Structural changes in the lectin domain of CD23, the low-affinity IgE receptor, upon calcium binding

CD23, the low-affinity receptor for IgE (Fc epsilonRII), regulates IgE synthesis and also mediates IgE-dependent antigen transport and processing. CD23 is a unique Fc receptor belonging to the C-type lectin-like domain superfamily and binds IgE in an unusual, non-lectin-like manner, requiring calcium but not carbohydrate. We have solved the high-resolution crystal structures of the human CD23 lectin domain in the presence and absence of Ca2+. The crystal structures differ significantly from a previously determined NMR structure and show that calcium binding occurs at the principal binding site, but not at an auxiliary site that appears to be absent in human CD23. Conformational differences between the apo and Ca2+ bound structures suggest how IgE-Fc binding can be both calcium-dependent and carbohydrate-independent.     

Seasonal distribution and fatty acid composition of littoral microalgae in the Yenisei River

We studied fatty acid (FA) composition of littoral microalgae in the fast-flowing oligotrophic river, the Yenisei, Siberia, monthly for 3 years. Seasonal dynamics of species composition had similar patterns in all the studied years. In springs, a pronounced dominance of filamentous green algae occurred, in summer and autumn diatoms were abundant, and in late autumn and winter epilithic biofilms consisted primarily of cyanobacteria and detritus. In general, FA composition of the algal periphytic community was dominated by 16:0, 16:1ω7, 20:5ω3, 14:0, and 18:3ω3 throughout the studied period. Several groups of FAs, which had peculiar seasonal dynamics, were differentiated by statistical analysis based on a method of correlation graphs. The seasonal changes in FA composition could be partly explained by the seasonal succession of species composition of the community. Besides, we found that populations of both diatom and green algae grown in summer at a higher water temperature were lower in polyunsaturated fatty acids than those in spring, at a lower temperature. Hence, we suppose that the regular seasonal dynamics of FA composition of the studied littoral microalgae was driven both by changes in species composition and by temperature adaptations of the algal populations. The highest content of essential polyunsaturated FAs, eicosapentaenoic and docosahexaenoic acids, in the spring “psychrophilic” populations of diatoms could make them of the higher nutritive value for zoobenthic primary consumers.     

Aromatic Amino Acid Auxotrophs Constructed by Recombinant Marker Exchange in Methylophilus methylotrophus AS1 Cells Expressing the aroP-Encoded Transporter of Escherichia coli

The isolation of auxotrophic mutants, which is a prerequisite for a substantial genetic analysis and metabolic engineering of obligate methylotrophs, remains a rather complicated task. We describe a novel method of constructing mutants of the bacterium Methylophilus methylotrophus AS1 that are auxotrophic for aromatic amino acids. The procedure begins with the Mu-driven integration of the Escherichia coli gene aroP, which encodes the common aromatic amino acid transporter, into the genome of M. methylotrophus. The resulting recombinant strain, with improved permeability to certain amino acids and their analogues, was used for mutagenesis. Mutagenesis was carried out by recombinant substitution of the target genes in the chromosome by linear DNA using the FLP-excisable marker flanked with cloned homologous arms longer than 1,000 bp. M. methylotrophus AS1 genes trpE, tyrA, pheA, and aroG were cloned in E. coli, sequenced, disrupted in vitro using a Km^r marker, and electroporated into an aroP carrier recipient strain. This approach led to the construction of a set of marker-less M. methylotrophus AS1 mutants auxotrophic for aromatic amino acids. Thus, introduction of foreign amino acid transporter genes appeared promising for the following isolation of desired auxotrophs on the basis of different methylotrophic bacteria.The nonpathogenic Gram-negative bacterium Methylophilus methylotrophus is able to grow efficiently using C₁ substrates (methanol, methylamine, or trimethylamine) as the sole source of carbon and energy, and it uses the ribulose monophosphate pathway for fixation of formaldehyde produced by the oxidation of methanol (36). Methanol has received considerable attention by the fermentation industry as an alternative substrate to the more generally used sugars from agricultural crops. It can be synthesized either from petrochemicals or renewable resources, such as biogas (48), and therefore the production of methanol does not compete directly with human food supplies. Methylotrophs can therefore be considered potentially useful strains for industrial biotechnology. M. methylotrophus AS1 is an obligate methylotroph originally isolated from activated sludge, and it has been deposited in the National Collections of Industrial, Marine and Food Bacteria (NCIMB; no. 10515). This organism was extensively studied in the 1970s and has been industrialized on a large scale for the manufacturing of single-cell proteins (SCP) from methanol (56, 63). During that period, a significant amount of research was conducted on the direct production of amino acids by fermentation from methanol (3, 58). Although initially promising, these efforts ultimately proved relatively unsatisfactory and impractical, due primarily to the rather poor set of genetic tools that had been developed for methylotrophs.Over the last 5 years, several genomes of methylotrophs have been sequenced (8, 20, 29, 37, 65, 67), and significant progress in elucidating their metabolism has been achieved (14). The number of tools available for the genetic and metabolic engineering of methylotrophic bacteria has been expanded greatly (1, 15, 21, 43), and strategies to produce fine and bulk chemicals by methylotrophs have been described (5, 42, 57, 61). All of these factors led to renewed interest in the construction of methylotrophic strain producers, and the larger knowledge base has enabled more targeted engineering of these bacteria (55).Although M. methylotrophus AS1 has been extensively studied with regard to the industrial scale production of SCP (56, 63) and the oxidation of methanol at the initial stages of carbon and energy metabolism (13, 28), there has been little metabolic analysis of amino acid biosynthesis in this organism. Moreover, selection of auxotrophic mutants of obligate methylotrophs for broadening convenient genetic tools remains a particularly complicated task (19). Although the isolation of several auxotrophs for M. methylotrophus AS1 has been described (6, 23, 40), their numbers are limited. Development of different methods for the isolation of the mutants did not lead to construction of a collection of auxotrophic mutants that could assist in the investigation of amino acid biosynthetic pathways in M. methylotrophus AS1.As for the l-lysine (Lys) synthesis, systematic research was carried out by specialists at Ajinomoto Co., Inc., Japan, beginning with the investigation of the Lys biosynthetic pathway in M. methylotrophus AS1 (23, 61) and continuing with the construction and improvement of a Lys producer (22, 24, 33, 34). This was followed by optimization of fed-batch fermentation for overproduction of Lys from methanol (35).The aim of our investigation was to generate strains based on M. methylotrophus AS1 with the potential for industrial production of aromatic amino acids (AroAAs). It is known that mutants with relaxed feedback inhibition of key biosynthetic enzymes should be isolated at the initial steps of the construction of the amino acid producers and that the relevant degradation pathways should be blocked due to selection of the corresponding auxotrophic strains (7, 31, 49).In this study, a novel method for the construction of AroAA auxotrophic mutants of M. methylotrophus AS1 is described. This method is based on the introduction of a foreign gene encoding a specific amino acid transporter into the genome of M. methylotrophus AS1. The resulting recombinant methylotrophic strain, which possesses increased permeability to the AroAAs and their analogues, was mutated by recombination-mediated substitution of the target chromosomal genes of aromatic pathways by a flippase recombinase (FLP)-excisable marker from artificial linear DNA. This approach led to the construction of a set of M. methylotrophus AS1 marker-less mutants with destroyed genes of AroAA biosynthesis. Thus, introduction of foreign amino acid transporter genes appeared promising for the following isolation of desired auxotrophs on the basis of different methylotrophic bacteria.     

Response of oat cultivars to <Emphasis Type="Italic">Fusarium</Emphasis> infection with a view to their suitability for food use

Oats as a source of antioxidants and complex polysaccharides are currently an important component in human nutrition. Producing healthy, safe and high-quality grain for this purpose depends upon growing oat cultivars with improved resistance to diseases caused by Fusarium spp. producing mycotoxins. Thirteen cultivars of naked (Ábel, Detvan, Izák and Avenuda) and covered (Zvolen, Auron, Atego, Flämingsstern, Kanton, Viktor, Zla?ák, Euro and Ardo) oats were inoculated with conidial suspensions of F. culmorum isolate in the field at flowering in 2006 and 2007. After harvest, reduction in thousand-kernel weight (R-TKW), reduction in panicle-kernel weight (R-PKW), and deoxynivalenol (DON) content in grain and hulls were determined. The ELISA immunochemical method was employed for the quantitative analyses of DON. Values of yield components (R-TKW; R-PKW) were 35.4% and 31.1% lower in dehulled covered oats than in naked oat cultivars. The DON accumulation was highest in hulls as compared with DON content in kernels of naked and covered oat cultivars. Accumulation of DON in dehulled covered cultivars was 34.4% lower than the average contamination in naked cultivars. When the cultivars were compared, there were positive correlations between R-TKW and R-PKW and also between DON content and R-PKW. With a view to growing oat cultivars for production of cereal foods, it was shown that dehulling of covered oat grain resulted in substantially reduced DON content.     

Cytoplasm and organelle transfer between mesenchymal multipotent stromal cells and renal tubular cells in co-culture

Cell-to-cell interactions of human mesenchymal multipotent stromal cells (MMSC) and rat renal tubular cells (RTC) were explored under conditions of co-cultivation. We observed formation of different types of intercellular contacts, including so called tunneling nanotubes. These contacts were shown to be able to provide transfer of cell's contents, including organelles. We documented intercellular exchange with fluorescent probes specific to cytosol, plasmalemma and mitochondria. Initial transport of cellular components was revealed after 3 h of co-culturing, and occurred in two directions—both direct and retrograde as referred to RTC. However, transport of probes toward MMSC was more efficient. One significant result of such transport was appearance of renal-specific Tamm-Horsfall protein in MMSC, indicating induction of their differentiation into kidney tubular cells. We conclude that transfer of cellular compartments between renal and stem cells could provide differentiation of MMSC when transplanted into kidney and result in therapeutic benefits in renal failure.     

Polymorphism of genes for catechol-O-methyltransferase (COMT) and hemochromatosis (HFE) in residents of radiocontaminated regions varying in chromosome aberration frequency

Biophysics - The association between polymorphisms in the COMT and HFE genes, involved in oxidative stress control, on the one hand, and chromosome aberration frequency in lymphocytes, on the other...     

Automated Removal of Noisy Data in Phylogenomic Analyses

Noisy data, especially in combination with misalignment and model misspecification can have an adverse effect on phylogeny reconstruction; however, effective methods to identify such data are few. One particularly important class of noisy data is saturated positions. To avoid potential errors related to saturation in phylogenomic analyses, we present an automated procedure involving the step-wise removal of the most variable positions in a given data set coupled with a stopping criterion derived from correlation analyses of pairwise ML distances calculated from the deleted (saturated) and the remaining (conserved) subsets of the alignment. Through a comparison with existing methods, we demonstrate both the effectiveness of our proposed procedure for identifying noisy data and the effect of the removal of such data using a well-publicized case study involving placental mammals. At the least, our procedure will identify data sets requiring greater data exploration, and we recommend its use to investigate the effect on phylogenetic analyses of removing subsets of variable positions exhibiting weak or no correlation to the rest of the alignment. However, we would argue that this procedure, by identifying and removing noisy data, facilitates the construction of more accurate phylogenies by, for example, ameliorating potential long-branch attraction artefacts.     

Polyphasic Characterization of a Thermotolerant Siderophilic Filamentous Cyanobacterium That Produces Intracellular Iron Deposits

Despite the high potential for oxidative stress stimulated by reduced iron, contemporary iron-depositing hot springs with circum-neutral pH are intensively populated with cyanobacteria. Therefore, studies of the physiology, diversity, and phylogeny of cyanobacteria inhabiting iron-depositing hot springs may provide insights into the contribution of cyanobacteria to iron redox cycling in these environments and new mechanisms of oxidative stress mitigation. In this study the morphology, ultrastructure, physiology, and phylogeny of a novel cyanobacterial taxon, JSC-1, isolated from an iron-depositing hot spring, were determined. The JSC-1 strain has been deposited in ATCC under the name Marsacia ferruginose, accession number BAA-2121. Strain JSC-1 represents a new operational taxonomical unit (OTU) within Leptolyngbya sensu lato. Strain JSC-1 exhibited an unusually high ratio between photosystem (PS) I and PS II, was capable of complementary chromatic adaptation, and is apparently capable of nitrogen fixation. Furthermore, it synthesized a unique set of carotenoids, but only chlorophyll a. Strain JSC-1 not only required high levels of Fe for growth (≥40 μM), but it also accumulated large amounts of extracellular iron in the form of ferrihydrite and intracellular iron in the form of ferric phosphates. Collectively, these observations provide insights into the physiological strategies that might have allowed cyanobacteria to develop and proliferate in Fe-rich, circum-neutral environments.Cyanobacteria inhabiting ferrous iron-rich hot springs with circum-neutral pH represent unique models for examining the mechanisms by which early organisms evolved to cope with such habitats common on early Earth. Such organisms have previously been shown to be resistant to Fe²⁺ (37) or Fe³⁺ (6, 7) at concentrations in the micromolar to millimolar range. Moreover, high Fe concentrations (apparent optimum of ∼0.5 mM) stimulated the growth of these cyanobacteria, which were described as siderophilic (having an affinity for iron) cyanobacteria (7).The cyanobacteria inhabiting the Chocolate Pots hot springs in Yellowstone National Park, Wyoming, were shown to have played at least a passive role in contributing to iron deposition by serving as nucleation sites for the accumulation of iron minerals and associated silica deposits (36, 38). The precipitation of external iron that encrusts the cyanobacterial cells inhabiting this hot spring appears to be dependent on the species composition and chemistry of the mat (36, 38); however, multiple anoxygenic phototrophs found in the Chocolate Pots hot springs (8) could also contribute to the formation of Fe oxides (21, 49). Therefore, only iron mineralization experiments with model cyanobacterial strains can demonstrate the role of siderophilic cyanobacteria in the formation of specific, iron-bearing minerals.An additional common feature of circum-neutral iron-depositing hot springs is elevated concentrations of hydrogen peroxide (50). Shcolnick and coauthors (41) showed that a wild type of Synechococcus sp. PCC 6803 was resistant to 8 mM H₂O₂ if grown with 0.3 μM Fe³⁺, while the same concentration of hydrogen peroxide completely inhibited the growth of this cyanobacterium if it was grown with 10 μM Fe³⁺. If a similar correlation between iron concentration and the magnitude of an externally applied oxidative stress were the case for siderophilic cyanobacteria, iron-depositing hot springs should be free of cyanobacteria. However, such springs are very rich with cyanobacteria (38, 7, 36), which suggests that siderophilic cyanobacteria may possess unusual mechanisms of iron homeostasis maintenance and oxidative stress mitigation. Additionally, understanding iron tolerance and phenomena associated with siderophily in oxygenic prokaryotes is also important because such siderophilic organisms might help us find applications for bioremediation of waters polluted with iron.The current work describes the morphology, ultrastructure, physiology, and phylogeny of a previously undescribed, siderophilic cyanobacterium. The results of this polyphasic characterization led to the conclusion that strain JSC-1 represents a new operational taxonomic unit (OTU). (The epithet for JSC-1, Marsacia ferruginose, was chosen in honor of Nicole Tandeau de Marsac.) Additionally, biomineralization of intracellular iron by a cyanobacterium is demonstrated for the first time.     

Natural ventilation effectively reduces hyperhydricity in shoot cultures of <Emphasis Type="Italic">Aloe polyphylla</Emphasis> Schönland ex Pillans

An investigation into the role of ventilation to reduce hyperhydricity in tissue cultures of Aloe polyphylla Schönland ex Pillans revealed that gaseous exchange between the in-vitro atmosphere and the outside environment is an essential prerequisite for controlling this disorder. In closed culture vessels, hyperhydricity affected as much as 84% of the newly-formed shoots on media gelled with gelrite. The leaves of hyperhydric shoots had a bright green colour, smooth epidermis and large, open stomata. Gaseous exchange was promoted by using modified lids with a hole covered with polyester or cotton mesh. In ventilated cultures, hyperhydricity was completely eliminated irrespective of the type of gelling agent used. Natural ventilation was further advantageous for the microplants in terms of leaf chlorophyll content as well as the deposition of epicuticular wax, indicating the onset of mechanisms that regulate water loss from the explants. Although culture ventilation was negatively correlated to the regeneration rate and shoot growth, it has the potential to control the appearance of abnormal phenotypes and can be easily adopted for routine A. polyphylla propagation in vitro.