Sebastian Dorawa  Olesia Werbowy  Magdalena Plotka  Anna-Karina Kaczorowska  Joanna Makowska  Lukasz P. Kozlowski  Olafur H. Fridjonsson  Gudmundur O. Hreggvidsson  Arnthr Aevarsson  Tadeusz Kaczorowski 《International journal of molecular sciences》2022,23(14)

We present a structural and functional analysis of the DNA polymerase of thermophilic Thermus thermophilus MAT72 phage vB_Tt72. The enzyme shows low sequence identity (<30%) to the members of the type-A family of DNA polymerases, except for two yet uncharacterized DNA polymerases of T. thermophilus phages: φYS40 (91%) and φTMA (90%). The Tt72 polA gene does not complement the Escherichia coli polA⁻ mutant in replicating polA-dependent plasmid replicons. It encodes a 703-aa protein with a predicted molecular weight of 80,490 and an isoelectric point of 5.49. The enzyme contains a nucleotidyltransferase domain and a 3′-5′ exonuclease domain that is engaged in proofreading. Recombinant enzyme with His-tag at the N-terminus was overproduced in E. coli, subsequently purified by immobilized metal affinity chromatography, and biochemically characterized. The enzyme exists in solution in monomeric form and shows optimum activity at pH 8.5, 25 mM KCl, and 0.5 mM Mg²⁺. Site-directed analysis proved that highly-conserved residues D15, E17, D78, D180, and D184 in 3′-5′ exonuclease and D384 and D615 in the nucleotidyltransferase domain are critical for the enzyme’s activity. Despite the source of origin, the Tt72 DNA polymerase has not proven to be highly thermoresistant, with a temperature optimum at 55 °C. Above 60 °C, the rapid loss of function follows with no activity > 75 °C. However, during heat treatment (10 min at 75 °C), trehalose, trimethylamine N-oxide, and betaine protected the enzyme against thermal inactivation. A midpoint of thermal denaturation at T_m = 74.6 °C (ΔH_cal = 2.05 × 10⁴ cal mol⁻¹) and circular dichroism spectra > 60 °C indicate the enzyme’s moderate thermal stability.  相似文献   

    

Wolfram Jger  Lukasz Drobiec 《Mauerwerk》2006,10(6):252-257

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Bartlomiej Czerwinski  Lukasz RzeznikRobert Paruch  Barbara J. GarrisonZbigniew Postawa 《Nuclear instruments & methods in physics research. Section B, Beam interactions with materials and atoms》2011,269(14):1578-1581

Molecular dynamics computer simulations have been used to investigate the effect of the cluster size on the sputtering yield dependence on the impact angle. Ar₃₆₆ and Ar₂₉₅₃ cluster projectiles with 14.75 keV of incident energy are directed at the surface of a solid benzene crystal described by a coarse-grained representation at angles between 0° and 70°. It is observed that the shape of the angular dependence of sputtering efficiency is strongly affected by the cluster size. For the Ar₃₆₆ cluster, the sputtering yield only slightly increases with the impact angle, has a broad maximum around 40°, and decreases at larger angles. For the Ar₂₉₅₃ cluster, the yield strongly increases with the impact angle, has a maximum around 45° followed by a steep decrease at larger angles. For both investigated cluster projectiles the primary energy is deposited so close to the surface so that the sputtering efficiency only weekly benefits from the shift of the deposited energy profile toward the surface which occurs at larger impact angles. In this study, molecular dynamics computer simulations are used to probe the effect of the impact angle on the efficiency of ejection molecules emitted from solid benzene by 14.75 keV Ar₃₆₆ and Ar₂₉₅₃ clusters.  相似文献   

    

Adriana Mika  Lukasz Kaska  Justyna Korczynska  Agnieszka Mirowska  Piotr Stepnowski  Monika Proczko  Krzysztof Ratnicki‐Sklucki  Elzbieta Goyke  Tomasz Sledzinski 《European Journal of Lipid Science and Technology》2015,117(7):926-932

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Joanna Polanska  Malgorzata Plechawska  Monika Pietrowska  Lukasz Marczak 《Expert Systems》2012,29(3):216-231

The article presents a method of protein matrix‐assisted laser desorption and ionization‐time of flight (TOF) spectra analysis. The method performs peaks detection. Spectra are analysed with Gaussian mixture decomposition. The results obtained are used for peaks identification purposes. The concept of the method is that a single peak is represented by one Gaussian distribution. The expectation‐maximization algorithm and maximum likelihood rule are used for spectra processing. The analysis can be done for a set of spectra with use of the mean spectrum, or it may be performed for a single spectrum at a time. The number of mixture‐model components is estimated by the Bayesian information criterion. Before the main analysis, a few pre‐processing steps need to be done. Spectra should be subjected to calibration, normalization, denoising, baseline correction, etc. The aim of the work is to identify peptides in the analysed sample on the basis of the parameters of the mixture model and to find differences between spectra in the analysed set.  相似文献   

    

Agnieszka Kij  Anna Bar  Kamil Przyborowski  Bartosz Proniewski  Lukasz Mateuszuk  Agnieszka Jasztal  Anna Kieronska-Rudek  Brygida Marczyk  Karolina Matyjaszczyk-Gwarda  Anna Tworzydlo  Camilla Enggaard  Pernille B. Lrkegaard Hansen  Boye Jensen  Maria Walczak  Stefan Chlopicki 《International journal of molecular sciences》2021,22(16)

Angiotensin II (Ang II) induces hypertension and endothelial dysfunction, but the involvement of thrombin in these responses is not clear. Here, we assessed the effects of the inhibition of thrombin activity by dabigatran on Ang II-induced hypertension and endothelial dysfunction in mice with a particular focus on NO- and 20-HETE-dependent pathways. As expected, dabigatran administration significantly delayed thrombin generation (CAT assay) in Ang II-treated hypertensive mice, and interestingly, it prevented endothelial dysfunction development, but it did not affect elevated blood pressure nor excessive aortic wall thickening. Dabigatran’s effects on endothelial function in Ang II-treated mice were evidenced by improved NO-dependent relaxation in the aorta in response to acetylcholine in vivo (MRI measurements) and increased systemic NO bioavailability (NO₂⁻ quantification) with a concomitant increased ex vivo production of endothelium-derived NO (EPR analysis). Dabigatran treatment also contributed to the reduction in the endothelial expression of pro-inflammatory vWF and ICAM-1. Interestingly, the fall in systemic NO bioavailability in Ang II-treated mice was associated with increased 20-HETE concentration in plasma (UPLC-MS/MS analysis), which was normalised by dabigatran treatment. Taking together, the inhibition of thrombin activity in Ang II-induced hypertension in mice improves the NO-dependent function of vascular endothelium and normalises the 20-HETE-depedent pathway without affecting the blood pressure and vascular remodelling.  相似文献   

    

Lukasz Bozycki  Joanna Mroczek  Laurence Bessueille  Saida Mebarek  Ren Buchet  Slawomir Pikula  Agnieszka Strzelecka-Kiliszek 《International journal of molecular sciences》2021,22(8)

The mineralization process is initiated by osteoblasts and chondrocytes during intramembranous and endochondral ossifications, respectively. Both types of cells release matrix vesicles (MVs), which accumulate P_i and Ca²⁺ and form apatites in their lumen. Tissue non-specific alkaline phosphatase (TNAP), a mineralization marker, is highly enriched in MVs, in which it removes inorganic pyrophosphate (PP_i), an inhibitor of apatite formation. MVs then bud from the microvilli of mature osteoblasts or hypertrophic chondrocytes and, thanks to the action of the acto-myosin cortex, become released to the extracellular matrix (ECM), where they bind to collagen fibers and propagate mineral growth. In this report, we compared the mineralization ability of human fetal osteoblastic cell line (hFOB 1.19 cells) with that of osteosarcoma cell line (Saos-2 cells). Both types of cells were able to mineralize in an osteogenic medium containing ascorbic acid and beta glycerophosphate. The composition of calcium and phosphate compounds in cytoplasmic vesicles was distinct from that in extracellular vesicles (mostly MVs) released after collagenase-digestion. Apatites were identified only in MVs derived from Saos-2 cells, while MVs from hFOB 1.19 cells contained amorphous calcium phosphate complexes. In addition, AnxA6 and AnxA2 (nucleators of mineralization) increased mineralization in the sub-membrane region in strongly mineralizing Saos-2 osteosarcoma, where they co-localized with TNAP, whereas in less mineralizing hFOB 1.19 osteoblasts, AnxA6, and AnxA2 co-localizations with TNAP were less visible in the membrane. We also observed a reduction in the level of fetuin-A (FetuA), an inhibitor of mineralization in ECM, following treatment with TNAP and Ca channels inhibitors, especially in osteosarcoma cells. Moreover, a fraction of FetuA was translocated from the cytoplasm towards the plasma membrane during the stimulation of Saos-2 cells, while this displacement was less pronounced in stimulated hFOB 19 cells. In summary, osteosarcoma Saos-2 cells had a better ability to mineralize than osteoblastic hFOB 1.19 cells. The formation of apatites was observed in Saos-2 cells, while only complexes of calcium and phosphate were identified in hFOB 1.19 cells. This was also evidenced by a more pronounced accumulation of AnxA2, AnxA6, FetuA in the plasma membrane, where they were partly co-localized with TNAP in Saos-2 cells, in comparison to hFOB 1.19 cells. This suggests that both activators (AnxA2, AnxA6) and inhibitors (FetuA) of mineralization were recruited to the membrane and co-localized with TNAP to take part in the process of mineralization.  相似文献   

    

Marcin Kolasa  Grzegorz Galita  Ireneusz Majsterek  Ewa Kucharska  Katarzyna Czerczak  Joanna Wasko  Angelika Becht  Justyna Fraczyk  Anna Gajda  Lukasz Pietrzak  Lukasz Szymanski  Agnieszka Krakowiak  Zbigniew Draczynski  Beata Kolesinska 《International journal of molecular sciences》2021,22(24)

The aim of the research was to check whether it is possible to use fragments of type IV collagen to obtain, as a result of self-assembling, stable spatial structures that could be used to prepare new materials useful in regenerative medicine. Collagen IV fragments were obtained by using DMT/NMM/TosO⁻ as a coupling reagent. The ability to self-organize and form stable spatial structures was tested by the CD method and microscopic techniques. Biological studies covered: resazurin assay (cytotoxicity assessment) on BJ, BJ-5TA and C2C12 cell lines; an alkaline version of the comet assay (genotoxicity), Biolegend Legendplex human inflammation panel 1 assay (SC cell lines, assessment of the inflammation activity) and MTT test to determine the cytotoxicity of the porous materials based on collagen IV fragments. It was found that out of the pool of 37 fragments (peptides 1–33 and 2.1–2.4) reconstructing the outer sphere of collagen IV, nine fragments (peptides: 2, 4, 5, 6, 14, 15, 25, 26 and 30), as a result of self-assembling, form structures mimicking the structure of the triple helix of native collagens. The stability of spatial structures formed as a result of self-organization at temperatures of 4 °C, 20 °C, and 40 °C was found. The application of the MST method allowed us to determine the K_d of binding of selected fragments of collagen IV to ITGα1β1. The stability of the spatial structures of selected peptides made it possible to obtain porous materials based on their equimolar mixture. The formation of the porous materials was found for cross-linked structures and the material stabilized only by weak interactions. All tested peptides are non-cytotoxic against all tested cell lines. Selected peptides also showed no genotoxicity and no induction of immune system responses. Research on the use of porous materials based on fragments of type IV collagen, able to form stable spatial structures as scaffolds useful in regenerative medicine, will be continued.  相似文献   

    

Jakub Cieslak  Lukasz Wodas  Alicja Borowska  Jan Sadoch  Piotr Pawlak  Kamila Puppel  Beata Kuczynska  Mariusz Mackowski 《Journal of the science of food and agriculture》2017,97(7):2174-2181

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Judy England  Lukasz Dobbek  Brishan Finn Leeming  Angela M. Gurnell  Geraldene Wharton 《Water and Environment Journal》2019,33(3):378-389

The installation of large wood and sediment berms to narrow the overwide channel of the River Bulbourne, Hertfordshire, aimed to restore geomorphological processes, improve channel habitat diversity and increase the amenity value of the park in which the river is located. The Modular River Survey provides a framework and suite of tools for river managers and volunteers to monitor and assess restoration activities. Applying this technique to the River Bulbourne before and after restoration demonstrated that the works increased physical habitat and vegetation complexity. The restored section was narrowed, substrate composition changed and the range of in‐stream vegetation morphotypes increased. The initial slight improvement in riparian habitat complexity immediately following the restoration is expected to increase further over time as the riparian vegetation develops and the restored section of channel matures. A public perception and recreational use survey reviewed how visitor experience and use of the park changed following restoration.  相似文献