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The Gridded Retarding Ion Drift Sensor (GRIDS) is a small sensor that will fly on the 6 U petitSat CubeSat. It is designed to measure the three-dimensional plasma drift velocity vector in the Earth's ionosphere. The GRIDS also supplies information about the ion temperature, ion density, and the ratio of light to heavy ions present in the ionospheric plasma. It utilizes well-proven techniques that have been successfully validated by similar instruments on larger satellite missions while meeting CubeSat-compatible requirements for low mass, size, and power consumption. GRIDS performs the functions of a Retarding Potential Analyzer (RPA) and an Ion Drift Meter (IDM) by combining the features of both types of instruments in a single package. The sensor alternates RPA and IDM measurements to produce the full set of measurement parameters listed above. On the petitSat mission, GRIDS will help identify and characterize a phenomenon known as plasma blobs (or enhancements).

Here we describe the first neutral wind sensor developed specifically for use on resource limited nano-satellite platforms. The instrument is a next generation redesign of the ram wind sensor flown on the Communications/Navigation Outage Forecasting System satellite for measurements of neutral velocity, temperature, and composition. Results of subsystem tests in vacuum conditions show low-power operation, promising design, and good resolution of measured parameters over the operational pressure and energy ranges expected in the low Earth orbit environment.

Alzheimer's disease (AD) and age-related cognitive decline represent a growing health burden and involve the hippocampus, a vulnerable brain region implicated in learning and memory. To understand the molecular effects of aging on the hippocampus, this study characterized the gene expression changes associated with aging in rodents using RNA-sequencing (RNA-seq). The glutamate modulator, riluzole, which was recently shown to improve memory performance in aged rats, prevented many of the hippocampal age-related gene expression changes. A comparison of the effects of riluzole in rats against human AD data sets revealed that many of the gene changes in AD are reversed by riluzole. Expression changes identified by RNA-Seq were validated by qRT-PCR open arrays. Riluzole is known to increase the glutamate transporter EAAT2's ability to scavenge excess glutamate, regulating synaptic transmission. RNA-seq and immunohistochemistry confirmed an increase in EAAT2 expression in hippocampus, identifying a possible mechanism underlying the improved memory function after riluzole treatment.

The design of the first retarding potential analyzer (RPA) built specifically for use on resource-limited cubesat platforms is described. The size, mass, and power consumption are consistent with the limitations of a nano-satellite, but the performance specifications are commensurate with those of RPAs flown on much larger platforms. The instrument is capable of measuring the ion density, temperature, and the ram component of the ion velocity in the spacecraft reference frame, while also providing estimates of the ion composition. The mechanical and electrical designs are described, as are the operating modes, command and data structure, and timing scheme. Test data obtained using an ion source inside a laboratory vacuum chamber are presented to validate the performance of the new design.

Thermal limits in ectotherms may arise through a mismatch between supply and demand of oxygen. At higher temperatures, the ability of their cardiac and ventilatory activities to supply oxygen becomes insufficient to meet their elevated oxygen demand. Consequently, higher levels of oxygen in the environment are predicted to enhance tolerance of heat, whereas reductions in oxygen are expected to reduce thermal limits. Here, we extend previous research on thermal limits and oxygen limitation in aquatic insect larvae and directly test the hypothesis of increased anaerobic metabolism and lower energy status at thermal extremes. We quantified metabolite profiles in stonefly nymphs under varying temperatures and oxygen levels. Under normoxia, the concept of oxygen limitation applies to the insects studied. Shifts in the metabolome of heat-stressed stonefly nymphs clearly indicate the onset of anaerobic metabolism (e.g., accumulation of lactate, acetate, and alanine), a perturbation of the tricarboxylic acid cycle (e.g., accumulation of succinate and malate), and a decrease in energy status (e.g., ATP), with corresponding decreases in their ability to survive heat stress. These shifts were more pronounced under hypoxic conditions, and negated by hyperoxia, which also improved heat tolerance. Perturbations of metabolic pathways in response to either heat stress or hypoxia were found to be somewhat similar but not identical. Under hypoxia, energy status was greatly compromised at thermal extremes, but energy shortage and anaerobic metabolism could not be conclusively identified as the sole cause underlying thermal limits under hyperoxia. Metabolomics proved useful for suggesting a range of possible mechanisms to explore in future investigations, such as the involvement of leaking membranes or free radicals. In doing so, metabolomics provided a more complete picture of changes in metabolism under hypoxia and heat stress.

The aims of this study were to characterise the major saturated and unsaturated lipid peaks in histologically normal cervical epithelium and stroma, dysplastic epithelium (low-grade cervical intraepithelial neoplasia, CIN) and cancer-containing tissue samples from patients with cervical cancer using diffusion-weighted (1) H high-resolution magic angle spinning MRS, to determine whether mobile lipid resonances (MLRs) distinguish tissue types and to test for a correlation between MLRs and the number of cytoplasmic lipid droplets. Diffusion-weighted spectra of tissue biopsies were acquired using a stimulated echo sequence with bipolar gradients. Major saturated and unsaturated MLRs were identified and multivariate analysis of peak combinations was used to determine the best separation between tissue classes. Lipid droplets were visualised with Nile red staining and fluorescence microscopy. Correlations of saturated lipid resonances (0.9 and 1.3 ppm), polyunsaturated resonances (2.8 ppm), triglycerides (4.3 ppm) and unsaturated resonances (5.3 ppm) with average droplet number (per image) were investigated using a Spearman rank test. A large heterogeneity in lipid content among samples was observed, resulting in no significant differences in MLR intensities of individual peaks between the three tissue classes. Linear discriminant analysis separated 'no cancer' from 'cancer' based on the intensities at 0.9, 1.3, 2.2 and 2.8 ppm [area under the curve (AUC) = 0.939, p < 0.001], 'low-grade CIN' from 'cancer' based on the intensities at 0.9, 4.1, 4.3 and 5.3 ppm (AUC = 0.987, p < 0.001) and 'no cancer' from 'low-grade CIN' based on intensities at 0.9, 2.2 and 4.3 ppm (AUC = 0.984, p < 0.001). The distribution of cytoplasmic lipid droplets was nonuniform and was not related to the presence of epithelial or stromal components. On average, there were more droplets visible in low-grade CIN and cancer-containing tissues. Significant correlations between MLR peaks and lipid droplet number were seen for 0.9 (p = 0.002), 1.3 (p = 0.003) and 2.8 ppm (p = 0.018). MLR combinations indicative of average lipid structure efficiently separated tissue classes. Increased lipid resonances correlated with increased numbers of cytoplasmic lipid droplets., (Copyright © 2013 John Wiley & Sons, Ltd.)

Direct infusion mass spectrometry (DIMS)-based untargeted metabolomics measures many hundreds of metabolites in a single experiment. While every effort is made to reduce within-experiment analytical variation in untargeted metabolomics, unavoidable sources of measurement error are introduced. This is particularly true for large-scale multi-batch experiments, necessitating the development of robust workflows that minimise batch-to-batch variation. Here, we conducted a purpose-designed, eight-batch DIMS metabolomics study using nanoelectrospray (nESI) Fourier transform ion cyclotron resonance mass spectrometric analyses of mammalian heart extracts. First, we characterised the intrinsic analytical variation of this approach to determine whether our existing workflows are fit for purpose when applied to a multi-batch investigation. Batch-to-batch variation was readily observed across the 7-day experiment, both in terms of its absolute measurement using quality control (QC) and biological replicate samples, as well as its adverse impact on our ability to discover significant metabolic information within the data. Subsequently, we developed and implemented a computational workflow that includes total-ion-current filtering, QC-robust spline batch correction and spectral cleaning, and provide conclusive evidence that this workflow reduces analytical variation and increases the proportion of significant peaks. We report an overall analytical precision of 15.9%, measured as the median relative standard deviation (RSD) for the technical replicates of the biological samples, across eight batches and 7 days of measurements. When compared against the FDA guidelines for biomarker studies, which specify an RSD of <20% as an acceptable level of precision, we conclude that our new workflows are fit for purpose for large-scale, high-throughput nESI DIMS metabolomics studies.

The purpose of this study was to implement a diffusion-weighted sequence for visualisation of mobile lipid resonances (MLR) using high resolution magic angle spinning (HR-MAS) (1)H MRS and to evaluate its use in establishing differences between tissues from patients with cervical carcinoma that contain cancer from those that do not. A stimulated echo sequence with bipolar gradients was modified to allow T(1) and T(2) measurements and optimised by recording signal loss in HR-MAS spectra as a function of gradient strength in model lipids and tissues. Diffusion coefficients, T(1) and apparent T(2) relaxation times were measured in model lipid systems. MLR profiles were characterised in relation to T(1) and apparent T(2) relaxation in human cervical cancer tissue samples. Diffusion-weighted (DW) spectra of cervical biopsies were quantified and peak areas analysed using linear discriminant analysis (LDA). The optimised sequence reduced spectral overlap by suppressing signals originating from low molecular weight metabolites and non-lipid contributions. Significantly improved MLR visualisation allowed visualisation of peaks at 0.9, 1.3, 1.6, 2.0, 2.3, 2.8, 4.3 and 5.3 ppm. MLR analysis of DW spectra showed at least six peaks arising from saturated and unsaturated lipids and those arising from triglycerides. Significant differences in samples containing histologically confirmed cancer were seen for peaks at 0.9 (p < 0.006), 1.3 (p < 0.04), 2.0 (p < 0.03), 2.8 (p < 0.003) and 4.3 ppm (p < 0.0002). LDA analysis of MLR peaks from DW spectra almost completely separated two clusters of cervical biopsies (cancer, 'no-cancer'), reflecting underlying differences in MLR composition. Generated Receiver Operating Characteristic (ROC) curves and calculated area under the curve (0.962) validated high sensitivity and specificity of the technique. Diffusion-weighting of HR-MAS spectroscopic sequences is a useful method for characterising MLR in cancer tissues and displays an accumulation of lipids arising during tumourigenesis and an increase in the unsaturated lipid and triglyceride peaks with respect to saturated MLR., (Copyright © 2009 John Wiley & Sons, Ltd.)

The most frequently observed mutations in ras oncogenes in solid human tumors are GC-->AT transitions at the 3' G residue of the GG doublet in codon 12 of these oncogenes. We had shown previously that mutagenesis by thymidine occurred with the same sequence specificity in mammalian cells, in that mutagenesis occurred preferentially at the 3' G of GG doublets. In this study, in vitro DNA synthesis experiments were carried out to assess the effect of local DNA sequence on base mispairing in order to determine the mechanism of sequence-directed mutagenesis by thymidine and its possible relationship to activating point mutations in N-, Ki- and Ha-ras oncogenes in solid human tumors. To avoid complicating the interpretation of the results because of the occurrence of mismatch repair as well as base misincorporation, the experiments were carried out in a repair-free environment with exonuclease-free Klenow polymerase. The results of these experiments showed that misincorporation of deoxyribosylthymine (dT) occurred with several-fold-greater efficiency opposite the 3' G compared to the 5' G of the GG doublet in codon 12 of human ras oncogenes. These results further demonstrated that the relative difference in the extent of dT misincorporation opposite the 3' G and the 5' G of GG doublets in codon 12 in the various ras oncogenes was affected by the base immediately upstream of the doublet. Within the GG doublet, it was seen that the 5' G and 3' G residues had an effect on the extent of dT misincorporation opposite each other. The 5' G was shown to have a stimulatory effect on dT misincorporation opposite the 3' G, while the 3' G was shown to have an inhibitory effect on dT misincorporation opposite the 5' G. Presumably, these mutual interactions within GG doublets are additive, such that the large differential in dT misincorporation observed between the 3' G and 5' G residues in GG doublets is the end result of the combined stimulatory and inhibitory effects within these doublets. Since the observed pattern of dT misincorporation within GG doublets corresponds to the most frequent mode of activation of ras oncogenes in solid human tumors, the results of these experiments suggest that sequence-directed dT misincorporation may be involved in the pattern of activation of human ras oncogenes, by causing GC-->AT transitions preferentially at the 3' G of the GG doublet in codon 12 of these oncogenes.

Severely comminuted fractures of the lateral tibial plateau with central defects of the articular cartilage have traditionally been treated with iliac crest or patellar autograft, with varying success. Arthrodesis or arthroplasty for late deformity or instability are not suitable for young, active patients. The use of the fibular head as a replacement for the tibial plateau obtained excellent or good functional results in five patients with these difficult fractures.

Whole cell hybrids and microcell hybrids between mouse fibroblasts and pigmented Syrian hamster melanoma cells were analyzed for coordinate regulation of melanocyte-specific gene products. Extinction of pigmentation was observed in whole-cell hybrids and in a microcell hybrid containing a single mouse chromosome (mouse chromosome 1). Analysis of melanocyte-specific transcripts using reverse transcription, combined with the polymerase chain reaction (RT-PCR), demonstrated that tyrosinase, TRP-1, TRP-2, and microphthalmia transcripts were all absent in unpigmented whole-cell hybrids and in the monochromosomal unpigmented microcell hybrid. A pigmented subclone of this microcell hybrid, however, re-expressed the tyrosinase, TRP-1, TRP-2, and microphthalmia genes. These data suggest that all of these genes are coordinately extinguished by a single fibroblast locus. Since the only fibroblast chromosome detected in the unpigmented microcell hybrid was mouse chromosome 1, these results also suggest that the extinguisher locus affecting the expression of the tyrosinase, TRP-1, TRP-2, and microphthalmia genes in hybrid cells is located on that mouse chromosome (or on a fragment of another chromosome present in the unpigmented monochromosomal microcell hybrid but undetected in our analyses). In contrast to the results with the melanocyte-specific genes mentioned above, transcripts for the melanocortin 1 receptor gene (MC1R) were present in the monochromosomal unpigmented microcell hybrid (although absent in the whole-cell hybrids). This suggests that regulation of MC1R gene expression is distinct from regulation of the other melanocyte-specific genes.

Human and mouse fibroblast chromosomes carrying tyrosinase or b-locus genes were introduced, by microcell hybridization, into pigmented Syrian hamster melanoma cells, and the microcell hybrids were tested for transactivation of the fibroblast tyrosinase and b-locus genes. By using species-specific PCR amplification to distinguish fibroblast and melanoma cDNAs, it was demonstrated that the previously silent fibroblast tyrosinase and b-locus genes were transactivated following chromosomal transfer into pigmented melanoma cells. However, transactivation of the mouse fibroblast tyrosinase gene was unstable in microcell hybrid subclones and possibly dependent on a second fibroblast locus that could have segregated in the subclones. This second locus was not necessary for transactivation of the fibroblast b-locus gene, thus demonstrating noncoordinate transactivation of fibroblast tyrosinase and b-locus genes. Transactivation of the fibroblast tyrosinase gene in microcell hybrids apparently is dependent on the absence of a putative fibroblast extinguisher locus for tyrosinase gene expression, which presumably is responsible for the extinction of pigmentation in hybrids between karyotypically complete fibroblasts and melanoma cells.

We have previously demonstrated that mutagenesis by bromodeoxyuridine (BrdU) and thymidine (dT) in mammalian cells occurs with a high degree of sequence specificity within runs of multiple adjacent guanine residues. To determine whether there is a structural component to this sequence specificity, we have analyzed stereochemical properties of guanine residues in different sequence contexts. Stereochemical differences were assessed by measuring the susceptibility of individual guanine residues to methylation by the agent dimethylsulfate (DMS). The results from this study suggest that there is a strong inverse correlation between susceptibility of various guanine residues to DMS methylation and the susceptibility of those residues to mutagenesis by BrdU and dT. These results suggest that the stereochemical attributes of guanine residues in different sequence contexts affect the susceptibility of those guanine residues to mutagenesis by BrdU and dT.

Loracarbef, the first carbacephem antibiotic to undergo clinical development, is excreted primarily unchanged in the urine (> 90%). Data analyzed from subjects with various degrees of renal dysfunction who were given single oral doses of loracarbef indicated a linear relationship between creatinine clearance (CLCR) and plasma clearance [CLP (L/hr) = 0.106.CLCR (ml/min/1.73 m2)]. The mean area under the plasma concentration-time curve in normal subjects and in patients with severe renal insufficiency (no dialysis/receiving dialysis) was 32 micrograms.hr/ml and 1085 micrograms.hr/ml/103 micrograms.hr/ml, respectively. Therefore, for individuals with moderate renal insufficiency (CLCR, 10 to 49 ml/min/1.73 m2), the dose should be halved or the dosing interval doubled; patients with severe renal insufficiency who are not receiving dialysis should be treated with the normal dose given once every 3 to 5 days. Loracarbef is readily cleared from plasma by hemodialysis; dosing should be repeated after a hemodialysis treatment.

5-Bromodeoxyuridine (BrdU), a thymidine analog, suppresses melanogenesis in Syrian hamster melanoma cells. Tyrosinase, which is the key enzyme for the synthesis of melanin, is suppressed by exposure to BrdU, and the drop in enzyme activity is correlated with a drop in tyrosinase mRNA level. In order to investigate whether suppression of tyrosinase mRNA by BrdU is due to BrdU substitution into coding sequences or upstream sequences of the tyrosinase gene, we carried out stable and transient transfection assays with constructs containing either the human tyrosinase cDNA sequence under the control of a nontyrosinase promoter or a chloramphenicol acetyltransferase (CAT) reporter gene under the control of 5' flanking sequences of the mouse tyrosinase gene. When the plasmid containing the tyrosinase cDNA was stably transfected into mouse fibroblasts, tyrosinase activity in the transfectants was not suppressed by BrdU. Since BrdU would be incorporated into the tyrosinase cDNA integrated in these transfectants, the results suggest that BrdU suppression of tyrosinase gene expression is not due to its incorporation into coding sequences of the tyrosinase gene. When plasmids with tyrosinase regulatory sequences were transfected into melanoma cells for transient expression assays, CAT gene expression was suppressed by BrdU. Because the CAT plasmids do not contain a mammalian origin of replication and should not replicate under the conditions of transient transfection, BrdU would not be incorporated into the DNA of those plasmids. Therefore, these results suggest that the suppression of tyrosinase gene expression by BrdU also is not due to the incorporation of BrdU into upstream sequences of the tyrosinase gene.

Imbalances in the intracellular nucleotide precursor pools in mammalian cells can result in the induction of mutations during the DNA replication process. By using a shuttle vector system developed in our laboratory, we have analyzed the sequence specificity of mutations induced in mouse A9 cells by exposure of the cells to a high concentration of thymidine. The target for mutagenesis in these studies was the bacterial gpt gene stably integrated into the chromosomal DNA of the mouse cells. Previous studies in this laboratory had generated a large panel of xanthine guanine phosphoribosyl-transferase (EC 2.4.2.22)-negative mutant lines that possess single-base mutations within the gpt coding sequence. This study utilized four xanthine guanine phosphoribosyltransferase-negative mutant lines to assess the frequency of mutation induced by thymidine at guanine residues in four sequence contexts: the 5' and 3' guanine residues of a GG doublet, the middle guanine residue of a GGG triplet, and the 3' guanine residue of a GGGG quartet. The results of this study demonstrate that treatment of cultured cells with a high concentration of thymidine can result in G.C----A.T transition mutations that occur preferentially at the 3' guanine residue of a run of two or more adjacent guanines. Guanine residues flanked on their 3' side by other guanine residues are severalfold less mutable by thymidine than are guanine residues flanked on their 3' side by a different base. This study demonstrates a sequence-specific mode for thymidine-induced mutations and suggests implications for mutagenesis in vivo.

For studies on molecular mechanisms of mutagenesis, it would be advantageous to transfer mutant genes with specific alterations into mammalian cells and use the transformed cells in reversion analyses. In the present paper, we describe an efficient method for analyzing reversion events occurring in cells that possess multiple copies of a mutational target gene. This method involves amplification of the chromosomally integrated target genes with the polymerase chain reaction (PCR) and restriction endonuclease digestion of the amplified product. Single reversion events that either create or destroy restriction endonuclease recognition sequences that encompass the site of the original mutation can be identified in a background of 10-20 copies of the gene that retain the mutant sequence. Using this method, we have analyzed revertants induced by 5-bromodeoxyuridine (BrdU) in a Chinese hamster ovary cell line that possesses multiple copies of a mutant bacterial gpt gene containing a specific alteration. The results of this study not only demonstrate the effectiveness of this method for analyzing reversion of a single gene copy in transfectants possessing multiple copies of a mutant target gene, but also demonstrate that the sequence specificity for BrdU-induced mutations is the same in Chinese hamster cells as previously observed with mouse cells.

The molecular mechanisms of incorporation-dependent, 5-bromodeoxyuridine (BrdU)-induced mutagenesis were analyzed in murine A9 cells that possess a single copy of the Escherichia coli gpt gene integrated into the chromosomal DNA as part of a shuttle vector. Four independently derived GPT- mutants with single base changes within the integrated gpt gene were utilized in BrdU-induced reversion analyses to test the relative mutability of guanine residues in four different settings: the 5' and 3' guanine residues of a GG doublet, the 3' guanine residue of a GGGG quartet, and the middle guanine residue of a GGG triplet. Two of the mutant lines possessed GG doublet sequences in which a GC----AT transition at either guanine residue of the doublet leads to restoration of GPT enzyme activity without restoring wild-type DNA sequence. Both lines were shown to be effectively reverted by BrdU incorporation-dependent mutagenesis, and sequencing of the gpt genes from numerous independently derived revertants of both lines demonstrated that greater than 90% of the revertants arose due to GC----AT transitions at the 3' guanine residue of the doublet. BrdU-induced reversion of two additional GPT- mutant lines demonstrated that the 3' guanine residue of a GGGG quartet is efficiently mutated, while the middle guanine residue of a GGG triplet sequence is at least 10-fold less mutable by BrdU incorporation-dependent mutagenesis than the 3' guanine residue of a GG doublet or GGGG quartet. All four mutant lines tested were equally revertible by treatment with the alkylating agent ethyl methane sulfonate. The results from this study define a sequence-specific mechanism for BrdU-induced, incorporation-dependent mutagenesis and demonstrate the use of reversion analysis for the determination of sequence specific effects at precise sites within a gene.

The thymidine analog 5-bromodeoxyuridine (BrdU) suppresses pigmentation and tyrosinase activity in Syrian hamster melanoma cells W1-1-1. Studies on the molecular mechanism of suppression of pigmentation indicated that BrdU treatment affects the level of tyrosinase gene transcripts. No detectable tyrosinase message was found by Northern blot analysis in cells cultured in the presence of BrdU at concentrations even as low as 0.2 microM. The level of tyrosinase mRNA was found to reflect the level of pigmentation and tyrosinase activity. Studies with dibutyryl cyclic AMP (cAMP) showed that it inhibited pigment synthesis in W1-1-1 cells. With increasing concentrations of cAMP ranging from 10 microM to 300 microM, pigmentation and tyrosinase activity decreased progressively. This inhibition was found to be associated with a corresponding decrease in the level of tyrosinase mRNA. W1-1-1 cells were found not to respond to melanocyte stimulating hormone (MSH). There was no change in pigmentation, tyrosinase activity, or tyrosinase mRNA level in W1-1-1 cells in the presence of MSH. Similarly, theophylline, a phosphodiesterase inhibitor, had no effect on pigmentation or tyrosinase activity in W1-1-1 cells.

The molecular mechanism of reversion induced by 5-bromodeoxyuridine (BrdU) replication-dependent mutagenesis in mammalian cells was studied. Murine cells with single mutant copies of the E. coli gpt gene integrated chromosomally as part of a shuttle vector were mutagenized with BrdU, and GPT+ revertants were selected. Thirteen mutant cell lines (each of which had a gpt gene that varied from the wild-type gene by a different GC----AT base transition in the coding region) were mutagenized, and only four were found to be effectively reverted. All revertant gpt genes that were analyzed had reverted via AT----GC base transition at the original site of mutation, thus demonstrating that replication-dependent mutagenesis by BrdU causes AT----GC transitions. The nine cell lines that were nonrevertible by BrdU replication-dependent mutagenesis could be mutated by this protocol to ouabain resistance as effectively as the four revertible lines, indicating that the nonrevertible lines were susceptible to such mutagenesis. Thus, differences among the cell lines in frequencies of HATr revertants generated by BrdU replication-dependent mutagenesis could not be attributed to differences in general susceptibility of the lines to the mutagenic protocol. The revertible and nonrevertible lines could not be separated according to the position of the original GC----AT transition in the gpt coding region. However, there was evidence that the DNA base sequence flanking the site of mutation affected the susceptibility of that site to BrdU replication-dependent mutagenesis. For example, six of the cell lines tested had gpt genes in which the mutant T residue was immediately adjacent on its 3' side to an A residue, and all six were found to be nonrevertible by BrdU replication-dependent mutagenesis. Furthermore, a target AT base pair flanked by GC base pairs in opposite orientation and either immediately adjacent to or one base removed from the target site on both the 5' and 3' sides appeared to have an increased susceptibility to BrdU replication-dependent mutagenesis.

The molecular mechanisms of reversion in mammalian cells were studied utilizing the pZipGptNeo shuttle vector, with the bacterial gpt gene in the vector integrated into the chromosomal DNA of mouse cells. From mutant cell lines containing gpt genes with single base changes, revertants were selected for the reappearance of GPT activity. The copy number and expression of the gpt genes in such revertants were analyzed, and the GPT activity encoded by revertant genes in both mammalian cells and bacteria characterized. Revertants with wild-type amino acid sequence had, on average, the highest levels of GPT activity. Revertants with amino acid sequences different from the original mutants but not corresponding to wild-type had, on average, approximately half the level of GPT activity as wild-type revertants. Revertants that still contained the original mutation in the gpt gene had even lower levels of activity. These revertants were found to have amplified mutant gpt genes, which, when transferred into bacteria, were seen to encode for GPT polypeptides with partial enzymatic activity. A revertant in which the original mutation that destroyed the AUG translational start codon was retained but in which there was a secondary mutation upstream of the start codon also was characterized. The second mutation generated an in-frame CUG codon that apparently functioned as an alternative, upstream translational start codon.

We have analyzed the specificity of mutations induced by ethyl methanesulfonate (EtMes) in mouse cells carrying a selectable bacterial gene. The target gene was the Escherichia coli gpt gene contained within a retroviral shuttle vector integrated into mouse chromosomal DNA. Following mutagenesis by EtMes, cells with mutations in the gpt gene were selected as resistant to 6-thioguanine. Shuttle vector sequences were recovered from the mutant cell lines following fusion with monkey COS cells and introduced into bacteria as part of a bacterial plasmid. The DNA base sequences of the mutant genes were directly determined from plasmid DNA. All of the EtMes-induced mutations involving single base changes were found to be G:C to A:T transitions.

By using a shuttle vector system developed in our laboratory, we have carried out studies on the molecular mechanism by which 5-bromodeoxyuridine (BrdUrd) induces mutations in mammalian cells. The target for mutagenesis in these studies was the Escherichia coli gpt gene that was contained within a retroviral shuttle vector and integrated into chromosomal DNA in mouse A9 cells. Shuttle vector-transformed cells expressing the gpt gene were mutagenized with BrdUrd and cells with mutations in the gpt gene were selected. Shuttle vector sequences were recovered from the mutant cells, and the base sequence of the mutant gpt genes was determined. The great majority of the BrdUrd-induced mutations involving single-base changes were found to be G.C----A.T transitions. We have shown that mutagenesis by BrdUrd depends upon perturbation of deoxycytidine metabolism. Thus, the current results suggest that BrdUrd mutagenesis involves mispairing and misincorporation of BrdUrd opposite guanine in DNA, driven by nucleotide pool perturbation caused by BrdUrd and the resulting imbalanced supply of triphosphates available for DNA synthesis. The results also revealed a very high degree of sequence specificity for the BrdUrd mutagenesis. BrdUrd-induced G.C----A.T transitions occurred almost exclusively in sequences with two adjacent guanine residues. Furthermore, in approximately equal to 90% of the cases, the guanine residue involved in mutation was the one in the more 3' position.

Murine erythroleukemic cells in culture accumulate cytoplasmic globin mRNA during differentiation induced by dimethyl sulfoxide (DMSO)1. Chromatin was prepared from DMSO induced erythroleukemic cells that were transcribing globin RNA in order to determine whether in vitro synthesis of globin RNA sequences was possible from chromatin. RNA was synthesized in vitro using 5-mercuriuridine triphosphate and exogenous Escheria coli RNA polymerase. Newly synthesized mercurated RNA was purified from endogenous chromatin associated RNA by affinity chromatography on a sepharose sulfhydryl column, and the globin RNA sequence content of the mercurated RNA was assayed by hybridization to cDNA globin. The synthesis of globin RNA sequences was shown to occur and to be sensitive to actinomycin and rifampicin and insensitive to alpha-amanitin. In contrast, synthesis of globin RNA sequence synthesis was not detected in significant amounts from chromatin prepared from uninduced erythroleukemic cells, nor from uninduced cell chromatin to which globin RNA was added prior to transcription. Isolated RNA:cDNA globin hybrids were shown to contain mercurated RNA by affinity chromatography. These results indicated that synthesis of globin RNA sequences from chromatin can be performed by E. coli RNA polymerase.

The effects of 5-bromodeoxyuridine (BrdU) on pigmentation, contact inhibition, cell morphology, and tumorogenicity of Syrian hamster melanoma cells are inhibited in the presence of deoxycytidine (dC). The inhibition of these biological effects of BrdU by dC is correlated with a decrease in the incorporation of BrdU into nuclear DNA. The results suggest that the intracellular changes resulting from the addition of dC to cells in the presence of BrdU are comparable to those resulting from a decrease in the concentration of BrdU in the medium.

When thymidine kinase-deficient mouse cells "transformed" by in activated herpes simplex virus and expressing the viral thymidine kinase (TK) are grown in nonselective medium, there is an exponential decay in the proportion of cells that continue to express the viral enzyme. However, the viral TK can be reactivated at a frequency of approximately 1 cell in 10(6) in every population that has lost TK activity. When cells in which the viral TK has been reactivated are grown in nonselective medium, a decay in the expression of the viral enzyme occurs again at the same rate as in the initial transformed population. Studies on the reactivation of viral TK indicate that reappearance of the enzyme is not induced by the selective medium (HAT) used to detect cells in which the enzyme has reappeared. Furthermore, treatments known to induce latent viruses in other systems--eg, exposure of the cells to mutagens or cell fusion--do not affect the frequency with which viral TK is reactivated.

Mouse melanoma cells in culture respond to melanocyte-stimulating hormone (MSH) by demonstrating increased activity of tyrosinase, the rate-limiting enzyme for melanin synthesis. Because this stimulation is strictly dependent upon continued transcription and translation, we have carried out studies to determine if MSH increases the level of tyrosinase mRNA. The abundance of tyrosinase message levels in melanoma cells treated with either MSH or dibutyryl cAMP was determined by Northern blot analysis utilizing a 946 base pair mouse tyrosinase cDNA probe. The tyrosinase cDNA was isolated from a lambda gt11 expression library generated from mRNA isolated from theophylline-induced Cloudman melanoma cells. The abundance of tyrosinase mRNA was determined in an amelanotic cell clone (AM-7AS) and a melanotic cell clone (MEL-11AS). The melanotic cell line had five times as much tyrosinase activity and almost 10 times more tyrosinase mRNA than the amelanotic line. Tyrosinase activity and mRNA increased in both cell lines after MSH addition. The amelanotic line treated with MSH for three days showed a fivefold increase in tyrosinase activity and a twofold increase in tyrosinase mRNA. The melanotic cell line treated with MSH for three days showed a 3.7-fold increase in enzyme activity and an eightfold increase in the abundance of tyrosinase mRNA. Dibutyryl cAMP also stimulated tyrosinase activity and the accumulation of tyrosinase mRNA. The data suggest that MSH, acting through cAMP, promotes an accumulation of tyrosinase mRNA.

Analyses of the herpes simplex virus (HSV) DNA sequences which are present in three HSV thymidine kinase-transformed (HSVtk+) mouse cell lines have revealed that these cells contain relatively large and variable portions of the viral genome. Two of these cell lines do not contain the viral DNA sequences known to encode the early viral genes normally responsible for regulating tk gene expression during lytic HSV infections. This finding suggests that cell-associated viral tk gene expression may be regulated by cellular rather than viral control mechanisms. In addition, we have compared the viral DNA sequences present in one unstable HSVtk+ cell line to those present in tk- revertant and tk+ rerevertant cell lines sequentially derived from it. Our results have shown that within the limits of sensitivity of our mapping approach, these three related cell lines contain the same set of viral DNA sequences. Thus, gross changes in viral DNA content do not appear to be responsible for the different tk phenotypes of these cells.

A comparison of the sequence of terminal replication of DNA in human chromosomes has been made between skin fibroblasts and peripheral blood lymphocytes from the same adult male individual. The chromosomes were identified by quinacrine-banding, and the order of termination of replication was determined by autoradiography. Chromosomes 4, 5, 8, and 13 were found to terminate DNA replication later in lymphocytes than in fibroblasts, wheres chromosomes 15, 19, 20, and 22 were found to terminate their replication earlier in lymphocytes than in fibroblasts.

The recombinant shuttle vector pSV2gpt was introduced into V79 Chinese hamster cells, and stable transformants expressing the Escherichia coli gpt gene were selected. Two transformants carrying tandem duplications of the plasmid at a single site were identified and fused to simian COS-1 cells. Plasmid DNA recovered from the heterokaryons was used to transform a Gpt- derivative of E. coli HB101, and the relative frequency of plasmids carrying a mutation in the gpt gene was determined. The high frequency of Gpt- plasmids (ca. 1%) was similar to that observed when plasmid was recovered from COS-1 cells which had been transfected with pSV2gpt. Most of the mutant plasmids had rearrangements in the region containing the gpt gene.

Using a panel of monoclonal antibodies for the immunophenotyping of haematological malignancies, we have made a direct comparison of the usefulness of indirect immunofluorescence using flow cytometry (IFC) alongside the alkaline phosphatase-anti-alkaline phosphatase (APAAP) immunoenzyme method. Analysis of 400 consecutive patient samples (blood and bone marrow) over a 2-year period, resulted in unequivocal phenotyping agreement by both methods in 98 per cent of cases. The positive results accounting for 2 per cent discordance were obtained by the APAAP method. The value of the technique in the clinical management of patients with leukaemia is documented together with guidelines for clinical laboratories.

The relationship between bromodeoxyuridine (BrdUrd) mutagenesis in mammalian cells and the effects of BrdUrd on deoxyribonucleoside triphosphate pools was analyzed. It was found that the exposure of Syrian hamster melanoma cells to mutagenic concentrations of BrdUrd resulted in the formation of a large bromodeoxyuridine triphosphate (BrdUTP) pool, which remained at a high level for several days. In contrast, the size of the deoxycytidine triphosphate (dCTP) pool dropped rapidly after the addition of BrdUrd, reached a minimum at about 6 h, and then expanded gradually to nearly its original level over the next 3 days. The addition of lower concentrations of BrdUrd, which had less of a mutagenic effect, resulted in the formation of a smaller BrdUTP pool and a slightly smaller drop in the dCTP pool. When a high concentration of deoxycytidine was added at the same time as a normally mutagenic concentration of BrdUrd, the drop in the dCTP pool was prevented, as was BrdUrd mutagenesis. In all of these experiments, mutagenesis was related to the ratio of BrdUTP to dCTP in the cells. In addition, it was shown that mutagenesis occurred primarily during the first 24 h of BrdUrd exposure, when the BrdUTP/dCTP ratio was at its highest level. It appears that there is a critical ratio of BrdUTP to dCTP that must be attained for high levels of mutagenesis to occur and that the extent of mutagenesis is related to the ratio of the BrdUrd and dCTP pools.

The addition of deoxycytidine (dCyd) to the growth medium of cultured Syrian hamster melanoma cells causes a reversal of the toxic effects of 5-bromodeoxyuridine (BrdU) and a decrease in the extent of incorporation of BrdU into nuclear DNA. These effects of dCyd can be accounted for, in part, by the intracellular conversion of the exogenously supplied dCyd to thymidine (dThd) nucleotides which can compete with BrdU nucleotides for incorporation into DNA. To some extent, the conversion of dCyd to dThd nucleotides can be inhibited by increasing the concentration of BrdU in the growth medium. The conversion of dCyd to dThd nucleotides is inhibited completely by aminopterin (Apt), and Apt also prevents dCyd from reversing BrdU toxicity and from decreasing the level of BrdU incorporation into nuclear DNA. In a clone of Syrian hamster melanoma cells, increasing the concentration of dCyd in the growth medium from 1 micron to 1000 micron resulted in a progressive increase in the percentage of dThd residues in nuclear DNA being derived from the exogenous dCyd, until more than 90% of the dThd residues came from the exogenous dCyd. However, despite the increasing amount of dThd derived from exogenous dCyd, there was a plateau in the decrease in BrdU incorporation into nuclear DNA at concentrations of dCyd above 8 micron.

Lines of mouse L cells resistant to the fusion-inducing effect of polyethylene glycol (PEG) 1000 have been isolated by treating cultures with 50% PEG and selecting cells that remained unfused and able to proliferate. With repeated cycles of PEG treatment and selection, populations have been obtained that exhibit progressively increased resistance to the fusion-inducing effect of PEG. With the parental line, more than 90% of the cells fused when treated with 50% PEG. In contrast, with a population isolated after 24 cycles of PEG selection, fewer than 25% of the cells fused when treated with PEG. The decreased fusion response of the selected cells appeared to be stable for long periods of growth in the absence of PEG treatment, and the populations of selected cells seemed homogeneous in terms of the fusion response of individual subclones. Resistance to the fusion-inducing effect of PEG was concentration dependent, as the selected cells that fused poorly when treated with 50% PEG fused very efficiently when the PEG concentration was increased to 55%. The cells that were resistant to the fusion-inducing effect of 50% PEG also were more resistant to the toxic effect of PEG than were the parental cells.

We demonstrate previously that fibroblasts from cystic fibrosis (CF) patients are significantly more resistant to the toxic effects of ouabain than normal human fibroblasts are. Ouabain is generally assumed to act primarily at the level of the cell membrane and to cause killing of cells by inhibiting ion transport. We have therefore examined the ability of normal and CF cells to survive exposure to ethacrynic acid, another inhibitor of ion transport, and to survive colchicine and aminopterin, resistance to which has been associated with membrane alterations in other cells. The effect of dexamethasone, which has a sterol nucleus similar to that of ouabain but is thought to have a different site of cellular action, was also tested. Exposure of the cells to ethacrynic acid, colchicine, or aminopterin did not reveal any differences in survival between normal and CF fibroblasts. However, CF cells survived exposure to dexamethasone significantly better than normal cells did. These results suggest that normal and CF cells do not differ in terms of a generalized resistance to ion transport inhibitors or to drugs that must pass through the membrane to be active. Instead, the results raise the possibility that CF cells have an enhanced resistance to drugs that have the sterol nucleus found in ouabain and dexamethasone.

The relationship between plasminogen activator levels and the expression of the transformed phenotype was studied in a 5-bromodeoxyuridine (BrdUrd) dependent mutant of Syrian hamster melanoma cells. In terms of cell morphology and cellular interactions, the BrdUrd dependent cells resemble transformed cells when grown in the presence of BrdUrd but resemble untransformed cells when grown in the absence of BrdUrd. It was found that the BrdUrd dependent cells release significant levels of plasminogen activator only when cultured in the absence of BrdUrd. In the presence of BrdUrd, the release of plasminogen activator by the dependent cells is suppressed, and the decreased level of plasminogen activator released in the presence of BrdUrd seems to be due to the decreased production of active enzyme. Growth tests revealed that the BrdUrd dependent cells, when attached to a substrate, required BrdUrd in order to attain high densities. Furthermore, the cells are able to grow well in soft agar only in the presence of BrdUrd. These results suggest that the production and release of high levels of plasminogen activator are not related (either as cause or effect) to the expression of the transformed phenotype in the BrdUrd dependent cells. The effect of dog serum (as a plasminogen source) on the BrdUrd dependent cells also was tested. It was found that cells cultured in medium containing dog serum exhibit a morphological alteration, but only in the absence of brdUrd. The morphological response of the cells to dog serum resembles that previously observed with virus-transformed cells. In the BrdUrd dependent cells, the morphological response to dog serum appears correlated with the release of plasminogen activator but separated from other transformed characteristics.

The effects of polyethylene glycol (PEG) molecular weight and concentration on mammalian cell hybridization were studied. The peak hybridization-inducing activity with all grades of PEG from 400-6000 was found to occur in the concentration range of 50-55%. However, changes in concentration were seen to have different quantitative effects with different grades of PEG. For monolayer fusions, PEG 1000 at 50% seems to be the optimal combination of PEG molecular weight and concentration, in terms of both efficiency of hybridization and relative insensitivity to dilution effects.

These studies were undertaken to examine the relationship between the inhibition by 5-bromodeoxyuridine (BrdU) of erythroid differentiation in Friend erythroleukemia cells and the incorporation of BrdU into DNA. Experiments were carried out in which the incorporation of BrdU into DNA and the concentration of BrdU to which the cells were exposed were varied independently of each other. In addition, the ability of deoxycytidine (dC) to reverse the effects of BrdU on hemoglobin production and to reduce the amount of BrdU in DNA was analyzed. Under all the conditions tested, the effects of BrdU were correlated with the amount of BrdU incorporated into nuclear DNA. These results differ from those of recent studies on the inhibition of pigmentation and the induction of mutations by BrdU in Syrian hamster melanoma cells. The results suggest that BrdU may be producing its biological effects by a variety of different mechanisms.

A fluorescent staining technique has demonstrated a contralateral arrangement of fluorescent spots in the centromeric region of mouse metacentric chromosomes which have resulted from centric fusion. The results suggest that centric fusion involves the maintenance of DNA polaritv through the centromere and that the thymidine-rich chain of satellite DNA in the centromeric region is associated with the same DNA chain in every mouse autosome.

A bromodeoxyuridine(BUdr) dependent cell line, called B4, Which requires BUdr not only for optimal growth but also for the maintenance of the non-contact inhibited state was described previously. We have now shown that contact inhibition in the B4 cells in the absence of BUdr is associated with a marked decrease in the percent of cells synthesizing DNA. The transition to the contact inhibited state in the absence of BUdr does not seem to be due to changes in cyclic AMP levels. It has also been shown that several but not all of the characteristics which distinguish transformed from untransformed cells also distinguish B4 cells grown in the presence of BUdr from B4 cells grown in the absence of BUdr. In addition to being contact inhibited, B4 cells grown in the absence of BUdr have a higher serum requirement, grow less well in soft agar, and are less agglutinable by wheat germ agglutinin than B4 cells grown in the presence of BUdr. Agglutinability by concanavalin A, however, is the same for B4 cells grown in the presence and absence of BUdr. Dependent cells maintained in the presence of BUdr do not form tumors and it is not yet clear how the transformed characteristics of the dependent cells are related to malignancy.

A recombinant shuttle vector containing the entire bovine papillomavirus (BPV) genome, sequences from pBR322, and the Escherichia coli gpt gene was used to transform mouse C127 cells. Plasmid extracted from the transformed mouse cells was used to transform a Gpt- derivative of E. coli HB101, and the relative frequency of plasmids carrying a mutation in the gpt gene was determined. Approximate mutant frequencies of 3-16 X 10(-3) were observed for plasmid molecules which had been passaged through the mammalian cells. Restriction digest analysis indicated that most of the mutant plasmid molecules had gross rearrangements in their DNA structures.

Mutant cell lines resistant to hydroxyurea (HU), an inhibitor of the enzyme ribonucleotide reductase, were selected from a line of Syrian hamster melanoma cells. Mutant lines were selected for resistance to 0.3 mM HU, and from these lines, second-step mutants were selected for resistance to 1.9 mM HU. The HUr lines were tested ffor their responses to 5-bromodeoxyuridine (brdU), in terms of toxicity, mutagenesis, and incorporation of BrdU into DNA. All of the HUr lines showed increased resistance to the toxic effects of BrdU. in addition, the HUr lines all were resistant to BrdU mutagenesis. Overall, there was good correlation among the levels of resistance to HU toxicity, BrdU toxicity, and BrdU mutagenesis in the HUr lines. These tests were carried out under conditions such that the parental and HUr cells incorporated equal amounts of BrdU into nuclear DNA. Therefore, the resistance of the HUr cells to the effects of BrdU cannot be attributed to decreased incorporation of BrdU into DNA. These results suggest that the HUr cells have an lateration in ribonucleotide reductase activity that simultaneously confers resistance to HU and BrdU. The properties of the HUr cells suggest that the perturbation of deoxcytidine metabolism by BrdU. The properties of the HUr cells suggest that the perturbation of deoxcytidine metabolism by BrdU triphosphate inhibition of ribonucleotide reductase activity plays a key role in the toxic and mutagenic effects of BrdU in mammalian cells.

Modifications in the techniques for the induction of mammalian somatic cell hybridization by polyethylene glycol (PEG) have led to procedures that are rapid, simple, and effective. The basic improvements, for both monolayer and suspension fusions, are a short exposure to PEG and a rapid dilution of PEG following treatment. There is a marked effect of PEG concentration on cell hybridization, and there seem to be inherent differences between cells in terms of the extent of cell fusion induced by PEG.

The halogenated thymidine (dT) analogue, 5-bromodeoxy-uridine (BUdR), has a variety of effects on mammalian cells, including toxicity, suppression of differentiation, and mutagenesis. Although it is generally assumed that the effects of BUdR are due primarily to its presence in DNA, results from our laboratory have raised doubts about such assumptions. We have shown, for example, that BUdR mutagenesis in mammalian cells is determined by the concentration of BUdR in the medium rather than in DNA, and that mutagenesis can be suppressed by deoxycytidine (dC) without changing the amount of BUdR in DNA. BUdR has also been shown to induce sister chromatid exchanges (SCEs) in mammalian cells. Initial results suggested that the relationship between BUdR and SCEs might not be explained by a single factor, and various correlations between BUdR and SCEs have been proposed. However, the results to date have been inconclusive, because the experiments did not resolve as independent variables the concentration of BUdR in the medium and the amount of BUdR incorporated into nuclear DNA. We have now carried out experiments to resolve these two factors; the results indicate that the major factor in determining the frequency of SCEs is the concentration of BUdR in the medium.

The effects of purine deoxyribonucleosides on bromodeoxyurdine (BrdU) mutagenesis in Syrian hamster melanoma cells were determined. Both deoxyguanosine (dG) and deoxyadenosine (dA) were found to stimulate mutagenesis without changing the amount of BrdU in DNA. In addition, the stimulation of mutagenesis by dG and dA was suppressed by the addition of deoxycytidine (dC). These results suggest that BrdU mutagenesis involves the perturbation of dC metabolism, which perturbation is enhanced by dGTP and dATP. The mutagenic activity of dG in the absence of BrdU was tested, as was that of thymidine (dT), which we had shown previously to stimulate BrdU mutageneis. With dG alone, no increase above the spontaneous mutation frequency was detected. However, at extremely high concentration, dT in the absence of BrdU was slightly mutagenic, and the mutagenesis by dT was enhanced by dG and suppressed by dC.

A retroviral shuttle vector was constructed by introducing the Escherichia coli xanthine (guanine) phosphoribosyltransferase gene (gpt) into the pZip-NeoSV(X)1 vector [Cepko, C. L., Roberts, B. E. & Mulligan, R. C. (1984) Cell 37, 1053-1062]. This vector was packaged into infectious virus which then was used to infect a hypoxanthine (guanine) phosphoribosyltransferase-deficient mouse cell line. Cell lines that expressed the gpt gene were isolated, and it was found that these cells contained a single integrated copy of the vector in a proviral form. Treatment of these cell lines with either ethyl methanesulfonate or BrdUrd produced a greater than 10-fold increase in the frequency of 6-thioguanine-resistant (Sgur) mutants. Intact gpt genes have been recovered from a number of Sgur cell lines after COS cell fusion and introduced into E. coli as part of a plasmid. The complete DNA sequences of three mutant genes have been determined. Two of the mutant genes have a single base substitution, whereas the third has a 34-base-pair deletion. This system should be valuable for analyzing mutagenic specificity and the molecular mechanisms of chemical mutagenesis in mammalian cells. A potentially important feature of the system relative to other shuttle-vector systems is that the mutations are induced in genes integrated into mammalian chromosomes rather than in genes existing as part of autonomously replicating plasmids.