IKK-1 and IKK-2: cytokine-activated IkappaB kinases essential for NF-kappaB activation.

Activation of the transcription factor nuclear factor kappa B (NF-kappaB) is controlled by sequential phosphorylation, ubiquitination, and degradation of its inhibitory subunit IkappaB. A large multiprotein complex, the IkappaB kinase (IKK) signalsome, was purified from HeLa cells and found to contain a cytokine-inducible IkappaB kinase activity that phosphorylates IkappaB-alpha and IkappaB-beta. Two components of the IKK signalsome, IKK-1 and IKK-2, were identified as closely related protein serine kinases containing leucine zipper and helix-loop-helix protein interaction motifs. Mutant versions of IKK-2 had pronounced effects on RelA nuclear translocation and NF-kappaB-dependent reporter activity, consistent with a critical role for the IKK kinases in the NF-kappaB signaling pathway.

Treating cat allergy with monoclonal IgG antibodies that bind allergen and prevent IgE engagement.

Acute allergic symptoms are caused by allergen-induced crosslinking of allergen-specific immunoglobulin E (IgE) bound to Fc-epsilon receptors on effector cells. Desensitization with allergen-specific immunotherapy (SIT) has been used for over a century, but the dominant protective mechanism remains unclear. One consistent observation is increased allergen-specific IgG, thought to competitively block allergen binding to IgE. Here we show that the blocking potency of the IgG response to Cat-SIT is heterogeneous. Next, using two potent, pre-selected allergen-blocking monoclonal IgG antibodies against the immunodominant cat allergen Fel d 1, we demonstrate that increasing the IgG/IgE ratio reduces the allergic response in mice and in cat-allergic patients: a single dose of blocking IgG reduces clinical symptoms in response to nasal provocation (ANCOVA, p = 0.0003), with a magnitude observed at day 8 similar to that reported with years of conventional SIT. This study suggests that simply augmenting the blocking IgG/IgE ratio may reverse allergy.

IkappaB kinase (IKK)-associated protein 1, a common component of the heterogeneous IKK complex.

Activation of the transcription factor NF-kappaB is controlled by the sequential phosphorylation, ubiquitination, and degradation of its inhibitory subunit, IkappaB. We recently purified a large multiprotein complex, the IkappaB kinase (IKK) signalsome, which contains two regulated IkappaB kinases, IKK1 and IKK2, that can each phosphorylate IkappaBalpha and IkappaBbeta. The IKK signalsome contains several additional proteins presumably required for the regulation of the NFkappaB signal transduction cascade in vivo. In this report, we demonstrate reconstitution of IkappaB kinase activity in vitro by using purified recombinant IKK1 and IKK2. Recombinant IKK1 or IKK2 forms homo- or heterodimers, suggesting the possibility that similar IKK complexes exist in vivo. Indeed, in HeLa cells we identified two distinct IKK complexes, one containing IKK1-IKK2 heterodimers and the other containing IKK2 homodimers, which display differing levels of activation following tumor necrosis factor alpha stimulation. To better elucidate the nature of the IKK signalsome, we set out to identify IKK-associated proteins. To this end, we purified and cloned a novel component common to both complexes, named IKK-associated protein 1 (IKKAP1). In vitro, IKKAP1 associated specifically with IKK2 but not IKK1. Functional analyses revealed that binding to IKK2 requires sequences contained within the N-terminal domain of IKKAP1. Mutant versions of IKKAP1, which either lack the N-terminal IKK2-binding domain or contain only the IKK2-binding domain, disrupt the NF-kappaB signal transduction pathway. IKKAP1 therefore appears to mediate an essential step of the NF-kappaB signal transduction cascade. Heterogeneity of IKK complexes in vivo may provide a mechanism for differential regulation of NF-kappaB activation.

Inhibitors of NF-kappaB and AP-1 gene expression: SAR studies on the pyrimidine portion of 2-chloro-4-trifluoromethylpyrimidine-5-[N-(3', 5'-bis(trifluoromethyl)phenyl)carboxamide].

We investigated the structure-activity relationship studies of N-[3, 5-bis(trifluoromethyl)phenyl][2-chloro-4-(trifluoromethyl)pyrimidin-5 -yl]carboxamide (1), an inhibitor of transcription mediated by both NF-kappaB and AP-1 transcription factors, with the goal of improving its potential oral bioavailability. Compounds were examined for cell-based activity, were fit to Lipinski's rule of 5, and were examined for potential gastrointestinal permeability using the intestinal epithelial cell line, Caco-2. Selected groups were substituted at the 2-, 4-, and 5-positions of the pyrimidine ring using solution-phase combinatorial methodology. The introduction of a fluorine in the place of 2-chlorine of 1 resulted in a compound with comparable activity. However, other substitutions at the 2-position resulted in a loss of activity. The trifluoromethyl group at the 4-position could be replaced with a methyl, ethyl, chlorine, or phenyl without a substantial loss of activity. The carboxamide group at the 5-position is critical for activity. If it was moved to the 6-position, the activity was lost. The 2-methyl analogue of 1 (81) showed comparable in vitro activity and improved Caco-2 permeability compared to 1.

Glucocorticoid induction of tryptophan oxygenase. Attenuation by intragastrically administered carbohydrates and metabolites.

In vivo tryptophan 2,3-dioxygenase (TPO) activity in male rats was estimated from the rate of production of 14CO2 after intragastric administration of [14C-2]tryptophan. The synthetic glucocorticoids hydrocortisone-21-sodium succinate or Triamcinolone acetonide were injected to elevate hepatic TPO activity on an acute (1-6 hr) or chronic (24 hr) basis. Glucose, fructose, or glycerol was intragastrically intubated in doses ranging from 4 to 16 mmoles to assess their abilities to attenuate acute or chronic increases of TPO activity by these glucocorticoids. Hydrocortisone-21-sodium succinate at doses of 0, 25, and 50 mg/kg produced dose-dependent elevations of TPO. A 50 mg/kg dose produced a 3-fold elevation of enzyme activity when measured in vitro as product produced by liver homogenates and a 2-fold elevation when assessed from expired radioactive carbon dioxide from radiolabeled tryptophan in vivo. Enzyme activity measured by 14CO2 production reached peak values in 2-3 hr and returned to baseline in 5 hr. Glucose, fructose or glycerol completely prevented the rise in conversion of [14C-2]tryptophan produced by hydrocortisone hemisuccinate when administered at doses of 12 or 16 mmoles 0.5 hr before the steroid. Lower doses had less effect. The potencies of the compounds in inhibiting acute increases in TPO activity produced by hydrocortisone hemisuccinate were in the order glycerol greater than fructose greater than glucose. Chronic Triamcinolone treatment elevated in vivo TPO activity by 2.5-fold and in vitro TPO activity by 5-fold. The chronic elevation of in vivo TPO by Triamcinolone could be arrested within 1 hr by an intragastric fructose load. The present finding, that acute or chronic glucocorticoid-induced increases in in vivo TPO activity were rapidly blocked by intragastric carbohydrate loads, is consistent with the view that dietary carbohydrates modulate hepatic TPO activity via feedback repression and not by a cessation of TPO enzyme synthesis.

Persistence of supercompensated muscle glycogen in trained subjects after carbohydrate loading.

Several carbohydrate (CHO)-loading protocols have been used to achieve muscle glycogen supercompensation and prolong endurance performance. This study assessed the persistence of muscle glycogen supercompensation over the 3 days after the supercompensation protocol. Trained male athletes completed a 6-day CHO-loading protocol that included cycle ergometer exercise and dietary manipulations. The 3-day depletion phase began with 115 min of cycling at 75% peak oxygen uptake followed by 3 x 60-s sprints and included the subjects consuming a low-CHO/high-protein/high-fat (10:41:49%) diet. Subjects cycled 40 min at the same intensity for the next 2 days. During the 3-day repletion phase, subjects rested and consumed a high-CHO/low-protein/low-fat (85:08:07%) diet, including a glucose-polymer beverage. A 3-day postloading phase followed, which involved a moderately high CHO diet (60%) and no exercise. Glycogen values for vastus lateralis biopsies at baseline and postloading days 1-3 were 408 +/- 168 (SD), 729 +/- 222, 648 +/- 186, and 714 +/- 196 mmol/kg dry wt, respectively. The CHO-loading protocol increased muscle glycogen by 1.79 times baseline, and muscle glycogen remained near this level during the 3-day postloading period. Results indicate that supercompensated muscle glycogen levels can be maintained for at least 3 days in a resting athlete when a moderate-CHO diet is consumed.

Epidural analgesia and uterine function.

OBJECTIVE: To determine whether continuous epidural analgesia with bupivacaine and fentanyl affects the rate of cervical dilation and myometrial contractility. METHODS: In a 5-week period, 62 consecutive women who received standardized epidural analgesia were matched with the next two groups of 124 consecutive women of the same parity who did not receive epidural analgesia. The outcome variables were uterine activity, rate of cervical dilation, oxytocin therapy, and operative deliveries. RESULTS: Continuous epidural analgesia with bupivacaine and fentanyl did not result in a change in myometrial contractility in the first hour after the initiation of analgesia. However, despite more oxytocin therapy, the rate of cervical dilation was significantly lower in the epidural group than in the nonepidural group (1.9 versus 5.6 cm/hour, P < .001). Operative deliveries were more common in patients with epidural analgesia than in those without it (12 of 62 versus two of 124, P < .001). CONCLUSION: After epidural analgesia, myometrial contractility is maintained with oxytocin, but the ability of the uterus to dilate the cervix is reduced significantly.

A model lightning safety policy for athletics.

OBJECTIVE: The purpose of this paper is to present a model policy on lightning safety for athletic trainers. BACKGROUND: Among college athletic programs in the United States there is a serious lack of written policy on lightning safety. Available evidence shows that most National Collegiate Athletic Association (NCAA) Division I institutions, even though they are located in high lightning activity areas of the country, do not have formal, written lightning safety policies. CLINICAL ADVANTAGES/ RECOMMENDATIONS: The policy presented herein, which is at the forefront of such policies, is the lightning safety policy written as part of a policies and procedures manual for the division of sports medicine at a public NCAA Division I university. This is a policy based on practicality that utilizes the "flash-to- bang" method for determining the distance of lightning activity from the observer. The policy begins with the importance of prevention, including the daily monitoring of weather reports. The policy defines a "safe shelter" and specifies the chain of command for determining who removes a team or individuals from an athletic site in the event of dangerous lightning activity.

Physical fitness in a submarine community as determined by the U.S. Navy Health and Physical Readiness Test.

This study was conducted to test the hypothesis that personnel assigned to submarine duty would display less physical fitness as compared to shore-based personnel. A message was submitted to all naval activities at a Naval Submarine Base requesting individual test scores from the annual Health and Physical Readiness (H&PR) Test. The results from the statistical analysis showed that the majority of personnel in both populations were classified as "good," which is an average level of physical fitness as determined by the Navy's fitness classification table. In both populations, 1% were classified as "outstanding," while approximately 10% failed to meet the minimum physical fitness requirements. A body fat value greater than 22% was the cause for the majority of test failures. In conclusion, the results of the analysis of H&PR data do not support the widely held belief that submarine personnel are less physically fit than their shore-based counterparts.

Cardiorespiratory fitness and cognitive performance before and after confinement in a nuclear submarine.

The aim of this study was to further document physiological deconditioning from occupational exposure to submarines as described in a small number of reports and determine whether cognitive performance parallels the physiological changes associated with physical training and deconditioning. We examined cardiorespiratory fitness and cognitive performance in 14 male subjects during 70 d of confinement in a nuclear submarine. Maximal oxygen consumption (VO2max) and anaerobic threshold (AT) were assessed before and after confinement. Six exercising subjects (ES) cycled 4-7 times per week for 20 min at 75% max heart rate for 8 weeks. Eight control subjects (CS) did no exercise. Every 14 d of the patrol, cognitive performance was evaluated in both groups by administering a mental arithmetic and choice reaction time tests before cycling, during cycling, and post cycling. The cycle bout consisted of exercising at 75% VO2max for 15 min. After confinement, VO2max remained constant for ES but declined 7% statistically nonsignificant for the CS. AT expressed as a percentage of VO2max increased 15% (p less than 0.05) in the ES and decreased 20% (p less than 0.05) in the CS. The only significant effect in the cognitive tests was that both groups responded faster in the choice reaction time test during the exercise session. In conclusion, these data demonstrate a training effect for AT in the ES and a deconditioning response for AT and a statistically nonsignificant reduction in VO2max in the CS. Under the conditions of this experiment we could find no effects of physical training and deconditioning on the cognitive performance test employed here, although some trends suggest that exercisers out-performed the control group.

Physiological responses during shipboard firefighting.

This study determined the level of heat strain experienced by U.S. Navy personnel while combating fires aboard a damage control research ship. Male volunteers (n = 9), wearing the standard Navy firefighting ensemble, were recorded for core temperature (Tre), skin temperatures (weighted mean, Tmsk), and heart rate (HR) during three fire test days. During the tests, air temperatures in the compartment containing the fire to be extinguished averaged 470 +/- 170 degrees C, while air temperatures in the compartment from which the fire was fought ranged from 40 to 125 degrees C. Dressing in the ensemble and execution of preliminary firefighting activities led to a gradual increase in Tre, Tmsk, and HR; while during active firefighting, Tre, Tmsk, and HR increased rapidly. For all tests, the rate of Tmsk rise (8.73 degrees C.h-1) exceeded the rate of Tre rise (2.95 degrees C.h-1), leading to convergence of these values. Average peak values for all tests were: Tre, 39.2 +/- 1.0 degrees C; Tmsk, 39.5 +/- 0.9 degrees C; body heat storage (HS), 2.02 +/- 0.77 kcal.kg-1; rate of HS during firefighting, 170 +/- 92 kcal.m-2.h-1; HR, 186 +/- 13 bpm. Our findings quantify the limits of tolerance of heat strain encountered during shipboard firefighting.

A pathogenic role for JNK signaling in experimental anti-GBM glomerulonephritis.

Activation of the c-Jun NH2-terminal kinase (JNK) signaling pathway is involved in the immune response; however, little is known of its role in immune-induced renal injury. In this study, we examine JNK signaling in the rat anti-glomerular basement membrane (GBM) disease model using CC-401, a specific JNK inhibitor. Animals were given CC-401, vehicle alone or no treatment starting before anti-GBM serum injection and continued treatment until killing. In acute disease, CC-401 blocked JNK signaling and reduced proteinuria in the first 24 h. The transient neutrophil influx seen at 3 h of disease was not affected, however. Continued CC-401 treatment suppressed glomerular and tubulointerstitial damage usually seen at 14 days. The protective effect may be due to modulation of macrophage activation, as CC-401 had no effect upon glomerular macrophage infiltration at day 14 despite the suppression of glomerular lesions and a marked reduction in renal tumor necrosis factor-alpha and inducible nitric oxide synthase messenger RNA levels. Treatment with CC-401 had no apparent effect on T cell or humoral immune responses. These studies suggest that JNK signaling promotes renal injury in acute and progressive rat anti-GBM disease. JNK inhibitors may be a novel therapeutic approach for the treatment of human glomerulonephritis.

c-Jun N-terminal kinase-dependent mechanisms in respiratory disease.

Respiratory diseases pose a multifaceted dilemma. Although the symptoms and pathology are obvious and provide multiple opportunities for therapeutic investigation, at the same time, the molecular complexities and prioritisation are overwhelming. Even within a disease such as asthma, the number of inducers, cell types, secondary mediators, chemical changes, immune responses and tissue modifications is remarkable. One means of therapeutically targeting this complexity is to identify individual factors responsible for regulating multiple disease processes. The mitogen-activated protein kinase family integrates multiple diverse stimuli, and, in turn, initiates a cell response by phosphorylating and thereby modulating the activity of many target proteins. The c-Jun N-terminal kinase is a critical regulator of pro-inflammatory genes, tissue remodelling and apoptosis, and, therefore, represents an attractive target for novel therapies. Pre-clinical and clinical investigation into the efficacy of c-Jun N-terminal kinase inhibitors has been ongoing since the late 1990s. Over the course of this work, hypotheses have shifted as to the role of c-Jun N-terminal kinase in the many processes that promote allergic, inflammatory, obstructive and fibrotic diseases of the lung. Inhibition of c-Jun N-terminal kinase may indeed provide a means of suppressing more pathological mechanisms in respiratory disease than first suspected.

Activation of c-Jun N-terminal kinase (JNK) signalling in experimentally induced gastric lesions in rats.

The c-Jun N-terminal kinase (JNK) participates in intracellular signalling cascades that mediate inflammatory responses. Therefore, the JNK signalling may be involved in gastric injury and inhibition of this pathway may form the basis of a new strategy for the treatment of gastric injury. The aim of this study was to determine whether JNK participates in the formation of gastric lesions in an experimental model. Acute gastric injury was induced in Sprague-Dawley rats by intragastric administration of 100% ethanol. The amount of phospho-JNK in the rat stomach was determined using immunohistochemistry and Western analysis. Animals received subcutaneous injections of a specific JNK inhibitor SP600125 or vehicle and the extent of mucosal damage in the stomach was determined. Western analysis revealed early phosphorylation of JNK and, to a lesser extent, p38 as well as late phosphorylation of the p42/44 extracellular signal-related kinases during the development of gastric lesions. JNK was phosphorylated in epithelial cells and in occasional mononuclear cells present at lesion sites. These cells were rarely found in samples from control specimens. Treatment with SP600125 significantly reduced the extent of gastric lesions. These findings indicate that experimental gastric injury is associated with activation of the JNK signalling pathway, and also suggest that JNK inhibitors may play a role in the treatment of gastric injury in humans.

Premature induction of hepatic tryptophan oxygenase.

Subcutaneous administration of hydrocortisone acetate to the newborn rat produces a premature induction of hepatic tryptophan oxygenase consisting of a transient rise in activity 6-8 h after treatment, followed by a second sustained rise beginning 40 h later, which plateaus at 10 days of age. Cycloheximide treatment at the midpoint of this second elevation inhibits protein synthesis, but not tryptophan oxygenase activity. In older animals, cycloheximide treatment does both. Tryptophan administration at this midpoint rapidly elevates tryptophan oxygenase activity. This elevation can be partially blocked by treatment with actinomycin D within 1 h of tryptophan administration, but not thereafter. Actinomycin treatment is ineffective in blocking the tryptophan-induced rise in older animals. Administration of hydrocortisone acetate to 5- and 10-day-old pups leads to a more rapid and sustained rise in tryptophan oxygenase activity without appearance of a transient induction phase. Neither tryptophan alone, δ-aminolevulinic acid alone, nor tryptophan plus δ-aminolevulinic acid prematurely induces tryptophan oxygenase in newborn or 5-day-old rats.