The spectrum of spontaneous mutations caused by deficiency in proteasome maturase Ump1 in Saccharomyces cerevisiae

Ump1 is responsible for maturation of the catalytic core of the 26S proteasome. Dysfunction of Ump1 causes an increase in the frequency of spontaneous mutations in Saccharomyces cerevisiae. In this study we analyze the spectrum of mutations occurring spontaneously in yeast deficient in Ump1 by use of the SUP4-o system. Single base substitutions predominate among the mutations analyzed (73 of the 91 alterations examined). Two major classes are GC to TA transversions and GC to AT transitions ( approximately 50 and approximately 30% of base substitutions, respectively). Besides base substitutions, almost all the major types of sequence alterations are represented. The specificity and distribution of mutations occurring in the ump1 strain are unique compared to the spectra previously established for other yeast mutators. However, the profile of mutations arising in this strain is similar to that observed in wild type. The same similarity has previously been reported for yeast deficient in Mms2, a protein involved in Rad6-dependent postreplication DNA repair (PRR). The specificity of the mutator effect caused by ump1 is discussed in light of the proposed role of the proteasome activity in the regulation of the PRR mechanisms.     

Nociceptive flexion reflex thresholds and pain during rest and computer game play in patients with hypertension and individuals at risk for hypertension

Supraspinal pain modulation may explain hypertensive hypoalgesia. We compared nociceptive flexion reflex (NFR) thresholds and pain during rest and computer game play in hypertensives and normotensives (Experiment 1) and normotensives with and without hypertensive parents (Experiment 2). The game was selected to modulate activity in pain pathways. NFR thresholds did not differ between groups during rest or game play. Pain ratings never differed between hypertensives and normotensives, whereas individuals with hypertensive parents reported less pain during the first two NFR assessments, compared to those without. NFR thresholds and pain were reduced by game play compared to rest. The failure of game play to differentially modulate NFR thresholds or associated pain reports between groups argues against enhanced supraspinal modulation of nociception and pain in hypertensives and those at increased risk for hypertension.     

Platelet-activating factor, a pleiotrophic mediator of physiological and pathological processes

Platelet-activating factor (PAF) is a potent proinflammatory phospholipid with diverse pathological and physiological effects. This bioactive phospholipid mediates processes as diverse as wound healing, physiological inflammation, apoptosis, angiogenesis, reproduction and long-term potentiation. Recent progress has demonstrated the participation of MAP kinase signaling pathways as modulators of the two critical enzymes, phospholipase A2 and acetyltransferase, involved in the remodeling pathway of PAF biosynthesis. The unregulated production of structural analogs of PAF by non-specific oxidative reactions has expanded this superfamily of signaling molecules to include "PAF-like" lipids whose mode of action is identical to that of authentic PAF. The action of members of this family is mediated by the PAF receptor, a G protein-coupled membrane-spanning molecule that can engage multiple signaling pathways in various cell types. Inappropriate activation of this signaling pathway is associated with many diseases in which inflammation is thought to be one of the underlying features. Inactivation of all members of the PAF superfamily occurs by a unique class of enzymes, the PAF acetylhydrolases, that have been characterized at the molecular level and that terminate signals initiated by both regulated and unregulated PAF production.     

Differential induction of apoptosis in undifferentiated and differentiated HL-60 cells by DNA topoisomerase I and II inhibitors

The effects of monocytic/macrophage and granulocytic differentiation induced by phorbol myristate acetate (TPA) and all-trans retinoic acid, respectively, were tested on the induction of apoptosis in human promyelocytic leukemia HL-60 cells treated with topoisomerase I and II inhibitors. Using a filter-binding assay, we observed a strong inhibition of DNA fragmentation induced by 3- and 24-hour continuous exposure to camptothecin, VP-16, VM-26, and m-AMSA in TPA- differentiated cells. The inhibition of the typical internucleosomal DNA fragmentation was confirmed by agarose gel electrophoresis. By contrast, drug-induced DNA fragmentation was not inhibited in retinoic acid-differentiated cells, and apoptosis occurred in these cells after 4 to 5 days in the absence of drug treatment. The TPA inhibitory effect was maximal after 24 hours of treatment and was correlated with differentiation, because phorbol dibutyrate ester was active, whereas 4- alpha-TPA, a nontumor promoter that does not induce differentiation, was not active. Using alkaline elution, we observed that TPA and retinoic acid differentiation were associated with changes in topoisomerase-mediated DNA breaks that were not correlated with their differential effects on drug-induced DNA fragmentation. Moreover, TPA also inhibited DNA fragmentation induced by vinblastine, cycloheximide, calphostin C, and x-rays. Using a cell-free system, we observed that DNA fragmentation was not inhibited in nuclei from TPA-differentiated cells. Rather, inhibition of apoptosis seemed to take place in the cytoplasm. We conclude that phenotypic changes associated with TPA- induced differentiation include inactivation of a cytoplasmic activity that can induce DNA fragmentation associated with apoptosis.     

Modulation of human upper intestinal nutrient transit by a beta adrenoreceptor mediated pathway.

To explore the role played by beta adrenoreceptor mediated pathways on human upper gut function a series of studies were conducted into the effects of beta adrenoreceptor agonists and antagonists on orocaecal and duodenocaecal transit and on antral and duodenal motor activity. Under control conditions orocaecal transit was consistent within individuals (mean coefficient of variation (18.0%) but varied widely between individuals (median transit 63 minutes, range 33-164). Prior administration of the non-selective beta adrenergic antagonist propranolol consistently hastened orocaecal transit (median transit 51:25-93, v control p < 0.005). The selective beta-1 antagonist, atenolol, also hastened transit (median transit 50:35-93 minutes, v control p < 0.01). The magnitude of an individual's response to beta blockade correlated closely with the orocaecal transit (Tau = 0.54, p < 0.01). Duodenocaecal transit was also hastened by propranolol from control values of 66:45-107 minutes to 50:16-62 minutes, p < 0.025). In contrast neither duodenal nor antral motility were consistently altered by beta blockade. The beta adrenoreceptor agonist, isoprenaline, delayed both orocaecal transient (97:55-178 minutes, v control p < 0.005) and also duodenocaecal transit (160:45-215 minutes, v 73:40-133) (p < 0.025). Isoprenaline also reduced antral motility by an effect which appeared to occur predominantly through a reduction in contraction amplitude (from a median amplitude of 27:5.39 mm Hg to 14:3-24 mm Hg, p < 0.03) rather than an effect on the interval between contractions. No effect on either amplitude or frequency of duodenal motor activity was observed. A beta adrenoreceptor mediated pathway thus appears to exert a biologically relevant effect on gut function not only under conditions of sympathetic stimulation, but also at rest when a basal beta adrenergic tone appears to influence the speed of nutrient transit through the human upper gut.