Effect of climate change on sporulation of the teleomorphs of <Emphasis Type="Italic">Leptosphaeria</Emphasis> species causing stem canker of brassicas

Leptosphaeria maculans and L. biglobosa are closely related sibling fungal pathogens that cause phoma leaf spotting, stem canker (blackleg) and stem necrosis of oilseed rape (Brassica napus). The disease is distributed worldwide, and it is one of the main causes of considerable decrease in seed yield and quality. Information about the time of ascospore release at a particular location provides important data for decision making in plant protection, thereby enabling fungicides to be used only when necessary and at optimal times and doses. Although the pathogens have been studied very extensively, the effect of climate change on the frequencies and distributions of their aerially dispersed primary inoculum has not been reported to date. We have collected a large dataset of spore counts from Poznan, located in central-west part of Poland, and studied the relationships between climate and the daily concentrations of airborne propagules over a period of 17 years (1998–2014). The average air temperature and precipitation for the time of development of pseudothecia and ascospore release (July–November), increased during the years under study at the rates of 0.1 °C and 6.3 mm per year. The day of the year (DOY) for the first detection of spores, as well as the date with maximum of spores, shifted from 270 to 248 DOY, and from 315 to 265 DOY, respectively. The acceleration of the former parameter by 22 days and the latter by 50 days has great influence on the severity of stem canker of oilseed rape.     

Classification of plasmid vectors using replication origin, selection marker and promoter as criteria

Although plasmid DNA vectors have been extensively applied in biotechnology, there is still a lack of standard plasmid vector classification. Here, we propose a classification method for commonly used plasmid vectors. Plasmid vectors were classified into different classes based on their replication origin, selection marker and promoter information. The replication origins of plasmid vectors were classified as: prokaryotic replication origin, eukaryotic replication origin and viral replication origin. Selection markers of plasmid vectors were mainly classified as ampicillin, kanamycin, neomycin, chloramphenicol, gentamycin, tetracycline, erythromycin, streptomycin, vancomycin and spectinomycin resistance gene markers. Promoter sequences were also classified as prokaryotic, eukaryotic and viral promoters. Finally, the nomenclature of common plasmid vectors has three determinants. We believe that the classification of plasmid vectors can provide useful information for researchers employing molecular cloning procedures. A web service of the plasmid classification was established and it is available from http://www.computationalmedicalbiology.org/plasclas.aspx.     

Knock-down of protein phosphatase 2A subunit B’γ promotes phosphorylation of CALRETICULIN 1 in Arabidopsis thaliana

Different types of plant pathogens may cause enormous losses in agriculture and also have an ecological impact in the nature. On molecular level, disease resistance is acquired through the action of tightly interconnected signaling pathways that may induce highly specific immune reactions in plant cells. Controlled protein dephosphorylation through protein phosphatase 2A activity is emerging as a crucial mechanism that regulates diverse signaling events in plants. PP2A is predominantly trimeric, and consists of a catalytic subunit, a scaffold subunit A, and a variable regulatory subunit B, which determines the target specificity of the PP2A holoenzyme.¹ Recently, we uncovered a specific role for a regulatory subunit B’γ of PP2A as a negative regulator of immune reactions in Arabidopsis thaliana (hereafter Arabidopsis).² Knock-down pp2a-b’γ mutants show constitutive activation of defense related genes, imbalanced antioxidant metabolism and premature disintegration of chloroplasts upon ageing. Proteomic analysis of soluble leaf extracts further revealed that the constitutive defense response in pp2a-b’γ leaves associates with increased levels of Cu/Zn superoxide dismutase, aconitase as well as components of the methionine-salvage pathway, suggesting PP2A-B’γ modulates methionine metabolism in leaves.     

A RecA Protein Surface Required for Activation of DNA Polymerase V

DNA polymerase V (pol V) of Escherichia coli is a translesion DNA polymerase responsible for most of the mutagenesis observed during the SOS response. Pol V is activated by transfer of a RecA subunit from the 3''-proximal end of a RecA nucleoprotein filament to form a functional complex called DNA polymerase V Mutasome (pol V Mut). We identify a RecA surface, defined by residues 112-117, that either directly interacts with or is in very close proximity to amino acid residues on two distinct surfaces of the UmuC subunit of pol V. One of these surfaces is uniquely prominent in the active pol V Mut. Several conformational states are populated in the inactive and active complexes of RecA with pol V. The RecA D112R and RecA D112R N113R double mutant proteins exhibit successively reduced capacity for pol V activation. The double mutant RecA is specifically defective in the ATP binding step of the activation pathway. Unlike the classic non-mutable RecA S117F (recA1730), the RecA D112R N113R variant exhibits no defect in filament formation on DNA and promotes all other RecA activities efficiently. An important pol V activation surface of RecA protein is thus centered in a region encompassing amino acid residues 112, 113, and 117, a surface exposed at the 3''-proximal end of a RecA filament. The same RecA surface is not utilized in the RecA activation of the homologous and highly mutagenic RumA''₂B polymerase encoded by the integrating-conjugative element (ICE) R391, indicating a lack of structural conservation between the two systems. The RecA D112R N113R protein represents a new separation of function mutant, proficient in all RecA functions except SOS mutagenesis.     

Long-term changes in abundance of bats as revealed by their frequency in tawny owls’ diet

Percentage of bats in tawny owls’ diet was compared in three periods: I — before 1976, II — 1976–1992, III — 1993–2009, by using the published and unpublished material from Poland (only samples over 100 vertebrate prey items). This species of owl showed an opportunistic predation on bats and took them more frequently in periods of higher abundance. Before the mass use of toxic pesticides in Poland, in the period I bats constituted more than 2% of vertebrates in four out of five diet samples (median 2.4%). The lowest bat abundance occurred in Poland in the 1980s and resulted in the lowest percentage of bats taken by owls in the period II (n = 11, median 0.2%). Due to the recovery of bat populations in the period III, the percentage of bats in tawny owls’ diet increased (n = 23, median 0.7%). In large samples (over 200 vertebrate items, n = 21) collected in central and north-eastern Poland the percentage of bats increased from 1980 to 2009 (the estimated average value at the end of that period slightly exceeded 1%). Samples collected at the same five sites in 1975–1992 and again in 2000–2009, confirmed the increasing trend in percentage of bats captured by tawny owls noted in last years.     

Measurement verification of dose distributions in pulsed-dose rate brachytherapy in breast cancer

Aim

The aim of the study was to verify the dose distribution optimisation method in pulsed brachytherapy.

Background

The pulsed-dose rate brachytherapy is a very important method of breast tumour treatment using a standard brachytheraphy equipment. The appropriate dose distribution round an implant is an important issue in treatment planning. Advanced computer systems of treatment planning are equipped with algorithms optimising dose distribution.

Materials and methods

The wax-paraffin phantom was constructed and seven applicators were placed within it. Two treatment plans (non-optimised, optimised) were prepared. The reference points were located at a distance of 5 mm from the applicators’ axis. Thermoluminescent detectors were placed in the phantom at suitable 35 chosen reference points.

Results

The dosimetry verification was carried out in 35 reference points for the plans before and after optimisation. Percentage difference for the plan without optimisation ranged from −8.5% to 1.4% and after optimisation from −8.3% to 0.01%. In 16 reference points, the calculated percentage difference was negative (from −8.5% to 1.3% for the plan without optimisation and from −8.3% to 0.8% for the optimised plan). In the remaining 19 points percentage difference was from 9.1% to 1.4% for the plan without optimisation and from 7.5% to 0.01% for the optimised plan.No statistically significant differences were found between calculated doses and doses measured at reference points in both dose distribution non-optimised treatment plans and optimised treatment plans.

Conclusions

No statistically significant differences were found in dose values at reference points between doses calculated by the treatment planning system and those measured by TLDs. This proves the consistency between the measurements and the calculations.     

The molecular chaperone, ClpA, has a single high affinity peptide binding site per hexamer

Substrate recognition by Clp chaperones is dependent on interactions with motifs composed of specific peptide sequences. We studied the binding of short motif-bearing peptides to ClpA, the chaperone component of the ATP-dependent ClpAP protease of Escherichia coli in the presence of ATPgammaS and Mg2+ at pH 7.5. Binding was measured by isothermal titration calorimetry (ITC) using the peptide, AANDENYALAA, which corresponds to the SsrA degradation motif found at the C terminus of abnormal nascent polypeptides in vivo. One SsrA peptide was bound per hexamer of ClpA with an association constant (K(A)) of 5 x 10(6) m(-1). Binding was also assayed by changes in fluorescence of an N-terminal dansylated SsrA peptide, which bound with the same stoichiometry of one per ClpA hexamer (K(A) approximately 1 x 10(7) m(-1)). Similar results were obtained when ATP was substituted for ATPgammaS at 6 degrees C. Two additional peptides, derived from the phage P1 RepA protein and the E. coli HemA protein, which bear different substrate motifs, were competitive inhibitors of SsrA binding and bound to ClpA hexamers with K(A)' > 3 x 10(7) m(-1). DNS-SsrA bound with only slightly reduced affinity to deletion mutants of ClpA missing either the N-terminal domain or the C-terminal nucleotide-binding domain, indicating that the binding site for SsrA lies within the N-terminal nucleotide-binding domain. Because only one protein at a time can be unfolded and translocated by ClpA hexamers, restricting the number of peptides initially bound should avoid nonproductive binding of substrates and aggregation of partially processed proteins.     

Bone marrow angiogenesis and proliferation in B-cell chronic lymphocytic leukemia

OBJECTIVE: To assess angiogenesis and the proliferative activity of bone marrow in patients with chronic lymphocytic leukemia (CLL) in relation to the bone marrow infiltration pattern. STUDY DESIGN: Bone marrow samples were obtained by trephine biopsy from 46 patients with B-cell CLL (B-CLL). Infiltration pattern was diffuse in 20 patients and nondiffuse--i.e., nodular, interstitial or mixed--in the remaining 26 patients. Ten normal bone marrow samples were used as a control group. Studies were carried out by immunohistochemical staining of paraffin-embedded bone marrow samples. Angiogenesis was assessed in the zones of highest vascular density (hot spots), visualized by the expression of endothelial antigen CD34 and expressed as a number of microvessels per high-powerfield (hpf) (final magnification, 400x). Proliferative activity was estimated by the expression of nuclear protein Ki-67, cyclin A and mean number of nucleolar organizer regions (AgNORs). RESULTS: Microvessel density was higher in B-CLL marrow than in normal marrow (30.1 and 16.44 per hpf, respectively) and was higher in the diffuse than nondiffuse pattern (33.6 and 27.5 per hpf, respectively). B-CLL bone marrow also showed higher proliferative activity, as assessed by mean number of AgNORs, than did normal marrow (1.52 and 1.25, respectively) and a higher mean percentage of cyclin A-positive cells (7.5 and 6.8, respectively). In contrast, mean Ki-67 expression was similar in B-CLL and the control group. Mean AgNORs number, Ki-67 and cyclin A-positive cell percentage were significantly higher in B-CLL marrow with a diffuse as compared to nondiffuse involvement pattern (AgNORs, 1.75 and 1.35; cyclin A, 9.27% and 3.95%; Ki-67, 34.9% and 23.3%, respectively). CONCLUSION: Our results indicate enhancement of bone marrow angiogenesis in B-CLL and a relationship between microvessel density and the bone marrow infiltration pattern. The study points also to a possible relationship between the bone marrow infiltration pattern and proliferative activity of bone marrow cells.     

Human CD5 protects circulating tumor antigen-specific CTL from tumor-mediated activation-induced cell death

We previously characterized several tumor-specific T cell clones from PBL and tumor-infiltrating lymphocytes of a lung cancer patient with identical TCR rearrangements and similar lytic potential, but with different antitumor response. A role of the TCR inhibitory molecule CD5 to impair reactivity of peripheral T cells against the tumor was found to be involved in this process. In this report, we demonstrate that CD5 also controls the susceptibility of specific T cells to activation-induced cell death (AICD) triggered by the tumor. Using a panel of tumor-infiltrating lymphocytes and PBL-derived clones expressing different levels of CD5, our results indicate that T lymphocyte AICD in response to the cognate tumor is inversely proportional to the surface expression level of CD5. They also suggest a direct involvement of CD5 in this process, as revealed by an increase in tumor-mediated T lymphocyte AICD following neutralization of the molecule with specific mAb. Mechanistically, our data indicate that down-regulation of FasL expression and subsequent inhibition of caspase-8 activation are involved in CD5-induced T cell survival. These results provide evidence for a role of CD5 in the fate of peripheral tumor-specific T cells and further suggest its contribution to regulate the extension of CTL response against tumor.