DNA variation within Juncaceae: comparison of impact of organelle regions on phylogeny

A phylogenetic analysis of the Juncaceae was conducted to assess relationships among the genera Juncus, Luzula and five other small South American genera (Distichia, Marsippospermum, Oxychloë, Patosia and Rostkovia). We examined parallel datasets from organelles (mtDNA: atp1 gene, cpDNA: trnL intron, trnL-F intergenic spacer, rbcL gene) with respect to qualities relevant to the phylogenetic analysis of the Juncaceae. The main aim of our work was to produce a robust phylogeny of the Juncaceae validated by data from both organelles. Our data confirm the monophyly of the genus Luzula, but do not provide support for monophyly of the genus Juncus. The majority of taxa clustered within two subgenera, Agathryon and Juncus, morphologically supported by the presence or absence of bracteoles and cymose or racemose inflorescences, respectively. The subgenus Juncus is divided into two separate clades, the first closely related to the subgenus Agathryon and the second in the most basal part of the tree. Moreover, small South American genera clustered together with Juncus sect. Graminifolii and also with Juncus sect. Juncus. In fact, comparison of results from separate analyses of mitochondrial and plastome genes demonstrates that the general resolution of main topology of the atp1 tree is similar to the separate rbcL tree; the genus Juncus is better resolved, but the genus Luzula remains mainly polytomic.     

Cohesin axis maturation and presence of RAD51 during first meiotic prophase in a true bug

We have analyzed in a true bug, Graphosoma italicum (Pentatomidae, Hemiptera), the temporal and functional relationships between recombination events, synapsis progression, and SMC1α and SMC3 cohesin axis maturation throughout the male first meiotic prophase. The localization of the histone variant histone H3 trimethylated at lysine 9 at chromosome ends has allowed us to determine the association of these heterochromatic domains through prophase I stages. Results highlighted that cohesins provide to be good markers for synapsis progression since the formation, morphology, and development of the SMC1α and SMC3 cohesin axes resemble the synaptonemal complex dynamics and, also, that in this species the initiation of recombination precedes synapsis. In addition, we have carried out an accurate cytological characterization of the diffuse stage, which takes place after pachytene, and also analyzed the presence of the cohesin subunits, SMC1α and SMC3, and the recombinase RAD51 at this stage. The mechanisms underlying the absence of SMC1α and SMC3 axes from the diffuse stage onwards are discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Head and neck squamous cancer stromal fibroblasts produce growth factors influencing phenotype of normal human keratinocytes

Epithelial–mesenchymal interaction between stromal fibroblasts and cancer cells influences the functional properties of tumor epithelium, including the tumor progression and spread. We compared fibroblasts prepared from stroma of squamous cell carcinoma and normal dermal fibroblasts concerning their biological activity toward normal keratinocytes assessed by immunocytochemistry and profiling of gene activation for growth factors/cytokines by microarray chip technology. IGF-2 and BMP-4 were determined as candidate factors responsible for tumor-associated fibroblast activity that influences normal epithelia. This effect was confirmed by addition of recombinant IGF-2 and BMP4, respectively, to the culture medium. This hypothesis was also verified by inhibition experiments where blocking antibodies were employed in the medium conditioned by cancer-associated fibroblast. Presence of these growth factors was also detected in tumor samples.     

Tyrosine 39 of GH13 <Emphasis Type="Italic">α</Emphasis>-amylase from <Emphasis Type="Italic">Thermococcus hydrothermalis</Emphasis> contributes to its thermostability

The presented work is focused on the naturally thermostable α-amylase from the archaebacterium Thermococcus hydrothermalis. From the evolutionary point of view, the archaeal α-amylases are most closely related to plant α-amylases. In a wider sense, especially when the evolutionary trees are based on the less conserved part of their amino acid sequences (e.g. domain C succeeding the catalytic TIM-barrel), also the representatives of bacterial liquefying (Bacillus licheniformis) and saccharifying (Bacillus subtilis) α-amylases as well as the one from Thermotoga maritima should be included into the relatedness with the archaeal and plant α-amylases. Based on the bioinformatics analysis of the α-amylase from T. hydrothermalis, the position of tyrosine 39 (Y16 if the putative 23-residue long signal peptide is considered) was mutated to isoleucine (present in the α-amylase from T. maritima) by the in vitro mutagenesis. The biochemical characterization of the wild-type α-amylase and its Y39I mutant revealed that: (i) the specific activity of both enzymes was approximately equivalent (0.55 ± 0.13 U/mg for the wild-type and 0.52 ± 0.15 U/mg for the Y39I); (ii) the mutant exhibited decreased temperature optimum (from 85°C for the wild-type to 80°C for the Y39I); and (iii) the pH optimum remained the same (pH 5.5 for both enzymes). The remaining activity of the α-amylases was also tested by one-hour incubation at 80°C, 85°C, 90°C and 100°C. Since the wild-type α-amylase lost only 13% of its activity after one-hour incubation at the highest tested temperature (100°C), whereas 27% decrease was seen for the mutant Y39I under the same conditions, it is possible to conclude that the position of tyrosine 39 could contribute to the thermostability of the α-amylase from T. hydrothermalis.     

Bringing the hospital to the patient: first treatment of stroke patients at the emergency site

Background

Early treatment with rt-PA is critical for favorable outcome of acute stroke. However, only a very small proportion of stroke patients receive this treatment, as most arrive at hospital too late to be eligible for rt-PA therapy.

Methods and Findings

We developed a “Mobile Stroke Unit”, consisting of an ambulance equipped with computed tomography, a point-of-care laboratory system for complete stroke laboratory work-up, and telemedicine capabilities for contact with hospital experts, to achieve delivery of etiology-specific and guideline-adherent stroke treatment at the site of the emergency, well before arrival at the hospital. In a departure from current practice, stroke patients could be differentially treated according to their ischemic or hemorrhagic etiology even in the prehospital phase of stroke management. Immediate diagnosis of cerebral ischemia and exclusion of thrombolysis contraindications enabled us to perform prehospital rt-PA thrombolysis as bridging to later intra-arterial recanalization in one patient. In a complementary patient with cerebral hemorrhage, prehospital diagnosis allowed immediate initiation of hemorrhage-specific blood pressure management and telemedicine consultation regarding surgery. Call-to-therapy-decision times were 35 minutes.

Conclusion

This preliminary study proves the feasibility of guideline-adherent, etiology-specific and causal treatment of acute stroke directly at the emergency site.     

The combined luminol/isoluminol chemiluminescence method for differentiating between extracellular and intracellular oxidant production by neutrophils

To address the question why isoluminol, but not luminol, failed to detect oxidants produced intracellularly, differences between these luminophores were investigated with respect to physicochemical parameters and the character of chemiluminescence signal. Our results showed the isoluminol molecule to be more polar, more hydrophilic and possessing lower ability to form intramolecular bonds than the luminol molecule. Therefore, isoluminol: (i) only slightly pervaded biological membranes; (ii) depended essentially on extracellular peroxidase; (iii) did not produce chemiluminescence in the presence of extracellular scavengers; and (iv) it could be considered a specific detector of extracellular radicals. On the other hand, the physicochemical parameters of luminol and partial resistance of its chemiluminescence to the effect of extracellular inhibitors proved the lipo/hydrophilic character of this luminophore and thus its ability to interact with radicals both outside and inside of cells. The luminol chemiluminescence measured in the presence of extracellular scavengers and the isoluminol chemiluminescence were used with the intention to differentiate the effects of two antihistamine drugs on intra- and extracellular radical formation. In activated human neutrophils, brompheniramine inhibited the extracellular and potentiated the intracellular part of chemiluminescence signal, whereas a reducing effect of loratadine was observed in both compartments.     

Inhibitory effect of stobadine on FMLP-induced chemiluminescence in human whole blood and isolated polymorphonuclear leukocytes.

The chemiluminescence (CL) technique with luminol and isoluminol was used to characterize the effect of stobadine on reactive oxygen metabolites (ROM) generation in human whole blood and in isolated polymorphonuclear leukocytes (PMNL) stimulated with N-formyl-methionyl-leucyl-phenyl-alanine (FMLP). In whole blood and in isolated PMNL, stobadine in the concentrations of 1, 10 and 100 micromol/L significantly inhibited the CL signal after FMLP, which activated predominantly extracellular generation of ROM. The same concentrations of stobadine were effective on CL in a cell-free system. On the other hand, myeloperoxidase (MPO) liberation was decreased by stobadine only in the concentration of 100 micromol/L. The results showed stobadine to act as a potent inhibitor/scavenger of extracellularly produced ROM in human PMNL and indicated interference of stobadine with ROM as well as with signalling events resulting in NADPH-oxidase activation and MPO liberation.     

Disruption of the<Emphasis Type="Italic">RAD51</Emphasis> gene sensitizes<Emphasis Type="Italic">S. cerevisiae</Emphasis> cells to the toxic and mutagenic effects of hydrogen peroxide

The RAD51 gene was disrupted in three different parental wild-type strains to yield three rad51 null strains with different genetic background. The rad51 mutation sensitizes yeast cells to the toxic and mutagenic effects of H2O2, suggesting that Rad51-mediated repair, similarly to that of RecA-mediated, is relevant to the repair of oxidative damage in S. cerevisiae. Moreover, pulsed-field gel electrophoresis analysis demonstrated that increased sensitivity of the rad51 mutant to H2O2 is accompanied by its decreased ability to repair double-strand breaks induced by this agent. Our results show that ScRad51 protects yeast cells from H2O2-induced DNA double-strand breakage.