Recombinant expression and initial characterization of the putative human enteric ferric reductase Dcytb

Recent studies of mutant mice with compromised ability to absorb dietary iron have identified involvement of two integral membrane proteins in the intestinal epithelial lining in iron uptake, a divalent metal ion transporter and a ferric reductase. The current study concerns the recombinant expression, purification, and initial spectroscopic characterization of a recombinant form of the human ferric reductase that was expressed and purified as the apoprotein from Escherichia coli. Reconstitution of the recombinant protein with ferriprotoporphyrin IX produced a red product with Soret (Fe3+, lambdamax 413.5 nm; Fe2+, lambdamax = 426 nm) and visible absorption maxima indicative of bisimidazole axial coordination. This observation was confirmed by electron paramagnetic resonance and magnetic circular dichroism spectroscopy. Titration of apo-Dcytb with ferriprotoporphyrin IX was consistent with the binding of two heme groups to the protein as predicted by the phylogenetic relationship of this protein to the cytochrome b561 family. Similar titrations and spectroscopic studies of two double variants of Dcytb, each lacking a pair of histidyl residues (H50 and H120 or H86 and H159) proposed on the basis of sequence alignment with other members of the cytochrome b561 family to provide axial ligands to bound heme, indicated that these variants were able to bind just one heme group each.     

Past and future approaches to ischemia-reperfusion lesion associated with liver transplantation

Ischemia-reperfusion (I/R) injury associated with liver transplantation remains a serious complication in clinical practice, in spite of several attempts to solve the problem. The present review focuses on the complexity of I/R injury, summarizing conflicting results obtained from the literature about the mechanisms responsible for it. We also review the therapeutic strategies designed in past years to reduce I/R injury, attempting to explain why most of them have not been applied clinically. These strategies include improvements in pharmacological treatments, modifications of University of Wisconsin (UW) preservation solution based on a variety of additives, and gene therapy. Finally, we will consider new potential protective strategies using trimetazidine, 5-amino-4-imidazole carboxamide riboside (AICAR), melatonin, modulators of the renin-angiotensin system (RAS) and the phosphatidylinositol-3-OH kinase (PI3K)-Akt and the p42/p44 extracellular signal-regulated kinases (Erk 1/2) pathway. These strategies have shown promising results for I/R injury but have not been tested in experimental liver transplantation to date. Moreover, we will review ischemic preconditioning, taking into account the recent clinical studies that suggest that this surgical strategy could be appropriate for liver transplantation.     

<Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>, <Emphasis Type="Italic">Cryptococcus gattii</Emphasis>: Serotypes in Venezuela

Mycoses due to yeasts belonging to other genera than Candida have become common in the last years especially in immuno-compromised patients. Species of the anamorphic basidiomycetous yeast genus Trichosporon are such opportunistic human pathogenic yeasts which cause several diseases. In this study, Trichosporon faecale is reported in Germany for the first time. The isolate was taken from a human foot, where it was associated with a tinea pedis. The fungal isolate was identified by investigating the morphology, physiology by a commercial API 32 C-set and molecular data of SSU and LSU rDNA as well as the ITS region.     

Couch potatoes do better: Delayed dispersal and territory size affect the duration of territory occupancy in a monogamous mammal

In territorial, socially monogamous species, the establishment and defense of a territory are an important strategy to maximize individual fitness, but the factors responsible for the duration of territory occupancy are rarely studied, especially in long‐lived mammals. A long‐term monitoring program in southeast Norway spanning over 18 years allowed us to follow the individual life histories of Eurasian beavers (Castor fiber) from adolescence in their natal family group to dispersal and territory establishment until the end of territory occupancy. We investigated whether territory size, resource availability, population density, and dispersal age could explain the duration of territory occupancy, which ranged from 1 to 11 years. The duration of territory occupancy was positively related to dispersal age, suggesting that individuals that delayed dispersal had a competitive advantage due to a larger body mass. This is in support with the maturation hypothesis, which states that an animal should await its physical and behavioral maturation before the acquisition of a territory. Further, we found that individuals that established in medium‐sized territories occupied them longer as compared to individuals in small or large territories. This suggests that large territories are more costly to defend due to an increased patrolling effort, and small territories might not have sufficient resources. The lifetime reproductive success ranged from zero to six kits and generally increased with an increasing duration of territory occupancy. Our findings show the importance of holding a territory and demonstrate that dispersal decisions and territory selection have important consequences for the fitness of an individual.     

Impaired cognitive performance in neuronal nitric oxide synthase knockout mice is associated with hippocampal protein derangements

Nitric oxide is implicated in modulation of memory and pharmacological as well as genetic inhibition of neuronal nitric oxide synthase (nNOS) leads to impaired cognitive function. We therefore decided to study learning and memory functions and cognitive flexibility in the Morris water maze (MWM) in 1-month-old male mice lacking nNOS (nNOS KO). Hippocampal protein profiling was carried out to possibly link protein derangement to impaired cognitive function. Two-dimensional gel electrophoresis with in-gel digestion of spots and subsequent MALDI-TOF identification of proteins and quantification of proteins using specific software was applied. In the memory as well as in the relearning task of the MWM, most of the nNOS KO failed to find the submerged platform within a given time. Proteomic evaluation of hippocampus, the main anatomical structure computing cognitive functions, revealed aberrant expression of a synaptosomal associated protein of the exocytotic machinery (NSF), glycolytic enzymes, chaperones 78 kDa glucose-regulated protein, T-complex protein 1; the signaling structure guanine nucleotide-binding protein G(I)/G(S)/G(T) and heterogeneous nuclear ribonucleoprotein H of the splicing machinery. We conclude that nNOS knockout mice show impaired spatial performance in the MWM, a finding that may be either linked to direct effects of nNOS/NO and/or to specific hippocampal protein derangements.     

Experimental inoculation of conventional pigs with porcine reproductive and respiratory syndrome virus and porcine circovirus 2

Postweaning multisystemic wasting syndrome (PMWS) is a disease of nursery and fattening pigs characterized by growth retardation, paleness of the skin, dyspnea, and increased mortality rates. Porcine circovirus 2 (PCV2) has been demonstrated to be the cause of PMWS. However, other factors are needed for full development of the syndrome, and porcine reproductive and respiratory syndrome virus (PRRSV) infection has been suggested to be one of them. Twenty-four conventional 5-week-old pigs were distributed in four groups: control (n = 5), PRRSV inoculated (n = 5), PCV2 inoculated (n = 7), and PRRSV and PCV2 inoculated (n = 7). The two groups inoculated with PRRSV showed growth retardation. Pigs inoculated with both PRRSV and PCV2 had increased rectal temperature. One of these pigs developed wasting, had severe respiratory distress, and died. The most important microscopic lesion in pigs inoculated with PCV2 was lymphocyte depletion with histiocytic infiltration of the lymphoid organs, more severe and in a wider range of tissues in doubly inoculated pigs. Interstitial pneumonia was observed in the three inoculated groups. PCV2 nucleic acid was found by in situ hybridization in larger amounts and in a wider range of lymphoid tissues in PRRSV- and PCV2-inoculated than in PCV2-inoculated pigs. TaqMan PCR was performed to quantify the PCV2 loads in serum during the experiment. PCV2 loads were higher in doubly inoculated pigs than in pigs inoculated with PCV2 alone. These findings indicate that severe disease can be reproduced in conventional 5-week-old pigs by inoculation of PRRSV and PCV2. Moreover, these results support the hypothesis that PRRSV infection enhances PCV2 replication.     

A modified host-cell reactivation assay to quantify DNA repair capacity in cryopreserved peripheral lymphocytes

The host-cell reactivation assay (HCRA) is a functional assay that allows the identification of the genes responsible for DNA repair-deficient syndromes, such as Xeroderma pigmentosum, by cross-complementation experiments. It has also been used in molecular epidemiology studies to correlate the low nucleotide excision repair pathway function in peripheral blood lymphocytes with an increased risk of bladder, head and neck, skin and lung cancers. Herein, we present the technical validation of a newly modified HCRA, where nucleofection is used for the transfection of the pmaxGFP plasmid into cryopreserved peripheral blood lymphocytes (PBLs) or lymphoblastoid cell lines. In each sample, 20-24h after transfection, the relative DNA repair capacity (DRC) was quantified by flow cytometry, comparing the transfection efficiency of nucleoporated cells with undamaged plasmid to those transfected with UV-light damaged plasmid in the seven cell lines that were characterized by different DNA repair phenotypes. Dead cells were excluded from the analysis. We observed a high reproducibility of the relative DRC, transfection efficiency and cell viability. The inter-experimental normalization of the flow cytometry resulted in an increased data accuracy and reproducibility. The amount of cells required for each transfection reaction was reduced fourfold, without affecting the final relative DRC. Furthermore, our HCRA demonstrated strong discrimination power in the UV-light dose-response, both in lymphoblastoid cell lines and cryopreserved PBLs. We also observed a strong correlation of the relative DRC data, when samples were measured against two independent batches of both damaged and undamaged plasmid DNA. The relative DRC variable shows a normal distribution when analyzed in the cryopreserved PBLs from a cohort of 35 lung cancer patients and a 5.59-fold variation in the relative DRC is identified among our patients. The mitotic dynamic was discarded as a confounding factor for the relative DRC measurement in this cohort of patients. The results indicate that our method is highly sensitive, reliable and reproducible, and thus, it suitable for population-based studies to quantify in vitro DNA-repair deficiencies.     

Insights into the historical construction of species-rich Mesoamerican seasonally dry tropical forests: the diversification of Bursera (Burseraceae, Sapindales)

? Mesoamerican arid biomes epitomize neotropical rich and complex biodiversity. To document some of the macroevolutionary processes underlying the vast species richness of Mesoamerican seasonally dry tropical forests (SDTFs), and to evaluate specific predictions about the age, geographical structure and niche conservatism of SDTF-centered woody plant lineages, the diversification of Bursera is reconstructed. ? Using a nearly complete Bursera species-level phylogeny from nuclear and plastid genomic markers, we estimate divergence times, test for phylogenetic and temporal diversification heterogeneity, test for geographical structure, and reconstruct habitat shifts. ? Bursera became differentiated in the earliest Eocene, but diversified during independent early Miocene consecutive radiations that took place in SDTFs. The late Miocene average age of Bursera species, the presence of phylogenetic geographical structure, and its strong conservatism to SDTFs conform to expectations derived from South American SDTF-centered lineages. ? The diversification of Bursera suggests that Mesoamerican SDTF richness derives from high speciation from the Miocene onwards uncoupled from habitat shifts, during a period of enhanced aridity resulting mainly from global cooling and regional rain shadows.