Evaluation of ion gradient-dependent H+ transport systems in isolated enterocytes from the chick

Summary The experiments reported here evaluate the capability of isolated intestinal epithelial cells to accomplish net H⁺ transport in response to imposed ion gradients. In most cases, the membrane potential was kept constant by means of a K⁺ plus valinomycin voltage clamp in order to prevent electrical coupling of ion fluxes. Net H⁺ flux across the cellular membrane was examined at pH 6.0 (the physiological lumenal pH) and at pH 7.4 using methylamine distribution or recordings of changes in media pH. Results from both techniques suggest that the cells have an Na⁺/H⁺ exchange system in the plasma membrane that is capable of rapid and sustained changes in intracellular pH in response to an imposed Na⁺ gradient. The kinetics of the Na⁺/H⁺ exchange reaction at pH 6.0 [K _t for Na⁺=57mm,V _max=42 mmol H⁺/liter 3OMG (3-O-methylglucose) space/min] are dramatically different from those at pH 7.4 (K _t for Na⁺=15mm,V _max=1.7 mmol H⁺/liter 3OMG space/min). Experiments involving imposed K⁺ gradients suggest that these cells have negligible K⁺/H⁺ exchange capability. They exhibit limited but measurable H⁺ conductance. Anion exchange for base equivalents was not detected in experiments performed in media nominally free of bicarbonate.     

Suppression of cyclic AMP-induced cell excitation in Dictyostelium discoideum by inhibitors of eicosanoid oxidation

Abstract Receptor-mediated stimulation of Dictyostelium cells by the aggregative chemoattractant cyclic AMP leads to a complex excitatory response resulting in chemotaxis and the synthesis and release of cyclic AMP as the relayed chemotactic signal. However, the mechanism of this stimulus-response coupling is not well understood. In this study, we show that a number of compounds, best known as inhibitors of cyclooxygenase activity in mammalian cells, prevent cyclic AMP receptor-mediated cell excitation and cyclic AMP accumulation in aggregation-competent Dictyostelium cells. These observations suggest that some eicosanoid-like compound(s) may be involved in stimulus-response coupling in this organism, as is the case in higher eukaryotic cells.     

Some factors affecting the in vitro invasion of HeLa cells by Trypanosoma cruzi

The penetration of metacyclic forms of Trypanosoma cruzi into HeLa cells after different treatments was studied. When cell development was synchronized by two different processes, maximum rates of parasitization occurred during the S phase of cell cycle (29.48 and 24.3%). However, when cells were treated with trypsin (0.1%), parasitization rates appeared to be lower than controls, reaching values similar to controls 14 h after the beginning of the treatment. Infection values remained unaltered after treatment with colcemid (0.6 μg ml^?1). Cell treatment either with valinomycin (1 μg ml^?1) or with actinomycin D (250 μg ml^?1) caused a marked decrease in the percentage of parasitization. When cells were treated and infected in the presence of tunicamycin (100 ng ml^?1), parasitization rates were increased (14.7%) compared to control cells (6%). On the other hand, no differences in parasitization rates were observed when cells were treated with cycloheximide (100 μg ml^?1). Infection in a low redox medium (?100 mV) resulted in considerable increase in parasitization.     

High-frequency1H NMR studies of the effects of growth factors and phorbol esters on normal syrian hamster diploid fibroblast cells

The nuclear magnetic resonance (NMR) parameters, spin-lattice (T₁), and spin-spin (T₂) relaxation time, are usually longer for neoplastic cells than for normal cells of the same cell type. This has generally been true at low NMR frequencies (≤100 MHz) when comparisons have been made between normal and neoplastic cells that have both spent a short time in culture. We have previously demonstrated that although the T₁ values of paired normal and neoplastic Syrian hamster (SH) fibroblastic cells in culture are not significantly different when measured at 300 MHz, the 300 MHz T₂ values for the neoplastic cells are smaller than those of the normal cells. (Xin et al. (1986),Cell Biophysics 8, 213.) Since treatment of normal diploid cells with polypeptide growth factors or tumor promoters frequently results in reversible expression of neoplasia-associated phenotypes, T₁ and T₂ were obtained at 300 MHz for treated and untreated SH cells to see if these compounds could also produce smaller 300 MHz T₂ values. Secondary culture SH fetal fibroblast cells were treated with epidermal growth factor (EGF), fibroblast growth factor (FGF), phorbol-12,13-didecanoate (PDD) and 4-α-phorbol-12,13-didecanoate (4αPDD). Treatment with either growth factor resulted in smaller T₂ values, but a statistically significant decrease was not observed for PDD or 4αPDD. The observed reductions in T₂ values were correlated with the morphological and growth-stimulatory effects of these compounds on the cells.     

Radical Cations in Aromatic Hydrocarbon Carcinogenesis

Most carcinogens, including polycyclic aromatic hydrocarbons (PAH), require metabolic activation to produce the ultimate electrophilic species that bind covalently with cellular macromolecules to trigger the cancer process. Metabolic activation of PAH can be understood in terms of two main pathways: one-electron oxidation to yield reactive intermediate radical cations and monooxygenation to produce bay-region diol epoxides. The reason we have postulated that one-electron oxidation plays an important role in the activation of PAH derives from certain common characteristics of the radical cation chemistry of the most potent carcinogenic PAH. Two main features common to these PAH are: 1) a relatively low ionization potential, which allows easy metabolic removal of one electron, and 2) charge localization in the PAH radical cation that renders this intermediate specifically and efficiently reactive toward nucleophiles. Equally important, cytochrome P-450 and mammalian peroxidases catalyze one-electron oxidation. This mechanism plays a role in the binding of PAH to DNA. Chemical, biochemical and biological evidence will be presented supporting the important role of one-electron oxidation in the activation of PAH leading to initiation of cancer.     

Two classes of continuous cell lines established from Syrian hamster 9 day gestation embryos: Preneoplastic cells and progenitor cells

Summary Primary cultures of 9-d-gestation Syrian hamster embryo (E9) cells are distinct from primary cultures of later gestational age in terms of their growth and differentiation. First, primary E9 cell cultures express multiple mesenchymal differentiation lineages (e.g., adipocyte, myoblast) only rarely seen in cultures of 13-d-gestation fetal (F13) cells. Second, although most primary E9 cultures have a limited in vitro proliferative life span and exhibit cellular senescence similar to primary cultures of F13 cells, E9 cultures seem to have higher frequency of escape from senescence and conversion to continuous cell lines compared to F13 cells. Moreover, this frequency can be further increased 4- to 5-fold by continuous exposure of the E9 cells to tumor promoters or epidermal growth factor. Eleven continuous cell lines have been isolated from unreated, promoter-treated, or epidermal growth factor-treated primary E9 cultures. Seven of these are neoplastic or preneoplastic. However, the remaining four do not show any evidence of being in neoplastic progression and three of these continue to express the same differentiated phenotype observed in ther parental primary cell cultures. These studies were supported in part by grants from the National Institutes of Health (AG 01998), Bethesda, MD, and the U.S. Department of Energy (DE-A-C02-76-EVO-3280), Washington, DC.     

Omega-3 fatty acids and the peroxisome

The interactions between the omega-3 unsaturated fatty acids and peroxisomal function have been reviewed, in order to update and integrate knowledge in this area. Following a brief retrospective of the major clinical involvements of these fatty acids, the participation of the peroxisome in their metabolism has been appraised - the peroxisome being shown to exert a major influence on both the synthesis and degradation of the omega-3 fatty acids, with these effects flowing on to the widespread physiological implications of the derivative eicosanoids. Interactions between the omega-3 and omega-6 families of fatty acids have been discussed, as have the interdependent phenomena of peroxisome proliferation, membrane remodelling and cellular signalling. Amongst the signalling involvements covered were those of steroid hormone receptor superfamily, the phosphatidy1choline cycle, and the regulatory influences of oxygen free radicals. Comment has also been included on the separate biological roles of the individual omega-3 fatty acids, their influence on differential gene function, and on the molecular mechanisms of their pharmacological effects. It is concluded that the peroxisome is intimately involved in directing the metabolism and physiological influence of the omega-3 unsaturated fatty acids, and that this organelle merits much greater emphasis in future research aimed at unravelling the profound biological effects of these unique and multipotent compounds.     

Large scale production of recombinant mouse and rat growth hormone by fed-batch GS-NSO cell cultures

Investigations of biological effects of prolonged elevation of growth hormone in animals such as mice and rats require large amounts of mouse and rat growth hormone (GH) materials. As an alternative to scarce and expensive pituitary derived materials, both mouse and rat GH were expressed in NSO murine myeloma cells transfected with a vector containing the glutamine synthetase (GS) gene and two copies of mouse or rat GH cDNA. For optimal expression, the mouse GH vector also contained sequences for targeting integration by homologous recombination. Fed-batch culture processes for such clones were developed using a serum-free, glutamine-free medium and scaled up to 250 L production scale reactors. Concentrated solutions of proteins, amino acids and glucose were fed periodically to extend cell growth and culture lifetime, which led to an increase in the maximum viable cell concentration to 3.5×10⁹ cells/L and an up to 10 fold increase in final mouse and rat rGH titers in comparison with batch cultures. For successful scale up, similar culture environmental conditions were maintained at different scales, and specific issues in large scale reactors such as balancing oxygen supply and carbon dioxide removal, were addressed. Very similar cell growth and protein productivity were obtained in the fed-batch cultures at different scales and in different production runs. The final mouse and rat rGH titers were approximately 580 and 240 mg/L, respectively. During fed-batch cultures, the cell growth stage transition was accompanied by a change in cellular metabolism. The specific glucose consumption rate decreased significantly after the transition from the growth to stationary stage, while lactate was produced in the exponential growth stage and became consumed in the stationary stage. This was roughly coincident with the beginning of ammonia and glutamate accumulation at the entry of cells into the stationary stage as the result of a reduced glutamine consumption and periodic nutrient additions.     

Effect of anti-herpes specific transfer factor

Using a blood cell separator, lymphocytes were collected from otherwise healthy convalescents suffering from herpetic infections. A specific anti-herpes dialysate (AH-DLE) was prepared from the lymphocytes, using standard procedures. Patients with recurrent herpetic infections were treated with a single dose of the dialysate, at the initial signs of herpetic infection (group A), with two doses (group B) or with three doses (group C). A total number of 37 patients (29 women, 8 men, age range 15–73 years) were treated. No improvement was observed in 7 patients (18.9%), whilst 7 patients did not manifest any exacerbation of their herpetic infection in the course of the one-year follow-up. The remaining 62.2% of the patients showed a marked improvement: decrease of the frequency and/or duration or relapses. Before AH-DLE administration, the mean number of herpes relapses in this group of patients was 12 p.a.. After therapy, the number of relapses decreased to 3.5 p.a.. No statistically significant difference was observed between groups A and B. The least favourable results were registered in group C. However, this group included 6 female patients extremely resistant to the previously therapeutic attempts, including inosiplex, non-specific DLE or acyclovir. Thus, even in this group, the therapy was successful in 50% of the patients.