Open Preperitoneal Inguinal Hernia Repair, TREPP Versus TIPP in a Randomized Clinical Trial.

OBJECTIVE: The aim of this study was to compare chronic postoperative inguinal pain (CPIP) in patients with an inguinal hernia after the TransREctus Sheath PrePeritoneal (TREPP) and the TransInguinal PrePeritoneal Technique (TIPP). BACKGROUND: The preperitoneal mesh position for inguinal hernia repair showed beneficial results regarding CPIP with low recurrence rates. Two open preperitoneal techniques, TREPP and TIPP, were compared in a randomized clinical trial with the hypothesis of fewer patients with CPIP after TREPP due to complete avoidance of nerve contact. METHODS: Adult patients with a primary unilateral inguinal hernia were randomized to either TREPP or TIPP in four hospitals. Before the trial's start the study protocol was ethically approved and published. Outcomes included CPIP after 1 year (primary outcome) and recurrence rates, adverse events, and health-related quality of life (secondary outcomes). Follow-up was performed at 2 weeks, 6 months, and 1 year. RESULTS: Baseline characteristics were comparable in both groups. Pain was less often present after TREPP at 2 weeks and 6 months, but CPIP at rest at 1 year was comparable: 1.9% after TREPP vs 1.4% after TIPP, P = 0.535). The overall recurrence rate was higher in the TREPP group, 8.9% vs 4.6%, P = 0.022). Corrected for a learning curve for TREPP, no significant difference could be assessed (TREPP 5.7% and TIPP 4.8%, P = 0.591). CONCLUSION: Both the TREPP and TIPP technique resulted in a low incidence of CPIP after 1-year follow-up. The TREPP method can be considered a solid method for inguinal hernia repair if expertise is present. The learning curve of the TREPP techniques needs further evaluation. TRIAL REGISTRATION: ISRCTN18591339.

Identification of IgG1 isotype phosphorylcholine antibodies for the treatment of inflammatory cardiovascular diseases.

BACKGROUND: Phosphorylcholine (PC) is an important pro-inflammatory damage-associated molecular pattern. Previous data have shown that natural IgM anti-PC protects against cardiovascular disease. We aimed to develop a monoclonal PC IgG antibody with anti-inflammatory and anti-atherosclerotic properties. METHODS: Using various techniques PC antibodies were validated and optimized. In vivo testing was performed in a femoral artery cuff model in ApoE3*Leiden mice. Safety studies are performed in rats and cynomolgus monkeys. RESULTS: A chimeric anti-PC (PC-mAb(T15), consisting of a human IgG1 Fc and a mouse T15/E06 Fab) was produced, and this was shown to bind specifically to epitopes in human atherosclerotic tissues. The cuff model results in rapid induction of inflammatory genes and altered expression of genes associated with ER stress and choline metabolism in the lesions. Treatment with PC-mAb(T15) reduced accelerated atherosclerosis via reduced expression of endoplasmic reticulum stress markers and CCL2 production. Recombinant anti-PC Fab fragments were identified by phage display and cloned into fully human IgG1 backbones creating a human monoclonal IgG1 anti-PC (PC-mAbs) that specifically bind PC, apoptotic cells and oxLDL. Based on preventing macrophage oxLDL uptake and CCL2 production, four monoclonal PC-mAbs were selected, which to various extent reduced vascular inflammation and lesion development. Additional optimization and validation of two PC-mAb antibodies resulted in selection of PC-mAb X19-A05, which inhibited accelerated atherosclerosis. Clinical grade production of this antibody (ATH3G10) significantly attenuated vascular inflammation and accelerated atherosclerosis and was tolerated in safety studies in rats and cynomolgus monkeys. CONCLUSIONS: Chimeric anti-PCs can prevent accelerated atherosclerosis by inhibiting vascular inflammation directly and through reduced macrophage oxLDL uptake resulting in decreased lesions. PC-mAb represents a novel strategy for cardiovascular disease prevention.

Coding and long non-coding RNAs provide evidence of distinct transcriptional reprogramming for two ecotypes of the extremophile plant Eutrema salsugineum undergoing water deficit stress.

BACKGROUND: The severity and frequency of drought has increased around the globe, creating challenges in ensuring food security for a growing world population. As a consequence, improving water use efficiency by crops has become an important objective for crop improvement. Some wild crop relatives have adapted to extreme osmotic stresses and can provide valuable insights into traits and genetic signatures that can guide efforts to improve crop tolerance to water deficits. Eutrema salsugineum, a close relative of many cruciferous crops, is a halophytic plant and extremophyte model for abiotic stress research. RESULTS: Using comparative transcriptomics, we show that two E. salsugineum ecotypes display significantly different transcriptional responses towards a two-stage drought treatment. Even before visibly wilting, water deficit led to the differential expression of almost 1,100 genes for an ecotype from the semi-arid, sub-arctic Yukon, Canada, but only 63 genes for an ecotype from the semi-tropical, monsoonal, Shandong, China. After recovery and a second drought treatment, about 5,000 differentially expressed genes were detected in Shandong plants versus 1,900 genes in Yukon plants. Only 13 genes displayed similar drought-responsive patterns for both ecotypes. We detected 1,007 long non-protein coding RNAs (lncRNAs), 8% were only expressed in stress-treated plants, a surprising outcome given the documented association between lncRNA expression and stress. Co-expression network analysis of the transcriptomes identified eight gene clusters where at least half of the genes in each cluster were differentially expressed. While many gene clusters were correlated to drought treatments, only a single cluster significantly correlated to drought exposure in both ecotypes. CONCLUSION: Extensive, ecotype-specific transcriptional reprogramming with drought was unexpected given that both ecotypes are adapted to saline habitats providing persistent exposure to osmotic stress. This ecotype-specific response would have escaped notice had we used a single exposure to water deficit. Finally, the apparent capacity to improve tolerance and growth after a drought episode represents an important adaptive trait for a plant that thrives under semi-arid Yukon conditions, and may be similarly advantageous for crop species experiencing stresses attributed to climate change.

Blood pressure-lowering effect of the sodium glucose co-transporter-2 inhibitor ertugliflozin, assessed via ambulatory blood pressure monitoring in patients with type 2 diabetes and hypertension.

This study compared the blood pressure-lowering effect of ertugliflozin (1, 5, 25 mg), hydrochlorothiazide (HCTZ; 12.5 mg) and placebo in 194 patients with type 2 diabetes mellitus and hypertension for 4 weeks using ambulatory blood pressure monitoring. Endpoints (change from baseline to week 4) were: 24-h mean systolic blood pressure (SBP; primary); daytime, night-time, seated predose SBP, 24-h, daytime, night-time, seated predose diastolic blood pressure, 24-h urinary glucose excretion and fasting plasma glucose (FPG; secondary). Safety and tolerability were monitored. Significant decreases in placebo-corrected 24-h mean SBP (-3.0 to -4.0 mmHg) were recorded for all doses of ertugliflozin (for HCTZ, this was -3.2 mmHg). Daytime, but not night-time SBP was consistently reduced. Ertugliflozin produced dose-dependent significant decreases in FPG and increases in urinary glucose excretion. No notable changes in plasma renin activity or urinary aldosterone were seen. The most common adverse events were urinary tract infection, genital fungal infection, upper respiratory tract infection and musculoskeletal pain.

Exposure of two Eutrema salsugineum (Thellungiella salsuginea) accessions to water deficits reveals different coping strategies in response to drought.

Eutrema salsugineum is an extremophile related to Arabidopsis. Accessions from Yukon, Canada and Shandong, China, were evaluated for their tolerance to water deficits. Plants were exposed to two periods of water deficit separated by an interval of re-watering and recovery. All plants took the same time to wilt during the first drought exposure but Yukon plants took 1 day longer than Shandong plants following the second drought treatment. Following re-watering and turgor recovery, solute potentials of Shandong leaves returned to predrought values while those of Yukon leaves were lower than predrought levels consistent with having undergone osmotic adjustment. Polar metabolites profiled in re-watered plants showed that different metabolites are accumulated by Yukon and Shandong plants recovering from a water deficit with glucose more abundant in Yukon and fructose in Shandong leaves. The drought-responsive expression of dehydrin genes RAB18, ERD1, RD29A and RD22 showed greater changes in transcript abundance in Yukon relative to Shandong leaves during both water deficits and recovery with the greatest difference in expression appearing during the second drought. We propose that the initial exposure of Yukon plants to drought renders them more resilient to water loss during a subsequent water deficit leading to delayed wilting. Yukon plants also established a high leaf water content and increased specific leaf area during the second deficit. Shandong plants undergoing the same treatment regime do not show the same beneficial drought tolerance responses and likely use drought avoidance to cope with water deficits.

Peroxide-Mediated Release of Organophosphates from Boron-Containing Phosphotriesters: A New Class of Organophosphate Prodrugs.

Phosphate mono- and diesters can be liberated efficiently from boryl allyloxy (BAO) and related phosphotriesters by H2O2. This protocol was applied to the release of a phosphorylated serine derivative and the nucleotide analogue AZT monophosphate. Nucleotide release in the presence of ATP and a kinase provides a diphosphate, demonstrating that this method can be applied to biological processes.

Identification of phosphomethylethanolamine N-methyltransferase from Arabidopsis and its role in choline and phospholipid metabolism.

Three sequential methylations of phosphoethanolamine (PEA) are required for the synthesis of phosphocholine (PCho) in plants. A cDNA encoding an N-methyltransferase that catalyzes the last two methylation steps was cloned from Arabidopsis by heterologous complementation of a Saccharomyces cerevisiae cho2, opi3 mutant. The cDNA encodes phosphomethylethanolamine N-methyltransferase (PMEAMT), a polypeptide of 475 amino acids that is organized as two tandem methyltransferase domains. PMEAMT shows 87% amino acid identity to a related enzyme, phosphoethanolamine N-methyltransferase, an enzyme in plants that catalyzes all three methylations of PEA to PCho. PMEAMT cannot use PEA as a substrate, but assays using phosphomethylethanolamine as a substrate result in both phosphodimethylethanolamine and PCho as products. PMEAMT is inhibited by the reaction products PCho and S-adenosyl-l-homocysteine, a property reported for phosphoethanolamine N-methyltransferase from various plants. An Arabidopsis mutant with a T-DNA insertion associated with locus At1g48600 showed no transcripts encoding PMEAMT. Shotgun lipidomic analyses of leaves of atpmeamt and wild-type plants generated phospholipid profiles showing the content of phosphatidylmethylethanolamine to be altered relative to wild type with the content of a 34:3 lipid molecular species 2-fold higher in mutant plants. In S. cerevisiae, an increase in PtdMEA in membranes is associated with reduced viability. This raises a question regarding the role of PMEAMT in plants and whether it serves to prevent the accumulation of PtdMEA to potentially deleterious levels.

Live predator stress in adolescence results in distinct adult behavioral consequences and dorsal diencephalic brain activation patterns.

Exposure to traumatic events during childhood increases the risk of adult psychopathology, including anxiety, depression, alcohol use disorders and their co-morbidity. Early life trauma also results in increased symptom complexity, treatment resistance and poor treatment outcomes. The purpose of this study was to establish a novel rodent model of adolescent stress, based on an ethologically relevant life-threatening event, live predator exposure. Rats were exposed to a live predator for 10 min. at three different time points (postnatal day (PND)31, 46 and 61). Adult depression-, anxiety-like behaviors and ethanol consumption were characterized well past the last acute stress event (two weeks). Behavioral profiles across assessments were developed to characterize individual response to adolescent stress. CNS activation patterns in separate groups of subjects were characterized after the early (PND31) and last predator exposure (PND61). Subjects exposed to live-predator adolescent stress generally exhibited less exploratory behavior, less propensity to venture into open spaces, a decreased preference for sweet solutions and decreased ethanol consumption in a two-bottle preference test. Additional studies demonstrated blunted cortisol response and CNS activation patterns suggestive of habenula, rostromedial tegmental (RMTg), dorsal raphe and central amygdala involvement in mediating the adult consequences of adolescent stress. Thus, adolescent stress in the form of live-predator exposure results in significant adult behavioral and neurobiological disturbances. Childhood trauma, its impact on neurodevelopment and the subsequent development of mood disorders is a pervasive theme in mental illness. Improving animal models and our neurobiological understanding of the symptom domains impacted by trauma could significantly improve treatment strategies.

Corpus callosum volume and interhemispheric transfer in multiple sclerosis.

BACKGROUND: The corpus callosum (CC) is frequently compromised in patients with multiple sclerosis (MS). Structural and functional measurements of the CC may be useful to monitor the progression of the disease. The aim of this pilot study was to determine if bimanual tactile temporal thresholds correlates with CC volume. A tactile temporal threshold is the longest temporal interval that separates the onsets of two tactile stimuli when they are judged by the observer as simultaneous. Judgments to bimanual stimulations require interhemispheric transfer via the CC. METHODS: Thresholds were examined in MS patients and matched controls. Magnetic resonance (MR) images were acquired on a 3T MR system within 48 hours of clinical assessment and measurement of thresholds. RESULTS: Corpus callosum volume was assessed by using a semiautomatic livewire algorithm. The CC volume was smaller (by 21% on average, p < 0.01) and thresholds were higher (by 49% on average, p < 0.03) in MS patients when compared to controls. A significant correlation (r = -0.66, p = 0.01) between CC volume and thresholds emerged for the MS patients. CONCLUSION: Measuring treatment benefits of neuroprotective and repair therapies is a well recognized challenge in MS research. The overall findings of this study suggest that these measurements, which involve the transfer of information interhemispherically via the CC, may be promising outcome measures that warrant further scientific exploration to develop a model to measure recovery.

T2 MRI texture analysis is a sensitive measure of tissue injury and recovery resulting from acute inflammatory lesions in multiple sclerosis.

T2 hyperintensity is pathologically non-specific in multiple sclerosis (MS) yet lesion analysis remains an important disease outcome. Texture analysis based on the polar Stockwell Transform (PST) was performed on twelve acute T2 lesions before, during, and after the development of gadolinium-enhancement. When regular myelin structure is disrupted coarse texture increases because tissue becomes disorganized. Coarse texture was quantified as the sum of low frequency energy (sumLFE). Matching regions of contralateral and general normal appearing white matter (NAWM) and chronic T2 lesions were analyzed in parallel as controls. Coarse texture increased in acute lesions during enhancement (p<0.05) then variably recovered. It remained stable in NAWM and tended to increase in chronic T2 lesions. Seven of twelve acute lesions persisted visually at 8 months and the sumLFE was higher in these visually persistent lesions (p<0.05) than in resolved lesions. The sumLFE at month 8 correlated with that in pre-lesional NAWM and in acute lesions (p<0.05) and was independent of lesion volume, signal intensity (SI), and location. This study suggests that PST texture analysis extracts more information about tissue integrity than conventional MRI analysis that relies on lesion size and SI. Texture analysis also appears to identify abnormalities in pre-lesional NAWM, to measure tissue injury in acute lesions, predict poor recovery, and detect mild ongoing tissue injury in chronic T2 lesions. PST texture analysis using conventional MRI may therefore provide valuable new insights into lesion pathology by measuring tissue integrity. This small longitudinal study supports further validation of the PST technique.

Isoniazid and iproniazid: activation of metabolites to toxic intermediates in man and rat.

Acetylhydrazine, a metabolite of isoniazid, a widely used antituberculosis drug, and isopropylhydrazine, a metabolite of iproniazid, an antidepressant removed from clinical use because of high incidence of liver injury, were oxidized by cytochrome P-450 enzymes in human and rat liver microsomes to highly reactive acylating and alkylating agents. Covalent binding of these metabolites to liver macromolecules paralleled hepatic cellular necrosis. The metabolites formed from these and probably other monosubstituted hydrazines are reactive electrophiles.

Accurate Patient-Specific Machine Learning Models of Glioblastoma Invasion Using Transfer Learning.

BACKGROUND AND PURPOSE: MR imaging-based modeling of tumor cell density can substantially improve targeted treatment of glioblastoma. Unfortunately, interpatient variability limits the predictive ability of many modeling approaches. We present a transfer learning method that generates individualized patient models, grounded in the wealth of population data, while also detecting and adjusting for interpatient variabilities based on each patient's own histologic data. MATERIALS AND METHODS: We recruited patients with primary glioblastoma undergoing image-guided biopsies and preoperative imaging, including contrast-enhanced MR imaging, dynamic susceptibility contrast MR imaging, and diffusion tensor imaging. We calculated relative cerebral blood volume from DSC-MR imaging and mean diffusivity and fractional anisotropy from DTI. Following image coregistration, we assessed tumor cell density for each biopsy and identified corresponding localized MR imaging measurements. We then explored a range of univariate and multivariate predictive models of tumor cell density based on MR imaging measurements in a generalized one-model-fits-all approach. We then implemented both univariate and multivariate individualized transfer learning predictive models, which harness the available population-level data but allow individual variability in their predictions. Finally, we compared Pearson correlation coefficients and mean absolute error between the individualized transfer learning and generalized one-model-fits-all models. RESULTS: Tumor cell density significantly correlated with relative CBV (r = 0.33, P < .001), and T1-weighted postcontrast (r = 0.36, P < .001) on univariate analysis after correcting for multiple comparisons. With single-variable modeling (using relative CBV), transfer learning increased predictive performance (r = 0.53, mean absolute error = 15.19%) compared with one-model-fits-all (r = 0.27, mean absolute error = 17.79%). With multivariate modeling, transfer learning further improved performance (r = 0.88, mean absolute error = 5.66%) compared with one-model-fits-all (r = 0.39, mean absolute error = 16.55%). CONCLUSIONS: Transfer learning significantly improves predictive modeling performance for quantifying tumor cell density in glioblastoma.

Regulation of hepatic glutathione turnover in rats in vivo and evidence for kinetic homogeneity of the hepatic glutathione pool.

The intracellular distribution of glutathione into kinetically distinct pools and the determinants of glutathione turnover were examined in vivo. Glutathione turnover was measured in individual, restrained rats with a biliary fistula by administration of acetaminophen to trap the previously labeled hepatic glutathione as an excretable acetaminophen adduct. Fasting for 48 h resulted in a decrease of hepatic glutathione from 4.7+/-0.9 to 3.6+/-0.8 mumol/g liver and a marked increase in the fractional rate of glutathione turnover from 0.19+/-0.04 to 0.43+/-0.07/h. Within 6 h following refeeding, the rate of glutathione turnover and the hepatic glutathione concentration returned to normal. The simultaneously determined specific activities of free intrahepatic glutathione and the acetaminophen-glutathione adduct in bile were identical, indicating that the hepatic glutathione pool is kinetically homogeneous. The synthesis of glutathione could, therefore, be estimated from the rate constant and the intrahepatic glutathione concentration. During fasting hepatic synthesis of glutathione increased from 0.86+/-0.17 to 1.50+/-0.23 mumol/g per h. In fed animals the administration of dibutyryl cyclic adenosine monophosphate and theophylline stimulated the rate of hepatic glutathione turnover similar to fasting. In contrast, glucose given intraduodenally to fasted animals decreased the rate of glutathione turnover. These data are consistent with the view that the increased glutathione turnover that occurs during fasting results from two mechanisms. Because of a decrease in the intrahepatic free glutathione/mixed disulfide ratio, which is apparently mediated by cyclic adenosine monophosphate, the free glutathione pool contracts and turns over more rapidly in order to maintain glutathione synthesis. In addition, glutathione consumption via the gamma-glutamyl cycle apparently is increased, which may be related to the increased uptake of amino acids for gluconeogenesis during fasting.

Reactive oxygen species during ischemia-reflow injury in isolated perfused rat liver.

The hypothesis that intracellular generation of reactive oxygen species in hepatocytes or reticuloendothelial cells may cause ischemia-reperfusion injury was tested in isolated perfused livers of male Fischer rats. GSSG was measured in perfusate, bile, and tissue as a sensitive index of oxidative stress. After a preperfusion phase of 30 min, the perfusion was stopped (global ischemia) for various times (30, 120 min) and the liver was reperfused for another 60 min. The bile flow (1.48 +/- 0.17 microliters/min X gram liver weight), the biliary efflux of total glutathione (6.54 +/- 0.94 nmol GSH eq/min X g), and GSSG (1.59 +/- 0.23 nmol GSH eq/min X g) recovered to 69-86% after short-term ischemia and to 36-72% after 2 h of ischemia when compared with values obtained from control livers perfused for the same period of time. During reperfusion, the sinusoidal efflux of total glutathione (16.4 +/- 2.1 nmol GSH eq/min X g) and GSSG (0.13 +/- 0.05 nmol GSH eq/min X g) did not change except for an initial 10-30-s increase during reperfusion washout. No increased GSSG secretion into bile was detectable at any time during reperfusion. The liver content of total glutathione (32.5 +/- 3.5 nmol GSH eq/mg protein) and GSSG (0.27 +/- 0.09 nmol GSH eq/mg protein) did not change significantly during any period of ischemia or reperfusion. We conclude, therefore, that at most only a minor amount of reactive oxygen species were generated during reperfusion. Thus, reactive oxygen species are unlikely to cause ischemia/reperfusion injury in rat liver by lipid peroxidation or tissue thiol oxidation.

Mechanism of action of N-acetylcysteine in the protection against the hepatotoxicity of acetaminophen in rats in vivo.

N-Acetylcysteine is the drug of choice for the treatment of an acetaminophen overdose. It is thought to provide cysteine for glutathione synthesis and possibly to form an adduct directly with the toxic metabolite of acetaminophen, N-acetyl-p-benzoquinoneimine. However, these hypothese have not been tested in vivo, and other mechanisms of action such as reduction of the quinoneimine might be responsible for the clinical efficacy of N-acetylcysteine. After the administration to rats of acetaminophen (1 g/kg) intraduodenally (i.d.) and of [(35)S]-N-acetylcysteine (1.2 g/kg i.d.), the specific activity of the N-acetylcysteine adduct of acetaminophen (mercapturic acid) isolated from urine and assayed by high pressure liquid chromatography averaged 76+/-6% of the specific activity of the glutathione-acetaminophen adduct excreted in bile, indicating that virtually all N-acetylcysteine-acetaminophen originated from the metabolism of the glutathione-acetaminophen adduct rather than from a direct reaction with the toxic metabolite. N-Acetylcysteine promptly reversed the acetaminophen-induced depletion of glutathione by increasing glutathione synthesis from 0.54 to 2.69 mumol/g per h. Exogenous N-acetylcysteine did not increase the formation of the N-acetylcysteine and glutathione adducts of acetaminophen in fed rats. However, when rats were fasted before the administration of acetaminophen, thereby increasing the stress on the glutathione pool, exogenous N-acetylcysteine significantly increased the formation of the acetaminophen-glutathione adduct from 57 to 105 nmol/min per 100 g. Although the excretion of acetaminophen sulfate increased from 85+/-15 to 211+/-17 mumol/100 g per 24 h after N-acetylcysteine, kinetic simulations showed that increased sulfation does not significantly decrease formation of the toxic metabolite. Reduction of the benzoquinoneimine by N-acetylcysteine should result in the formation of N-acetylcysteine disulfides and glutathione disulfide via thiol-disulfide exchange. Acetaminophen alone depleted intracellular glutathione, and led to a progressive decrease in the biliary excretion of glutathione and glutathione disulfide. N-Acetylcysteine alone did not affect the biliary excretion of glutathione disulfide. However, when administered after acetaminophen. N-acetylcysteine produced a marked increase in the biliary excretion of glutathione disulfide from 1.2+/-0.3 nmol/min per 100 g in control animals to 5.7+/-0.8 nmol/min per 100 g. Animals treated with acetaminophen and N-acetylcysteine excreted 2.7+/-0.8 nmol/min per 100 g of N-acetylcysteine disulfides (measured by high performance liquid chromatography) compared to 0.4+/-0.1 nmol/min per 100 g in rats treated with N-acetylcysteine alone. In conclusion, exogenous N-acetylcysteine does not form significant amounts of conjugate with the reactive metabolite of acetaminophen in the rat in vivo but increases glutathione synthesis, thus providing more substrate for the detoxification of the reactive metabolite in the early phase of an acetaminophen intoxication when the critical reaction with vital macromolecules occurs.

Biliary excretion of glutathione and glutathione disulfide in the rat. Regulation and response to oxidative stress.

Regulation of the biliary excretion of reduced glutathione (GSH) and glutathione disulfide (GSSG) and responses to selected model toxins were examined in male Sprague-Dawley rats. In control and phenobarbital-pretreated rats in which the intrahepatic concentration of GSH was modulated by the administration of diethyl maleate or acetaminophen, the biliary concentration of GSH was consistently lower than, but directly proportional to, the intrahepatic concentration of GSH. Furthermore, increments in bile flow produced by the infusion of sulfobromophthalein (BSP)-glutathione were associated with proportional increases in the biliary excretion of GSH, suggesting that GSH passes into bile passively along a concentration gradient. In contrast, GSSG appears to be secreted into bile against a steep concentration gradient. An increased hepatic production and biliary excretion of GSSG resulted from the administration of t-butyl hydroperoxide. Measurement of biliary GSSG and BSP during a constant infusion of the GSH adduct of BSP indicated that GSSG shares a common excretory mechanism with GSH adducts. Diquat, nitrofurantoin, and paraquat also markedly stimulated the biliary excretion of GSSG. On a molar basis, these compounds generated much more GSSG than a direct substrate for glutathione peroxidase such as t-butyl hydroperoxide, indicating that the compounds undergo redox-cycling with concomitant production of hydrogen peroxide. Aminopyrine (0.8 mmol/kg) also significantly increased biliary GSSG. This increase, however, was associated with a proportional increase in bile flow and in the biliary excretion of GSH such that the GSSG/GSH ratio in bile did not change. Acetaminophen and chloroform, two compounds generating electrophilic metabolites that deplete intrahepatic GSH, led to a progressive decrease in the biliary excretion of GSH and GSSG. Furosemide and dimethylnitrosamine, the electrophilic metabolites of which do not deplete hepatic GSH, minimally altered biliary GSH and GSSG. Similarly, carbon tetrachloride and iproniazid, which yield organic radical metabolites that can peroxidize membrane lipids, did not increase the biliary excretion of GSSG. This finding indicates that membrane-bound lipid hydroperoxides may not be good substrates for glutathione peroxidases. The measurement of the biliary excretion of GSSG and of the GSSG/GSH ratio in bile is a sensitive index of oxidative stress in vivo and thus complements other in vivo parameters for the study of reactive intermediates of xenobiotics such as the determination of covalent binding, the formation of lipid hydroxy acids, and the depletion of intracellular GSH.

Guanethidine and related agents. 3. Antagonism by drugs which inhibit the norepinephrine pump in man.

Antagonism of the antihypertensive action of guanethidine by the tricyclic antidepressants, desipramine and protriptyline, has been demonstrated in controlled studies. These antidepressants also prevent the effect of the related ring-substituted guanidinium adrenergic neuron blockers, bethanidine and debrisoquin. That the rise in blood pressure when desipramine is added to guanethidine therapy is not due simply to a pressor action of the two drugs in combination was demonstrated by the lack of an increase in blood pressure when guanethidine was added to desipramine therapy. Investigations were conducted to determine whether antagonism of guanethidine's clinical effect could result from blockade by the tricyclic antidepressants of the norepinephrine pump in the adrenergic neuron membrane, thereby preventing the uptake of guanethidine into the neuron by this pump. Like guanethidine, the indirectly acting pressor amine, tyramine, enters the neuron via the norepinephrine pump. Desipramine, protriptyline, and amitriptyline in clinical doses all were found to block the pressor action of tyramine while potentiating the pressor effect of norepinephrine. The amino acid, methyldopa, does not enter the neuron via the norepinephrine pump, and its antihypertensive action is not altered by concomitant administration of tricyclic antidepressants. It is concluded from the evidence in this investigation together with the results of previous studies in experimental animals that clinical doses of desipramine-like drugs inhibit the norepinephrine pump in the peripheral adrenergic neuron in man and thereby prevent uptake of guanethidine to its site of action.

Effect of aminoglutethimide on blood pressure and steroid secretion in patients with low renin essential hypertension.

An inhibitor of adrenal steroid biosynthesis, aminoglutethimide, was administered to seven patients with low renin essential hypertension, and the antihypertensive action of the drug was compared with its effects on adrenal steroid production. In all patients aldosterone concentrations in plasma and urine were within normal limits before the study. Mean arterial pressure was reduced from a pretreatment value of 117+/-2 (mean+/-SE) mm Hg to 108+/-3 mm Hg after 4 days of aminoglutethimide therapy and further to 99+/-3 mm Hg when drug administration was stopped (usually 21 days). Body weight was also reduced from 81.6+/-7.2 kg in the control period to 80.6+/-7.0 kg after 4 days of drug treatment and to 80.1+/-6.7 kg at the termination of therapy. Plasma renin activity was not significantly increased after 4 days of treatment but had risen to the normal range by the termination of aminoglutethimide therapy. Mean plasma concentrations of deoxycorticosterone and cortisol were unchanged during aminoglutethimide treatment whereas those of 18-hydroxydeoxycorticosterone, progesterone, 17alpha-hydroxyprogesterone, and 11-deoxycortisol were increased as compared to pretreatment values. In contrast, aminoglutethimide treatment reduced mean plasma aldosterone concentrations to about 30% of control values. Excretion rates of 16beta-hydroxydehydroepiandrosterone, 16-oxo-androstenediol, 17-hydroxycorticosteroids and 17-ketosteroids, and the secretion rate of 16beta-hydroxydehydroepiandrosterone were not significantly altered by aminoglutethimide treatment whereas the excretion rate of aldosterone was reduced from 3.62+/-0.5 (mean+/-SE) in the control period to 0.9+/-0.2 mug/24 h after 4 days and to 1.1+/-0.3 mug/24 h at the termination of aminoglutethimide treatment. The gradual lowering of blood pressure and body weight during aminoglutethimide therapy is consistent with the view that the antihypertensive effect of the drug is mediated through a reduction in the patients' extracellular fluid volume, probably secondary to the persistent decrease in aldosterone production. The observation that chronic administration of aminoglutethimide lowered blood pressure in these patients and elevated their plasma renin activity to the normal range without decreasing production of the adrenal steroids, deoxycorticosterone, 18-hydroxydeoxycorticosterone, and 16beta-hydroxydehydroepiandrosterone, makes it unlikely that these steroids are responsible either for the decreased renin or the elevated blood pressure in patients with low renin essential hypertension.

A box H/ACA small nucleolar RNA-like domain at the human telomerase RNA 3' end.

Simple sequence repeat telomeric DNA is maintained by a specialized reverse transcriptase, telomerase. The integral RNA subunit of telomerase contains a template region that determines the sequence added to chromosome ends. Aside from providing the template, little is known about the role of the telomerase RNA. In addition, no hypotheses have been suggested to account for the striking evolutionary divergence in size and sequence between telomerase RNAs of ciliates, yeasts, and mammals. We show that the two- to threefold increase in size of the mammalian telomerase RNAs relative to ciliate telomerase RNAs is due to the presence of an extra domain resembling a box H/ACA small nucleolar RNA (snoRNA). The human telomerase RNA (hTR) H/ACA domain is essential in vivo for hTR accumulation, hTR 3' end processing, and telomerase activity. By substituting the U64 box H/ACA snoRNA for the hTR H/ACA domain, we demonstrate that a heterologous snoRNA can function to promote chimeric RNA accumulation and 3' end processing but not telomerase activity. In addition, we show that maturation of full-length hTR and its assembly into active telomerase occur from an mRNA promoter-driven RNA polymerase II transcript but not from a U6 snRNA promoter-driven RNA polymerase III transcript. Finally, we show that a small percentage of hTR is associated with nucleoli. These results have implications for the biogenesis and structure of hTR and the human telomerase ribonucleoprotein complex. They also expand the structural and functional diversity of the box H/ACA snoRNA motif.

RNA binding domain of telomerase reverse transcriptase.

Telomerase is a ribonucleoprotein reverse transcriptase that extends the ends of chromosomes. The two telomerase subunits essential for catalysis in vitro are the telomerase reverse transcriptase (TERT) and the telomerase RNA. Using truncations and site-specific mutations, we identified sequence elements of TERT and telomerase RNA required for catalytic activity and protein-RNA interaction for Tetrahymena thermophila telomerase. We found that the TERT amino and carboxyl termini, although evolutionarily poorly conserved, are nonetheless important for catalytic activity. In contrast, high-affinity telomerase RNA binding requires only a small region in the amino terminus of TERT. Surprisingly, the TERT region necessary and sufficient for telomerase RNA binding is completely separable from the reverse transcriptase motifs. The minimal Tetrahymena TERT RNA binding domain contains two sequence motifs with ciliate-specific conservation and one TERT motif with conservation across all species. With human TERT, we demonstrate that a similar region within the TERT amino terminus is essential for human telomerase RNA binding as well. Finally, we defined the Tetrahymena telomerase RNA sequences that are essential for TERT interaction. We found that a four-nucleotide region 5' of the template is critical for TERT binding and that the 5' end of telomerase RNA is sufficient for TERT binding. Our results reveal at least one evolutionarily conserved molecular mechanism by which the telomerase reverse transcriptase is functionally specialized for obligate use of an internal RNA template.