PssP2 Is a Polysaccharide Co-Polymerase Involved in Exopolysaccharide Chain-Length Determination in Rhizobium leguminosarum

Production of extracellular polysaccharides is a complex process engaging proteins localized in different subcellular compartments, yet communicating with each other or even directly interacting in multicomponent complexes. Proteins involved in polymerization and transport of exopolysaccharide (EPS) in Rhizobium leguminosarum are encoded within the chromosomal Pss-I cluster. However, genes implicated in polysaccharide synthesis are common in rhizobia, with several homologues of pss genes identified in other regions of the R. leguminosarum genome. One such region is chromosomally located Pss-II encoding proteins homologous to known components of the Wzx/Wzy-dependent polysaccharide synthesis and transport systems. The pssP2 gene encodes a protein similar to polysaccharide co-polymerases involved in determination of the length of polysaccharide chains in capsule and O-antigen biosynthesis. In this work, a mutant with a disrupted pssP2 gene was constructed and its capabilities to produce EPS and enter into a symbiotic relationship with clover were studied. The pssP2 mutant, while not altered in lipopolysaccharide (LPS), displayed changes in molecular mass distribution profile of EPS. Lack of the full-length PssP2 protein resulted in a reduction of high molecular weight EPS, yet polymerized to a longer length than in the RtTA1 wild type. The mutant strain was also more efficient in symbiotic performance. The functional interrelation between PssP2 and proteins encoded within the Pss-I region was further supported by data from bacterial two-hybrid assays providing evidence for PssP2 interactions with PssT polymerase, as well as glycosyltransferase PssC. A possible role for PssP2 in a complex involved in EPS chain-length determination is discussed.     

Abiotic Determinants of the Historical Buildings Biodeterioration in the Former Auschwitz II – Birkenau Concentration and Extermination Camp

The paper presents the results of a study conducted at the Auschwitz-Birkenau State Museum in Oświęcim on the occurrence of biodeterioration. Visual assessment of the buildings revealed signs of deterioration of the buildings in the form of dampness, bulging and crumbling plaster, and wood fiber splitting. The external surfaces, and especially the concrete strips and ground immediately adjoining the buildings, were colonized by bryophytes, lichens, and algae. These organisms developed most intensively close to the ground on the northern sides of the buildings. Inside the buildings, molds and bacteria were not found to develop actively, while algae and wood-decaying fungi occurred locally. The factors conducive to biological corrosion in the studied buildings were excessive dampness of structural partitions close to the ground and a relative air humidity of above 70%, which was connected to ineffective moisture insulation. The influence of temperature was smaller, as it mostly affected the quantitative composition of the microorganisms and the qualitative composition of the algae. Also the impact of light was not very strong, but it was conducive to algae growth.     

Differences between TNM classification and 2-[18F]FDG PET parameters of primary tumor in NSCLC patients

BackgroundThe aim of the study was to compare the TNM classification with 2-[¹⁸F]FDG PE T biological parameters of primary tumor in patients with NSCLC.Materials and methodsRetrospective analysis was performed on a group of 79 newly diagnosed NSCLC patients. PET scans were acquired on Gemini TF PET/CT scanner 60–70 min after injection of 2-[¹⁸F]FDG with the mean activity of 364 ± 75 MBq, with the area being examined from the vertex to mid-thigh. The reconstructed PET images were evaluated using MIM 7.0 Software for SUV_max, MTV and TLG values.ResultsThe analysis of the cancer stage according to TNM 8^th edition showed stage IA2 in 8 patients, stage IA3 — 6 patients, stage IB — 4 patients, IIA — 3 patients, 15 patients with stage IIB, stage IIIA — 17 patients, IIIB — 5, IIIC — 5, IVA in 7 patients and stage IVB in 9 patients. The lowest TLG values of primary tumor were observed in stage IA2 (11.31 ± 15.27) and the highest in stage IIIC (1003.20 ± 953.59). The lowest value of primary tumor in SUV_max and MTV were found in stage IA2 (6.8 ± 3.8 and 1.37 ± 0.42, respectively), while the highest SUV_max of primary tumor was found in stage IIA (13.4 ± 11.4) and MTV in stage IIIC (108.15 ± 127.24).ConclusionTNM stages are characterized by different primary tumor 2-[¹⁸F]FDG PET parameters, which might complement patient outcome.     

A cooperative mechanism drives budding yeast kinetochore assembly downstream of CENP-A

Kinetochores are megadalton-sized protein complexes that mediate chromosome–microtubule interactions in eukaryotes. How kinetochore assembly is triggered specifically on centromeric chromatin is poorly understood. Here we use biochemical reconstitution experiments alongside genetic and structural analysis to delineate the contributions of centromere-associated proteins to kinetochore assembly in yeast. We show that the conserved kinetochore subunits Ame1^CENP-U and Okp1^CENP-Q form a DNA-binding complex that associates with the microtubule-binding KMN network via a short Mtw1 recruitment motif in the N terminus of Ame1. Point mutations in the Ame1 motif disrupt kinetochore function by preventing KMN assembly on chromatin. Ame1–Okp1 directly associates with the centromere protein C (CENP-C) homologue Mif2 to form a cooperative binding platform for outer kinetochore assembly. Our results indicate that the key assembly steps, CENP-A recognition and outer kinetochore recruitment, are executed through different yeast constitutive centromere-associated network subunits. This two-step mechanism may protect against inappropriate kinetochore assembly similar to rate-limiting nucleation steps used by cytoskeletal polymers.     

Ascorbate Efflux as a New Strategy for Iron Reduction and Transport in Plants

Iron (Fe) is essential for virtually all living organisms. The identification of the chemical forms of iron (the speciation) circulating in and between cells is crucial to further understand the mechanisms of iron delivery to its final targets. Here we analyzed how iron is transported to the seeds by the chemical identification of iron complexes that are delivered to embryos, followed by the biochemical characterization of the transport of these complexes by the embryo, using the pea (Pisum sativum) as a model species. We have found that iron circulates as ferric complexes with citrate and malate (Fe(III)₃Cit₂Mal₂, Fe(III)₃Cit₃Mal₁, Fe(III)Cit₂). Because dicotyledonous plants only transport ferrous iron, we checked whether embryos were capable of reducing iron of these complexes. Indeed, embryos did express a constitutively high ferric reduction activity. Surprisingly, iron(III) reduction is not catalyzed by the expected membrane-bound ferric reductase. Instead, embryos efflux high amounts of ascorbate that chemically reduce iron(III) from citrate-malate complexes. In vitro transport experiments on isolated embryos using radiolabeled ⁵⁵Fe demonstrated that this ascorbate-mediated reduction is an obligatory step for the uptake of iron(II). Moreover, the ascorbate efflux activity was also measured in Arabidopsis embryos, suggesting that this new iron transport system may be generic to dicotyledonous plants. Finally, in embryos of the ascorbate-deficient mutants vtc2-4, vtc5-1, and vtc5-2, the reducing activity and the iron concentration were reduced significantly. Taken together, our results identified a new iron transport mechanism in plants that could play a major role to control iron loading in seeds.     

Bidirectional Modulation of Thermal and Chemical Sensitivity of TRPM8 Channels by the Initial Region of the N-terminal Domain

TRPM8, a nonselective cation channel activated by cold, voltage, and cooling compounds such as menthol, is the principal molecular detector of cold temperatures in primary sensory neurons of the somatosensory system. The N-terminal domain of TRPM8 consists of 693 amino acids, but little is known about its contribution to channel function. Here, we identified two distinct regions within the initial N terminus of TRPM8 that contribute differentially to channel activity and proper folding and assembly. Deletion or substitution of the first 40 residues yielded channels with augmented responses to cold and menthol. The thermal threshold of activation of these mutants was shifted 2 °C to higher temperatures, and the menthol dose-response curve was displaced to lower concentrations. Site-directed mutagenesis screening revealed that single point mutations at positions Ser-26 or Ser-27 by proline caused a comparable increase in the responses to cold and menthol. Electrophysiological analysis of the S27P mutant revealed that the enhanced sensitivity to agonists is related to a leftward shift in the voltage dependence of activation, increasing the probability of channel openings at physiological membrane potentials. In addition, we found that the region encompassing positions 40–60 is a key element in the proper folding and assembly of TRPM8. Different deletions and mutations within this region rendered channels with an impaired function that are retained within the endoplasmic reticulum. Our results suggest a critical contribution of the initial region of the N-terminal domain of TRPM8 to thermal and chemical sensitivity and the proper biogenesis of this polymodal ion channel.     

Oospore variation in <Emphasis Type="Italic">Nitella gracilis</Emphasis> and <Emphasis Type="Italic">Nitella mucronata</Emphasis> (Charales,Charophyceae) from Poland

The determination of the members of Charales (Charophyceae) is not easy. The main reason of this is a wide range of variation in vegetative characters within most of the species. Many features such as the extent of branchlet cortication, the degree of incrustation, length and number of bract cells, spine cells, stipulodes, diameter of the main axis and oospore size and color do not have universal taxonomic significance. In similar, many Nitella species characters such as: general habitus, branchlets and presence of the top segment (mucro), are helpful in determination, however they are often not developed. In this case, the features of oospore wall ornamentation revealed by light and scanning microscopy can be important taxonomic characters of the Charales. The present study shows interspecific variations of the oospore in two Nitella species from Poland which can be helpful in correct determination of both species. Some of the oospore features like number of striae and fossa width can not be used, as they appeared statistically unimportant, and cannot be used in distinguishing between both species. The presented results are the first detailed report of oospore wall ornamentation pattern from Poland for the species studied. Oospore features are presented in detail on the background of some additional tips and among them particularly: length and width of the oospore, ISI idex, width of fossa, width of striae and number of striae.     

Prognostic value of S-100 immunostaining in tumour cells of non-small cell lung cancer

S-100 protein expression is present in various malignant tissues, yet its prognostic relevance is debatable. The aim was to assess in non-small cell lung cancer (NSCLC) patients' prognostic value of S-100 protein considered alone or in relation with other variables. Tumour samples taken from 86 NSCLC patients during resection were assayed for S-100 protein expression with the use of polyclonal DAKO ZO311 antibody. S-100 expression was found in 32 cases (37%). Positive staining was not correlated with clinical characteristics including age, sex, pathology type of tumour, stage and cigarette smoking. There was a tendency for simultaneous expression of S-100 and P53 protein (p=0.06). A median survival rate for the entire group was 2.3 years (95% CI, 0.9-3.6 years). The median and 5-year survival of patients with positive staining for S-100 protein was 1.5 years and 25%, respectively, compared with 3.0 years and 35%, respectively, in the S-100 negative group (p=0.17). In the final model of a multivariate analysis, S-100 protein expression in tumour cells was associated with significantly decreased survival (p=0.005). S-100 protein expression in tumour cells seems to be an independent predictor of poor prognosis in NSCLC patients.