DNA transfection to study translational control in mammalian cells

Mammalian cells respond to changes in their environment by rapid and reversible covalent modification of the translational machinery. In most cases, these modifications involve the phosphorylation and dephosphorylation of translation initiation factors (for review see Ref. 1). The modification of translation initiation factors may affect translational activity of either specific mRNAs or general cellular mRNAs. To study the effect of a particular factor or its modification on the translational capacity of an mRNA, there are a number of potential approaches that include in vitro translation reactions as well as in vivo experiments. Generally, experiments initially report a covalent modification that correlates with altered translational capacity of either a specific or a general class of mRNAs. The modification and the particular amino acid residue involved are then identified. Then mutations are made at the modified residue to prevent modification (for example, a serine-to-alanine mutation to prevent phosphorylation) and the effect of the mutant factor on the translation of a target mRNA is tested. The most convenient method for monitoring the effect of a mutant translation factor on translation is the use of transient DNA transfection. However, in certain situations it is desirable to isolate stably transfected cell lines to study the effect of overexpression, underexpression, or expression of a particular mutant translation factor. This article reviews two methods that are routinely used to study translational control that involve either transient or stable DNA transfection.     

Ovarian control of gonadotropin hormone-releasing hormone pulse generator activity in the rhesus monkey: duration of the associated hypothalamic signal

The activity of the GnRH pulse generator in the rhesus monkey is associated with abrupt increases in multiunit electrical activity (MUA) volleys recorded from the mediobasal hypothalamus that precede each pulse of LH in the peripheral circulation. In long-term ovariectomized animals the duration of these MUA volleys is 10-25 min and consists of a brief initial 'overshoot' followed by a plateau phase that ends in a rapid decline to baseline activity. In intact monkeys, however, the MUA volley lasts only 1-3 min, a duration equivalent to the overshoot in ovariectomized animals. In addition, the maximal frequency of neuronal activity during each MUA volley is reduced in normal animals when compared to castrates. As shown in earlier studies, estradiol given to ovariectomized monkeys causes a reduction in the duration of MUA volleys to that characteristic of intact animals within 3-5 h. In contrast to this acute effect of estradiol, the increase in MUA volley duration following ovariectomy is a gradual phenomenon, 4-6 weeks being required to achieve the MUA volley duration observed in long-term ovariectomized monkeys. A similar slow time course was observed for the increase in maximal neuronal frequency during each MUA volley. This protracted effect of ovariectomy on MUA volley duration and firing rate may be the consequence of hypothalamic remodelling but this consideration must be tempered by the observation that estradiol reverses these phenomena within hours.     

Histone inhibition of DNA synthesis in eukaryotic cells permeable to macromolecules

Mouse L Cells, grown in suspension culture can be rendered permeable to exogenous deoxynucleoside triphosphates by a cold shock in a near isotonic buffer system. These cells use the deoxynucleotides to synthesize DNA in a semiconservative fashion. The addition of 0.05% Triton X-100 to this system increases the permeability of the cells so that exogenously supplied macromolecules gain access to the DNA. When DNAase and phosphodiesterase are added to the detergent-permeabilized cells, the cell DNA is rapidly degraded, demonstrating that the enzymes reach the DNA within the first 2 min of the incubation period. Addition of whole calf thymus histone or histone fractions to the detergent-permeabilized cells inhibits DNA synthesis. The lysine-rich histone, F is a more effective inhibitor than the arginine-rich histone, F3. The other histone fractions including the slightly lysine-rich fractions, F2a and F2b, are intermediate between F1 and F3 as inhibitors of DNA- synthesis. Kinetic analysis demonstrates that the added histones increase apparent Km and reduce V of DNA synthesis in the permeabilized cells. These studies suggest the possibility that histones alter the association of the DNA replication complex and the DNA template in a manner that reduces the rate of DNA synthesis.     

Induction of tolerance to hypothermia and hyperthermia by a common mechanism in mammalian cells

Pretreatment by hypothermic (25 degrees C) cycling (PHC) of attached exponential-phase V79 Chinese hamster cells by Method 4 (24 hr at 25 degrees C + 1.5 hr at 37 degrees C + 24 hr at 25 degrees C + trypsin + 3 hr at 37 degrees C) or by Method 3 (48 hr at 25 degrees C + trypsin + 3 hr at 37 degrees C) make mammalian V79 cells significantly more resistant to 43 degrees C hyperthermia. There is no significant difference in the 43 degrees C curves whether Method 3 or 4 is used for pre-exposure. If pre-exposure at 15 or 10 degrees C, the resistance to hyperthermia is significantly reduced. PHC by Method 4 significantly increases survival of cells exposed to 5 degrees C and, to a lesser extent, to 10 degrees C. The increase in hyper- and hypothermic survival after PHC cannot be accounted for by changes in cell cycle distribution. Heat-shock protein synthesis is not induced by PHC; hence, protection does not result from newly synthesized proteins. When cells are made tolerant to hyperthermia by a pretreatment in 2% DMSO for 24 hr at 37 degrees C (Method 8), the cells are not more resistant to subsequent exposures to hypothermia, either at 5 or 10 degrees C. The results imply that there may be two mechanisms of inducing resistance to hyperthermia, only one of which also confers resistance to hypothermia.     

A system for mutation measurement in mammalian cells: application to gamma-irradiation

Monitoring of mutagenesis by environmental agents for the purpose of preventing genetic disease including cancer must include quantitation of cell killing, sensitive measurement of mutation production by appropriate doses of each agent, and assessment of mutation repair effects in mammalian cells. A four-step procedure, in the presence and absence of a repair suppressor, is proposed: (i) determination of the survival curve; (ii) measurement of the mitotic index in cells collected after treatment with colcemid; (iii) construction of a mutagenesis yield curve in the presence and absence of a repair suppressor, like caffeine; and (iv) assessment of the effect of test agents on the repair of mutations produced by other mutagens. The procedure is quantitative, reproducible, and reasonably rapid. It involves measurement of mutations causing visible chromosomal aberrations. Numerical parameters are proposed defining quantitatively mutation, cell killing, and mutation repair capacity. The procedure is applied to gamma-irradiation and can detect the effects of doses as low as 2-5 cGy. Theoretical analysis of the underlying processes is presented, using the concept of D(0)E, the effective dose of mutagen after repair mechanisms and neutralizing agents have acted. Microscopically visible chromosome aberrations are due to mutations that distort the process of mitotic chromosome condensation, with or without actual chromosome breakage.     

Chromosomal response of insect and mammalian cells to streptonigrin: a comparative study

Respiratory syncytial virus (RSV) infections are characterized by upper or lower respiratory tract symptoms including bronchiolitis and pneumonia. Apnoea may be the first sign of disease in children with RSV infection. The aims of this study were the identification of independent risk factors for RSV associated apnoea and the prediction of the risk for mechanical ventilation in children with RSV associated apnoea. Medical records of children younger than 12 months of age admitted with RSV infection between 1992 and 1995 to the Sophia Children's Hospital, were reviewed. Demographic parameters, clinical features and laboratory parameters (SaO2, pCO2 and pH) were obtained upon admission and during hospitalization. Children with and without apnoea were compared using univariate and multivariate logistic and linear regression analysis. One hundred and eighty-five patients with RSV infection were admitted of whom 38 (21%) presented with apnoea. Patients with apnoea were significantly younger, had a significantly lower temperature, higher pCO2 and lower pH and had on chest radiographs also more signs of atelectasis. The number of patients admitted to the ICU because of mechanical ventilation and oxygen administration was significantly higher in children with RSV associated apnoea. Apnoea at admission was a strong predictor for recurrent apnoea. The relative risk for mechanical ventilation increased with the number of episodes of apnoea: 2.4 (95% CI 0.8-6.6) in children with one episode of apnoea (at admission) versus 6.5 (95% CI 3.3-12.9) in children with recurrent episodes of apnoea. CONCLUSIONS: Age below 2 months is the strongest independent risk factor for RSV associated apnoea. Apnoea at admission increases the risk for recurrent apnoea. The risk for mechanical ventilation significantly increases in children who suffer from recurrent apnoea.     

Molecular biology of mutagenesis of mammalian cells by ionizing radiation

Although the genotoxic potential of styrene is known, very limited information is available regarding its dose-dependent genotoxic response to human blood lymphocytes and how such response correlates with different metabolic events in whole blood lymphocytes. The present study was therefore carried out to study such a relationship using in vitro human blood lymphocytes from healthy volunteers. To study genotoxic response to styrene, sister chromatid exchanges (SCEs), cell cycle, and cell survival were analyzed. Lymphocytes were cultured for 72 hr in the presence of different concentrations of styrene (0-1,000 microM). Twenty-four hr before harvest, BrdU (5 micrograms/ml) was added to assess the increase in SCEs and cell cycle delay. Both the SCE frequency and the cell cycle length were increased linearly with increasing concentrations of styrene up to 200 microM, without addition of any exogenous metabolizing system. Above 200 microM, no further increase in genotoxic response occurred. The range of concentrations (10-200 microM) at which increase of cell cycle length due to styrene was observed did not impair the viability of the cells, suggesting that such cell cycle delay is a genotoxic-related event and not caused by cytotoxicity. In vitro metabolic transformation of styrene in whole-blood lymphocyte cultures without the presence of any exogenous metabolic activation system showed the formation of a reactive intermediate, styrene 7,8-oxide, to be capacity-limited, as verified from a nonlinear increase in the formation of styrene glycol. The value of such metabolic parameter reached a plateau above 200 microM styrene. The same phenomenon of saturation has also been observed with regard to other metabolic effects due to styrene in whole blood lymphocytes in culture, such as dose-dependent increase in lipid peroxidation and depletion of blood lymphocyte glutathione. Based on the relationship between the formation of different metabolic events and the genotoxicity of styrene, it may be possible that the genotoxic properties of styrene in human blood lymphocytes may be mediated initially not only by the formation of the presumably reactive styrene 7,8-oxide, but also by that of a reactive oxygen species as well. However, the present data are not sufficient enough to definitely identify the role of reactive oxygen species in such toxicity and therefore it warrants further study.     

Chromosomal mutations induced by triplex-forming oligonucleotides in mammalian cells

Specific recognition of a region of duplex DNA by triplex-forming oligonucleotides (TFOs) provides an attractive strategy for genetic manipulation. Based on this, we have investigated the ability of the triplex-directed approach to induce mutations at a chromosomal locus in living cells. A mouse fibroblast cell line was constructed containing multiple chromosomal copies of the lambdasupFG1 vector carrying the supFG1 mutation-reporter gene. Cells were treated with specific (psoAG30) or control (psoSCR30) psoralen-conjugated TFOs in the presence and absence of UVA irradiation. The results demonstrated a 6- to 10-fold induction of supFG1 mutations in the psoAG30-treated cells as compared with psoSCR30-treated or untreated control cells. Interestingly, UVA irradiation had no effect onthe mutation frequencies induced by the psoralen-conjugated TFOs, suggesting a triplex-mediated but photoproduct-independent process of mutagenesis. Sequencing data were consistent with this finding since the expected T.A-->A.T transversions at the predicted psoralen crosslinking site were not detected. However, insertions and deletions were detected within the triplex binding site, indicating a TFO-specific induction of mutagenesis. This result demonstrates the ability of triplex-forming oligonucleotides to influence mutation frequencies at a specific site in a mammalian chromosome.     

Signal transduction by protein kinase C in mammalian cells

1. The past two decades have witnessed great advances in our understanding of the role of protein kinase C (PKC) in signal transduction. The Ca(2+)-activated, phospholipid-dependent protein kinase discovered by Nishizuka's group in 1977 is now a family of at least 11 isoforms. Protein kinase C isoforms exist in different proportions in a host of mammalian cells and each isoform has a characteristic subcellular distribution in each cell type. 2. Stimulation of a specific PKC isoform often causes redistribution of the isoform from one subcellular compartment to another compartments where it complexes with and phosphorylates a specific protein substrate. 3. The interaction of a specific PKC isoform with its protein substrate may directly activate a specific function of the cell or may trigger a cascade of protein kinases that ultimately stimulates a specific response in differentiated cells or regulates growth and proliferation in undifferentiated cells.     

Stimulation of tetrapyrrole synthesis in mammalian epithelial cells in culture by exposure to aminolaevulinic acid

Tetrapyrrole synthesis in CNCM-1221 cells exposed to 0.6 mM aminolaevulinic acid (ALA) was found to be approximately linear over a 6-h period of incubation. The rate was not significantly affected by cell density over a range of 0.015 to 0.15 x 10(6) cells cm(-2) (final cell density). Tetrapyrrole synthesis was not affected by GABA or glutamic acid in concentrations up to 6 mM and 2.72 mM respectively, suggesting that these amino acids, which are similar in structure to ALA, do not competitively inhibit the ALA uptake pathway in these cells. Pre-exposure to haem arginate (up to 100 microM) was inhibitory, presumably by suppression (through the inhibition of ALA synthase) of an endogenous component of the response. The ALA-stimulated response was not modified by co-exposure to AIA (up to 100 mg ml(-1)). Despite significant reduction of protein synthesis, the porphyrinogenic response of cells exposed to ALA was unaffected by cycloheximide (10 microg ml(-1)) or actinomycin D (10 microg ml(-1)) even when cells were preincubated with these agents for 3 h before ALA exposure. Fetal bovine serum (10%) inhibited tetrapyrrole synthesis by 30% but increased the rate of porphyrin export by cells by a factor of 1.5. The uptake of [14C]ALA was shown to be strongly influenced by the density of the cultures. In dense cultures (final cell density of approximately 0.15 x 10(6) cells cm(-2)), the ALA uptake rate was less than 0.8 compared with a maximum rate of 4.2 fmol per cell h(-1) at a cell density of 0.02 x 10(6) cells cm(-2). Since tetrapyrrole synthesis is less affected than ALA uptake by cell density, the resultant discrepancy in ALA incorporation occurring in dense cultures implies that endogenous ALA synthesis is induced in these cells. ALA uptake was not affected by cycloheximide or actinomycin D in serum-free conditions. However, fetal bovine serum decreased external ALA uptake by about 50%. This effect was abrogated by preincubation with cycloheximide.     

Metabolism of caffeine to nucleic acid precursors in mammalian cells

The mission of this study was to determine whether or not arteriovenous connections, indicative of a "closed" type of circulation, existed in the human spleen. Spleens from four patients requiring therapeutic splenectomy were the basis for this report. Scanning electron microscopy of plastic corrosion casts, prepared from these four spleens, revealed direct vascular conduits between splenic pulp arteries or arterial capillaries and the venous sinuses in the red pulp. Also demonstrated were a few arteriovenous shunts between pulp arteries or arterial capillaries and pulp or trabecular veins. Inclusion of sized microspheres in low-viscosity perfusion plastic illustrated that some diameters of the connecting shunts were 7-10 mum, with other shunts even smaller. Not only do arteriovenous connections exist in human spleens, but their frequency, as revealed by methods accentuating three-dimensional aspects of the splenic microcirculation, justify future reconsiderations of the functional significance of this closed type of circulation. Examination of samples of the same intact spleens, prepared by freeze-fracture and conventional critical-point drying, also revealed an "open" type circulation structure, namely, pore-patterned sinus walls that could facilitate blood cell movement from pulp cords into venous sinuses. Scanning electron microscopy thus has provided direct evidence that human spleens have both "open" and "closed" circulatory pathways in their microvasculature.     

Effect of pulse duration and size of interelectrode interval on electric-spark spraying. I. Effect of pulse duration and size of interelectrode interval on rate of electric-spark transfer

The influence exerted by electric-spark spraying on the kinetics of mass transfer and the physicomechanical properties of coatings is investigated. It is shown that electric-spark spraying is determined by the dynamic properties of the cathode jets, which depend on the electrical parameters of the spark discharge, the size of the interelectrode interval, and the physical properties of the coatings, which vary during spraying.     

Effect of pulse duration on wound healing using a CO2 laser

Epidemiological data strongly implicate sunlight as the principal environmental cause of melanoma; however, critical molecular targets for ultraviolet (UV)-induced melanoma remain to be identified. The p53 tumor suppressor gene is one possible target, being abnormally expressed in 20-40% of primary melanomas. We undertook a population-based molecular epidemiological study with the aim of determining the environmental and phenotypic factors associated with p53-positive and p53-negative melanomas. One hundred fifty cases of melanoma were randomly ascertained from the Queensland Cancer Registry and matched to 150 electoral roll controls. Data on environmental and phenotypic exposures were collected through interviews and physical examination of all participants. Sections of tumor tissue were obtained from 134 (89%) cases and stained with the anti-p53 DO-7 monoclonal antibody (MAb) following microwave antigen retrieval. Of 121 useable sections, 22 tumors (18%) had more than 1% cells with positive staining consistent with abnormalities in p53 expression. Strongest predictors of p53-positive melanoma were inability to tan [odds ratio (OR) 6.8], history of non-melanoma skin cancer (OR 3.2) and site of melanoma: head/neck (OR 2.2) and lower limbs (OR 2.3). In contrast, factors such as nevus density and freckling propensity were strongly associated only with p53-immunonegative melanoma (OR 8.6 for >25 moles; OR 3.0 for heavy facial freckling). Overall, the determinants of p53-positive and p53-negative melanomas were independent and complementary, the former being associated with features of sun-sensitivity and chronic sun exposure, the latter with phenotypic markers of melanocytic proliferation. Our findings are consistent with at least 2 independent pathways in the pathogenesis of melanoma, characterized by environmental induction and p53 overexpression on the one hand and pigment cell instability on the other.     

Induction of diplochromosomes in mammalian cells by inhibitors of topoisomerase II

Diplochromosomes, consisting of four chromatids lying side-by-side, instead of the normal two, are produced when cells go through two rounds of DNA replication without separation of chromatids. They are thus an indication of the failure of the normal chromosome separation mechanism. In the present experiments, induction of diplochromosomes by inhibitors of topoisomerase II (Topo II) was used to provide further evidence that Topo II is required for separation of daughter chromosomes. Actively growing cultures of CHO cells were treated with Colcemid, and separated into metaphase and interphase fractions, each of which was treated for 2 h with the Topo II inhibitor being tested. The cells were then cultivated in fresh medium without inhibitor for periods of between 18 and 44 h, and metaphase cells once again accumulated by treatment with Colcemid. Chromosome preparations were made in the standard way and stained with Giemsa. Up to 2,000 metaphases were counted from each culture, and the proportion with diplochromosomes calculated. At appropriate concentrations, the Topo II inhibitors etoposide and mitoxantrone induced substantial levels of metaphases with diplochromosomes in cultures that had been treated when the cells were in interphase (up to 30% and 11%, respectively). Amsacrine, however, only produced a smaller proportion (4.7%) of metaphases with diplochromosomes after a much longer culture period following treatment. All the inhibitors caused severe chromosome damage. When used to treat metaphase cells, mitoxantrone and amsacrine only induced diplochromosomes after prolonged culture, although a small number of diplochromosomes were seen after etoposide treatment and a shorter period of culture. Results with cells treated in metaphase might indicate that Topo II is, in fact, not required for anaphase chromosome separation, although it is clearly important for segregation of newly replicated DNA.     

High-frequency transfer of cloned herpes simplex virus type 1 sequences to mammalian cells by protoplast fusion

The protoplast fusion technique of Schaffner (W. Schaffner, Proc. Natl. Acad. Sci. U.S.A. 77:2163-2167, 1980) has been adapted to introduce cloned herpes simplex virus genes into cultured mammalian cells. The technique involves digesting bacterial cell walls with lysozyme to produce protoplasts and then fusing the protoplasts to mammalian cells by treatment with polyethylene glycol. For monitoring transfer, protoplasts were labeled with the fluorescent dye fluorescein isothiocyanate before fusion. After fusion, greater than 50% of the mammalian cells were fluorescent, demonstrating that bacterial material was transferred with high frequency. Transfer of plasmid pBR325 occurred at frequencies of 1 to 2%, as measured by in situ hybridization. Fusion transfer of a chimeric plasmid consisting of the herpes simplex virus type 1 (strain KOS) EcoRI fragment F in pBR325 resulted in expression of some viral genomic sequences in about 5% of the mammalian cells, as detected by indirect immunofluorescence. One Ltk- cell in 300 to 500 was transformed to the TK+ phenotype after fusion with protoplasts carrying the chimeric plasmid pX1, which consists of pBR322 and the BamHI fragment coding for the herpes simplex virus type 1 thymidine kinase gene.     

Functional complementation by electroporation of human BACs into mammalian fibroblast cells

Bacterial Artificial Chromosomes (BACs) have been used to complement a metabolic defect and to transfer a drug resistance marker into mammalian cells by electroporation. The selectable markers are stable and the recipient cells have BAC DNA integrated into the chromosomes as shown by fluorescent in situ hybridization, PCR and Southern hybridization.     

Wortmannin sensitizes mammalian cells to radiation by inhibiting the DNA-dependent protein kinase-mediated rejoining of double-strand breaks

STATEMENT OF PROBLEM: The cone-screw abutment has been shown to diminish micromovement by reducing the burden of component loosening and fracture. However, anecdotal concern for cold welding of cone-screw joints in implant design has been identified as a potential source for lack of retrievability. PURPOSE: This comparative study evaluated the loosening torque, as a percentage of tightening torque, for the ITI Straumann and Astra Tech (3.5 and 4.0 mm diameters) implant systems, which use an 8-degree and 11-degree internal cone, respectively. MATERIAL AND METHODS: Implants and abutments from each system were mounted in a torque device, and a range of tightening torques was applied. Loosening torques were then measured, and the influence of conus angle, interfacial surface area, saliva contamination, and time delay to loosening were all assessed. RESULTS: The loosening torque only exceeded tightening torque at the highest levels, just before component failure, when plastic deformation was expected. For all clinically relevant levels of torque, both in a dry environment and with components bathed in artificial saliva at 37 degrees C, loosening torque was always seen to be 80% to 90% of tightening torque, demonstrating that cold welding does not occur. There was a high correlation between loosening and tightening torque for all systems tested, but no statistical difference when comparing wet versus dry or comparing individual data for each system. CONCLUSIONS: It can be concluded that for clinically relevant levels of tightening torque, no problems are anticipated with respect to retrievability.