Activity of the antimutagenic enzyme 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) in cultured chinese hamster ovary cells: effects of cell cycle, proliferation rate, and population density.

Mammalian 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolases (8-oxo-dGTPases), such as MTH1, are believed to play the same antimutagenic role as their bacterial homologues, like MutT. Both decompose promutagenic 8-oxo-dGTP, a product of active oxygen's attack on dGTP. It is not known how 8-oxo-dGTPase expression and function are regulated.

Crystal structure of RNA 3'-terminal phosphate cyclase, a ubiquitous enzyme with unusual topology.

Background: RNA cyclases are a family of RNA-modifying enzymes that are conserved in eucarya, bacteria and archaea. They catalyze the ATP-dependent conversion of the 3'-phosphate to the 2',3'-cyclic phosphodiester at the end of RNA, in a reaction involving formation of the covalent AMP-cyclase intermediate. These enzymes might be responsible for production of the cyclic phosphate RNA ends that are known to be required by many RNA ligases in both prokaryotes and eukaryotes.

The aspartic proteinase from Saccharomyces cerevisiae folds its own inhibitor into a helix.

Aspartic proteinase A from yeast is specifically and potently inhibited by a small protein called IA3 from Saccharomyces cerevisiae. Although this inhibitor consists of 68 residues, we show that the inhibitory activity resides within the N-terminal half of the molecule. Structures solved at 2.

Theoretical calculations of glycine and alanine gas-phase acidities.

The gas-phase acidities of glycine and alanine were determined by using a variety of high level theoretical methods to establish which of these would give the best results with accessible computational efforts. MP2, MP4, QCISD, G2 ab initio procedures, hybrid Becke3-LYP (B3LYP) and gradient corrected Becke-Perdew (BP) and Perdew-Wang and Perdew (PWP) nonlocal density functionals were used for the calculations. A maximum deviation of approximately 13 and 18 kJ/mol from experimental data was observed for the computed delta Hacid and delta Gacid values, respectively.

Stem Trace: an interactive visual tool for comparative RNA structure analysis.

Motivation: Stem Trace is one of the latest tools available in STRUCTURELAB, an RNA structure analysis computer workbench. The paradigm used in STRUCTURELAB views RNA structure determination as a problem of dealing with a database of a large number of computationally generated structures. Stem Trace provides the capability to analyze this data set in a novel, visually driven, interactive and exploratory way.

Expression of dominant negative Erk2 inhibits AP-1 transactivation and neoplastic transformation.

The mitogen activated protein (MAP) kinases or extracellular signal-regulated kinases (Erks) are activated in response to Ras expression or exposure to tumor promoters or to growth factors, and have been implicated in AP-1 transactivation in some models. We have shown that tumor promoter induced activation of the transcription factor AP-1 is required for induced neoplastic transformation in the Balb/C JB6 cell model. Jun and Fos family protein levels have been found not to be limiting for AP-1 response.

Telomere dynamics in HIV-1 infected and uninfected chimpanzees measured by an improved method based on high-resolution two-dimensional calibration of DNA sizes.

We developed an improved method for accurately measuring telomere lengths based on two-dimensional calibration of DNA sizes combined with pulsed field electrophoresis and quantitative analysis of high-resolution gel images. This method was used to quantify the length of telomeres in longitudinal samples of peripheral blood mononuclear cells (PBMCs) from five chimpanzees infected with human immunodeficiency virus type 1 (HIV-1) and three uninfected animals, 14 to 27 years of age. The average length of the telomere restriction fragments (TRF) of infected and uninfected chimpanzees were 11.

Long-term telomere dynamics: modest increase of cell turnover in HIV-infected individuals followed for up to 14 years.

To quantify the long-term dynamics of telomere lengths and the effect of HIV infection on lymphocyte turnover rates, we measured in a blinded study longitudinal samples from 6 individuals using a highly accurate method based on two-dimensional calibration of DNA sizes. For two uninfected controls followed 8 and 10 years the average telomeric terminal restriction fragment (TRF) shortening rate in peripheral blood mononuclear cells (PBMCs) was 50 and 60 bp/year, respectively, in agreement with previous measurements of cross-sectional samples. The TRF lengths of PBMCs from two slow progressors followed for 14 years declined by a rate of 120 +/-10 bp/year, i.

Determination of flavopiridol (L86 8275; NSC 649890) in human plasma by reversed-phase liquid chromatography with electrochemical detection.

Purpose: Flavopiridol is a flavone which inhibits several cyclin-dependent kinases, and exhibits potent growth-inhibitory activity against a number of human tumor cell lines both in vitro, and when grown as xenografts in mice. It is currently being evaluated in a phase I clinical trial at the National Cancer Institute. The objective of this project was to develop and validate an analytical method for the assay of flavopiridol in human plasma, with sufficient sensitivity to permit the plasma pharmacokinetics of flavopiridol to be studied during clinical trials.

A novel assay of 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) activity in cultured cells and its use for evaluation of cadmium(II) inhibition of this activity.

8-Oxo-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) is a product of oxidative modification of dGTP, thatcan be misincorporated into DNA, causing AT-->CG mutations. Cells are protected against 8-oxo-dGTP by 8-oxo-dGTP 5'-pyrophosphohydrolases (8-oxo-dGTP-ases) that convert it to 8-oxo-dGMP. Thus, inhibition of 8-oxo-dGTPases may lead to cancer.

Development and applications of enhanced green fluorescent protein mutants.

The introduction of several mutations resulted in the generation of improved mutants of the green fluorescent protein (GFP). A strong green (GFPsg25) and blue (BFPsg50) fluorescent protein, gave 50-fold-100-fold brighter fluorescence compared to wild-type GFP and BFP (Tyr66His), respectively, upon expression in mammalian cells. GFPsg25 and BFPsg50 have different excitation and emission maxima.

Recombinant production of cyanovirin-N, a potent human immunodeficiency virus-inactivating protein derived from a cultured cyanobacterium.

Here we describe the recombinant production and purification of a novel anti-human immunodeficiency virus (HIV) protein, cyanovirin-N (CV-N), in Escherichia coli. Initial attempts to express CV-N using a vector containing an ompA signal peptide sequence resulted in production of an intractable mixture of the full-length (101 amino acid residue) protein and a truncated form lacking the first two N-terminal amino acids. The truncated protein was observed regardless of the host cell line, culture conditions, or induction time.

Preclinical evaluation of 9-chloro-2-methylellipticinium acetate alone and in combination with conventional anticancer drugs for the treatment of human brain tumor xenografts.

Some ellipticine derivative salts, including 9-chloro-2-methylellipticinium (CME), have been found to have a marked selectivity against all eight brain tumor cell lines of the U.S. National Cancer Institute's disease-oriented in vitro screen.

Sensitivity of Escherichia coli (MutT) and human (MTH1) 8-oxo-dGTPases to in vitro inhibition by the carcinogenic metals, nickel(II), copper(II), cobalt(II) and cadmium(II).

The toxicity of Ni(II), Co(II) and Cu(II) in animals, and that of Cd(II) in cultured cells, has been associated with generation of the promutagenic lesion 8-oxo-7,8-dihydroguanine (8-oxoguanine) in DNA, among other effects. One possible source of this base may be 8-oxo-7,8-dihydro-2'-deoxyguanosine-5'-triphosphate (8-oxo-dGTP), a product of oxidative damage to the nucleotide pool, from which it is incorporated into DNA. To promote such incorporation, the metals would have to inhibit specific cellular 8-oxo-dGTPases that eliminate 8-oxo-dGTP from the nucleotide pool.

Analysis of sequence requirements for biological activity of cyanovirin-N, a potent HIV (human immunodeficiency virus)-inactivating protein.

Site-directed mutagenesis of DNA constructs coding for the novel, HIV-inactivating proteins cyanovirin-N (CV-N) and FLAG-cyanovirin-N (F-CV-N) was performed using mutagenic oligonucleotide primers in the polymerase chain reaction or by a restriction site elimination maneuver. The mutant constructs were expressed in Escherichia coli and the recombinant protein products were tested for binding to the HIV surface envelope glycoprotein gp 120 and for antiviral activity against infectious HIV. Results showed an overall very high correlation (r2 > 0.

Effects of glutathione depletion on cadmium-induced metallothionein synthesis, cytotoxicity, and proto-oncogene expression in cultured rat myoblasts.

Cadmium (Cd) is a highly toxic metal and a known carcinogen. Although the carcinogenic mechanism of action is unknown, Cd will induce transcriptional activation of c-myc and c-jun. We have previously found that the extent of Cd-induced oncogene expression is limited by the presence of cellular metallothionein (MT) in rat L6 myoblasts.

ERF: genomic organization, chromosomal localization and promoter analysis of the human and mouse genes.

ERF (Ets2 Repressor Factor) is a ubiquitously expressed ets-domain protein that exhibits strong transcriptional repressor activity, has been shown to suppress ets-induced transformation and has been suggested to be regulated by MAPK phosphorylation. We report here the sequence of the mouse gene, the genomic organization of the human and the mouse genes, their chromosomal position and the analysis of the promoter region. Genomic clones encompassing either the human ERF or the mouse Erf gene were isolated and utilized to define their molecular organization.

Lack of p53 and ras mutations in Helicobacter hepaticus-induced liver tumors in A/JCr mice.

Helicobacter hepaticus is a recently discovered bacterium that invades mouse liver causing chronic active hepatitis followed by development of preneoplastic hepatocellular foci, hepatocellular adenomas and carcinomas. This establishes a unique animal model for study of the mechanisms of cancer development due to a chronic bacterial infection. A possible mechanism of bacteria-associated tumorigenesis is mutation of oncogenes or tumor suppressor genes.

Interferon expression by B cells.

Interferons have effects on many facets of immune system development, maturation, and function. B cells are a target for the interferons, causing alterations in cell differentiation and gene expression. However, production of interferons by B cells has not been widely studied.

The Ly-49D receptor activates murine natural killer cells.

Proteins encoded by members of the Ly-49 gene family are predominantly expressed on murine natural killer (NK) cells. Several members of this gene family have been demonstrated to inhibit NK cell lysis upon recognizing their class I ligands on target cells. In this report, we present data supporting that not all Ly-49 proteins inhibit NK cell function.

Mechanistic studies of the inhibition of MutT dGTPase by the carcinogenic metal Ni(II).

Promutagenic 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) levels are increased in DNA of animals exposed to carcinogenic metals, such as Ni(II). Besides being generated directly in genomic DNA, 8-oxo-dG may be incorporated there from 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP), a product of oxidative damage to the nucleotide pool. The Escherichia coli dGTPase MutT, and analogous dGTPases in rats and humans, have been suggested as a defense against such incorporation because they hydrolyze 8-oxo-dGTP to 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-monophosphate (8-oxo-dGMP).

All-atom models for the non-nucleoside binding site of HIV-1 reverse transcriptase complexed with inhibitors: a 3D QSAR approach.

Several molecular modeling techniques were used to generate an all-atom molecular model of a receptor binding site starting only from Ca atom coordinates. The model consists of 48 noncontiguous residues of the non-nucleoside binding site of HIV-1 reverse transcriptase and was generated using a congeneric series of nevirapine analogs as structural probes. On the basis of the receptor-ligand atom contacts, the program HINT was used to develop a 3D quantitative structure activity relationship that predicted the rank order of binding affinities for the series of inhibitors.

ETS1 and ETS2 in p53 regulation: spatial separation of ETS binding sites (EBS) modulate protein: DNA interaction.

p53 is an extensively studied tumor suppressor gene implicated in the genesis of a large number of varied tumors. However, the pathways of regulation for the wild-type p53 gene and its product are as yet unknown. In situ hybridization analyses of ETS1 and ETS2 expression during mouse embryogenesis, have shown a pattern similar to that of p53 gene expression.

Evolutionary histories of highly repeated DNA families among the Artiodactyla (Mammalia).

Six highly repeated DNA families were analyzed using Southern blotting and fluorescence in situ hybridization in a comparative study of 46 species of artiodactyls belonging to seven of the eight extant taxonomic families. Two of the repeats, the dispersed bovine-Pst family and the localized 1.715 component, were found to have the broadest taxonomic distributions, being present in all pecoran ruminants (Giraffidae, Cervidae, Antilocapridae, and Bovidae), indicating that these repeats may be 25-40 million years old.