Thermodynamic and kinetic characterisation of individual haems in multicentre cytochromes c3

The characterisation of individual centres in multihaem proteins is difficult due to the similarities in the redox and spectroscopic properties of the centres. NMR has been used successfully to distinguish redox centres and allow the determination of the microscopic thermodynamic parameters in several multihaem cytochromes c(3) isolated from different sulphate-reducing bacteria. In this article we show that it is also possible to discriminate the kinetic properties of individual centres in multihaem proteins, if the complete microscopic thermodynamic characterisation is available and the system displays fast intramolecular equilibration in the time scale of the kinetic experiment. The deconvolution of the kinetic traces using a model of thermodynamic control provides a reference rate constant for each haem that does not depend on driving force and can be related to structural factors. The thermodynamic characterisation of three tetrahaem cytochromes and their kinetics of reduction by sodium dithionite are reported in this paper. Thermodynamic and kinetic data were fitted simultaneously to a model to obtain microscopic reduction potentials, haem-haem and haem-proton interacting potentials, and reference rate constants for the haems. The kinetic information obtained for these cytochromes and recently published data for other multihaem cytochromes is discussed with respect to the structural factors that determine the reference rates. The accessibility for the reducing agent seems to play an important role in controlling the kinetic rates, although is clearly not the only factor.     

Response to Fusarium culmorum inoculation in barley

In field tests replicated in 2004 and 2005, 32 cultivars of spring barley were assessed for resistance to Fusarium head blight (FHB) by single floret inoculation and spray inoculation with Fusarium culmorum (W. G. Smith) Sacc. It was found that the weather conditions in individual years affect to a large extent the progression of FHB and production of mycotoxin deoxynivalenol (DON). At the same time, in both years the cultivars reacted to F. culmorum infection similarly with respect to areas under disease progress curve (AUDPC) values and content of mycotoxin DON. Spraying inoculation led to stronger infection. The biggest differences in AUDPC values were observed between the cultivars Brise and Celinka, and weak reaction was found in the cultivars Kompakt and Madonna. The cultivars Kompakt and Tolar were most resistant towards FHB. In both monitored years the variety Ludan contained the lowest amounts of mycotoxin DON. Cultivars with high infection and low DON content (r = 0.78) showed weak positive relationship between resistance to FBH and accumulation of DON (concentration 70–200 mg/kg). This is the first information on FHB and in vivo concentrations of DON in certificated barley cultivars in Slovakia.     

Thermodynamic redox behavior of the heme centers of cbb3 heme-copper oxygen reductase from Bradyrhizobium japonicum

A comprehensive study of the thermodynamic redox behavior of the hemes from the cbb3 oxygen reductase from Bradyrhizobium japonicum was performed. This enzyme is a member of the C-type heme-copper oxygen reductase superfamily and has three subunits with six redox centers: four low-spin hemes and a high-spin heme and one copper ion, composing the site where oxygen is reduced. In this analysis, the visible spectra and redox properties of the five heme centers were deconvoluted. Their redox profiles and the pH dependence of the midpoint reduction potentials (redox-Bohr effect) were investigated. The reference reduction potentials (defined for a state where all centers are reduced) and homotropic interaction potentials were determined in the framework of a model of pairwise interacting redox centers. At pH 7.7, the reference reduction potentials for the three hemes c are 390, 300, and 220 mV, with low interaction potentials between them, weaker than -15 mV. For hemes b and b3, reference reduction potentials of 375 and 290 mV, respectively, were obtained; these two redox centers show an interaction potential weaker than -60 mV. The midpoint reduction potentials of all five hemes are pH-dependent. The study of these thermodynamic parameters is important in understanding the coupling mechanism of the redox and chemical processes during oxygen reduction. The analysis of the thermodynamic redox behavior of the cbb3 oxygen reductase contributes to the investigation of the mechanism of electron transfer and proton translocation by heme-copper oxygen reductases in general and indicates a thermodynamic coupling for the electron and proton transfer mechanisms.     

Phylogeography of Mabuya maculilabris (Reptilia) from São Tomé Island (Gulf of Guinea) inferred from mtDNA sequences

The pattern of genetic variation of the lizard Mabuya maculilabris from S?o Tomé Island (Gulf of Guinea) was investigated using a combination of three mitochondrial DNA gene fragments. Forty-eight haplotypes were recovered among 66 individuals covering the whole island. The genealogy inferred from the most parsimonious network of haplotypes allows us to detect two main and long branches departing from the putative group of oldest haplotypes. The tips of these branches exhibit star-like phylogenies, which may indicate of recently expanded populations, most probably from a small number of founders. A nested clade analysis suggests a complex pattern of past events that gave rise to the extant geographical pattern found in the haplotype distribution: past and allopatric fragmentation, range expansion, restricted gene flow and long-distance dispersal. These results are consistent with the complex geological history of the island where important volcanic activity with extensive lava flows has occurred during several periods. Mismatch-distribution analysis and AMOVA also support these conclusions. Substantial genetic structuring among these lizards was detected as well as high levels of differentiation between the southern edge populations (particularly those from the Rolas Islet) and the remaining ones. However, variation is low relative to the geological age of the island. Our results indicate that patterns of variation observed in reptiles in other oceanic islands are not indicative of those observed in the islands of the Gulf of Guinea.     

Protein folding cooperativity: basic insights from minimalist models

Basic concepts about two-state, cooperative protein folding and its relation to first-order phase transitions are reviewed. Minimalist models capable of reproducing the required free energy barrier between folded and unfolded macroscopic states are described. A significantly more restrictive "calorimetric" criterion is also discussed, which is based on direct comparison between model and experimental heat capacities with additional assumptions about conformational enthalpy variation in the unfolded state.     

Evaluation of 16S rDNA- and gyrB-DGGE for typing members of the genus Aeromonas

The DNA sequence of the gyrB gene is a suitable phylogenetic marker for bacterial systematics. In this study, the diversity of Aeromonas spp. present in environmental samples was assessed by a PCR combined with DGGE approach. PCR primers targeting the gyrB gene of aeromonads were designed and the resulting amplicons were analyzed by DGGE. The gyrB-DGGE analysis was evaluated with Aeromonas isolates and reference strains allowing discrimination of the majority of strains. The gyrB-DGGE analysis is a powerful tool to monitor the presence and evaluate the diversity of aeromonads in complex samples, as is the case of water from a wastewater treatment plant.     

Characterization of Sparus aurata osteonectin cDNA and in silico analysis of protein conserved features: evidence for more than one osteonectin in Salmonidae

Osteonectin is a matricellular protein involved in various cellular mechanisms but its exact function remains unclear despite numerous studies. We present here the cloning of Sparus aurata partial osteonectin cDNA and the reconstruction of 15 other sequences from both vertebrates and invertebrates, almost doubling the set of available sequences (a total of 35 sequences is now available). Taking advantage of the resulting large amount of data, we have created multiple sequence alignments and identified osteonectin putative conserved features (intra- and inter-disulfide bonds, collagen- and calcium-binding domains and phosphorylation sites) likely to be important for protein structure and function. This work also provides the first evidence for the presence of more than one osteonectin in some species. Finally, S. aurata osteonectin gene expression has been shown to initiate during larval development shortly after gastrulation, and to be high in bone-derived cell lines while down-regulated during extracellular matrix mineralization, further emphasizing the important role of osteonectin in skeletal development and bone formation.     

The Le Douarin phenomenon: a shift in the paradigm of developmental self-tolerance

Ever since the foundations of Immunology, "self-tolerance" has remained a central issue in this field, pertaining to basic and clinical questions alike. Burnet and Medawar shared the Nobel Prize in 1960 for proposing that tolerance is induced by tissue antigens, if present during the development of the immune system during the embryonic/neonatal period. Very elegant experiments by Le Douarin and colleagues in the 1980's demonstrated that this is not the case; rather, the establishment of tolerance to peripheral tissues requires thymic epithelium which selects CD4 T lymphocytes mediating "dominant tolerance". The recent wealth of work on "regulatory T cells", as well as observations on the selective regulation of "tissue-specific" gene expression in thymic epithelial cells, confirm the critical relevance of those seminal findings in modern immunology.     

Intraspecific comparative analysis of the species Salinibacter ruber

Salinibacter ruber is the first extremely halophilic member of the Bacteria domain of proven environmental relevance in hypersaline brines at or approaching NaCl saturation, that has been brought to pure culture. A collection of 17 strains isolated from five different geographical locations (Mallorca, Alicante, Ebro Delta, Canary Islands, and Peruvian Andes) were studied following the currently accepted taxonomic approach. Additionally, random amplification of genomic DNA led to the phenetic analysis of the intraspecific diversity. Altogether the taxonomic study indicated that S. ruber remained highly homogeneous beyond any geographical barrier. However, genomic fingerprints indicated that populations from different isolation sites could still be discriminated.     

African female heritage in Iberia: a reassessment of mtDNA lineage distribution in present times

The Iberian peninsula is a peripheral region of Europe in close proximity to Africa. Its inhabitants have an overall mtDNA genetic landscape typical of European background, although with signs of some African influence, whose features we deemed to disclose by analyzing available mtDNA HVRI distributions and new data. We analyzed 1,045 sequences. The most relevant results are the following: (1) North African sequences (haplogroup U6) present an overall frequency of 2.39%, and sub-Saharan sequences reach 3.83%, values that are, in both cases, much higher than those generally observed in Europe; and (2) there is a substantial geographic heterogeneity in the distribution of these lineages (haplogroup L being the most frequent in the south, whereas haplogroup U6 is generally more common in the north). The analysis of the observed diversity within each haplogroup strongly suggests that both were recently introduced (in historical times). Although for haplogroup U6 the documented event that is demographically compatible is the Islamic period (beginning of the 8th century to the end of the 15th century), for haplogroup L the most probable origin is the modern slave trade (mid 15th century to the end of the 18th century). However, the observed geographic structuring for one of the haplogroups does not fit the expected distribution provided by simplistic historical considerations. In fact, although for haplogroup L the north-south increasing frequency is corroborated by historical data, the opposite trend, observed for haplogroup U6, is more difficult to reconcile with the magnitude and time span of the Islamic political and cultural influence, which lasted longer and was more intense in the south. To clarify this conundrum, we need not only a substantial increase in the amount of mtDNA data (particularly for North Africa) but also new historical data and interpretations.