The molecular features of non-peptidic nucleophilic substrates and acceptor proteins determine the efficiency of sortagging.

Sortase A-mediated ligation (SML) or "sortagging" has become a popular technology to selectively introduce structurally diverse protein modifications. Despite the great progress in the optimization of the reaction conditions and design of miscellaneous C- or N-terminal protein modification strategies, the reported yields of conjugates are highly variable. In this study, we have systematically investigated C-terminal protein sortagging efficiency using a combination of several rationally selected and modified acceptor proteins and a panel of incoming surrogate non-peptidic amine nucleophile substrates varying in the structural features of their amino linker parts and cargo molecules.

Synthetic Amine Linkers for Efficient Sortagging.

Enzymatic site-specific bioconjugation techniques, in particular sortase-mediated ligation, are increasingly used to generate conjugated proteins for a wide array of applications. Extension of the utility and practicality of sortagging for diverse purposes is critically dependent on further improvement of the efficiency of sortagging reactions with a wider structural variety of substrates. We present a comprehensive comparative mass spectrometry screening study of synthetic nonpeptidic incoming amine nucleophile substrates of Sortase A enzyme.

Genotoxic Effects of Exposure to Water-Soluble Fraction of Diesel Fuel in Sand Dollar Gametes.

Pollution of marine areas with oil and oil products is steadily growing. As part of this connection, the study of the impact of petroleum hydrocarbons on marine hydrobionts is an urgent issue of modern ecotoxicology. In our study, the genotoxic effect of the water-soluble fraction of diesel fuel at different concentrations on the gametes of the sand dollar was evaluated.

Exposure of adult sand dollars (Scaphechinus mirabilis) (Agassiz, 1864) to copper oxide nanoparticles induces gamete DNA damage.

The increase in the number of products containing nanoparticles (NPs) poses a real threat to the environment. Recently, more evidence has been added to predictive models about the presence of NPs in various natural and anthropogenic systems. The acute toxicity of most aquatic NPs has now been well documented.

Strained contacts with the cell membrane may influence ligand affinity to G protein coupled receptors: a case of free fatty acid receptor 1 agonists.

A set of 1,3,4-thiadiazole-2-carboxamides bearing a substituted biphenyl in the amide portion was synthesised and tested for agonistic activity towards free fatty acid receptor 1 (FFA1). The observed activity trends were impossible to rationalised based solely on the docking energy scores of Glide SP. On the contrary, when the phospholipid cell membrane bilayer was reconstructed around FFA1, it became apparent that inactive compounds displayed significant strained contacts with the membrane while for active compounds the strain was noticeably lower.

Exploration of the nitrogen heterocyclic periphery around the core of the advanced FFA1 agonist fasiglifam (TAK-875).

Three types of heterocyclic moieties-piperidines fused to a heteroaromatic moiety-were explored as potential periphery motifs for the pharmacophoric core of fasiglifam (TAK-875), with fasiglifam being the most advanced agonist of free fatty acid receptor 1, a promising target for therapeutic intervention in type 2 diabetes. Several observed structure-activity relationship trends were corroborated by in silico docking results. Balanced selection based on potency and Caco-2 permeability advanced six compounds to cellular efficacy tests (glucose-stimulated insulin secretion in rat insulinoma INS1E cells).

Sea Urchin Embryogenesis as Bioindicators of Marine Pollution in Impact Areas of the Sea of Japan/East Sea and the Sea of Okhotsk.

The embryogenesis of the sea urchin sand dollar Scaphechinus mirabilis was used as bioindicators of seawater quality from the impact areas of the Sea of Japan/East Sea (Peter the Great Bay) and the Sea of Okhotsk (northwestern shelf of Sakhalin Island and western shelf of Kamchatka Peninsula). Fertilization membrane formation, first cleavage, blastula formation, gastrulation, and 2-armed and 4-armed pluteus formation have been analyzed and a number of abnormalities were calculated. Number of embryogenesis anomalies in sand dollar larvae exposed to sea water from different stations in Peter the Great Bay corresponds to pollution level at each area.

Involvement of human beta-defensin-2 in regulation of malignant potential of cultured human melanoma cells.

Background And Aim: Human beta-defensin-2 (hBD-2) is an antimicrobial cationic peptide capable to control human carcinoma cell growth via cell cycle regulation. The present study was aimed on determination of hBD-2 influence on the growth patterns and malignant potential of cultured human melanoma cells.

Methods: The study was performed on cultured human melanoma cells of mel Z and mel Is lines treated with recombinant hBD-2 (rec-hBD-2); cell viability, proliferation, cell cycle distribution, and anchorage-independent growth were analyzed using MTT test, direct cell counting, flow cytometry, and colony forming assay respectively.

Biologic activities of recombinant human-beta-defensin-4 toward cultured human cancer cells.

Aim: The aim of the study was in vitro analysis of biological activity of recombinant human beta-defensin-4 (rec-hBD-4).

Methods: hBD-4 cDNA was cloned into pGEX-2T vector, and recombinant plasmid was transformed into E. coli BL21(DE3) cells.

Human beta-defensin-2 controls cell cycle in malignant epithelial cells: in vitro study.

Aim: In the present research we analyze the mechanism of human beta-defensin-2 (hBD-2) influence on cultured malignant epithelial cell growth.

Materials And Methods: The analysis of a concentration-dependent effect of recombinant hBD-2 (rec-hBD-2) on cell growth patterns and cell cycle distribution has been performed in vitro with 2 cell lines (human lung adenocarcinoma A549 cells and human epidermoid carcinoma A431 cells) using MTT test, flow cytometry and direct cell counting. To study intracellular localization of hBD-2 immunocytofluorescent and immunocytochemical analyses were applied, and effect of hBD-2 on signal cascades involved in cell cycle regulation has been studied by Western blotting.

Influence of water pollution on the growth and pigment concentration of the microalgae Phaeodactylum tricornutum Bohlin (Bacillariophyta).

The aim of the work was to study the influence of the water taken from one of the most polluted parts of the Peter the Great Bay (the Japan Sea), the Nakhodka Bay, on the growth and chlorophyll a concentration in the cells of microalga P. tricornutum Bohlin (Bacillariophyta). The estimation of the dynamics of cell number growth and chlorophyll a concentration in the cells of microalga grown in the water from the Nakhodka Bay was made.

Immunohistochemical analysis of beta-defensin-2 expression in human lung tumors.

Aim: The present research was directed on analysis of the expression patterns of human beta-defensin-2 (hBD-2) in human lung tumors.

Materials And Methods: Specimens of surgically resected human lung tumors (n = 31) of different histological type (1 case of small cell lung cancer, and 30 cases of non-small cell lung cancer (1 case of clear cell carcinoma, 9 cases of squamous cell carcinoma (SCC), and 20 cases of adenocarcinoma (AC)) were analyzed for expression of hBD-2 with the use of immunohistochemical analysis.

Results: Immunohistochemical analysis has revealed that all lung tumor samples independently on their histological type express hBD-2 peptide, however at different levels (from < 5% to 100% cells).

Administration of vitamin D3 improves antimetastatic efficacy of cancer vaccine therapy of Lewis lung carcinoma.

Aim: To analyze antitumor efficacy of experimental cancer vaccine therapy combined with introduction of vitamin D3 (VD3) for treatment of Lewis lung carcinoma (3LL).

Materials And Methods: Cancer vaccines composed from recombinant murine beta-defensin-2 (mBD-2) and 3LL cell lysate, or DNA, coding for mBD-2-Muc1 fusion construct cloned in pcDNA3+ vector, were prepared and used for intradermal vaccination. Experimental cancer vaccines introduced i.

Expression patterns of murine beta-defensin-2 mRNA in Lewis lung carcinoma cells in vitro and in vivo.

Aim: To evaluate the anti-tumor activity of murine beta-defensin-2 (mBD-2) expression in vitro and in vivo.

Materials And Methods: Based on pcDNA3 vector, constructs containing mBD-2 cDNA coding mature defensin molecule (pcDNA3-mBD2), and Igk-mBD-2 insertion, coding secretory sequence plus mature defensin molecule (pcDNA3-Igk-mBD-2) were generated. Lewis lung carcinoma (3LL) cells were transfected in vitro with these plasmids and with blank pcDNA3 vector, and the proliferative rate and clonogenic ability of obtained cell lines cultivated in vitro were analyzed using (3)H-incorporation technique and colony formation in semi-soft medium, respectively.

Expression of human beta-defensins-1, 2 and 4 mRNA in human lung tumor tissue: a pilot study.

Aim: To analyze the patterns of human beta-defensin-1, 2, 4 (hBDs) expression in human lung tumors.

Materials And Methods: Tissue samples of surgically resected human lung tumors (squamous cell carcinoma (SCC), n=10; adenocarcinoma (AC), n=10) paired with conditionally normal tissue samples were analyzed for expression of hBD-1, 2, 4 mRNA by semiquantitative RT-PCR.

Results: In a number of studied lung cancer tissue samples, overexpression of defensin mRNA was registered: hBD-1 mRNA (50% of SCC and 60% AC), hBD-2 mRNA (60% of SCC and 50% of AC) or hBD-4 (40% of SCC and 20% AC).

Regulated expression of human beta-defensin-2 leads to altered phenotype and growth patterns of cultured human embryonal kidney cells.

Aim: To create cell line with regulated expression of human beta-defensin-2 (hBD-2) and evaluate the influence of expressed peptide on its phenotypic and growth patterns.

Materials And Methods: Using cloning techniques, on the base of human embryonic kidney cells of HEK293T line, stable T-rex HEK-hBD2-m cell subline expressing mature biologically active hBD-2 molecule upon the presence of tetracycline in culture medium was generated. The morphological patterns, growth characteristics and colony forming activity of these cells were studied using routine techniques.

Involvement of human beta-defensin-2 in intracellular signaling: in vitro study.

Aim: To analyze involvement of human beta-defensin-2 (hBD-2) in intracellular signaling in vitro.

Materials And Methods: A431cells were cultured in the presence of 1 microg/ml of recombinant hBD-2 and/or 10 ng/ml EGF. For evaluation of expression of mRNAs for p70S6 kinase, isoforms alpha and beta, RT-PCR analysis was applied.

Involvement of human beta-defensin-2 in proliferation of transformed cells of human cervix.

Aim: To evaluate the influence of human beta-defensin-2 (hBD-2) on viability and proliferation of cultured human epithelial cells and the patterns of hBD-2 expression in normal tissues and early-stage human cervical neoplasia in the relation to proliferative state of these cells.

Materials And Methods: The influence of recombinant hBD-2 on viability and proliferation of cultured cells of A431 and M-HeLa lines in vitro was performed by MTT-test, 3H-thymidine incorporation and cell counting techniques. Immunohistochemical analysis of expression of hBD-2 and PCNA in tissue samples (10 normal cases (control), 30 carcinomas of the cervix uteri: 15 - squamous cell carcinoma in situ (Stage 0), and 15 squamous cell carcinoma (Stage Ia)) was performed with the use of anti-hBD-2 and anti-PCNA-mAbs, respectively.

[Interlocus associations and their variability in cattle].

The results of analysis of interloci associations between two pairs of syntenic loci (transferrin and ceruloplasmin, receptor for vitamin D and kappa-casein) and two non-syntenic ones (amylase-1 and post-transferrin 2) in two cattle groups of Red Steppe breed (infected and uninfected by bovine leukosis virus) and in two groups of Black-and-White Holsteins (from relatively "pure" zone and from the 10 km zone of Chernobyl NPP) were presented. It is found that "linkage disequilibrium" between loci is observed independent of their synteny. The data obtained allowed the authors to suppose, that the interloci associations are rather controlled by different factors of artificial and natural selection than by the genetic linkages between genes.