Characterization of a nucleotide kinase encoded by bacteriophage t7

Gene 1.7 protein is the only known nucleotide kinase encoded by bacteriophage T7. The enzyme phosphorylates dTMP and dGMP to dTDP and dGDP, respectively, in the presence of a phosphate donor. The phosphate donors are dTTP, dGTP, and ribo-GTP as well as the thymidine and guanosine triphosphate analogs ddTTP, ddGTP, and dITP. The nucleotide kinase is found in solution as a 256-kDa complex consisting of ～12 monomers of the gene 1.7 protein. The two molecular weight forms co-purify as a complex, but each form has nearly identical kinase activity. Although gene 1.7 protein does not require a metal ion for its kinase activity, the presence of Mg(2+) in the reaction mixture results in either inhibition or stimulation of the rate of kinase reactions depending on the substrates used. Both the dTMP and dGMP kinase reactions are reversible. Neither dTDP nor dGDP is a phosphate acceptor of nucleoside triphosphate donors. Gene 1.7 protein exhibits two different equilibrium patterns toward deoxyguanosine and thymidine substrates. The K(m) of 4.4 × 10(-4) m obtained with dTTP for dTMP kinase is ～3-fold higher than that obtained with dGTP for dGMP kinase (1.3 × 10(-4) m), indicating that a higher concentration of dTTP is required to saturate the enzyme. Inhibition studies indicate a competitive relationship between dGDP and both dGTP, dGMP, whereas dTDP appears to have a mixed type of inhibition of dTMP kinase. Studies suggest two functions of dTTP, as a phosphate donor and a positive effector of the dTMP kinase reaction.     

Ecological variation between marginal and central populations of Potamogeton polygonifolius,a rare and endangered species in Central Europe

The present study is focused on Potamogeton polygonifolius – the species considered to be endangered in Germany, the Czech Republic, Poland and Switzerland. Field studies were carried out in Lower Silesia and Lusatia in southwestern Poland in order to verify the present state of the marginal populations of P. polygonifolius, to determine the habitat preferences of the species in the study area, to compare the ecological data between marginal and central populations and finally to recognise the factors constraining the easternmost limit of the P. polygonifolius. In total, 18 populations were examined, and 11 relevés were collected. The relevés gathered were compared with 95 relevés made in northwestern Germany, and with 10 relevés collected in the German part of Lusatia. There were no significant differences between the ecology of populations of P. polygonifolius on the eastern edge of its range and those in the central part. P. polygonifolius was found growing both in natural habitats like unspoiled peat bogs, and in anthropogenic habitats such as exploited peat bogs and drainage ditches. In both types of habitats its populations were abundant and competent in terms of generative propagation. The lack of geographical barriers as well as biology and fitness of P. polygonifolius individuals from the study area suggest that marginal populations are possibly a part of a large metapopulation, covering the whole (Polish and German) area of Lusatia, however, this hypothesis was not tested in detail. The main factor shaping the easternmost limit of the general range of P. polygonifolius is most likely climate, which prevents P. polygonifolius from spreading farther eastwards.     

The influence of the fungal pathogen Mycocentrospora acerina on the proteome and polyacetylenes and 6-methoxymellein in organic and conventionally cultivated carrots (Daucus carota) during post harvest storage

Many carrots are discarded during post harvest cold storage due to development of fungal infections, caused by, e.g., Mycocentrospora acerina (liquorice rot). We compared the susceptibility of carrots grown under conventional and organic agricultural practices. In one year, organically cultivated carrots showed 3 × to 7 × more symptoms than conventionally cultivated, when studying naturally occurring disease at 4 and 6 months, respectively. On the other hand, we have developed a bioassay for infection studies of M. acerina on carrots and observed that organic roots were more susceptible after one month of storage than conventional ones, but no differences were apparent after four or six months storage. Levels of polyacetylenes (falcarinol, falcarindiol and falcarindiol-3-acetate) did not change, whereas the isocoumarin phytoalexin (6-methoxymellein) accumulated in infected tissue as well as in healthy tissue opposite the infection. The proteomes of carrot and M. acerina were characterized, the intensity of 33 plant protein spots was significantly changed in infected roots including up regulation of defence and stress response proteins but also a decrease of proteins involved in energy metabolism. This combined metabolic and proteomic study indicates that roots respond to fungal infection through altered metabolism: simultaneous induction of 6-methoxymellein and synthesis of defence related proteins.     

DNA damage and repair in gastric cancer--a correlation with the hOGG1 and RAD51 genes polymorphisms

Aristolochic acid (AA) is a potent nephrotoxin and carcinogen and is the causative factor for Chinese herb nephropathy. AA has been associated with the development of urothelial cancer in humans, and kidney and forestomach tumors in rodents. To investigate the molecular mechanisms responsible for the tumorigenicity of AA, we determined the DNA adduct formation and mutagenicity of AA in the liver (nontarget tissue) and kidney (target tissue) of Big Blue rats. Groups of six male rats were gavaged with 0, 0.1, 1.0 and 10.0 mg AA/kg body weight five times/week for 3 months. The rats were sacrificed 1 day after the final treatment, and the livers and kidneys were isolated. DNA adduct formation was analyzed by ³²P-postlabeling and mutant frequency (MF) was determined using the λ Select-cII Mutation Detection System. Three major adducts (7-[deoxyadenosin-N⁶-yl]-aristolactam I, 7-[deoxyadenosin-N⁶-yl]-aristolactam II and 7-[deoxyguanosin-N²-yl]-aristolactam I) were identified. There were strong linear dose-responses for AA-induced DNA adducts in treated rats, ranging from 25 to 1967 adducts/10⁸ nucleotides in liver and 95–4598 adducts/10⁸ nucleotides in kidney. A similar trend of dose-responses for mutation induction also was found, the MFs ranging from 37 to 666 × 10⁻⁶ in liver compared with the MFs of 78–1319 × 10⁻⁶ that we previously reported for the kidneys of AA-treated rats. Overall, kidneys had at least two-fold higher levels of DNA adducts and MF than livers. Sequence analysis of the cII mutants revealed that there was a statistically significant difference between the mutation spectra in both kidney and liver of AA-treated and control rats, but there was no significant difference between the mutation spectra in AA-treated livers and kidneys. A:T → T:A transversion was the predominant mutation in AA-treated rats; whereas G:C → A:T transition was the main type of mutation in control rats. These results indicate that the AA treatment that eventually results in kidney tumors in rats also results in significant increases in DNA adduct formation and cII MF in kidney. Although the same treatment does not produce tumors in rat liver, it does induce DNA adducts and mutations in this tissue, albeit at lower levels than in kidney.     

Cytocidal effect of interleukin 1 (IL-1) on HeLa cells is mediated by both soluble and transmembrane tumor necrosis factor (TNF)

Interleukin 1 (IL-1) is a pleiotropic cytokine able to induce cytocidal effect. The aim of the presented work was to analyze the mechanism of IL-1-induced cytocidal effect in HeLa cells in the presence of cycloheximide (CHX). We found that the pattern of IL-1-induced cell death shares significant similarities with the effect of tumor necrosis factor (TNF) in these cells. Subsequently, we identified IL-1 cytotoxicity as an indirect effect. The supernatant collected from the cells treated with IL-1 and CHX showed toxic activity towards IL-1-resistant while TNF-sensitive A9 cells. Furthermore, antibodies neutralizing TNF blocked HeLa cell death induced by IL-1/CHX. TNF was then detected in HeLa cells by means of flow cytometry, fluorescence microscopy and ELISA of detergent-soluble cell extracts. In the presence of an inhibitor of TNF sheddase (TACE), the cytotoxic effect of IL-1/CHX and the amount of TNF protein in detergent-soluble cell extracts were enhanced. These results suggest that in response to interleukin 1/CHX, the amount of transmembrane TNF is increased. Taken together, we demonstrated that the mechanism of IL-1 cytotoxic activity in HeLa cells in the presence of CHX depends on the function of soluble and transmembrane TNF.     

Peptides with 6-Aminohexanoic Acid: Synthesis and Evaluation as Plasmin Inhibitors

Fifteen new peptide derivatives of ?-aminocaproic acid (EACA) containing the known fragment –Ala–Phe–Lys– with an affinity for plasmin were synthesised in the present study. The synthesis was carried out a solid phase. The following compounds were synthesised: H–Phe–Lys–EACA–X, H–d-Ala–Phe–Lys–EACA–X, H–Ala–Phe–Lys–EACA–X, H–d-Ala–Phe–EACA–X and H–Ala–Phe–EACA–X, where X = OH, NH₂ and NH–(CH₂)₅–NH₂. All peptides, except for those containing the sequence H–Ala–Phe–EACA–X, displayed higher inhibitory activity against plasmin than EACA. The most active and selective inhibitor of plasmin was the compound H–d-Ala–Phe–Lys–EACA–NH₂ which inhibited the amidolytic activity of plasmin (IC₅₀ = 0.02 mM), with the antifibrinolytic activity weaker than EACA. The resulting peptides did not affect the viability of fibroblast cells, colon cancer cell line DLD-1, breast MCF-7 and MDA-MB-231 cell lines.     

Data integration from pathology slides for quantitative imaging of multiple cell types within the tumor immune cell infiltrate

Background

Immune cell infiltrates (ICI) of tumors are scored by pathologists around tumor glands. To obtain a better understanding of the immune infiltrate, individual immune cell types, their activation states and location relative to tumor cells need to be determined. This process requires precise identification of the tumor area and enumeration of immune cell subtypes separately in the stroma and inside tumor nests. Such measurements can be accomplished by a multiplex format using immunohistochemistry (IHC).

Method

We developed a pipeline that combines immunohistochemistry (IHC) and digital image analysis. One slide was stained with pan-cytokeratin and CD45 and the other slide with CD8, CD4 and CD68. The tumor mask generated through pan-cytokeratin staining was transferred from one slide to the other using affine image co-registration. Bland-Altman plots and Pearson correlation were used to investigate differences between densities and counts of immune cell underneath the transferred versus manually annotated tumor masks. One-way ANOVA was used to compare the mask transfer error for tissues with solid and glandular tumor architecture.

Results

The overlap between manual and transferred tumor masks ranged from 20%–90% across all cases. The error of transferring the mask was 2- to 4-fold greater in tumor regions with glandular compared to solid growth pattern (p < 10^?6). Analyzing data from a single slide, the Pearson correlation coefficients of cell type densities outside and inside tumor regions were highest for CD4 + T-cells (r = 0.8), CD8 + T-cells (r = 0.68) or CD68+ macrophages (r = 0.79). The correlation coefficient for CD45+ T- and B-cells was only 0.45. The transfer of the mask generated an error in the measurement of intra- and extra- tumoral CD68+, CD8+ or CD4+ counts (p < 10^?10).

Conclusions

In summary, we developed a general method to integrate data from IHC stained slides into a single dataset. Because of the transfer error between slides, we recommend applying the antibody for demarcation of the tumor on the same slide as the ICI antibodies.