (L-PGDS). Northern and Western blot analysis revealed that L-PGDS was induced both by acute and chronic estrogen treatment. Northern blot analysis, using estrogen receptor (ER)-disrupted mice, suggests that L-PGDS is specifically induced by ERbeta in vivo. In further support of ERbeta-selective regulation, we identify a functional estrogen-responsive element in the L-PGDS promoter, the activity of which is up-regulated by ERbeta, but not by ERalpha. We demonstrate that a one-nucleotide change (A to C) in the L-PGDS estrogen-responsive element affects receptor selectivity.     

Gene expression in autumn leaves

Two cDNA libraries were prepared, one from leaves of a field-grown aspen (Populus tremula) tree, harvested just before any visible sign of leaf senescence in the autumn, and one from young but fully expanded leaves of greenhouse-grown aspen (Populus tremula x tremuloides). Expressed sequence tags (ESTs; 5,128 and 4,841, respectively) were obtained from the two libraries. A semiautomatic method of annotation and functional classification of the ESTs, according to a modified Munich Institute of Protein Sequences classification scheme, was developed, utilizing information from three different databases. The patterns of gene expression in the two libraries were strikingly different. In the autumn leaf library, ESTs encoding metallothionein, early light-inducible proteins, and cysteine proteases were most abundant. Clones encoding other proteases and proteins involved in respiration and breakdown of lipids and pigments, as well as stress-related genes, were also well represented. We identified homologs to many known senescence-associated genes, as well as seven different genes encoding cysteine proteases, two encoding aspartic proteases, five encoding metallothioneins, and 35 additional genes that were up-regulated in autumn leaves. We also indirectly estimated the rate of plastid protein synthesis in the autumn leaves to be less that 10% of that in young leaves.     

Localization of dibenzodioxocin substructures in lignifying Norway spruce xylem by transmission electron microscopy–immunogold labeling

The lignification process in mature Norway spruce [Picea abies (L.) H. Karsten] xylem cell walls was studied using transmission electron microscopy (TEM)–immunogold detection with a polyclonal antibody raised against a specific lignin substructure, dibenzodioxocin. The study reveals for the first time the exact location of this abundant eight-ring structure in the cell wall layers of wood. Spruce wood samples were collected in Southern Finland at the time of active growth and lignification of the xylem cell walls. In very young tracheids where secondary cell wall layers were not yet formed, the presence of the dibenzodioxocin structure could not be shown at all. During secondary cell wall thickening, the dibenzodioxocin structure was more abundant in the secondary cell wall layers than in the middle lamella. The highest number of gold particles revealing dibenzodioxocin was in the S2+S3 layer. Statistically significant differences were found in the frequency of gold particles present in various cell wall layers. For comparison, wood sections were also cut with a cryomicrotome for light and fluorescence microscopy.