Kinetic analysis of T4 polynucleotide kinase via isothermal titration calorimetry.

T4 polynucleotide kinase (T4 PNK) phosphorylates the 5'-terminus of DNA and RNA substrates. It is widely used in molecular biology. Single nucleotides can serve as substrates if a 3'-phosphate group is present.

VirB, a key transcriptional regulator of virulence, requires a CTP ligand for its regulatory activities.

species cause bacillary dysentery, the second leading cause of diarrheal deaths worldwide. There is a pressing need to identify novel molecular drug targets. virulence phenotypes are controlled by the transcriptional regulator, VirB.

VirB, a key transcriptional regulator of virulence, requires a CTP ligand for its regulatory activities.

Unlabelled: The VirB protein, encoded by the large virulence plasmid of spp., is a key transcriptional regulator of virulence genes. Without a functional gene, cells are avirulent.

Aqueous solubility of beryllium(II) at physiological pH: effects of buffer composition and counterions.

Beryllium ion elicits p53-mediated cell cycle arrest in some types of human cancer cells, and it is a potent inhibitor of GSK3 kinase activity. Paradoxically, Be is regarded to have almost negligible aqueous solubility at physiological pH, due to precipitation as Be(OH). This study demonstrates that the interaction of Be with serum proteins greatly increases its effective solubility.

The GSK3 kinase inhibitor lithium produces unexpected hyperphosphorylation of β-catenin, a GSK3 substrate, in human glioblastoma cells.

Lithium salt is a classic glycogen synthase kinase 3 (GSK3) inhibitor. Beryllium is a structurally related inhibitor that is more potent but relatively uncharacterized. This study examined the effects of these inhibitors on the phosphorylation of endogenous GSK3 substrates.

Site-directed mutants of human RECQ1 reveal functional importance of the zinc binding domain.

RecQ helicases are a highly conserved family of ATP-dependent DNA-unwinding enzymes with key roles in DNA replication and repair in all kingdoms of life. The RECQ1 gene encodes the most abundant RecQ homolog in humans. We engineered full-length RECQ1 harboring point mutations in the zinc-binding motif (amino acids 419-480) within the conserved RecQ-specific-C-terminal (RQC) domain known to be critical for diverse biochemical and cellular functions of RecQ helicases.

Estimation of alkane-water logP for neutral, acidic, and basic compounds using an alkylated polystyrene-divinylbenzene high-performance liquid chromatography column.

Reliable HPLC methods are available to estimate octanol-water partition coefficients, but there is no comparable method for alkane-water partition coefficients that is accurate and applicable across a broad span of logP(alk). This study describes a high-throughput method for determining HPLC-logP(alk), a chromatographic parameter closely related to logP(alk), using an alkylated polystyrene-divinylbenzene column and fast acetonitrile gradient. A structurally diverse set of neutral, acidic, and basic compounds was analyzed under ionization-suppressing pH conditions.

RECQ1 interacts with FEN-1 and promotes binding of FEN-1 to telomeric chromatin.

RecQ helicases are a family of highly conserved proteins that maintain genomic stability through their important roles in replication restart mechanisms. Cellular phenotypes of RECQ1 deficiency are indicative of aberrant repair of stalled replication forks, but the molecular functions of RECQ1, the most abundant of the five known human RecQ homologues, have remained poorly understood. We show that RECQ1 associates with FEN-1 (flap endonuclease-1) in nuclear extracts and exhibits direct protein interaction in vitro.

Beryllium is an inhibitor of cellular GSK-3β that is 1,000-fold more potent than lithium.

Glycogen synthase kinase 3β (GSK-3β) is a key regulator in signaling networks that control cell proliferation, metabolism, development, and other processes. Lithium chloride is a GSK-3 family inhibitor that has been a mainstay of in vitro and in vivo studies for many years. Beryllium salt has the potential to act as a lithium-like inhibitor of GSK-3, but it is not known whether this agent is effective under physiologically relevant conditions.

Alzheimer's disease drug development: translational neuroscience strategies.

Alzheimer's disease (AD) is an urgent public health challenge that is rapidly approaching epidemic proportions. New therapies that defer or prevent the onset, delay the decline, or improve the symptoms are urgently needed. All phase 3 drug development programs for disease-modifying agents have failed thus far.

Beryllium sulfate induces p21 CDKN1A expression and a senescence-like cell cycle arrest in susceptible cancer cell types.

In fibroblasts, beryllium salt causes activation of the p53 transcription factor and induction of a senescence-like state. It is not known whether Be(2+) can affect the proliferation of cancer cells, which are generally unsusceptible to senescence. A172 glioblastoma and RKO colon carcinoma cell lines each have wildtype p53, so these cell types have the potential to be responsive to agents that activate p53.

Physical and functional interaction between human oxidized base-specific DNA glycosylase NEIL1 and flap endonuclease 1.

The S phase-specific activation of NEIL1 and not of the other DNA glycosylases responsible for repairing oxidatively damaged bases in mammalian genomes and the activation of NEIL1 by proliferating cell nuclear antigen (PCNA) suggested preferential action by NEIL1 in oxidized base repair during DNA replication. Here we show that NEIL1 interacts with flap endonuclease 1 (FEN-1), an essential component of the DNA replication. FEN-1 is present in the NEIL1 immunocomplex isolated from human cell extracts, and the two proteins colocalize in the nucleus.

Beryllium induces premature senescence in human fibroblasts.

After cells have completed a sufficient number of cell divisions, they exit the cell cycle and enter replicative senescence. Here, we report that beryllium causes proliferation arrest with premature expression of the principal markers of senescence. After young presenescent human fibroblasts were treated with 3 microM BeSO(4) for 24 h, p21 cyclin-dependent kinase inhibitor mRNA increased by >200%.

The interaction site of Flap Endonuclease-1 with WRN helicase suggests a coordination of WRN and PCNA.

Werner and Bloom syndromes are genetic RecQ helicase disorders characterized by genomic instability. Biochemical and genetic data indicate that an important protein interaction of WRN and Bloom syndrome (BLM) helicases is with the structure-specific nuclease Flap Endonuclease 1 (FEN-1), an enzyme that is implicated in the processing of DNA intermediates that arise during cellular DNA replication, repair and recombination. To acquire a better understanding of the interaction of WRN and BLM with FEN-1, we have mapped the FEN-1 binding site on the two RecQ helicases.

The p53 inhibitor pifithrin-alpha forms a sparingly soluble derivative via intramolecular cyclization under physiological conditions.

The transcription factor p53 coordinates cell cycle arrest and apoptosis in response to DNA damage. Pifithrin-alpha (PFT-alpha) is a small molecule inhibitor of p53 activity that is frequently used in cell culture studies of p53 function. Here we report an investigation of the stability of this compound.

Quantitative assay of senescence-associated beta-galactosidase activity in mammalian cell extracts.

Senescence-associated beta-galactosidase activity is a widely used biomarker for assessing replicative senescence in mammalian cells. This enzymatic activity has generally been measured by staining cells with the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside (X-gal) at pH 6.0, a reaction condition that suppresses lysosomal beta-galactosidase activity sufficiently to ensure that most nonsenescent cells will appear unstained.