15 research outputs found
Intracellular nitrate storage by diatoms can be an important nitrogen pool in freshwater and marine ecosystems
Identifying and quantifying nitrogen pools is essential for understanding the nitrogen cycle in aquatic ecosystems. The ubiquitous diatoms represent an overlooked nitrate pool as they can accumulate nitrate intracellularly and utilize it for nitrogen assimilation, dissipation of excess photosynthetic energy, and Dissimilatory Nitrate Reduction to Ammonium (DNRA). Here, we document the global co-occurrence of diatoms and intracellular nitrate in phototrophic microbial communities in freshwater (n = 69), coastal (n = 44), and open marine (n = 4) habitats. Diatom abundance and total intracellular nitrate contents in water columns, sediments, microbial mats, and epilithic biofilms were highly significantly correlated. In contrast, diatom community composition had only a marginal influence on total intracellular nitrate contents. Nitrate concentrations inside diatom cells exceeded ambient nitrate concentrations ∼100–4000-fold. The collective intracellular nitrate pool of the diatom community accounted for <1% of total nitrate in pelagic habitats and 65–95% in benthic habitats. Accordingly, nitrate-storing diatoms are emerging as significant contributors to benthic nitrogen cycling, in particular through Dissimilatory Nitrate Reduction to Ammonium activity under anoxic conditions
The role of the oncoprotein DEK in DNA replication stress and damage repair
The DEK oncoprotein is a chromatin architectural factor that has essential functions in the maintenance of heterochromatin integrity. It is an abundant and unique chromatin constituent showing no sequence homology to any other known protein, and is highly conserved among multicellular eukaryotes. DEK binds to DNA, RNA, and interacts with various chromatin components including histones. The affinity of DEK to its binding partners is determined by posttranslational modifications, predominantly by phosphorylation as well as poly(ADP-ribosyl)ation. When interacting with DNA, DEK preferentially binds to cruciform DNA structures which arise during perturbed DNA replication and the repair of DNA strand breaks.Several lines of evidence, among others the frequent overexpression in highly malignant tumors and the positive correlation between DEK expression levels and chemoresistance, have led to the definition of DEK as a bona fide oncogene. On the other hand, investigation of a potential role of this protein in DNA repair revealed that DNA strand breaks are repaired less efficiently when DEK expression is downregulated leading to hypersensitivity towards genotoxic insults. DEK’s function as a positive regulator of DNA repair has been difficult to reconcile with a genuine tumor promoting activity, since cancer development is most often characterized by defects in DNA repair and genomic instability. This thesis aimed at elucidating this apparent conundrum by a detailed investigation of the impact of DEK on DNA damage susceptibility. The study focused on DNA replication stress as a characteristic source of DNA damage in hyperproliferating tumors.The data obtained show that DEK renders cells less sensitive to DNA replication stress counteracting accumulation of replication stress-induced DNA damage. DEK facilitates replication fork progression, in particular under conditions of mild but prolonged replication stress, as occurring at the early stages of transformation. For this function, DEK was shown to depend on PARP1/2 activity. DEK’s proliferation promoting activity was also confirmed in the human cancer model of c-myc-induced replication stress. Furthermore, DEK was demonstrated to reduce damage transmission through mitosis to the next cell generation. Evidence for a role of DEK in the regulation of mitosis progression and chromosome congression was also gained. Finally, DEK localization was shown to be drastically and persistently affected by DNA replication stress, an effect that might be mediated by SUMO1-modification.In sum, the study presented here proposes a model for DEK´s tumorigenic activity in which this protein contributes to circumvent the cell´s intrinsic barrier against cancerogenesis imposed by the DNA damage response thereby enabling proliferation under stress and eventually tumor growth and malignancy. This study discloses a novel mode of action of this protein frequently deregulated in aggressive tumors that bears the potential for new therapeutic approaches
Discrimination of cell cycle phases in PCNA-immunolabeled cells
BackgroundProtein function in eukaryotic cells is often controlled in a cell cycle-dependent manner. Therefore, the correct assignment of cellular phenotypes to cell cycle phases is a crucial task in cell biology research. Nuclear proteins whose localization varies during the cell cycle are valuable and frequently used markers of cell cycle progression. Proliferating cell nuclear antigen (PCNA) is a protein which is involved in DNA replication and has cell cycle dependent properties. In this work, we present a tool to identify cell cycle phases and in particular, sub-stages of the DNA replication phase (S-phase) based on the characteristic patterns of PCNA distribution. Single time point images of PCNA-immunolabeled cells are acquired using confocal and widefield fluorescence microscopy. In order to discriminate different cell cycle phases, an optimized processing pipeline is proposed. For this purpose, we provide an in-depth analysis and selection of appropriate features for classification, an in-depth evaluation of different classification algorithms, as well as a comparative analysis of classification performance achieved with confocal versus widefield microscopy images.ResultsWe show that the proposed processing chain is capable of automatically classifying cell cycle phases in PCNA-immunolabeled cells from single time point images, independently of the technique of image acquisition. Comparison of confocal and widefield images showed that for the proposed approach, the overall classification accuracy is slightly higher for confocal microscopy images.ConclusionOverall, automated identification of cell cycle phases and in particular, sub-stages of the DNA replication phase (S-phase) based on the characteristic patterns of PCNA distribution, is feasible for both confocal and widefield images
Clinical outcome in patients with cranial or maxillofacial bone defects reconstructed with bone stimulating implants
DNA replication stress is a major source of DNA strand breaks and genomic instability, and a hallmark of precancerous lesions. In these hyperproliferative tissues, activation of the DNA damage response results in apoptosis or senescence preventing or delaying their development to full malignancy. In cells, in which this antitumor barrier is disabled by mutations (for example, in p53), viability and further uncontrolled proliferation depend on factors that help to cope with replication-associated DNA damage. Replication problems preferentially arise in chromatin regions harboring complex DNA structures. DEK is a unique chromatin architectural factor which binds to non-B-form DNA structures, such as cruciform DNA or four-way junctions. It regulates DNA topology and chromatin organization, and is essential for the maintenance of heterochromatin integrity. Since its isolation as part of an oncogenic fusion in a subtype of AML, DEK has been consistently associated with tumor progression and chemoresistance. How DEK promotes cancer, however, is poorly understood. Here we show that DEK facilitates cellular proliferation under conditions of DNA replication stress by promoting replication fork progression. DEK also protects from the transmission of DNA damage to the daughter cell generation. We propose that DEK counteracts replication stress and ensures proliferative advantage by resolving problematic DNA and/or chromatin structures at the replication fork.Oncogene advance online publication, 27 October 2014; doi:10.1038/onc.2014.346
Argonaute Family Protein Expression in Normal Tissue and Cancer Entities.
The members of the Argonaute (AGO) protein family are key players in miRNA-guided gene silencing. They enable the interaction between small RNAs and their respective target mRNA(s) and support the catalytic destruction of the gene transcript or recruit additional proteins for downstream gene silencing. The human AGO family consists of four AGO proteins (AGO1-AGO4), but only AGO2 harbors nuclease activity. In this study, we characterized the expression of the four AGO proteins in cancer cell lines and normal tissues with a new mass spectrometry approach called AGO-APP (AGO Affinity Purification by Peptides). In all analyzed normal tissues, AGO1 and AGO2 were most prominent, but marked tissue-specific differences were identified. Furthermore, considerable changes during development were observed by comparing fetal and adult tissues. We also identified decreased overall AGO expression in melanoma derived cell lines compared to other tumor cell lines and normal tissues, with the largest differences in AGO2 expression. The experiments described in this study suggest that reduced amounts of AGO proteins, as key players in miRNA processing, have impact on several cellular processes. Deregulated miRNA expression has been attributed to chromosomal aberrations, promoter regulation and it is known to have a major impact on tumor development and progression. Our findings will further increase our basic understanding of the molecular basis of miRNA processing and its relevance for disease
Argonaute protein distribution in melanoma and non-melanoma cell lines.
<p>(A) AGO protein percentage distribution of each AGO to total AGO protein amount in non-melanoma cell lines CaCo2, HepG2, SW1353, MCF7, HeLa and melanoma cell lines Mel Ju, Mel Im, Mel Wei, Mel Ei and Mel Ho derived from AGO-APP. (B) Total protein amount determination of each AGO enriched by AGO-APP in CaCo2, HepG2, SW1353, MCF7, HeLa, Mel Ju, Mel Im, Mel Wei, Mel Ei and Mel Ho. (C) Average AGO protein amount of each AGO in non-melanoma compared to melanoma cell lines. The reduced AGO concentration in melanoma cell lines compared to non-melanoma cell lines is only significant for AGO2. ** = p<0.01 (D) AGO1 western blot analysis and corresponding (E) Western blot quantification of melanoma cell lines (Mel Ju, Mel Im, Mel Wei, Mel Ei, Mel Ho) and non-melanoma cell lines (CaCo2, SW1353, MCF7, HeLa, HepG2). The two additional values in the AGO1 western blot quantification illustrate the average AGO1 concentration in melanoma and non-melanoma cell lines. Quantification was done relative to Actin in the respective blot.</p
Argonaute gene expression in different melanoma and non-melanoma cell lines.
<p>Relative (A) AGO1, (B) AGO2, (C) AGO3 and (D) AGO4 mRNA expression in melanoma cell lines derived from primary tumors (Mel Ei, Mel Juso, Mel Ho and Mel Wei) and metastases (Mel Ju, Mel Im, SkMel28 and Hmb2) and other non-melanoma cell lines (HeLa, CaCo2, PLC, Jurkat, Hep3b, SW1353 and MCF7). Each point shows the measurement of one independently derived cDNA sample. Bars show mean and S.D. (E) The compilation of percentage distribution of each AGO to the aggregate AGO amount. (F) Entire mRNA expression of all four AGOs compared to actin in melanoma cell lines derived from primary tumors or metastases and in other non-melanoma cell lines.</p
Oligonucleotide sequences and qRT-PCR conditions.
<p>Oligonucleotide sequences and qRT-PCR conditions.</p
Argonaute gene expression in different human healthy tissues compared to NHEMs.
<p>Relative (A) AGO1, (B) AGO2, (C) AGO3 and (D) AGO4 mRNA expression in NHEMs (derived from different cultivation passages (P4-P6) from three different donors respectively), skin (derived from three different tissue samples) and heart, kidney, bone marrow, fetal brain, bowel, thymus, uterus, trachea, brain, muscle, bone marrow, liver, fetal liver, brain and fetal brain (derived from a total RNA bank and shown as technical replicates). (E) The compilation of percentage distribution of each AGO to the total AGO amount in the respective cell line or tissue.</p