Long-term follow-up with stress echocardiograms of patients with Kawasaki's disease

Patients with a history of Kawasaki's disease (KD), particularly those not treated with intravenous gamma-globulin, are at risk of coronary artery aneurysms and later obstruction. Twenty-eight patients with a history of KD (4 had coronary artery aneurysms) were examined with stress echocardiograms. Fourteen patients received gamma-globulin < or =10 days of the onset, 8 patients received gamma-globulin >10 days and 6 received no gamma-globulin. The mean age at diagnosis was 7.2 +/- 4.1 years; the median follow-up was 8.0 +/- 7.4 years. All tests were negative. Using a binomial model, a power of 0.80, a sensitivity of each test of 80% and assuming uniform risk, the individual rate of failure to detect was <7%. At least 640 patients in each group would be needed to detect a difference of 3.5% vs. 7.0% and 184 in each group would be needed to detect a difference of 1.5% vs. 7.5%. We conclude that the probability of an abnormal stress echo in asymptomatic patients with a history of KD is at most 7% and that a more precise determination of the risk of an abnormal stress echo in KD requires a much larger study.     

Molecular cloning of Brevundimonas diminuta for efficacy assessment of reverse osmosis devices

Brevundimonas diminuta is the test organism specified in the United States Environmental Protection Agency's (USEPA) reverse osmosis (RO) treatment device verification protocol. As non-selective growth medium is employed, enumeration of B. diminuta may be impaired due to interference by indigenous heterotrophic bacteria. Thus the microbial removal capability of the filtration system may be incorrectly assessed. As these treatment devices are used in emergency situations, the health of the public could be compromised. The objective of this study was to develop selective approaches for enumerating viable B. diminuta in test water. Two molecular approaches were investigated: expression of a kanamycin resistance gene and expression of a fluorescent protein gene. The USEPA protocol specifies a 0.3 μm cell size, so the expression of the selective markers were assessed following growth on media designed to induce this small cell diameter. The kan(R) strain was demonstrated to be equivalent to the wild type in cell dimension and survival following exposure to the test water. The kan(R) strain showed equivalent performance to the wild type in the RO protocol indicating that it is a viable alternative surrogate. By utilizing this strain, a more accurate validation of the RO system can be achieved.     

TCDD alters pituitary-adrenal function I: Adrenal responsiveness to exogenous ACTH

Plasma ACTH concentrations in 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD)-treated rats (50 μg/kg; single, oral dose) were 2.1-, 2.1-, 2.9-, 1.7-, 1.5-, 2.0- and 3.0-fold greater than control values, respectively, at days 1, 3, 5, 7, 10, and 14. At days 1 and 5 plasma corticosterone concentrations were increased 5.1- and 8.0-fold, respectively; whereas, at days 10 and 14 they were depressed to values of 50% and 39% of controls, respectively. Adrenal glands were exised from rats treated with TCDD and corticosterone production was assessed. Basal corticosterone concentrations produced by treated adrenals were depressed to 81%, 72%, and 71% of control values at days 5, 7, and 14, respectively. Corticosterone secretion by ACTH stimulated adrenals was equivalent to controls. These findings suggest that TCDD exposure decreases the bioactivity of the ACTH secreted by the anterior pituitary.     

Inactivation of ethanol-inducible cytochrome P450 and other microsomal P450 isozymes by trans-4-hydroxy-2-nonenal, a major product of membrane lipid peroxidation.

Of the microsomal P450 cytochromes, the ethanol-inducible isoform, P450 2E1, is believed to be predominant in leading to oxidative damage, including the generation of radical species that contribute to lipid peroxidation, and in the reductive beta-scission of lipid hydroperoxides to give hydrocarbons and aldehydes. In the present study, the sensitivity of a series of P450s to trans-4-hydroxy-2-nonenal (HNE), a known toxic product of membrane lipid peroxidation, was determined. After incubation of a purified cytochrome with HNE, the other components of the reconstituted system (NADPH-cytochrome P450 reductase, phosphatidylcholine, and NADPH) were added, and the rate of oxygenation of 1-phenylethanol to yield acetophenone was assayed. Inactivation occurs in a time-dependent and HNE concentration-dependent manner, with P450s 2E1 and 1A1 being the most sensitive, followed by isoforms 1A2, 3A6, and 2B4. At an HNE concentration of 0.24 microM, which was close to the micromolar concentration of the enzyme, four of the isoforms were significantly inhibited, but not P450 2B4. In other experiments, the reductase was shown to be only relatively weakly inactivated by HNE. P450s 2E1 and 2B4 in microsomal membranes from animals induced with acetone or phenobarbital, respectively, are as readily inhibited as the purified forms. Evidence was obtained that the P450 heme is apparently not altered and the sulfur ligand is not displaced, that substrate protects against HNE, and that the inactivation is reversed upon dialysis. Higher levels of reductase or substrate do not restore the activity of inhibited P450 in the catalytic assay. Our results suggest that the observed inhibition of the various P450s is of sufficient magnitude to cause significant changes in the metabolism of foreign compounds such as drugs and chemical carcinogens by the P450 oxygenase system at HNE concentrations that occur in biological membranes. In view of the known activities of P450 2E1 in generating lipid hydroperoxides and in their beta-scission, its inhibition by this product of membrane peroxidation may provide a negative regulatory function.     

Stimulation by voluntary exercise of adrenal glucocorticoid secretion in mature female hamsters.

The possibility that habitual voluntary running induces a chronic change in adrenal glucocorticoid synthesis and secretion was examined in freely running mature female hamsters, in whom this behavior accelerates growth, reduces body fat levels, and elevates core temperature. Hamsters were free to run on horizontal discs or in vertical wheels between 32 and 80 days, in 14L:10D or in 10L:14D photoperiods, and at the end of this period, corticosterone and cortisol steroidogenesis and serial plasma corticosterone concentrations during day and night were used as measures of the chronic stimulation of adrenal cortical activity. Habitual voluntary running significantly increased steroidogenesis of both glucocorticoids and plasma corticosterone concentrations and alone accounted for all the variance in enhanced synthesis and secretion of corticosterone. Acute exercise and/or the nocturnal phase of circadian period enhanced the chronic stimulatory effects of exercise on cortisol. Despite its voluntary and apparently stress-free nature, running induces chronic increases in basal glucocorticoid secretion in mature female hamsters. Putative oversecretion of corticotropin releasing factor in freely running hamsters could account for increased steroidogenesis, acceleration of growth, reduced body fat levels, and core temperature elevation.     

Tetrachlorodibenzo-p-dioxin alters rat hypothalamic endorphin and mu opioid receptors.

The present study was undertaken to assess if hypothalamic beta-endorphin (beta E) and/or brain mu opioid receptors are associated with 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) (50 micrograms/kg)-induced hypophagia and body weight decline in rats. Hypothalamic beta E concentrations were initially increased to 166% of controls on day 1, and then were depressed to 39% and 49% of control values on days 2 and 3, respectively. Brain mu opioid receptor number was increased 60% in TCDD-treated rats at day 3 without a change in the binding affinity. Food-restricted rats did not exhibit changes in hypothalamic beta E concentrations or brain mu opioid receptor number. These results indicate that TCDD causes early perturbations in hypothalamic beta E concentrations and brain mu receptor number, which may contribute to the mechanisms by which TCDD leads to decreased food intake and progressive weight loss.     

Human placental lipid peroxidation--II. NADPH and iron dependent stimulation of microsomal lipid peroxidation by paraquat

Paraquat, a widely used herbicide, was found to cause a marked stimulation of lipid peroxidation in the human placental microsomes in vitro. Both NADPH and chelated iron were necessary to observe paraquat-stimulated lipid peroxidation. The malondialdehyde accumulation in the incubation medium increased with increase in time, protein and paraquat concentration. The reaction did not exhibit the initial lag phase suggesting that endogenous membrane-bound antioxidants in human placental microsomes are either absent or present in extremely small quantities.     

Inhibition of gastrin-stimulated cell proliferation by the CCK-B/gastrin receptor ligand CI-988.

The gastrointestinal hormone gastrin functions as a trophic factor for oxyntic mucosa as well as a secretagogue for gastric acid. In preclinical toxicology studies CI-988, a peptoid cholecystokinin (CCK) ligand with nanomolar affinity for the CCK-B/gastrin receptor, caused gastric gland degeneration and mucosal atrophy in cynomolgus monkeys, perhaps consistent with an expected pharmacological outcome of inhibition of the trophic effect of gastrin on stomach mucosa. Because of the expense and difficulty associated with experimental use of non-human primates, we investigated the effects of CI-988 on signal transduction pathways associated with gastrin-stimulated cell proliferation using the AR42J rat pancreatic tumour cell line as a model. The AR42J cell line was selected because it is known to express the CCK-B/gastrin receptor and because it is responsive to the growth promoting effects of gastrin in vitro. Gastrin-17 at 1 nM stimulated proliferation of AR42J cells 26% and 104% above control after 24 and 96 hours, respectively. CI-988 at 1 nM had no apparent effect on basal cell proliferation rates, but decreased gastrin-17 stimulated cell proliferation 13% and 47%, respectively, after 24 and 96 hours of treatment, consistent with competitive antagonism at the gastrin receptor. Because the trophic effect of gastrin towards AR42J cells has been linked to intracellular calcium ([Ca2+]i) mobilization and/or cyclic AMP, the effect of CI-988 on these second messengers were also investigated. Gastrin-17 at 10 nM stimulated both ([Ca2+]i) and cAMP, while CI-988 alone at 100 nM had no effect, but blocked the gastrin-stimulated increases in both mediators. Therefore, using the AR42J pancreatic tumour cell line as a model, the dipeptoid CCK-B/gastrin receptor ligand CI-988 behaves as an antagonist towards gastrin receptor-stimulated signal transduction pathways and cell proliferation in vitro.     

In Vitro 2,3,7,8-Tetrachlorodibenzo-p-dioxin Interference with the Anterior Pituitary Hormone Adrenocorticortropin

Treatment of male Sprague-Dawley rats with a single oral doseof 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shownto increase serum adrenocorticotropin (ACTH) and decrease serumcorticosterone. The present in vitro study was designed to assesswhether TCDD has a direct effect on the anterior pituitary underbasal and stimulated conditions. Primary anterior pituitarycell cultures were prepared from normal 180- to 220-g male Sprague-Dawleyrats and the cultures treated with 10^–9–10^–19M TCDD. Maximal secretion of ACTH occurred between 10^–11and 10^–15 M TCDD for both medium (2-fold) and intracellular(1.5-fold) concentrations after 24 h TCDD exposure. TCDD treatmentalso caused an early (6 h) and persistent (10 days) increasein basal medium (1.4- to 2.8-fold) and intracellular (1.1- to1.7-fold) ACTH concentrations. However, while stimulation withcorticotropin-releasing hormone (CRH) increased intracellularACTH 1.5- to 1.7-fold in pituitary cells treated for 24 h with10^–9–10^–13 M TCDD, ACTH secreted into themedia was decreased by 30–50% compared with controls.Lastly, the secretagogue arginine-8-vaso-pressin (AVP), didnot increase the amount of ACTH secreted above levels observedwith basal TCDD exposure. From this study, it appears that TCDDstimulates in vitro synthesis and secretion of ACTH by the anteriorpituitary under basal conditions, but decreases the pituitary'sresponsiveness to CRH and AVP stimulation.