Cell cycle regulation of redoxyendonuclease activity in human neuroblastoma cells.

Redoxyendonuclease activity was detected in extracts of human neuroblastoma cells using a base-release assay specific for thymine glycol in DNA. The level of redoxyendonuclease activity was more than 2-fold higher in dividing cells compared to quiescent cells, suggesting that quiescent cells may have a reduced capacity to repair oxidative DNA base damages. Cells were synchronized by serum deprivation and then stimulated to enter the cell cycle by the addition of serum to determine enzyme activity at different stages of the cell cycle. The redoxyendonuclease activity was regulated in a biphasic manner with a peak in early G1 and a peak in S phase. This suggests that at specific times during the cell cycle actively growing cells may be more resistant to oxidative DNA damage due to increased repair capacity. The repair capacity of neuroblastoma cells was quantified as the decrease in enzyme-sensitive sites determined by alkaline sucrose density gradient centrifugation following treatment with the oxidant osmium tetroxide. Actively dividing cells repaired the oxidative damage in approximately 24 hours, while the quiescent cells failed to excise the damaged sites and subsequently died. These results indicate that non-dividing cells do not effectively repair oxidative DNA damage, as compared to the dividing cells. Similarly, quiescent cells, treated with osmium tetroxide and fed a serum-enriched media, failed to re-enter the cell cycle and did not repair the oxidative damage. The data indicate that non-dividing cells, such as neurons, do not have the capacity to repair excess oxidative damage and may suffer the biological consequences of DNA damage accumulation, including cellular death, mutagenesis or carcinogenesis. When synchronized cells were damaged with osmium tetroxide, there were differential DNA repair rates, depending on the stage of the cell cycle. These DNA repair rates coordinated with the redoxyendonuclease activity profile. The results of the studies described contribute to a further understanding of the DNA repair pathways which are interlinked with complex processes of cell cycle regulation

Multiplex Cytological Profiling Assay to Measure Diverse Cellular States

Computational methods for image-based profiling are under active development, but their success hinges on assays that can capture a wide range of phenotypes. We have developed a multiplex cytological profiling assay that “paints the cell” with as many fluorescent markers as possible without compromising our ability to extract rich, quantitative profiles in high throughput. The assay detects seven major cellular components. In a pilot screen of bioactive compounds, the assay detected a range of cellular phenotypes and it clustered compounds with similar annotated protein targets or chemical structure based on cytological profiles. The results demonstrate that the assay captures subtle patterns in the combination of morphological labels, thereby detecting the effects of chemical compounds even though their targets are not stained directly. This image-based assay provides an unbiased approach to characterize compound- and disease-associated cell states to support future probe discovery

High-throughput luminescent reporter of insulin secretion for discovering regulators of pancreatic beta-cell function

Defects in insulin secretion play a central role in the pathogenesis of type 2 diabetes, yet the mechanisms driving beta-cell dysfunction remain poorly understood, and therapies to preserve glucose-dependent insulin release are inadequate. We report a luminescent insulin secretion assay that enables large-scale investigations of beta-cell function, created by inserting Gaussia luciferase into the C-peptide portion of proinsulin. Beta-cell lines expressing this construct cosecrete luciferase and insulin in close correlation, under both standard conditions or when stressed by cytokines, fatty acids, or ER toxins. We adapted the reporter for high-throughput assays and performed a 1,600-compound pilot screen, which identified several classes of drugs inhibiting secretion, as well as glucose-potentiated secretagogues that were confirmed to have activity in primary human islets. Requiring 40-fold less time and expense than the traditional ELISA, this assay may accelerate the identification of pathways governing insulin secretion and compounds that safely augment beta-cell function in diabetes

Stable interaction between the products of the BRCA1 and BRCA2 tumor suppressor genes in mitotic and meiotic cells

BRCA1 and BRCA2 account for most cases of familial, early onset breast and/or ovarian cancer and encode products that each interact with hRAD51. Results presented here show that BRCA1 and BRCA2 coexist in a biochemical complex and colocalize in subnuclear foci in somatic cells and on the axial elements of developing synaptonemal complexes. Like BRCA1 and RAD51, BRCA2 relocates to PCNA+ replication sites following exposure of S phase cells to hydroxyurea or UV irradiation. Thus, BRCA1 and BRCA2 participate, together, in a pathway(s) associated with the activation of double-strand break repair and/or homologous recombination. Dysfunction of this pathway may be a general phenomenon in the majority of cases of hereditary breast and/or ovarian cancer

A small-molecule inducer of PDX1 expression identified by high-throughput screening

Pancreatic and duodenal homeobox 1 (PDX1), a member of the homeodomain-containing transcription factor family, is a key transcription factor important for both pancreas development and mature β cell function. The ectopic overexpression of Pdx1, Neurog3, and MafA in mice reprograms acinar cells to insulin-producing cells. We developed a quantitative PCR-based gene expression assay to screen more than 60,000 compounds for expression of each of these genes in the human PANC-1 ductal carcinoma cell line. We identified BRD7552, which upregulated PDX1 expression in both primary human islets and ductal cells, and induced epigenetic changes in the PDX1 promoter consistent with transcriptional activation. Prolonged compound treatment induced both insulin mRNA and protein and also enhanced insulin expression induced by the three-gene combination. These results provide a proof of principle for identifying small molecules that induce expression of transcription factors to control cellular reprogramming

Taloushallinnon opas voimisteluseuralle : Bounce Espoo ry

Opinnäytetyön tarkoituksena oli luoda taloushallinnon opas voimisteluseura Bounce Espoo ry:lle. Bounce Espoo ry on espoolainen trampoliinivoimisteluun erikoistunut seura. Opinnäytetyö toimii ohjeena nykyiselle tai mahdolliselle uudelle henkilölle, joka hoitaa yhdistyksen taloushallintoa. Opinnäytetyö aihe tuli toimeksiantona Bounce Espoo ry:ltä. Opas on toteutettu yhdistyksen tarpeiden mukaan. Opas hyödyttää toimeksiantajayhdistyksen lisäksi myös muita pieniä yhdistyksiä. Opinnäytetyö on toiminnallinen, ja se koostuu raporttiosuudesta sekä oppaasta. Opinnäytetyön teoriaosuudessa käsiteltiin yhdistyksen koko taloushallintoa ja oppaassa keskityttiin kirjanpitoon. Teoria pohjautuu yleisesti yhdistyksen toimintaan, taloushallintoon ja juridiikkaan. Tekijät laativat yhdistystä hyödyttävän oppaan, jota yhdistys voi hyödyntää usean vuoden ajan, ottaen huomioon muuttuvan lainsäädännön ja valtion asettamat säännökset. Opas selventää taloushallintoa teoreettisesti ja käytännön esimerkkien avulla.The purpose of this thesis was to create a financial administration manual for gymnastics club Bounce Espoo ry. Bounce Espoo ry is a gymnastics club that specializes in trampoline gymnastics. The thesis is a guide for the current or prospective future employee who takes care of the financial management of the association. The thesis was commissioned by Bounce Espoo ry. The guide has been drawn out according to the needs of the association. In addition to Bounce Espoo, the guide will benefit other small associations, too. Our thesis is functional and consists of a report part and a manual. The theoretical part of the thesis discusses the entire financial management of the association and the guide focuses on the accounts. The theory is based on the practices, financial and legal affairs of the association in general. We made a guide which benefits the association. The association can use the guide for several years, but they must take notice of potential changes in state laws and regulations. The guide clarifies financial management theoretically and with practical provides