Proteomic identification of in vivo interactors reveals novel function of skin cornification proteins.

Protection against injurious external insults and loss of vital fluids is essential for life and is in all organisms, from bacteria to plants and humans, provided by some form of barrier. Members of the small proline-rich (SPRR) protein family are major components of the cornified cell envelope (CE), a structure responsible for the barrier properties of our skin. These proteins are efficient reactive oxygen species (ROS) quenchers involved not only in the establishment of the skin's barrier function but also in cell migration and wound healing.

Activity-based profiling reveals reactivity of the murine thymoproteasome-specific subunit beta5t.

Epithelial cells of the thymus cortex express a unique proteasome particle involved in positive T cell selection. This thymoproteasome contains the recently discovered beta5t subunit that has an uncharted activity, if any. We synthesized fluorescent epoxomicin probes that were used in a chemical proteomics approach, entailing activity-based profiling, affinity purification, and LC-MS identification, to demonstrate that the beta5t subunit is catalytically active in the murine thymus.

Accurate non-invasive image-based cytotoxicity assays for cultured cells.

Background: The CloneSelect Imager system is an image-based visualisation system for cell growth assessment. Traditionally cell proliferation is measured with the colorimetric MTT assay.

Results: Here we show that both the CloneSelect Imager and the MTT approach result in comparable EC50 values when assaying the cytotoxicity of cisplatin and oxaliplatin on various cell lines.

Relevance of the leaving group for antitumor activity of new platinum(II) compounds containing anthracene derivatives as a carrier ligand.

A new anticancer-active platinum(II) compound [Pt(A9pyp)(dmso)(cbdca)], containing the E-1-(9-anthryl)-3-(2-pyridyl)-2-propenone ligand (abbreviated as A9pyp) has been synthesized by the replacement of the anionic chloride ligands in cis-[Pt(A9pyp)(dmso)Cl(2)] by the dianionic chelating cyclobutanedicarboxylate ligand (abbreviated as cbdca). The in vitro relevance of the leaving group of these new platinum(II) compounds has been investigated. Measurements of the time-dependent intracellular accumulation of both compounds in human ovarian carcinoma cell lines show that the leaving group affects their cellular uptake.

Cytotoxic activity and cellular processing in human ovarian carcinoma cell lines of a new platinum(II) compound containing a fluorescent substituted propylene diamine ligand.

A new fluorescent platinum(II) compound containing the N,N'-bis-(anthracen-9-ylmethyl)propane-1,3-diamine as a carrier ligand has been designed, synthesized and characterized. High cytotoxic activity of cis-[Pt(bapda)Cl2] is observed in A2780 and A2780R cells (human ovarian carcinoma sensitive and cisplatin-resistant, respectively). Nevertheless, cross-resistance to platinum from cis-[Pt(bapda)Cl2] in the A2780R cells was found.

Cellular accumulation and DNA platination of two new platinum(II) anticancer compounds based on anthracene derivatives as carrier ligands.

The anticancer properties of two new fluorescent platinum(II) compounds, cis-[Pt(A9opy)Cl(2)] and cis-[Pt(A9pyp)(dmso)Cl(2)] are described. These compounds are highly active against several human tumor cell lines, including human ovarian carcinoma sensitive and cisplatin-resistant cell lines (A2780 and A2780R). To study the cellular processing of these new compounds, a series of in vitro studies have been performed, including the investigation of intracellular platinum accumulation and DNA-platination experiments in A2780 and A2780R cells.

Synthesis, crystal structure, studies in solution and cytotoxicity of two new fluorescent platinum(II) compounds containing anthracene derivatives as a carrier ligand.

Two new cytotoxic fluorescent platinum(II) compounds, cis-[Pt(A9opy)Cl2] (1) and cis-[Pt(A9pyp)(DMSO)Cl2] (2),have been designed, synthesized, and characterized by IR, 1H NMR, and 195Pt NMR spectroscopy; electrospray ionization mass spectrometry (ESI-MS); and single-crystal X-ray diffraction. The carrier ligands selected for thesynthesis of these fluorescent platinum(II) compounds are E-2-[1-(9-anthryl)-3-oxo-3-prop-2-enylpyridine] (abbreviatedas A9opy) and E-1-(9-anthryl)-3-(2-pyridyl)-2-propenone (abbreviated as A9pyp). The compound cis-[Pt(A9opy)Cl2](1) comprises a peculiar cis-platinum(II) organometallic compound, in which the platinum(II) ion is bound to the photoisomerizable carbon-carbon double bond of the carrier ligand.

The NER protein Rad33 shows functional homology to human Centrin2 and is involved in modification of Rad4.

In the yeast Saccharomyces cerevisiae the Rad4-Rad23 complex is implicated in the initial damage recognition of the Nucleotide Excision Repair (NER) pathway. NER removes a variety of lesions via two subpathways: Transcription Coupled Repair (TCR) and Global Genome Repair (GGR). We previously showed that the new NER protein Rad33 is involved in both NER subpathways TCR and GGR.

The synthesis, chemical and biological properties of dichlorido(azpy)gold(III) chloride (azpy=2-(phenylazo)pyridine) and the gold-induced conversion of the azpy ligand to the chloride of the novel tricyclic pyrido[2,1-c][1,2,4]benzotriazin-11-ium cation.

A new Au(III) coordination compound with the ligand 2-(phenylazo)pyridine has been synthesized and fully characterized by means of elemental analysis, IR, UV-visible, conductivity measurements, NMR, electrospray ionization (ESI-MS) and inductively coupled plasma optical emission spectrometry (ICP-OES). The chemical stability of the cation in this compound, [Au(azpy)Cl(2)](+) (abbreviated: Au-azpy), was analyzed by means of several physicochemical methods. While stable in the solid state, stability studies performed with the gold compound in solution showed an unexpected and unprecedented reactivity.

ZM241385, DPCPX, MRS1706 are inverse agonists with different relative intrinsic efficacies on constitutively active mutants of the human adenosine A2B receptor.

The human adenosine A(2B) receptor belongs to class A G protein-coupled receptors (GPCRs). In our previous work, constitutively active mutant (CAM) human adenosine A(2B) receptors were identified from a random mutation bank. In the current study, three known A(2B) receptor antagonists, 4-{2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-yl-amino]ethyl}phenol (ZM241385), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and N-(4-acetylphenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]acetamide (MRS1706) were tested on wild-type and nine CAM A(2B) receptors with different levels of constitutive activity in a yeast growth assay.

Rad33, a new factor involved in nucleotide excision repair in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae the Rad4-Rad23 complex is involved in initial damage recognition and responsible for recruiting the other NER proteins to the site of the lesion. The Rad4-Rad23 complex is essential for both NER subpathways, Transcription Coupled Repair (TCR) and Global Genome Repair (GGR). Previously, we reported on the role of the Rad4 homologue YDR314C in NER.

New antitumour active platinum compounds containing carboxylate ligands in trans geometry: synthesis, crystal structure and biological activity.

New asymmetric trans-platinum(II) complexes, composed of an isopropylamine, an azole and two carboxylate leaving groups, are presented. The crystal and molecular structures of one of the complexes has been determined and the cytotoxicity and reactivity with 5'-guanosine monophosphate is reported. The complexes show a reduced reactivity, but no decrease in cytotoxic activity compared to their chloro-counterparts.

Repair characteristics and differentiation propensity of long-term cultures of epidermal keratinocytes derived from normal and NER-deficient mice.

Epidermal keratinocytes constitute the most relevant cellular system in terms of DNA damage because of their continuous exposure to UV light and genotoxic chemicals from the environment. Here, we describe the establishment of long-term keratinocyte cultures from the skin of wild-type and nucleotide excision repair (NER) deficient mouse mutants. The use of media with a lowered calcium concentration and the inclusion of keratinocyte growth factor (KGF) permitted repeated passaging of the cultures and resulted in the generation of stable cell lines that proliferated efficiently.

Solution structure of the second PDZ domain of the neuronal adaptor X11alpha and its interaction with the C-terminal peptide of the human copper chaperone for superoxide dismutase.

Protection against reactive oxygen species is provided by the copper containing enzyme superoxide dismutase 1 (SOD1). The copper chaperone CCS is responsible for copper insertion into apo-SOD1. This role is impaired by an interaction between the second PDZ domain (PDZ2alpha) of the neuronal adaptor protein X11alpha and the third domain of CCS (McLoughlin et al.

Combinatorial discovery of new asymmetric cis platinum anticancer complexes is made possible with solid-phase synthetic methods.

To efficiently access asymmetric cis platinum (II) complexes for biological evaluation, a new solid-phase synthesis was designed. This synthesis was used for the preparation of a small library of platinum compounds. Several compounds from this library revealed promising activity during a cytotoxicity screen.

The Rad4 homologue YDR314C is essential for strand-specific repair of RNA polymerase I-transcribed rDNA in Saccharomyces cerevisiae.

Summary The Saccharomyces cerevisiae protein Rad4 is involved in damage recognition in nucleotide excision repair (NER). In RNA polymerase II-transcribed regions Rad4 is essential for both NER subpathways global genome repair (GGR) and transcription coupled repair (TCR). In ribosomal DNA (rDNA), however, the RNA polymerase I-transcribed strand can be repaired in the absence of Rad4.

Homologous recombination is a highly conserved determinant of the synergistic cytotoxicity between cisplatin and DNA topoisomerase I poisons.

Phase I and II clinical trails are currently investigating the antitumor activity of cisplatin and camptothecins (CPTs; DNA topoisomerase I poisons), based on the dramatic synergistic cytotoxicity of these agents in some preclinical models. However, the mechanistic basis for this synergism is poorly understood. By exploiting the evolutionary conservation of DNA repair pathways from genetically tractable organisms such as budding and fission yeasts to mammalian cells, we demonstrate that the synergism of CPT and cisplatin requires homologous recombination.

Dinuclear platinum complexes with N, N'-bis(aminoalkyl)-1,4-diaminoanthraquinones as linking ligands. Part I. Synthesis, cytotoxicity, and cellular studies in A2780 human ovarian carcinoma cells.

A series of N, N'-bis(aminoalkyl)-1,4-diaminoanthraquinones (aminoalkyl=2-aminoethyl, 3-aminoprop-1-yl and 4-aminobut-1-yl) was functionalized with trans-platinum DNA-binding moieties. Cytotoxicity testing in A2780 human ovarian carcinoma cells revealed high anticancer activity of the formed cationic dinuclear platinum complexes. The cationic dinuclear platinum complexes with the shortest aminoalkyl chain were shown to be the most active, which agrees with the structure-activity relationship found for the corresponding free ligands without platinum.

Random mutagenesis of the human adenosine A2B receptor followed by growth selection in yeast. Identification of constitutively active and gain of function mutations.

To gain insight in spontaneous as well as agonist-induced activation of the human adenosine A2B receptor, we applied a random mutagenesis approach in yeast to create a large number of receptor mutants and selected mutants of interest with a robust screening assay based on growth. The amino acid sequence of 14 mutated receptors was determined. All these mutated receptors displayed constitutive activity.

Anticancer drug resistance induced by disruption of the Saccharomyces cerevisiae NPR2 gene: a novel component involved in cisplatin- and doxorubicin-provoked cell kill.

The therapeutic potential of antitumor drugs is seriously limited by the manifestation of cellular drug resistance. We used the budding yeast Saccharomyces cerevisiae as a model system to identify novel mechanisms of resistance to one of the most active anticancer agents, cisplatin. We pinpointed NPR2 (nitrogen permease regulator 2) as a gene whose disruption conferred resistance to cisplatin.

Extending solid-phase methods in inorganic synthesis: the first dinuclear platinum complex synthesised via the solid phase.

A method for obtaining potentially anti-tumour active dinuclear platinum coordination compounds via solid-phase inorganic synthesis is described for the first time.

Transcription elongation factor Spt4 mediates loss of phosphorylated RNA polymerase II transcription in response to DNA damage.

Previously, we found that Rad26, the yeast Cockayne syndrome B homolog and the transcription elongation factor Spt4 mediate transcription-coupled repair of UV-induced DNA damage. Here we studied the effect of DNA damage on transcription by directly analyzing the RNA polymerase II localization at active genes in vivo. A rad26 defect leads to loss of Ser5 phosphorylated RNA polymerase II localization to active genes, while localization is only transiently diminished in wild type cells.

Glutathione induces cellular resistance against cationic dinuclear platinum anticancer drugs.

The sulfur-containing tripeptide glutathione (GSH) is one of the most abundant molecules in cells. Elevated levels of GSH render some types of cancer cells resistant against well-known platinum anti-cancer drugs such as cisplatin and carboplatin. Platinum complexes are often very reactive towards the cysteine residue of GSH, which detoxifies these compounds by a rapid binding mechanism.

Defective nucleotide excision repair in yeast hpr1 and tho2 mutants.

Nucleotide excision repair (NER) and transcription are intimately related. First, TFIIH has a dual role in transcription initiation and NER and, secondly, transcription leads to more efficient repair of damage present in transcribed sequences. It is thought that elongating RNAPII, stalled at a DNA lesion, is used for the loading of the NER machinery in a process termed transcription-coupled repair (TCR).