Characterization of Exiguobacterium isolates from the Siberian permafrost. Description of Exiguobacterium sibiricum sp. nov.

Three Gram-positive bacterial strains, 7-3, 255-15 and 190-11, previously isolated from Siberian permafrost, were characterized and taxonomically classified. These microorganisms are rod-shaped, facultative aerobic, motile with peritrichous flagella and their growth ranges are from -2.5 to 40 degrees C. The chemotaxonomic markers indicated that the three strains belong to the genus Exiguobacterium. Their peptidoglycan type was A3alpha L-Lys-Gly. The predominant menaquinone detected in all three strains was MK7. The polar lipids present were phosphatidyl-glycerol, diphosphatidyl-glycerol and phosphatidyl-ethanolamine. The major fatty acids were iso-C13:0, anteiso-C13:0, iso-C15:0, C16:0 and iso-C17:0. Phylogenetic analysis based on 16S rRNA and six diverse genes, gyrB (gyrase subunit B), rpoB (DNA-directed RNA polymerase beta subunit), recA (homologous recombination), csp (cold shock protein), hsp70 (ClassI-heat shock protein-chaperonin) and citC (isocitrate dehydrogenase), indicated that the strains were closely related to Exiguobacterium undae (DSM 14481(T)) and Exiguobacterium antarcticum (DSM 14480(T)). On the basis of the phenotypic characteristics, phylogenetic data and DNA-DNA reassociation data, strain 190-11 was classified as E. undae, while the other two isolates, 7-3 and 255-15, comprise a novel species, for which the name Exiguobacterium sibiricum sp. nov. is proposed.     

Environmental factors influence on chemical polymorphism of the essential oils of Lychnophora ericoides

Lychnophora ericoides is a Brazilian medicinal plant used in folk medicine as an anti-inflammatory and analgesic agent. The essential oils from leaves of two populations with and without scent, collected at 2-month intervals during an 1-year period, were analysed by GC-MS. The results were submitted to principal component and cluster analysis which allowed two groups of essential oils to be distinguished with respect to sampling site and scent: cluster I (Vianópolis site, with specimens exhibiting an aromatic scent) containing a high percentage of alpha-bisabolol (44.7-76.4%) and alpha-cadinol (10.9-23.5%), and cluster II (Cristalina site, with specimens without scent) characterised by a high content of (E)-nerolidol (31.3-47.1%) and ar-dihydro-turmerone (4.8-15.4%). The canonical discriminant analysis showed that using the data set of the seven sampling months and (E)-nerolidol and alpha-bisabolol as predictable variables, it was possible to distinguish between the samples harvested according to Cerrado seasons, dry winter (May-September) and humid summer (November-March). In addition, canonical correlation analysis between the soil sampling sites and the populations revealed a significant relationship between oil components and edaphic factors. Oxygenated sesquiterpenes and potential acidity, Al saturation, cationic exchange capacity, silt, and sand load as the first canonical variate were fairly strongly related to samples collected in Vianópolis site. On the other hand, monoterpenes and sesquiterpene hydrocarbons were strongly related to chemical balance in soils (organic matter, P and base saturation), which is related to samples at the Cristalina site. The chemovariation observed appears to be environmentally determined.     

Influence of Hydrological Pulse on Bacterial Growth and DOC Uptake in a Clear-Water Amazonian Lake

This study was conducted to evaluate: (1) the bacterial growth and the dissolved organic carbon (DOC) uptake in an Amazonian lake (Lake Batata) at high-water and low-water periods of the flood pulse; (2) the influence of nitrogen and phosphorus (NP) additions on bacterial growth and DOC uptake in Lake Batata at two flood pulse periods; and (3) the bioavailability of the main DOC sources in Lake Batata. Lake Batata is a typical clear-water Amazonian lake, located in the watershed of Trombetas River, Central Amazon, Brazil. Bacterial batch cultures were set up with 90% 0.2-μm filtered water and 10% inoculum from Lake Batata. N-NH₄NO₃ and P-KH₂PO₄, with final concentrations of 50 and 5 μM, respectively, were added to the cultures, except for controls. Extra sources of DOC (e.g., algal lysate, plant leachates) were added to constitute six distinct treatments. Bacterial response was measured by maximum bacterial abundance and rates of bacterial production, respiration, DOC uptake, and bacterial growth efficiency (BGE). Bacterial growth and DOC uptake were higher in NP treatments than in controls, indicating a consistent nutrient limitation in Lake Batata. The composition of DOC also seems to be an important regulating factor of bacterial growth in Lake Batata. Seasonally, bacterial growth and DOC bioavailability were higher at low-water period, when the phytoplankton is a significant extra source of DOC, than at high-water period, when the forest is the main source of DOC. DOC bioavailability was better estimated based on the diversity and the diagenetic stage of carbon compounds than on single classes of labile compounds. Changes in BGE were better related to CNP stoichiometry in the water, and the “excess” of organic substrates was oxidized in catabolism, despite the quality of these compounds for bacterial growth. Finally, we conclude that bacterial growth and DOC uptake vary throughout the flood pulse in clear-water Amazonian ecosystems as a result of changes in nutrient concentration and in DOC composition.     

Fourier transform infrared spectroscopy provides a fingerprint for the tetramer and for the aggregates of transthyretin

Transthyretin (TTR) is an amyloidogenic protein whose aggregation is responsible for several familial amyloid diseases. Here, we use FTIR to describe the secondary structural changes that take place when wt TTR undergoes heat- or high-pressure-induced denaturation, as well as fibril formation. Upon thermal denaturation, TTR loses part of its intramolecular beta-sheet structure followed by an increase in nonnative, probably antiparallel beta-sheet contacts (bands at 1,616 and 1,686 cm(-1)) and in the light scattering, suggesting its aggregation. Pressure-induced denaturation studies show that even at very elevated pressures (12 kbar), TTR loses only part of its beta-sheet structure, suggesting that pressure leads to a partially unfolded species. On comparing the FTIR spectrum of the TTR amyloid fibril produced at atmospheric pressure upon acidification (pH 4.4) with the one presented by the native tetramer, we find that the content of beta-sheets does not change much upon fibrillization; however, the alignment of beta-sheets is altered, resulting in the formation of distinct beta-sheet contacts (band at 1,625 cm(-1)). The random-coil content also decreases in going from tetramers to fibrils. This means that, although part of the tertiary- and secondary-structure content of the TTR monomers has to be lost before fibril formation, as previously suggested, there must be a subsequent reorganization of part of the random-coil structure into a well-organized structure compatible with the amyloid fibril, as well as a readjustment of the alignment of the beta-sheets. Interestingly, the infrared spectrum of the protein recovered from a cycle of compression-decompression at pD 5, 37 degrees C, is quite similar to that of fibrils produced at atmospheric pressure (pH 4.4), which suggests that high hydrostatic pressure converts the tetramers of TTR into an amyloidogenic conformation.     

Water permeability of rat liver mitochondria: A biophysical study

The movement of water accompanying solutes between the cytoplasm and the mitochondrial spaces is central for mitochondrial volume homeostasis, an important function for mitochondrial activities and for preventing the deleterious effects of excess matrix swelling or contraction. While the discovery of aquaporin water channels in the inner mitochondrial membrane provided valuable insights into the basis of mitochondrial plasticity, questions regarding the identity of mitochondrial water permeability and its regulatory mechanism remain open. Here, we use a stopped flow light scattering approach to define the water permeability and Arrhenius activation energy of the rat liver whole intact mitochondrion and its membrane subcompartments. The water permeabilities of whole brain and testis mitochondria as well as liposome models of the lipid bilayer composing the liver inner mitochondrial membrane are also characterized. Besides finding remarkably high water permeabilities for both mitochondria and their membrane subcompartments, the existence of additional pathways of water movement other than aquaporins are suggested.     

Synthesis of galactofuranose-containing disaccharides using thioimidoyl-type donors

Four galactofuranose-containing disaccharides have been prepared utilising various thioimidates [Galf-SC(NR)XR'] and suitably protected acceptors as key precursors. We observed that the efficiency of the coupling reactions was particularly dependent on the aglycon present on the furanosyl donor when copper(II) ions were used as the promoter, and that activation could be correlated with the nature of the third heteroatom, X.     

Remodeling of the epitope repertoire of a candidate idiotype vaccine by targeting to lysosomal degradation in dendritic cells

The generation of efficacious vaccines against self-antigens expressed in tumor cells requires breakage of tolerance, and the refocusing of immune responses toward epitopes for which tolerance may not be established. While the presentation of tumor antigens by mature dendritic cells (mDC) may surpass tolerance, broadening of the antigenic repertoire remains an issue. We report that fusion of the candidate idiotype vaccine IGKV3-20 to the Gly-Ala repeat (GAr) of the Epstein-Barr virus nuclear antigen (EBNA)-1 inhibits degradation by the proteasome and redirects processing to the lysosome. mDCs transduced with a recombinant lentivirus encoding the chimeric idiotype efficiently primed CD4+ and CD8+ cytotoxic T-cell (CTL) responses that lysed autologous blasts expressing IGKV3-20 or pulsed with IGKV3-20 synthetic peptides, and HLA-matched IGKV3-20-positive tumor cell lines. Comparison of the cytotoxic response of CD4+ and CD8+ T lymphocytes activated by mDCs expressing the wild-type or chimeric IGKV3-20 reveled largely non-overlapping epitope repertoires in both CD4+ and CD8+ effectors. Thus, fusion to the GAr may provide an effective means to broaden the immune response to an endogenous protein by promoting the presentation of antigenic epitopes that require a lysosome-dependent processing step.     

Genetic and physiological analysis of Rht8 in bread wheat: an alternative source of semi-dwarfism with a reduced sensitivity to brassinosteroids

Over the next decade, wheat grain production must increase to meet the demand of a fast growing human population. One strategy to meet this challenge is to raise wheat productivity by optimizing plant stature. The Reduced height 8 (Rht8) semi-dwarfing gene is one of the few, together with the Green Revolution genes, to reduce stature of wheat (Triticum aestivum L.), and improve lodging resistance, without compromising grain yield. Rht8 is widely used in dry environments such as Mediterranean countries where it increases plant adaptability. With recent climate change, its use could become increasingly important even in more northern latitudes. In the present study, the characterization of Rht8 was furthered. Morphological analyses show that the semi-dwarf phenotype of Rht8 lines is due to shorter internodal segments along the wheat culm, achieved through reduced cell elongation. Physiological experiments show that the reduced cell elongation is not due to defective gibberellin biosynthesis or signalling, but possibly to a reduced sensitivity to brassinosteroids. Using a fine-resolution mapping approach and screening 3104 F(2) individuals of a newly developed mapping population, the Rht8 genetic interval was reduced from 20.5 cM to 1.29 cM. Comparative genomics with model genomes confined the Rht8 syntenic intervals to 3.3 Mb of the short arm of rice chromosome 4, and to 2 Mb of Brachypodium distachyon chromosome 5. The very high resolution potential of the plant material generated is crucial for the eventual cloning of Rht8.     

Mutations in SRCAP, encoding SNF2-related CREBBP activator protein, cause Floating-Harbor syndrome

Floating-Harbor syndrome (FHS) is a rare condition characterized by short stature, delayed osseous maturation, expressive-language deficits, and a distinctive facial appearance. Occurrence is generally sporadic, although parent-to-child transmission has been reported on occasion. Employing whole-exome sequencing, we identified heterozygous truncating mutations in SRCAP in five unrelated individuals with sporadic FHS. Sanger sequencing identified mutations in SRCAP in eight more affected persons. Mutations were de novo in all six instances in which parental DNA was available. SRCAP is an SNF2-related chromatin-remodeling factor that serves as a coactivator for CREB-binding protein (CREBBP, better known as CBP, the major cause of Rubinstein-Taybi syndrome [RTS]). Five SRCAP mutations, two of which are recurrent, were identified; all are tightly clustered within a small (111 codon) region of the final exon. These mutations are predicted to abolish three C-terminal AT-hook DNA-binding motifs while leaving the CBP-binding and ATPase domains intact. Our findings show that SRCAP mutations are the major cause of FHS and offer an explanation for the clinical overlap between FHS and RTS.     

Construction of engineered fructosyl peptidyl oxidase for enzyme sensor applications under normal atmospheric conditions

Current enzymatic methods for the analysis of glycated proteins use flavoenzymes that catalyze the oxidative deglycation of fructosyl peptides, designated as fructosyl peptidyl oxidases (FPOXs). However, as FPOXs are oxidases, the signals derived from electron mediator-type electrochemical monitoring based on them are affected by dissolved O₂. Improvement of dye-mediated dehydrogenase activity of FPOXs and its application to enzyme electrode construction were therefore undertaken. Saturation mutagenesis study on Asn56 of FPOX from Phaeosphaeria nodorum, produced mutants with marked decreases in the catalytic ability to employ O₂ as the electron acceptor, while showing higher dye-mediated dehydrogenase activity employing artificial electron acceptors than the parental enzyme. Thus constructed virtually fructosyl peptide dehydrogenase, Asn56Ala, was then applied to produce an enzyme electrode for the measurement of fructosyl-^α N-valyl-histidine (f-^αVal-His), the protease-digested product of HbA1c. The enzyme electrode could measure f-^αVal-His in the physiological target range in air.