Proteomic analysis of complexes formed by human topoisomerase I

Human topoisomerase I is a nuclear enzyme that catalyses DNA relaxation and phosphorylation of SR proteins. Topoisomerase I participates in several protein-protein interactions. We performed a proteomic analysis of protein partners of topoisomerase I. Two methods were applied to proteins of the nuclear extract of HeLa cells: a co-immunoprecipitation and an affinity chromatography combined with mass spectrometry. Complexes formed by topoisomerase I with its protein partners were immunoprecipitated by scleroderma anti-topoisomerase I antibodies. To identify binding sites for the protein partners, baits corresponding to fragments of topoisomerase I were constructed and used in the affinity chromatography. The N-terminal domain and the cap region of the core domain appeared to be the main regions that bound proteins. We identified 36 nuclear proteins that were associated with topoisomerase I. The proteins were mainly involved in RNA metabolism. We found 29 new and confirmed 7 previously identified protein partners of topoisomerase I. More than 40% proteins that associate with the cap region contain two closely spaced RRM domains. Docking calculations identified the RRM domains as a possible site for the interaction of these proteins with the cap region.     

Consumer Risk Assessment Associated with Intake of Pesticide Residues in Food of Plant Origin from the Retail Market in Poland

The aim of this study was to characterize short- and long-term risk for consumers associated with dietary intake of pesticide residues in fruits, vegetables, and other foodstuffs available on the Polish market based on 2010–2013 official surveillance results. Among 779 samples collected from 2010 to 2013 no pesticide residue was found in 39.7% samples while 58.5% contained residues at or below the EU Maximum Residue Levels (MRLs). Non-compliances (residues above the respective MRLs) were found in 14 samples (1.8%). Most of the estimated daily intakes were well below 1% of respective acceptable daily intake (ADI) values. The highest intake for children and adults was about 7% and 1.5% of ADI, respectively. For non-compliant results acute risk was characterized. Predicted short-term intakes for children and adults ranged from 0.7% to 425%, and from 0.2% to 100% of respective acute reference dose, respectively. Results of chronic risk characterization show that consumers in Poland are adequately protected; however, incidental cases where residue levels may potentially pose a threat to consumers’ health due to acute exposure cannot be excluded.     

Assessment of cartilage invasion in case of laryngeal cancer by means of longitudinal sectioning for histopathology – Clinical implications

AimThe aim of the study was to assess the accuracy of radiological diagnosis of laryngeal cartilage infiltration by histopathological examination of laryngeal specimen after total laryngectomy.BackgroundDespite the development of new medical technologies and significant clinical advances allowing early diagnosis and treatment of laryngeal cancer, mortality is still on the rise. Neoplastic infiltration of the laryngeal cartilages is the most common source of error in the assessment of cancer staging. Furthermore, cartilage invasion is listed as a contraindication to partial surgical techniques as well as radiotherapy.Materials and methodsThe study was carried out on 21 larynges following total laryngectomy. Before taking the decision to perform surgery, high-resolution CT scans were performed in all cases. An extended histopathological examination was conducted using a unique vertical cross-section of the whole larynx.ResultsPathology reported 2 cases of arytenoid cartilage invasion, 5 cases of cricoid cartilage invasion, 12 cases of thyroid cartilage penetration, 1 case of internal cortex invasion and 9 cases of extra-laryngeal spread. CT imaging identified 8 of 13 cases (61.5%) of pathologically proven invasion of thyroid cartilage and only 2 cases (2/9, 22%) of extra-laryngeal spread. According to CT results, arytenoid cartilage invasion was correctly identified in 2 cases, cricoid cartilage invasion in 4 (4/5, 80%). The positive predictive values for thyroid, cricoid and arytenoid cartilage invasion and penetration were 80%, 66.7% and 50%, respectively. In case of pre-laryngeal spread the positive predictive value was 100%.ConclusionDespite increasingly advanced methods involved in the diagnosis of laryngeal cancer, many discrepancies may be observed between the radiological and histopathological assessments. CT imaging has limitations especially in thyroid cartilage penetration and extra-laryngeal spread assessment in advanced laryngeal cancer cases. An extended histopathological examination, involving vertical cross-sections of the whole larynx is a very precise study that allows a precise determination of local cancer staging (T).     

Antiendotoxin activity of protegrin analog IB-367 alone or in combination with piperacillin in different animal models of septic shock

The therapeutic efficacy of protegrin peptide IB-367 was investigated in three rat models of septic shock: (i) rats injected intraperitoneally with 1mg Escherichia coli 0111:B4 lipopolysaccharide, (ii) rats given an intraperitoneal injection of 2 X 10(10) CFU of E. coli ATCC 25922, and (iii) rats in which intra-abdominal sepsis was induced via cecal ligation and puncture. All animals were randomized to receive parenterally isotonic sodium chloride solution, 1mg/kg of IB-367, 60mg/kg piperacillin and 1mg/kg of IB-367 plus 60mg/kg piperacillin. The peptide demonstrated lower level of antimicrobial activity than piperacillin, nevertheless it exhibited the dual properties of antimicrobial and antiendotoxin agent. Finally IB-367 and piperacillin association showed to be the most effective therapeutic approach.     

Fungi inhabiting knotwood of Pinus sylvestris infected by Porodaedalea pini

Abundance and diversity of fungi in naturally formed knots of Pinus sylvestris affected by Porodaedalea pini were investigated. Samples were taken from trees that were (i) affected, with internal heartwood decay and no conks, (ii) affected, with internal heartwood decay and conks and (iii) controls. The Illumina sequencing technology was used for amplification of DNA, sequencing and analysis. In total, 566,279 raw sequences were obtained from six samples. Sequences included 74% of culturable and 8.4% of non‐culturable fungi and 17.6% of organisms with no reference sequences in NCBI. Abundance of organisms in knotwood, measured as number of OTUs, ranged from 36,272 (29,506 for fungi) to 178,535 (177,484 for fungi) and differed significantly between two trees in a stand and between stands. The highest and lowest average number of fungal OTUs occurred in infected trees with no conks and in trees with conks, respectively. Number of taxa ranged from 171 to 415 and often differed significantly between two trees in one stand and between stands. Greatest diversity occurred in control trees. The number of fungal taxa shared by two trees in one stand was 67–152 and that shared by two stands was 51–141. The majority of fungi were Ascomycota. Those most common in pines affected by P. pini were Coniochaeta hoffmannii and C. fodinicola (19.65%–59.92%). Infundichalara microchona, Leotiomycetes spp. and Rhinocladiella atrovirens were also present. Another common species, Lecanora conizaeoides, occurred most often in control trees (0.30%–8.82%). Porodaedalea pini was detected only sporadically. Non‐culturable fungi were most frequent in the control trees. The greater average abundance and smaller average diversity of fungi in knots of trees infected by P. pini suggest that the pathogen successfully competes with some fungal species and does not inhibit the growth of survivors. Some fungi detected may contribute to production of natural biocides.     

Subtypes of cytotoxic lymphocytes and natural killer cells infiltrating cancer nests correlate with prognosis in patients with vulvar squamous cell carcinoma

Objective

Adaptive immune effectors do not influence prognosis in vulvar squamous cell carcinoma (vSCC). Therefore, we tried to clarify the prognostic role of innate immunity and granzyme B-dependent cytotoxicity as defined by intratumoral infiltrates of natural killer cells (CD56+) and lymphocytes expressing granzyme B (GrB+).

Methods

We analyzed 76 primary vSCCs and 35 lymph node metastases that were obtained from 76 patients with a full clinical history. The distribution and density of GrB+ and CD56+ cells within cancer tissues were evaluated by immunohistochemistry and correlated with clinicopathological features, commonly recognized prognostic factors and overall survival (OS).

Results

CD56+ cells were mostly detected within the cancer nests, while GrB+ cells were predominant in the tumor stroma. Intraepithelial (IE) CD56+ infiltrates at the primary site were correlated with depth of invasion (r = 0.339, p = 0.003) and recurrence (r = 0.295, p = 0.011), while IE GrB+ infiltrates were correlated with tumor grade (r = 0.304, p = 0.009) and age (r = 0.333, p = 0.004). The primary cancer nests of metastatic patients were infiltrated more by intraepithelial (IE) CD56+ cells than were those of the non-metastatic patients (p = 0.05). The median OS was 41.16 months (range 1.7–98.43). High IE GrB+ infiltrates predicted longer OS among patients without metastases (p = 0.028). High IE CD56+ infiltrates were correlated with longer OS in metastatic cases (p = 0.009).

Conclusion

The combined cytotoxicity of innate and adaptive immune effectors infiltrating cancer nests (IE GrB+) predicts an improved clinical outcome among non-metastatic vSCC patients. The functional status of prognostic IE CD56+ infiltrates in immune escaped (metastatic) tumors requires further investigation.     

Xanthophyll-induced aggregation of LHCII as a switch between light-harvesting and energy dissipation systems

The xanthophyll cycle pigments, violaxanthin and zeaxanthin, present outside the light-harvesting pigment-protein complexes of Photosystem II (LHCII) considerably enhance specific aggregation of proteins as revealed by analysis of the 77 K chlorophyll a fluorescence emission spectra. Analysis of the infrared absorption spectra in the Amide I region shows that the aggregation is associated with formation of intermolecular hydrogen bonding between the alpha helices of neighboring complexes. The aggregation gives rise to new electronic energy levels, in the Soret region (530 nm) and corresponding to the Q spectral region (691 nm), as revealed by analysis of the resonance light scattering spectra. New electronic energy levels are interpreted in terms of exciton coupling of protein-bound photosynthetic pigments. The energy of the Q excitonic level of chlorophyll is not high enough to drive the light reactions of Photosystem II but better suited to transfer excitation energy to Photosystem I, which creates favourable energetic conditions for the state I-state II transition. The lack of fluorescence emission from this energy level, at physiological temperatures, is indicative of either very high thermal energy conversion rate or efficient excitation quenching by carotenoids. Chlorophyll a fluorescence was quenched up to 61% and 34% in the zeaxanthin- and violaxanthin-containing samples, respectively, as compared to pure LHCII. Enhanced aggregation of LHCII, observed in the presence of the xanthophyll cycle pigments, is discussed in terms of the switch between light-harvesting and energy dissipation systems.     

Insights into Chi recognition from the structure of an AddAB-type helicase-nuclease complex

In bacterial cells, processing of double-stranded DNA breaks for repair by homologous recombination is dependent upon the recombination hotspot sequence Chi and is catalysed by either an AddAB- or RecBCD-type helicase-nuclease. Here, we report the crystal structure of AddAB bound to DNA. The structure allows identification of a putative Chi-recognition site in an inactivated helicase domain of the AddB subunit. By generating mutant protein complexes that do not respond to Chi, we show that residues responsible for Chi recognition are located in positions equivalent to the signature motifs of a conventional helicase. Comparison with the related RecBCD complex, which recognizes a different Chi sequence, provides further insight into the structural basis for sequence-specific ssDNA recognition. The structure suggests a simple mechanism for DNA break processing, explains how AddAB and RecBCD can accomplish the same overall reaction with different sets of functional modules and reveals details of the role of an Fe-S cluster in protein stability and DNA binding.     

Strong light-induced reorganization of pigment-protein complexes of thylakoid membranes in rye (spectroscopic study)

The supramolecular reorganization of LHCII complexes within the thylakoid membrane in Secale cereale leaves under low and high light condition was examined. Rye seedlings were germinated hydroponically in a climate chamber with a 16 h daylight photoperiod, photosynthetic photon flux density (PPFD) of 150 μmol m⁻² s⁻¹ and 24/16 °C day/night temperature. The influence of pre-illumination of the plants with high light intensity on the PSII antenna complexes was studied by comparison of the structure and function of the LHCII complexes and organization of thylakoid membranes isolated from 10-day-old plants illuminated with low (150 μmol m⁻² s⁻¹) or high (1200 μmol m⁻² s⁻¹) light intensity. Aggregated and trimeric with monomeric forms of LHCII complexes were separated from the whole thylakoid membranes using non-denaturing electrophoresis. Analyses of fluorescence emission spectra of these different LHCII forms showed that the monomer was the most effective aggregating antenna form. Moreover, photoprotection connected with LHCII aggregation was more effective upon LHCII monomers in comparison to trimer aggregation. Light stress induced specific organization of neighboring LHCII complexes, causing an increase in fluorescence yield of the long-wavelength bands (centered at 701 and 734 nm). The changes in the organization of the thylakoid membrane under light stress, observed by analysis of absorbance spectra obtained by Fourier transform infrared spectroscopy, also indicated light-induced LHCII aggregation.