Mutations resistant to bromodeoxyuridine in mouse lymphoma cells selected by repeated exposure to EMS characteristics of phenotypic instability and reversion to HAT resistance by 5-azacytidine

Mutations induced by repeated EMS treatments were investigated by using mouse L5178Y cells. The frequency of TG^r mutations increased linearly with the number of EMS treatments whereas the yield of BrdUrd^r mutations showed a curvilinear dose-response curve. The BrdUrd^r frequency was roughly proportional to the square of the TG^r frequency and the results were compatible with the hypothesis that BrdUrd^r cells were induced by two mutational events within a cell. Most of the BrdUrd^r colonies isolated after 6 EMS treatments, however, were unstable. When BrdUrd^r colonies that had arisen in BrdUrd medium after 2 weeks' incubation were isolated in normal medium, the descendant cells showed a nearly normal level of thymidine incorporation and low plating efficiencies of about 1% in BrdUrd medium. In contrast, after isolation of the same colonies in BrdUrd medium, a low level of thymidine incorporation and high plating efficiencies in BrdUrd medium were observed in the descendant cells.

Reverse selection from BrdUrd^r to HAT^r was accomplished with frequencies of 10⁻⁶−10⁻³ for the descendants grown in BrdUrd medium, and AzaCyd treatment drastically increased the reversion frequency to nearly 10⁻¹. Further re-revertants from HAT^r to BrdUrd^r were also found with frequencies of 10⁻³−10⁻² without treatment. These results indicate that the initial BrdUrd^r cells did not result from inactivation of the thymidine-kinase gene but that the mode of gene expression was altered in some way.     

1H nmr studies of complexes of ferric leghemoglobin with substituted pyridines and nicotinic acids

The ¹H nuclear magnetic resonance (nmr) spectra of complexes of soybean ferric leghemoglobin with 3-substituted pyridines and 5-substituted nicotinic acids have been recorded in order to determine the influence of axial ligands on heme electronic structure. The hyperfine shifted resonances of the heme group were assigned by analogy to previous assignments for the pyridine and nicotinic acid complexes of leghemoglobin. The spectra are characteristic of predominantly low-spin ferric heme complexes. For the pyridine complexes, the rate of ligand exchange was found to increase with decreasing ligand pK_A. For many of the complexes, optical and nmr spectra reveal the presence of an equilibrium mixture of high- and low-spin states of the iron atom. The percentage of high-spin component increases with decreasing ligand pK_A Smaller hyperfine shifts are noted for leghemoglobin complexes with ligands capable of weak ligand → metal π bonding. The pattern of hyperfine shifted resonances is similar for all complexes studied and indicates that the overall heme electronic structure is dominated by the bonding to the proximal histidine.     

Modulation of endogenous prostaglandins by thymosin-α1 in lymphocytes

The effects of thymosin-α₁ on the stimulation of specific release of prostaglandin E₂ (PGE₂) from splenic lymphocytes and thymocytes were studied. Experiments were also performed to study in parallel the absolute levels of thymosin-α₁ in the blood and the induction of serum FTS activity and of azathioprine sensitivity of spleen cells from adult thymectomized (ATx) mice. A significant difference in the release of PGE₂ between normal splenocytes and splenocytes from ATx mice was observed. Thymosin-α₁ at certain concentrations was able to stimulate PGE₂ release from lymphocytes of ATx mice while inhibiting release in lymphocytes of normal mice. Also, thymocytes were stimulated to release PGE₂ after incubation with α₁ in a manner similar to that seen in spleen cells of ATx mice. Approximately the same concentration of α₁ was found to also correct the low azathioprine sensitivity of splenocytes from ATx mice. Determinations of FTS-like activity in the blood and the pharmacokinetics of α₁ after administration of this synthetic molecule show a clear dissociation. A maximum peak of α₁ activity was obtained after 1 hr, while maximal FTS-like activity was observed after 24 hr. The inhibition of the induction by α₁ of FTS-like activity and of Thy 1.2 antigen by indomethacin suggests that the action of α₁ requires prostaglandin biosynthesis.     

Antioxidant Activity of 5-Hydroxytryptophan, 5-Hydroxyindole, and Dopa Against Microsomal Lipid Peroxidation and Its Dependence on Vitamin E

The antioxidant capacity of 5-hydroxy-tryptophan. 5-hydroxy-indole. and DOPA (3,4-dihydroxy-phenyI-alanine) was tested in the Fe-induced lipid peroxidation of liver microsomes of normal- and vitamin E-deficient rats, using ascorbate as a reductant. Lipid peroxidation was monitored as low-level chemilu-minescence, indicative of generation of electronically-excited states arising from the recombination of secondary lipid peroxyl radicals.     

Specific enhancement of LTD4-induced contractions of isolated guinea pig trachea by 5-hydroxyeicosatetraenoic acid (5-HETE)

In view of the likely production of monohydroxyeicosatetraenoic acid (HETE's) in bronchial asthma, the role of these lipoxygenase products in the development of a classical clinical element of airway disease, namely airway hyperreactivity, has been investigated. Tracheas removed from guinea-pigs actively sensitized to ovalbumin produced, upon antigenic challenge (0.01 μg/ml), a 17-fold increase (0.97 ± 0.34 ng/ml to 16.73 ± 1.58 ng/ml) in the amount of 5-hydroxyeicosatetraenoic acid (5-HETE) as measured by radioimmunoassay of the tissue-bath fluid, indicating that this tissue is capable of producing 5-HETE. While 5-HETE alone, at concentrations equal to or greater than those found during the above antigenic response (0.001 to 1.0 μM), failed to produce intrinsic contractions of normal, nonsensitized guinea-pig trachea, a 30 min pretreatment with 5-HETE (1.0 μM) enhanced subsequent LTD₄-induced contractions. Pretreatment with either 12- or 15-HETE, at similar concentrations and conditions, failed to potentiate LTD₄ concentration-response curves. The effect of 5-HETE was time-dependent, since pretreatment for either 15 or 60 min had little or no effect on subsequent LTD₄ responses. Also, the 5-HETE-induced enhancement seemed specific fot LTD₄, since contractions to LTC₄ (in the presence of l-serine borate), acetylcholine, histamine, PGD₂ or U-46619 were unaffected by 5-HETE. Therefore, 5-HETE may have a role in the development of airway hyperreactivity by interacting with released LTD₄ to exacerbate airway smooth muscle contraction in asthma.     

Inhibition of hexose transport in the human erythrocyte by 5, 5′-dithiobis(2-nitrobenzoic acid): Role of an exofacial carrier sulfhydryl group

Summary The sulfhydryl reagent 5, 5-dithiobis (2-nitrobenzoic acid) (DTNB) was used to study the functional role of an exofacial sulfhydryl group on the human erythrocyte hexose carrier. Above 1mm DTNB rapidly inhibited erythrocyte 3-O-methylglucose influx, but only to about half of control rates. Efflux was also inhibited, but to a lesser extent. Uptake inhibition was completely reversed by incubation and washing with 10mm cysteine, whereas it was only partially reduced by washing in buffer alone, suggesting both covalent and noncovalent interactions. The covalent thiol-reversible reaction of DTNB occurred on the exofacial carrier, since (i) penetration of DTNB into cells was minimal, (ii) blockade of potential uptake via the anion transporter did not affect DTNB-induced hexose transport inhibition, and (iii) DTNB protected from transport inhibition by the impermeant sulfhydryl reagent glutathione-maleimide-I. Maltose at 120mm accelerated the covalent transport inhibition induced by DTNB, whereas 6.5 m cytochalasin B had the opposite effect, indicating under the one-site carrier model that the reactive sulfhydryl is on the outward-facing carrier but not in the substrate-binding site. In contrast to glutathione-maleimide-I, however, DTNB did not restrict the ability of the carrier to reorient inwardly, since it did not affect equilibrium cytochalasin B binding. Thus, carrier conformation determines exposure of the exofacial carrier sulfydryl, but reaction of this group may not always lock the carrier in an outward-facing conformation.     

Entropy as a factor in the binding of γ-aminobutyric acid and nipecotic acid to the γ-aminobutyric acid transport system

Nipecotic acid is one of the most potent competitive inhibitors and alternative substrates for the high-affinity -aminobutyric acid transport system in neurons, but the structural basis of this potency is unclear. Because -aminobutyrate is a highly flexible molecule in solution, it would be expected to lose rotational entropy upon binding to the transport system, a change which does not favor binding. Nipecotic acid, in contrast, is a much less flexible molecule, and one would expect the loss of conformational entropy upon binding to be smaller thus favoring the binding of nipecotic acid over -aminobutyric acid. To investigate this possibility, the thermodynamic parameters, G°, H°, and S°, were determined for the binding of -aminobutyrate and nipecotic acid to the high affinity GABA transport system in synaptosomes. In keeping with expectations, the apparent entropy change for nipecotic acid binding (112±13 J·K^–1) was more favorable than the apparent entropy change for -aminobutyric acid binding (61.3±6.6 J·K^–1). The results suggest that restricted conformation per se is an important contributory factor to the affinity of nipecotic acid for the high-affinity transport system for -aminobutyric acid.This work was conducted when both authors were at the Department of Chemistry, University of Maryland, College Park.Special issue dedicated to Dr. Elling Kvamme.     

The influence of ovarian hormones on the rat oviductal and uterine concentration of noradrenaline and 5-hydroxytryptamine

This paper describes the effects of estradiol and progesterone on the concenirations of noradrenaline and 5-hydroxytryptamine in the Wistar rat oviduct and uterus. The levels of noradrenaline and 5-hydroxytryptamine are higher in the oviduct than in the uterus whereas p-tyrosine and tryptophan are similar in both tissues. Estradiol treatment reduced the oviductal concentration of noradrenaline but not 5-hydroxytryptamine in oviduct, while the concentrations of both noradrenaline and 5-hydroxytryptamine were reduced in uterine horn. The levels of noradrenaline in the oviduct and uterus in rats in estrus were lower than those of diestrous rats. Bilateral ovariectomy produced an increase in uterine noradrenaline and 5-hydroxytryptamine levels. These changes were reversed in the presence of ovarian hormones as indicated by experiments where unilateral ovariectomy was performed. Reserpine administration reduced noradrenaline concentration in both the oviduct and the uterus but did not change oviductal or uterine 5-hydroxytryptamine.These results indicate the existence of noradrenaline within postganglionic sympathetic nerve terminals and suggest that estrogens increase the utilization and the synthesis of noradrenaline in both the oviducts and the uterine horns. With respect to 5-hydroxytryptamine the data support the concept that it is mainly associated with mast cells.     

Alternating bolus and continuous infusion 5-fluorouracil: a strategy to overcome resistance to this fluoropyrimidine in advanced colorectal cancer patients

Focusing our effort on the importance of FUra scheduling we have tested the hypothesis that pulse and continuous infusion (CI) of the fluoropyrimidine have different mechanisms of cytotoxicity. Our initial approach was to compare the mechanism of resistance of a cell line resistant to a short term exposure to FUra (HCT-8/FU4hR) to that of a cell line resistant to a prolonged exposure to the fluoropyrimidine (HCT-8/FU7dR). Cytotoxicity studies showed that HCT-8/FU4hR cells were still sensitive to FUra given as a 7-d exposure, suggesting different mechanisms of resistance. Indeed, rapid recovery of TS activity after drug removal was evident in the HTC-8/FU7dR cell line while HCT-8/FU4hR cells were similar to the parental cell line with regard to both the degree of in situ TS inhibition by FUra and duration of inhibition after FUra removal. In contrast, labelling studies with [³H-6] FUra (4 h exposure, 100 M) showed that the incorporation of the fluoropyrimidine into RNA is significantly decreased in HCT-8/FU4hR cells as compared to parental HCT-8 cells.Given the lack of cross resistance between the two schedulesin vitro, a pilot trial was done on patients with colorectal cancer refractory to bolus FUra. On 15 patients failing after FUra+LV or FUra alone 1 PR, 3 MR, 3 SD and 8 P were observed, confirmng a certain degree of activity of CI FUra in patients clinically resistant to bolus FUra.Based on this rationale, a phase II trial of schedule-oriented biochemical modulation of FUra in advanced colorectal cancer patients was conducted, employing a hybrid regimen of 2 biweekly cycles of FUra bolus (600 mg/sqm), preceeded by (24 h interval) methotrexate, 200 mg/sqm (in order to maximize the RNA effect of the drug) alternating with FUra continuous infusion, 200 mg/sqm daily for 3 weeks, modulated by leucovorin, 20 mg/sqm weekly bolus (in order to maximize the DNA effect).Thirty-three consecutive patients (median ECOG PS 1) with advanced measurable colorectal cancer and no prior therapy for metastatic disease entered the study, from February 1992 to August 1993. Three complete and 13 partial responses were obtained among these 33 patients (RR=48%, 95% confidence limis, 31–66%). After a median follow-up time of 23 months, 16 patients are still alive. The median progression free survival and overall survival were 9.6 and 20.8 months, respectively. No toxic deaths or grade 4 toxicity occurred. The incidence of grade 3 toxicity per patient in any cycle was: mucositis 6%, diarrhea 3% and vomiting 3% for the bolus part and 21%, 3% and 6% respectively, for the continuous infusion part of the regimen. Hand-foot syndrome occurred in 27% of the patients treated with the continuous infusion regimen.In conclusion, this experimental and clinical project has generated a novel regimen of schedule oriented biochemical modulation that is twice as active and half as toxic compared to bolus FU+LV given with either the daily x 5 or the weekly schedule. This high clinical activity is very encouraging, especially considering that 1) consecutive patients were entered, 2) the responses were independently reviewed, 3) the progression free survival and survival were much longer than those actually reported for this disease, 4) the toxicity of the program, in particular the bolus regimen, was relatively low allowing further intensification.     

γ-Glutamyl conjugation of 5-hydroxytryptamine (serotonin) in the earthworm (Lumbricus terrestris)

The conversion of 5-hydroxytryptamine to several potential metabolites was examined in the annelid earthworm (Lumbricus terrestris). 5-hydroxytryptamine and some related amines were found to be present in several tissues of the earthworm. Injection of 5-hydroxytryptamine into the body cavity of the earthworm resulted in the production of a -glutamyl conjugate of 5-hydroxytryptamine. Incubations of the anterior nerve cord of the earthworm resulted in the accumlation of considerable amounts of 5-hydroxytryptamine and -glutamyl 5-hydroxytryptamine in the incubation medium. The earthworm did not produce any N-acetyl 5-hydroxytryptamine and only very little 5-hydroxyindoleacetic acid. Experiments involving the injection of radiolabeled 5-hydroxytryptamine or coninjection of radiolabeled glutamic acid with unlabeled 5-hydroxytryptamine into the earthworm resulted in the production of radiolabeled -glutamyl 5-hydroxytryptamine. This work demonstrates that the enzymatic conversion of 5-HT in the earthworm is markedly different from that of vertebrates and insects.