Modulation of restriction enzyme-induced damage by chemicals that interfere with cellular responses to DNA damage: a cytogenetic and pulsed-field gel analysis

The electroporation of restriction enzymes into mammalian cells results in DNA double-strand breaks that can lead to chromosome aberrations. Four chemicals known to interfere with cellular responses to DNA damage were investigated for their effects on chromosome aberrations induced by AluI and Sau3AI; in addition, the number of DNA double-strand breaks at various times after enzyme treatment was determined by pulsed-field gel electrophoresis (PFGE). The poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide (3AB) dramatically increased the yield of exchanges and deletions and caused a small but transitory increase in the yield of double-strand breaks induced by the enzymes. 1-beta-D-Arabinofuranosylcytosine, which can inhibit DNA repair either by direct action on DNA polymerases alpha and delta or by incorporation into DNA, potentiated aberration induction but to a lesser extent than 3AB and did not affect the amount of DNA double-strand breakage. Aphidicolin, which inhibits polymerases alpha and delta, had no effect on AluI-induced aberrations but did increase the aberration yield induced by Sau3AI. The postreplication repair inhibitor caffeine had no effect on aberration yields induced by either enzyme. Neither aphidicolin nor caffeine modulated the amount of DNA double-strand breakage as measured by PFGE. These data implicate poly(ADP-ribosyl)ation and polymerases alpha and delta as important components of the cellular processes required for the normal repair of DNA double-strand breaks with blunt or cohesive ends. Comparison of these data with the effect of inhibitors on the frequency of X-ray-induced aberrations leads us to the conclusion that X-ray-induced aberrations can result from the misjoining or nonrejoining of double-strand breaks, particularly breaks with cohesive ends, but that this process accounts for only a portion of the induced aberrations.     

Restoration of embryogenic competence in repeatedly subcultured carrot cell lines

Summary The production of somatic embryos from carrot suspension cultures invariably decreases through simple, repeated subculturing. Extracellular, concentrated compounds extracted from already established embryo culture not only recovered the embryogenic capability, but also accelerated the embryo production as much as two-fold (up to 1600 embryos/ml) compared with that of a control culture. Sugars, which were only a small portion of the total concentrate, were excluded as possible causative factors. It is likely that a protein fraction that is generated directly by competent, embryogenic cultures is important for the restoration of embryogenic potential.     

Randomised controlled trial of short term treatment to eradicate Helicobacter pylori in patients with duodenal ulcer.

OBJECTIVE--To determine whether one week''s drug treatment is sufficient to eradicate Helicobacter pylori in patients with duodenal ulcer. DESIGN--Single blind, randomised controlled trial. SETTING--Specialised ulcer clinic in a teaching hospital. PATIENTS--155 patients with H pylori and a duodenal ulcer verified endoscopically which had either bled within the previous 24 hours or was causing dyspepsia. INTERVENTIONS--Patients were allocated randomly to receive either omeprazole for four weeks plus bismuth 120 mg, tetracycline 500 mg, and metronidazole 400 mg (all four times a day) for the first week (n = 78), or omeprazole alone for four weeks (n = 77). Further endoscopy was performed four weeks after cessation of all drugs. MAIN OUTCOME MEASURES--Presence or absence of H pylori (by urease testing, microscopy, and culture of antral biopsy specimens), duodenal ulcer, and side effects. RESULTS--Eradication of H pylori occurred in 70 (95%) patients taking the four drugs (95% confidence interval 86% to 97%) compared with three (4%) patients taking omeprazole alone (1% to 11%). Duodenal ulcers were found in four (5%) patients taking the four drugs (2% to 12%) and in 16 (22%) patients taking omeprazole alone (14% to 32%). Mild dizziness was the only reported side effect (six patients in each group) and did not affect compliance. CONCLUSIONS--A one week regimen of bismuth, tetracycline, and metronidazole is safe and effective in eradicating H pylori and reduces the number of duodenal ulcers four weeks after completing treatment.     

Structure-activity relationships of cyclic β-casomorphin-5 analogues

Cyclic analogues of the β-casein-derived opioid peptide β-casomorphin-5 (H-Tyr-Pro-Phe-Pro-Gly-OH) were prepared through substitution of the Pro² residue with various ,ω-diamino acid residues (lysine, ornithine, 2,4-diaminobutyric acid) and cyclization of the ω-amino group to the C-terminal carboxyl function. Compounds of this type, with D-configuration at the 2-position residue, showed high opioid receptor affinity with some preference for μ receptors over δ receptors, high potency in the guinea pig ileum assay and considerable activity in the mouse vas deferens assay. Configurational inversion at the 4-position in these cyclic analogues resulted in enhanced affinity for both μ and δ receptors, whereas N-methylation of the Phe³ residue produced a potency decrease.     

Characterization of recombinant human renin: Kinetics,pH-stability, and peptidomimetic inhibitor binding

The kinetic behavior andpH-stability of recombinant human renin was analyzed using a new fluorogenic substrate based on the normal P₆-P₃ renin cleavage sequence in human angiotensinogen. The design of this fluorogenic substrate makes possible, for the first time, direct monitoring of the kinetics of proteolytic conversion of prorenin to renin. ThepH-stability profile for renin, measured with the substrate at 25°C, indicated a broad plateau of stability betweenpH 6.0 and 10.0. Analysis of thepH-activity profile of renin for the substrate indicated a minimumK _m (1.8 µM) atpH 7.4 and a maximumV _m betweenpH 7.4 and 8.0. The thermodynamics of the binding of a novel, soluble, peptidomimetic inhibitor to renin indicated it is possible to retain the tight-binding characteristics and enthalpy contributions to binding of larger peptide-derived inhibitors, while reducing inhibitor size and entropic contributions to binding. A novel derivative of the fluorogenic substrate, containing a 3-methyl histidine substitution at the P₂ site, was used to test the recent hypothesis that renin functions by virtue of substrate-directed catalysis.     

Dopamine acts through a D2-like receptor on a jellyfish motor neuron

Summary Dopamine, which is present in nerve-rich tissues of the hydromedusa Polyorchis penicillatus, produces membrane hyperpolarization in identified motor neurons from this jellyfish. In this study we demonstrate that the inhibitory action of dopamine is mediated by conventional drug-receptor interactions which are reversible, saturable and specific. When 10 M dopamine was applied by micro-spritzing onto voltage-clamped (holding potential, –20 mV), cultured swimming motor neurons, an outward current of about 1 nA was evoked. Using this technique, we established a potency order for several amines: dopaminenorepinephrine>tyramine >octopamine>-phenylethylamine. Dopamine is effective at concentrations betweeen 1 × 10^-8 and 1 × 10^-3 M. Several dopamine receptor blockers such as fluphenazine, haloperidol and spiperone reduced the dopamine-induced current in a concentration-dependent manner. Although propranolol, a -adrenergic blocker, reduced the dopamine response and SKF 83566, a D₁ blocker, increased the response, it appears that the dopamine receptors in these jellyfish neurons share pharmacological properties with mammalian D₂ dopamine receptors.     

T3 reverses the changes in met-enkephalin and beta-endorphin contents in the anterior lobe, but not the neuro-intermediate lobe of the pituitary of rats rendered hypothyroid by PTU-treatment

The changes in met-enkephalin and beta-endorphin contents in the pituitary in PTU-induced hypothyroidism were studied in the rat. After 2 weeks of PTU-treatment, both IR-met-enkephalin and IR-beta-endorphin contents in the pituitary were significantly reduced. Gel filtration chromatography followed by radioimmunoassay showed that the immunoactivities in the peaks of precursors, met-enkephalin, beta-lipotropin and beta-endorphin were all lower in the pituitaries from the PTU-treated rats. In another experiment, some of the PTU-treated rats were injected daily with 500 micrograms T3/kg b.w. In the hypothyroid rats, IR-met-enkephalin and IR-beta-endorphin contents were decreased in both the anterior and neurointermediate lobes. Only the changes in the anterior lobe were reversed by T3 treatment. In conclusion, while the effects on the anterior lobe are probably due to a deficiency in thyroid hormones, the mechanism for the decrease of opioid peptide contents in the neurointermediate lobe is still unclear.