Role for c-jun N-terminal kinase in treatment-refractory acute myeloid leukemia (AML): signaling to multidrug-efflux and hyperproliferation.

A relationship was proved between constitutive activity of leukemic cell c-jun-N-terminal kinase (JNK) and treatment failure in AML. Specifically, early treatment failure was predicted by the presence of constitutive JNK activity. The mechanistic origins of this association was sought. A multidrug resistant leukemic cell line, HL-60/ADR, characterized by hyperexpression of c-jun and JNK activity, was transfected with a mutant c-jun vector, whose substrate N-terminal c-jun serines were mutated. Down-regulated expression occurred of c-jun/AP-1-dependent genes, catalase and glutathione-S-transferase (GST) pi, which participate in cellular homeostasis to oxidative stress and xenobiotic exposure. MRP-efflux was abrogated in HL-60/ADR cells with dominant-negative c-jun, perhaps because MRP1 protein expression was also lost. Heightened sensitivity to daunorubicin resulted in cells subjected to this change. Biochemical analysis in 67 primary adult AML samples established a statistical correlation between cellular expression of c-jun and JNK activity, JNK activity with hyperleukocytosis at presentation of disease, and with exuberant MRP efflux. These findings reflect the survival role for c-jun/AP-1 and its regulatory kinase previously demonstrated for yeast in homeostatic response to oxidative stress and in operation of ATP-binding cassette efflux pumps, and may support evolutionary conservation of such function. Thus, JNK and c-jun may be salient drug targets in multidrug resistant AML.

Characterization and imaging of A6 epithelial cell clones expressing fluorescently labeled ENaC subunits.

A6 model renal epithelial cells were stably transfected with enhanced green fluorescent protein (EGFP)-tagged alpha- or beta-subunits of the epithelial Na(+) channel (ENaC). Transfected RNA and proteins were both expressed in low abundance, similar to the endogenous levels of ENaC in native cells. In living cells, laser scanning confocal microscopy revealed a predominantly subapical distribution of EGFP-labeled subunits, suggesting a readily accessible pool of subunits available to participate in Na(+) transport. The basal level of Na(+) transport in the clonal lines was enhanced two- to fourfold relative to the parent line. Natriferic responses to insulin or aldosterone were similar in magnitude to the parent line, while forskolin-stimulated Na(+) transport was 64% greater than control in both the alpha- and beta-transfected lines. In response to forskolin, EGFP-labeled channel subunits traffic to the apical membrane. These data suggest that channel regulators, not the channel per se, form the rate-limiting step in response to insulin or aldosterone stimulation, while the number of channel subunits is important for basal as well as cAMP-stimulated Na(+) transport.

A preliminary analysis of psychophysiological variables and nursing performance in situations of increasing criticality.

OBJECTIVE: To examine the relationship between psychological, physiological, and performance variables in intensive care unit (ICU) nurses in situations of increasing criticality. SUBJECTS AND METHODS: Psychophysiological variables and endotracheal suctioning performance were examined in a classroom, a skills laboratory, and an ICU. Situation-specific anxiety (state anxiety) and the predisposition to view situations as threatening (trait anxiety), cognitive appraisal, and heart rate were measured and compared with self-appraisal and a nurse instructor's ratings of suctioning performance. Baseline data were obtained during class on 45 novice ICU nurses. RESULTS: Twenty-six nurses provided complete data, which included being videotaped and monitored in the classroom, in the skills laboratory performing endotracheal suctioning, and in the ICU during suctioning. High state anxiety significantly predicted poor ICU suctioning performance (P<.04). Nurses high in state and trait anxiety, worry, and heart rate performed poorly compared with less anxious nurses. Nurses in this study who performed best had a mean heart rate of 94 beats/min. CONCLUSION: Those nurses who are high state anxious, high trait anxious, and worried and who had a faster heart rate performed less well than their more relaxed peers. Nurses with high state anxiety may be at risk for attrition, burnout, medical errors, and poor performance in other ICU nursing tasks.

Is there a role for the adrenals in the development of hypercholesterolemia in Zucker fatty rats?

We tested the hypothesis that hypercorticosteronemia causes the hypercholesterolemia in young developing "fatty" rats. Obesity induced increases in corticosterone. Insulin, glucose, body weight, average daily food intake, plasma triglyceride, plasma phospholipids, liver weight, liver triglyceride, various adipose tissue parameters, and liver hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase activity were all ameliorated by adrenalectomy. Adrenalectomy exacerbated the hypercholesterolemia in obese animals and induced it in lean rats. Changes or lack of change in hepatic microsomal cholesterol, HMG-CoA reductase, and 7 alpha-hydroxylase, combined with the adrenalectomy-induced curtailment of tissue storage of cholesterol in adipose tissue, all contribute to the hypercholesterolemia caused by adrenalectomy. We suggest a mechanism whereby this may be related to elevated hepatic very low-density lipoprotein secretion rates. The elevated HMG-CoA reductase activity in obese rats results from the lower liver microsomal free cholesterol content. We conclude that the absence of glucocorticoids does not directly reduce plasma cholesterol in obese Zucker rats. The surprising elevation of cholesterol by adrenalectomy is due to other prevailing mechanisms in liver and adipose tissue, which curtail their capacity to store cholesterol.

Various levels of maternal caffeine ingestion during gestation affects biochemical parameters of fetal rat brain differently.

Pregnant dams were divided into four groups on day 10 of gestation. Dams of group 1 were fed an 20% protein diet as controls. Dams of groups 2, 3 and 4 were fed a 20% protein diet supplemented with 0.5 mg, 1 mg and 2 mg caffeine/100 g body weight of dams, respectively. Pups were delivered surgically on day 22, and their brains were rapidly removed and analyzed for DNA, protein, cholesterol, zinc and alkaline phosphatase activity. The dams' brains were analyzed for the same parameters as those of the pups. Plasma and brain caffeine levels were also determined in caffeine-supplemented groups. The pups' brains in group 2 were heavier than those in group 4. The DNA concentration of group 2 was higher than that of the other groups. The protein concentration of group 4 was higher than that of the other groups. The cholesterol concentration of group 3 and 4 was less than that of the controls. The zinc concentration of group 4 was less than that of group 2. Alkaline phosphatase activity was decreased in groups 3 and 4 compared with either controls or group 2. Dams showed no significant difference among the groups in the same biochemical parameters except for cholesterol concentration that was higher in groups 2, 3 and 4 than in the controls. Plasma and brain caffeine levels of the fetuses and plasma caffeine of the dams in group 4 were higher than those of either group 2 or 3. It is concluded that various amounts of maternal caffeine intake exert different effects on fetal brain growth. In contrast, the effect of caffeine on the dams' brain is relatively minor.

Interaction between caffeine intake and nutritional status on growing brains in newborn rats.

The purpose of this study was to determine whether caffeine's effects on the growing brain in suckling pups are modified by the nutritional status of the dams. Upon delivery, 8 randomly selected pups were assigned to each dam. They were divided into four groups; group 1 was fed a 20% protein diet as a control; group 2 was fed a 6% protein diet as a malnourished group; group 3 was pair-fed to group 1, but the 20% protein diet was supplemented with caffeine (2 mg/100 g body weight of dams), and group 4 was pair-fed to group 2 with a 6% protein diet with caffeine. At day 15, pups were killed. Brains were removed, weighted and homogenized. Caffeine content of plasma, brain of the pups and maternal milk in groups 3 and 4 were determined. Brains were analyzed for zinc, alkaline phosphatase activity, DNA, protein, and cholesterol. Body weight and protein content of group 3 were greater than group 1, but the zinc contents and alkaline phosphatase activity of group 3 were less than group 1. DNA and cholesterol contents of group 4 were greater than group 2. Supplementation of caffeine to the maternal diet appeared to have various effects on the growing brains of the suckling pups. Caffeine's effects and nutritional status are closely interrelated.

Effects of different levels of caffeine supplemented to the maternal diet on the brains of newborn rats and their dams.

At birth, dams with 8 randomly assigned pups were divided into three groups. Dams of group 1 were fed a control diet. Dams of groups 2 and 3 were fed the control diet supplemented with caffeine (1 mg and 2 mg/100 g body weight, respectively). Pups were killed at day 15 and their brains removed. After weighing, brains were analyzed for DNA, protein, cholesterol, zinc and alkaline phosphatase activity. Brain and plasma caffeine levels were also determined on groups 2 and 3. The dams were milked to measure caffeine levels. The brains from the dams were analyzed for the same parameters as the pups. Caffeine levels in group 3 were consistently higher than in group 2. In the pups, body and brain weights were heavier in group 3 than in the controls. Protein and cholesterol concentrations in group 2 were less than either controls or group 3. Alkaline phosphatase activity in group 2 was higher than either controls or group 3. In the dams, DNA concentration in groups 2 and 3 was less than the controls. Protein and cholesterol concentration in group 2 was less than group 3. It was concluded that low levels of caffeine in the maternal diet during lactation could affect various parameters in the newborn brain. These effects were different from those when the dietary caffeine level was doubled. In contrast, the effects of caffeine on brains of the dams were relatively minor.

Chronic caffeine intake alters the composition of various parts of the brain in young growing rats.

Time-pregnant rats were fed a regular laboratory diet until delivery. Litters delivered within an 8-hour period were combined and 8 randomly selected pups were assigned to each dam. Dams were then divided into two groups. Group I received a 20% protein diet. Group II was pair-fed to group I with a 20% protein diet containing caffeine (1 mg/100 g body weight) until day 22 postpartum. On weaning (day 22), only male pups were selected and fed the 20% protein or 20% protein + caffeine diet according to which diet their respective dams had been fed. On day 43, rats were killed and the brains were divided into 6 different areas. DNA, RNA, protein and cholesterol contents were measured. Caffeine supplementation resulted in a decrease in the total brain weight, and DNA and RNA content. DNA value of cortex-midbrain was smaller in the caffeine group whereas the values of medulla oblongata and striatum were greater in comparison with the controls. Caffeine's effects on RNA included an increase in the cerebellum, cortex-midbrain and hippocampus and a decrease in the medulla oblongata. Protein content of cerebellum was increased and of hippocampus decreased due to caffeine supplementation. Cholesterol content of the medulla oblongata and hippocampus was less in the caffeine group than in the control group. The present study shows that chronic caffeine intake during rapid periods of growth influences various parts of the brain in entirely different biochemical manners.

Effect of chronic caffeine intake on myocardial function during early growth.

The purpose of these studies was to evaluate the effects of chronic caffeine ingestion on the myocardium during fetal and neonatal growth and development. The isolated perfused working heart preparation was used to evaluate cardiac function. During gestation and lactation, one group of dams consumed a caffeine supplemented diet (10 mg/kg/day). Their offspring were sacrificed and the hearts analyzed 50 days after birth. We found that the intake of caffeine by the dams resulted in significant increases in the offspring's coronary flow, peak systolic pressure, and myocardial work. A second group of dams ingested a diet containing caffeine (10 mg/kg/day) during lactation only. Their pups continued to consume the caffeine diet until 50 days. Pup hearts exhibited significant reductions in cardiac output, stroke volume, pressure development, myocardial work, and external efficiency when compared to controls. Caffeine did not affect body or heart weight or adipose size or number in these experiments. Thus, continued caffeine consumption following birth may alter cardiac performance of the offspring.

Chronic caffeine intake by rat dams during gestation and lactation affects various parts of the neonatal brain.

Timed-pregnant rats were randomly divided into 2 groups on day 13 of gestation. Group 1 received a 20% protein diet. Group 2 was pair-fed to group 1 with a 20% protein diet containing caffeine (1 mg/100 g body weight). At parturition, the dams of each group were continued on their respective diets until day 22 postpartum. At the time of weaning (day 22), only male rats were continued in the study. At this time, rats from both groups were fed the control diet containing 20% protein. On day 57 and 58, rats were killed, the brains divided into six areas, and DNA, RNA and protein contents were measured. In certain areas of the brain, weight, cholesterol, DNA, RNA and protein contents were different even long after returning to the caffeine-free control diet. The present study demonstrates that even if a relatively small amount of caffeine is taken during gestation and lactation, a time during which the growth rate is greater than in any other period of life, certain areas of the brain may be affected. These findings suggest that future central nervous system impairment may be expressed later in life in these offspring.

Adenohypophyseal response to hypophysiotropic hormones in male obese Zucker rats.

Description of the recessive, homozygote obese Zucker rat (fafa) includes disorders of growth and reproduction. The aim of this study was to compare responsiveness of adenohypophyseal cells, obtained from male fafa rats and from their lean siblings, to growth hormone-releasing factor (GRF) and to luteinizing hormone-releasing hormone (LHRH). Pituitary cells were cultured for 4 days and were then challenged with either GRF-29 (the NH2-terminal 29 amino acid GRF peptide that expresses full biological activity of its parent 44 amino acid molecule) or [D-Trp6]LHRH (LHRH-A, an LHRH agonist). Medium was assayed for growth hormone (GH), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) by radioimmunoassay. Dose-response curves were compared using the computer program ALLFIT. The median effective GRF-29 concentration (EC50) computed for hypophyseal cells cultured from lean animals (0.30 +/- 0.01 fM; means +/- SE of 4 experiments) was less (P less than 0.01) than that calculated for cells obtained from fafa rats (15.8 +/- 6.7 fM). In contrast, cells derived from lean littermates required a larger (EC50) concentration of LHRH-A than did gonadotrophs cultured from obese rats [58.2 +/- 1.2 vs. 10.7 +/- 1.2 pM (P less than 0.01) and 59.4 +/- 10.4 vs. 15.7 +/- 7.6 pM (P less than 0.05)] to secrete LH and FSH, respectively. Our data describe an attenuated pituitary response to GRF-29 and an enhanced response to LHRH-A in the fafa.

Effects of chronic caffeine ingestion on growth and myocardial function.

Experiments were conducted to determine whether chronic caffeine consumption during early growth and development affected cardiac performance and development of adipose tissue. Dams were fed a nutritionally complete diet with or without the addition of 10 mg/kg caffeine during lactation. After weaning, the pups were maintained on this diet until they were sacrificed at 88 days of age. Body weight at the time of sacrifice was comparable for both groups. The hearts from caffeine-fed animals were significantly (P less than 0.05) larger based on both dry and wet weights although the dry weight/wet weight ratios were similar. Ventricular function curves were generated on each heart using an isolated working heart preparation. The isolated hearts of caffeine-fed rats exhibited a significant reduction in cardiac output, stroke volume, mean aortic pressure, and estimated myocardial work when compared to controls. The rats fed caffeine had greater plasma triglyceride levels with no significant differences in adipocyte size or number in the epididymal and perirenal depots. It is concluded that chronic caffeine intake from birth may alter cardiac function of the offspring.

Adipocyte fatty acid mobilization in vivo: effects of age and anatomical location.

The objectives of the present study were to determine if adipocyte triglyceride fatty acid (TGFA) mobilization in vivo varied among the different adipose tissue depots and whether these rates were affected by age. In order to accomplish these objectives, two groups of rats were studied. The first group initially weighed 84 +/- 1 and the second group 333 +/- 2. Both groups were placed on a semisynthetic diet containing 6% corn oil (w/w) and 14% triundecanoin (w/w) for a period of 4 wk. Triundecanoin contains an 11-carbon (C-11) fatty acid (undecanoic acid) that was used to label the adipocyte TGFA. At the end of the 4-wk feeding period, triundecanoin was removed from the diet and replaced with an equivalent amount of corn oil. At this time and at weekly intervals for the next 4 wk, 5 rats from each age group were killed for the determination of TGFA composition in isolated adipocytes from the epididymal (Epi), perirenal (PR), subcutaneous (SC) and mesenteric (M) adipose tissue depots. When the content of C-11 was expressed as mole percent of the total fatty acids, mobilization was significantly more rapid from the PR and M depots than in the other two depots in the young rats. In the older rats mobilization was significantly slower in all depots compared to the younger group. The rates of mobilization were not different between the depots in the older animals. Since fat cell size continued to increase throughout the duration of the study, part of the decrease in C-11 content can be accounted for by dilution by newly acquired TGFA.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of orally administered caffeine on cellular response in protein energy-malnourished neonatal rat brain.

The purpose of this study was to determine the effects of caffeine on growing rats and how protein energy malnutrition can modify these potential effects. Caffeine (1,3,7-trimethylxanthine) is not only the most commonly consumed, neurally active stimulant in our daily lives, but it is widely used in the management of apnea in the premature neonate. One group of dams (20%) (n = 6) was begun on a 20% protein diet ad libitum. The second group (6%) (n = 4) was begun on a 6% protein diet. A third group (20% + C) (n = 4) was pair-fed to group 1 (20%) with the 20% protein diet, but beginning on day 3 the pups received 10 mg/kg body weight of caffeine via intragastric feeding needle every other day. The fourth group (6% + C) (n = 5) was pair-fed with group 2 (6%) with the 6% protein diet and the pups received 10 mg/kg of caffeine in the same manner as the 20% + C group from day 3. Although the 6% protein diet was associated with the expected reduced body and brain growth, there were no additional growth alterations associated with caffeine administration in either the 20% or 6% diet groups. This growth failure was accompanied by the expected reductions in total whole brain DNA, RNA, protein, and cholesterol content regardless of whether caffeine was received or not. Effects of caffeine which were similar in both diet groups included an increase in brain RNA content and concentration and an increase in the RNA/DNA ratio. However, there were differential effects of caffeine seen depending on diet group assignment.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose tissue and cholesterol metabolism.

Adipose tissue in man is a major site for cholesterol storage. In obesity over half of total body cholesterol may reside within this tissue; however, relatively little attention has been directed toward understanding the cholesterol metabolism and its relationship to whole body cholesterol homeostasis in this tissue. In this review the factors which influence cholesterol storage are discussed, with particular emphasis on the effects of diet and drug treatment in both animals and man. The uptake, synthesis, and mobilization of adipose tissue cholesterol appears to be mediated and/or regulated, as in other tissues, by the plasma lipoproteins, and these processes are examined with regard to both normal and pathologic states.

Accumulation of adipocyte cholesterol during hypolipidemic drug treatment in cholesterol-fed rats.

An attempt was made to mobilize adipose tissue cholesterol independently of triacylglycerol by feeding cholesterol to intact Fischer 344 rats to 'load' adipocytes followed by hypolipidemic drug treatment in order to lower plasma cholesterol and, hence, adipocyte cholesterol. In this strain of rat, body weight and adipocyte sizes remain relatively constant after 1 year of age. Therefore, alterations in adipocyte cholesterol can be ascribed to factors other than cell size. Both oxandrolone and combined cholestyramine/clofibrate treatment caused significant reductions in plasma cholesterol in cholesterol-fed rats, but cholesterol concentrations in liver were reduced only by cholestyramine/clofibrate treatment. Oxandrolone enhanced the development of liver fatty liver in the cholesterol-fed rats, but cholestyramine/clofibrate significantly reduced liver triacylglycerol concentrations. Adipocyte cholesterol in the epididymal depot was significantly elevated, not lowered, in both concentrations. Adipocyte cholesterol in the epididymal depot was significantly elevated, not lowered, in both groups of drug-treated animals. Subcutaneous adipocytes from rats receiving drug treatment also contained more cholesterol, especially in rats given oxandrolone. Increments in adipocyte cholesterol were associated with decreases in the absolute amounts of apolipoproteins, A-I and A-IV, as measured by densitometric scanning of electrophoretic gels. Under the present experimental conditions, changes in plasma cholesterol scanning of electrophoretic gels. Under the present experimental conditions, changes in plasma cholesterol concentration did not adequately reflect the cholesterol content of either liver or adipose tissue.

Lipoprotein lipase activities in adipose tissues and muscle in the obese Zucker rat.

Activity of lipoprotein lipase (LPL) was determined in whole adipose tissue and isolated adipocytes, and in heart and skeletal muscle that contained predominantly red or white fiber types in lean and obese Zucker rats. In rats of both sexes at 9-11 and 26-30 wk of age, no differences were observed between lean and obese rats when LPL activity of the perirenal (PR), subcutaneous (SC), and mesenteric (M) adipose tissues was expressed per millimole of tissue triglyceride. Within each sex, data relating the LPL content of isolated adipocytes to cell size was a linear function in which data for lean and obese rats fell on the same regression line. Measurement of the distribution of adipose tissue LPL activity between adipocytes and other tissue constituents showed no differences between lean and obese rats, a finding that is inconsistent with the hypothesis that obesity results in part by an alteration in adipose tissue enzyme distribution. Activity of LPL in the myocardium and red fiber types in the younger group of both sexes showed significant decreases in obese animals. This was also true for white fibers of males but not females. No differences in heart or muscle LPL between lean and obese rats were observed in the older group of either sex.

Metabolic fate of VLDL apolipoproteins B and E in hepatectomized rats.

The metabolic fate of VLDL apolipoproteins B and E was examined in functionally hepatectomized rats. 1 h after hepatectomy, there was almost complete absence of ultracentrifugally isolated VLDL lipid and protein, including apolipoproteins B and E. Analysis of apolipoprotein concentrations by electroimmunoassay showed hepatectomy did not affect the total serum concentrations of apolipoproteins B and E; thus, hepatectomy caused a redistribution of these apolipoproteins from VLDL to higher density lipoproteins. In the LDL (d = 1.03--1.063 g/ml) fraction, hepatectomy, increased the concentrations of free cholesterol (40%), esterified cholesterol (57%) and protein (18--67%), due to an increase in apolipoproteins B (22--48%) and E (250--300%). After hepatectomy, the HDL fraction accumulated the greatest total amount of apolipoprotein E. Since the majority of apolipoprotein E was isolated in the d greater than 1.21 g/ml fraction after sequential ultracentrifugation, the redistribution of apolipoproteins B and E was further defined by fractionation of serum on 5 M agarose columns. Electroimmunoassay of the column fractions showed that the apolipoprotein B peak eluted before the apolipoprotein E peak. Although a considerable portion of apolipoprotein E eluted with A-I, the peak of apolipoprotein E eluted before the A-I peak in both groups. These data suggest that a portion of apolipoprotein E is associated with particles which are similar than LDL but are larger than A-I-rich HDL. Hepatectomy caused an accumulation of apolipoprotein B in LDL, and apolipoprotein E and cholesterol in particles which were smaller than LDL and may represent LDL1. It is likely that under normal physiological conditions the liver plays a role in the removal of these apolipoprotein E-rich particles which are derived, at least in part, from the metabolism of VLDL.