TF — A novel cell-permeable and selective inhibitor of human protein kinase CK2 induces apoptosis in the prostate cancer cell line LNCaP

BackgroundAbnormally high activity of protein kinase CK2 is linked to various diseases including cancer. Therefore, the inhibition of CK2 is a promising therapeutic strategy to fight this disease.MethodsWe screened a library of synthetic molecules concerning their capacity to inhibit CK2. The activity of CK2 and their IC₅₀ and K_i values were determined by a capillary electrophoresis assay. The effects of the inhibitor in a cell culture model were analyzed by cell counting, a viability assay, cytofluorimetry and Western blot.ResultsThe best CK2 inhibitor found in this screen was 6,7-dichloro-1,4-dihydro-8-hydroxy-4-[(4-methylphenylamino)methylen]dibenzo [b,d]furan-3(2H)-one, which we refer to as “TF”. TF showed tight binding to CK2 with low IC₅₀ (29 nM) and K_i (15 nM) values. TF inhibited only seven out of 61 human kinases tested (> 70% inhibition). Incubation of LNCaP cells with 50 μM TF for 48 h decreased the intracellular CK2 activity by 50%, confirming that the inhibitor is membrane permeable. The decrease in activity was correlated with a severe reduction in cell viability. The reduction in cell viability is at least partly due to the induction of apoptosis.General significanceIn many cancers the protein kinase CK2 is significantly up-regulated and supports the neoplastic phenotype. New therapeutic strategies should be based on diverse reliable inhibitors to reverse the abnormal high levels to normal settings.     

Juxtaposition of System Dynamics and Agent-Based Simulation for a Case Study in Immunosenescence

Advances in healthcare and in the quality of life significantly increase human life expectancy. With the aging of populations, new un-faced challenges are brought to science. The human body is naturally selected to be well-functioning until the age of reproduction to keep the species alive. However, as the lifespan extends, unseen problems due to the body deterioration emerge. There are several age-related diseases with no appropriate treatment; therefore, the complex aging phenomena needs further understanding. It is known that immunosenescence is highly correlated to the negative effects of aging. In this work we advocate the use of simulation as a tool to assist the understanding of immune aging phenomena. In particular, we are comparing system dynamics modelling and simulation (SDMS) and agent-based modelling and simulation (ABMS) for the case of age-related depletion of naive T cells in the organism. We address the following research questions: Which simulation approach is more suitable for this problem? Can these approaches be employed interchangeably? Is there any benefit of using one approach compared to the other? Results show that both simulation outcomes closely fit the observed data and existing mathematical model; and the likely contribution of each of the naive T cell repertoire maintenance method can therefore be estimated. The differences observed in the outcomes of both approaches are due to the probabilistic character of ABMS contrasted to SDMS. However, they do not interfere in the overall expected dynamics of the populations. In this case, therefore, they can be employed interchangeably, with SDMS being simpler to implement and taking less computational resources.     

Ds excision from extrachromosomal geminivirus vector DNA is coupled to vector DNA replication in maize

Analysis of transposition products generated after Activator (Ac) excision from the P locus in maize suggest that Ac excises either during or after replication of the P locus. The frequency of excision of the non-autonomous Ac derivative, Dissociation ( Ds ), from extrachromosomal replicating and nonreplicating vector DNAs in transfected black mexican sweet maize protoplasts was compared to assess directly a role of extrachromosomal vector DNA replication in Ds excision. Replicating (rep⁺) and non-replicating (rep⁻) vector DNAs comprised a Ds element that harbored a geminivirus, wheat dwarf virus (WDV), origin of replication and WDV genes required for viral DNA replication (rep⁺) or mutant, inactive derivatives of these genes (rep⁻). Excision of Ds was detected only in those cell nuclei co-transfected with the replicating Ds -vector DNA and a transposase expression vector. Quantitative reconstruction experiments showed that Ds excised at least 3 × 10⁵-fold more frequently from replicating vector DNA as compared with nonreplicating vector DNA. Therefore, these results provide direct evidence for a coupling of Ds excision from extrachromosomal vector DNA to vector DNA replication in maize.     

Lipase of Pseudomonas cepacia for biotechnological purposes: purification,crystallization and characterization

Commercial lipase (triacylglycerol lipase, EC 3.1.1.3) of Pseudomonas cepacia (Amano) has been purified to homogeneity by a single chromatography on phenyl Sepharose. The eluted lipase crystallized spontaneously at 4°C in the eluent, containing 58–69% 2-propanol. The yield of the lipase was 87–100% and the specific activity during the hydrolysis of triolein 5800 U/mg protein. This protein has a molecular weight of 34.1 kDa as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Its purity was determined by SDS-Page and capillary zone electrophoresis to be ≥ 99%. Immobilization on Sepharose increased its stability in organic solvents. This lipase of P. cepacia differs from that of other Pseudomonas strains in respect of substrate specificity and during crystallization. It exhibits a high stability in organic solvents and supercritical carbon dioxide.     

Oligonucleotide Directed Mutagenesis of the E. coli hisS Gene: Introduction of an Ncoi Restriction Site at the Initiating GTG and Cloning of the Mutated Gene to the Expression Vector pKK 233–2

Abstract

Site directed mutagenesis of the E. coli his gene with a double mismatch primer changed the initiation codon GTG to ATG and introduced an Ncol restriction site at the start codon. The promoter-deleted structural gene was cloned to the expression vector pKK 233–2.     

Zur Spezifität des histochemischen Carboanhydratase-Nachweises im inselorgan der Bauchspeicheldrüse

Zusammenfassung Die enzymatische Aufspaltung von Kobalthydrogenkarbonaten durch das Ferment CAH führt beim Häuslerschen Fermentnachweis über eine sekundäre Visualisation der Kobaltkarbonatniederschläge durch Umwandlung in Kobaltsulfid zur Markierung der Fermentaktivität am Schnitt. Untersuchungen am Pankreas zeigen, daß die Aussagekraft fermentmarkierender Niederschläge durch eine unspezifische Fällung zweiwertiger, nichtfermentgebundener Zinkionen beeinträchtigt wird. Das inkretorische Parenchym des Pankreas enthält keine CAH, wird aber intensiv imprägniert durch seinen Reichtum an nichtfermentgebundenen Zinkionen. Nach Abfangen dieser Metallionen durch Metallchelatbildner (Dithizon, NDDC) fehlt eine Kobaltsulfidschwärzung der Inselzellen.Im exkretorischen Parenchym führt dagegen die Bebrütung von Kryostatschnitten zu spezifischer Fermentmarkierung. Aus sterischen Gründen (tertiäre Bindung des zweiwertigen Metalls im Enzymmolekül) ist das CAH-Zink einer Chelatbindung mit Dithizon oder NDDC nicht zugänglich, die Aktivität der CAH in den Gangepithelien der Bauchspeicheldrüse wird durch Chelatbildner nicht beeinflußt.Die Spezifität des Häuslerschen Fermentnachweises ist unbestritten. Zu fordern ist aufgrund der vorliegenden Ergebnisse aber eine Kontrolle der Fermentreaktion durch Chelatbildner, um eine gleichzeitige unspezifische Imprägnation zweiwertiger Metallionen (insbesondere des Zinks) im Kryostatschnitt auszuschließen.

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Summary In the Häusler incubation method the demonstration of the catalytic activity of carbonic anhydrase in tissue sections involves an enzymatic splitting of cobalt hydrogencarbonates. The resulting carbonate precipitates are secondarily visualized by being transformed into cobalt sulfide. Examination of the pancreas by the modified Häusler reaction indicates that the specificity of the precipitates marking the enzyme is masked by an unspecific precipitation of bivalent zinc ions that are not bound to the enzyme. The endocrine parenchyma of the pancreas does not contain any carbonic anhydrase, but is intensely impregnated because of its abundance of zinc ions bound to insulin or glucagon. If these metallic ions are sequestered by the use of metal chelating agents (dithiozone, sodium diethyldithiocarbamate), the unspecific precipitates in the islets of Langerhans are eliminated.In the exocrine parenchyma, however, the incubation of cryostat sections results in a specific demonstration of the enzyme. The steric arrangement of zinc ions within the enzymic molecules (trivalent complexes of bivalent metal ions) prevents a complex linkage of dithiozone or sodium diethyldithiocarbamate to carbonic anhydrase. The activity of carbonic anhydrase in the epithelium of the ducts is not at all inhibited by chelating agents.The specificity of the Häusler incubation method for demonstrating carbonic anhydrase is uncontested. The results show, however, that in histochemical studies of the enzyme the use of chelating agents is necessary as a control to exclude a simultaneous unspecific precipitation of bivalent metal ions (especially zinc) in cryostat sections.

     

Non-condensation of one segment of a chromosome No. 2 in a male with an otherwise normal karyotype (and severe hypospadias)

Summary A child with severe hypospadias is presented, whose karyotype showed in about 11% of mitoses of peripheral blood one member of chromosome pair No. 2 with a non-condensed region near the centromere. The non-condensed segment does not show late replication, however, it is situated very close to the late replicating segment of the long arms of chromosome No. 2. The nature and possible implications of this kind of aberration are discussed. It is held that non-condensation can produce localized chromosome breaks by a mechanism possibly different from any of the classical breakage mechanisms.