An in situ assay for induced diphtheria-toxin-resistant mutants of diploid human fibroblasts

The sensitivity of diploid human fibroblasts to the cytotoxic effects of diphtheria toxin (DT) depended on the cell growth status. Exponentially growing cells treated with 10^?3-1 lethal flocculating units (LF) of DT/ml for 4 days survived with a frequency of 4 × 10^?4. However, the DT-resistant phenotype of colonies isolated under these conditions was not stable. When the growth of the cells had been arrested by confluence or deprivation of serum growth factors prior to treatment with DT (4 days, 10^?3-0.6 LF/ml), the survival decreased to 2 × 10^?6 and the resistance of isolated colonies was stable. An in situ assay for induced DT-resistant mutants was developed in order to avoid problems associated with the possible reduced viability of the mutants relative to that of wild-type cells. A reproducible and linear dose response was obtained for the induction of DT-resistant mutants by ethylnitrosourea. The mutants were induced with high frequency by this compound (e.g., 10^?3 mutants/viable cell at a 37% survival dose); complete expression of the mutant phenotype occurred after 6 generations of growth under nonselective conditions. Isolated mutant colonies showed stable resistance to DT and were cross-resistant to Pseudomonas aeruginosa exotoxin A.     

Effect of DNA repair on the cytotoxicity and mutagenicity of polycyclic hydrocarbon derivatives in normal and xeroderma pigmentosum human fibroblasts

The cytotoxicity of the “K-region” epoxides as well as several other reactive metabolites or chemical derivatives of polycyclic hydrocarbons was compared in normally-repairing human diploid skin fibroblasts and in fibroblasts from a classical xeroderma pigmentosum (XP) patient (XP2BE) whose cells have been shown to carry out excision repair of damage induced in DNA by ultraviolet (UV) radiation at a rate approx. 20% that of normal cells. Each compound tested exhibited a 2- to 3-fold greater cytotoxicity in this XP strain than in the normal strain. To determine whether this difference in survival reflected a difference in the capacity of the strains to repair DNA damage caused by such hydrocarbon derivatives, we compared the cytotoxic effect of several “K-region” epoxides in two additional XP strains, each with a different capacity for repair of UV damage. The ration of the slopes of the survival curves for each of the XP strains to that of the normal strain, following exposure to each epoxide, was very similar to that which we had previously determined for their respective UV curves, suggesting that human cells repair damage induced in DNA by exposure to hydrocarbon derivatives with the same system used for UV-induced lesions.To determine whether the deficiency in rate of excision repair in this classical XP strain (XP2BE) causes such cells to be abnormally susceptible to mutations induced by “K-region” epoxides of polycyclic hydrocarbons, we compared them with normal cells for the frequency of induced mutations to 8-azaguanine resistance. The XP cells were two to three times more susceptible to mutations induced by the “K-region” epoxide of benzo(a)pyrene (BP), 7,12-dimethylbenz(a)anthracene (DMBA), and dibenz(a,h)anthracene (DBA). Evidence also was obtained that cells from an XP variant patient are abnormally susceptible to mutations induced by hydrocarbon epoxides and, as is the case following exposure to UV, are abnormally slow in converting low molecular weight DNA, synthesized from a template following exposure to hydrocarbon epoxides, into large-size DNA.     

A region of the broad-host-range plasmid RK2 causes stable in planta inheritance of plasmids in Rhizobium meliloti cells isolated from alfalfa root nodules.

We demonstrate for the first time that the broad-host-range stabilization loci from plasmid RK2 cause total retention of plasmids in cells of Rhizobium meliloti during symbiosis with alfalfa. Two derivatives of plasmid RK2, pRK290 and a 7.3-kb mini-RK2 plasmid, were stabilized in R. meliloti cells isolated from root nodules by the insertion of a 3.2-kb DNA fragment or a smaller 0.8-kb DNA fragment derived from the RK2 stabilization region.     

Long range electron transfer between tyrosine and tryptophan in hen egg-white lysozyme

The azide, dibromide and dichloride radicals oxidize one or more tryptophan side chains in hen egg-white lysozyme. The indolyl radical produced in this second-order 1-electron oxidation subsequently oxidizes a tyrosine side chain to the phenoxy radical in an intramolecular reaction with a rate constant of 130 +/- 10 s-1 at pH 7, 25 degrees C. The final indolyl and phenoxy equilibrium mixture then decays with a t1/2 approximately 2 s. The faster intramolecular reaction exhibits a pH dependence; on decreasing the pH from 9 the first-order rate constant increases to a maximum near pH 5.4 and then declines as the pH is lowered further. In contrast, the first-order rate constant for the intramolecular electron transfer between the tyrosine and tryptophan of the peptide trpH-pro-tyrOH remains unchanged between approx. pH 11 and 6.5 and then increases as the pH is lowered further. This difference in the observed pH dependence suggests that changes in structure or ionization state influence the protein electron transfer rate. We also discuss the radiation inactivation of lysozyme in light of these observations.     

Control of melanin synthesis and secretion by B16/C3 melanoma cells

In culture, B16/C3 murine melanoma cells grown in the presence of serum undergo melanogenesis at a specific time after plating. At this time, melanin is synthesized intracellularly and then secreted into the extracellular culture fluid. We have found that melanin secretion is dependent on the presence of serum in the growth medium. When confluent cultures are deprived of serum, that is, refed with serum-free medium, cells remain viable but do not undergo melanogenesis. Addition of serum-free medium supplemented with either melanocyte-stimulating hormone (MSH) or dibutyryl cAMP induced melanogenesis in these cells but did not result in melanin secretion. Furthermore, when B16/C3 cells are grown in serum-free, hormone-supplemented medium, they also undergo melanogenesis but fail to release melanin. The addition of serum, however, to B16/C3 cells induced to undergo melanogenesis with MSH, dibutyryl cAMP, or hormone-supplemented medium promotes melanin secretion. Fractionation studies hence revealed that serum contains specific factors capable of inducing melanin secretion. These results demonstrate that factors that regulate melanin synthesis are distinct from those that induce cells to release melanin into their extracellular environment. Furthermore, the ability to induce melanogenesis with single factors will permit us to study the precise sequence of events leading to differentiation in B16/C3 cells under chemically defined conditions.     

Potentiation of arginine-induced glucagon secretion by adenosine

Adenosine and the synthetic adenosine agonists 2-chloroadenosine and N⁶-(L-2-phenylisopropyl)-adenosine were tested for effects on hormone secretion from the rat isolated perfused pancreas. These nucleosides, at concentrations of 5 μM, markedly potentiated both phases of arginine-induced glucagon release; the two synthetic agonists were more effective than adenosine. In the absence of arginine, each of the nucleosides induced a transient burst of glucagon. In contrast, adenosine and both synthetic agonists inhibited arginine-induced insulin secretion to varying degrees and caused only negligible insulin release when perfused without arginine. The adenosine antagonist 8-($\text{p}$-sulfophenyl)-theophylline prevented the actions of adenosine on hormone release from the pancreas. Our data suggest that adenosine potentiation of arginine-induced glucagon release may be mediated via adenosine receptors on alpha cell membranes; such a mechanism could provide an important endogenous control over glucagon secretion.     

Tumor promoters and epidermal growth factor stimulate anchorage-independent growth of adenovirus-tranformed rat embryo cells.

Previous studies indicated that the potent tumor promoter 12--0--tetradecanoyl-phorbol-13-acetate (TPA) enhances transformation of rat embryo cells (2 degrees RE) by a mutant of human Ad5 (H5ts125). This study examines the effect of TPA, its structural analogs and epidermal growth factor (EGF) on anchorage-independent growth of a cloned population of H5ts125-transformed 2 degrees RE cells (clone E11). Both TPA and EGF (approximately 10(-8) M) induced a 3--5 fold increase in agar cloning efficiency of E11 cells. In addition, macroscopic colonies appeared earlier and were larger and more diffuse. The TPA analogs phorbol--12,13--didecanoate (PDD) and ingenol--3,20--dibenzoate also enhanced growth in agar of E11 cells, whereas phorbol, 4 alpha PDD and 4--0--meTPA, which are inactive as tumor promoters, failed to enhance agar growth. In contrast to the results obtained with E11 cells, TPA, PDD or ingenol--3,20--bidenzoate failed to induce growth in agar of normal 2 degrees RE cells. Dexamethasone (10(-5)--10(-6) M), trans retinoic acid (10(-5)--10(-6) M) and the protease inhibitors leupeptin, antipain and elastatinol did not inhibit the ability of TPA to enhance the growth of E11 cells in agar. The TPA-enhanced anchorage independence was a stable property, since subclones of E11 cells isolated from TPA-agar plates had a higher agar cloning efficiency than the parental E11 cells when retested in the absence of TPA. This effect of TPA does not appear to reflect simple selection of a subpopulation of cells. When the parental E11 cells were first cloned in monolayer culture in the absence of TPA, all ten randomly picked clones showed enhanced growth in agar in the presence of TPA. In addition, prior growth of E11 cells in monolayer culture in the presence of TPA did not enhance their subsequent growth in agar. This system therefore provides an example in which TPA appears to enhance the acquisition of a stable cell property, and thus may be a useful model for studying mechanisms of tumor promotion and progression.     

A scanning electron microscopy study of micro-arterial damage and repair.

The effects of microvascular clamps on the femoral vessels of rats were studied, using the SEM. The early changes observed were (1) local fusiform dilatation of the area secondary to necrosis of the muscular wall, (2) flattening of the longitudinal ridges in the endothelial (3) loss of laminar flow, (4) endothelial sloughing, (5) platelet aggregation, and (6) leukocyte adherence and diapedesis. The repair of the endothelium occurred by an early replication of the adjacent undamaged endothelial cells -- with their subsequent migration across a platelet bed. The coverage was complete in one week, although reorientation of the neo-endothelial cells took longer. On the basis of this study and our clinical experience, we think the ideal microvascular clamp would possess the following characteristics: small size, light weight, mechanical simplicity, flat jaws (one to two mm in diameter) coated with a non-slip surface, and calibrated to produce a pressure less than 30 gm per mm2. In addition the clamp should be unaffected by blood, autoclaving, or repeated use. No such clamp is commercially available now, but we hope that one will be available in the near future.     

Topological constraints in nucleic acid hybridization kinetics

A theoretical examination of kinetic mechanisms for forming knots and links in nucleic acid structures suggests that molecules involving base pairs between loops are likely to become topologically trapped in persistent frustrated states through the mechanism of ‘helix-driven wrapping’. Augmentation of the state space to include both secondary structure and topology in describing the free energy landscape illustrates the potential for topological effects to influence the kinetics and function of nucleic acid strands. An experimental study of metastable complementary ‘kissing hairpins’ demonstrates that the topological constraint of zero linking number between the loops effectively prevents conversion to the minimum free energy helical state. Introduction of short catalyst strands that break the topological constraint causes rapid conversion to full duplex.