Toxicology and carcinogenesis studies of a nondecolorized [corrected] whole leaf extract of Aloe barbadensis Miller (Aloe vera) in F344/N rats and B6C3F1 mice (drinking water study).

BACKGROUND: Extracts from the leaves of the Aloe vera plant (Aloe barbadensis Miller) have long been used as herbal remedies and are also now promoted as a dietary supplement, in liquid tonics, powders or tablets, as a laxative and to prevent a variety of illnesses. We studied the effects of Aloe vera extract on rats and mice to identify potential toxic or cancer-related hazards. METHODS: We gave solutions of nondecolorized extracts of Aloe vera leaves in the drinking water to groups of rats and mice for 2 years. Groups of 48 rats received solutions containing 0.5%, 1% or 1.5% of Aloe vera extract in the drinking water, and groups of mice received solutions containing 1%, 2%, or 3% of Aloe vera extract. Similar groups of animals were given plain drinking water and served as the control groups. At the end of the study tissues from more than 40 sites were examined for every animal. RESULTS: In all groups of rats and mice receiving the Aloe vera extract, the rates of hyperplasia in the large intestine were markedly increased compared to the control animals. There were also increases in hyperplasia in the small intestine in rats receiving the Aloe vera extract, increases in hyperplasia of the stomach in male and female rats and female mice receiving the Aloe vera extract, and increases in hyperplasia of the mesenteric lymph nodes in male and female rats and male mice receiving the Aloe vera extract. In addition, cancers of the large intestine occurred in male and female rats given the Aloe vera extract, though none had been seen in the control groups of rats for this and other studies at this laboratory. CONCLUSIONS: We conclude that nondecolorized Aloe vera caused cancers of the large intestine in male and female rats and also caused hyperplasia of the large intestine, small intestine, stomach, and lymph nodes in male and female rats. Aloe vera extract also caused hyperplasia of the large intestine in male and female mice and hyperplasia of the mesenteric lymph node in male mice and hyperplasia of the stomach in female mice.

Effect of Aloe vera whole leaf extract on short chain fatty acids production by Bacteroides fragilis, Bifidobacterium infantis and Eubacterium limosum.

AIMS: To investigate the effect of Aloe vera whole leaf extract on pure and mixed human gut bacterial cultures by assessing the bacterial growth and changes in the production of short chain fatty acids. METHODS AND RESULTS: Bacteroides fragilis, Bifidobacterium infantis, and Eubacterium limosum were incubated with Aloe vera extracts [0%, 0.5%, 1%, 1.5% and 2%; (w/v)] for 24 and 48 h. Short chain fatty acids production was measured by gas chromatography/mass spectrometry analyses. A significant linear increase in growth response to Aloe vera supplementation was observed at 24 h for each of the bacterial cultures; however, only B. infantis and a mixed bacterial culture showed a significant positive linear dose response in growth at 48 h. In pure bacteria cultures, a significantly enhanced dose response to Aloe vera supplementation was observed in the production of acetic acid by B. infantis at 24 h and of butyric acid by E. limosum at 24 and 48 h. In the mixed bacterial culture, the production of propionic acid was reduced significantly at 24 and 48 h in a dose-dependent fashion, whereas butyric acid production showed a significant linear increase. CONCLUSIONS: The results indicated that Aloe vera possessed bacteriogenic activity in vitro and altered the production of acetic, butyric and propionic acids by micro-organisms selected for the study. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of the study suggest that consumption of a dietary supplement, Aloe vera, may alter the production of short chain fatty acids by human intestinal microflora.

Modelling of aircrew radiation exposure from galactic cosmic rays and solar particle events.

Correlations have been developed for implementation into the semi-empirical Predictive Code for Aircrew Radiation Exposure (PCAIRE) to account for effects of extremum conditions of solar modulation and low altitude based on transport code calculations. An improved solar modulation model, as proposed by NASA, has been further adopted to interpolate between the bounding correlations for solar modulation. The conversion ratio of effective dose to ambient dose equivalent, as applied to the PCAIRE calculation (based on measurements) for the legal regulation of aircrew exposure, was re-evaluated in this work to take into consideration new ICRP-92 radiation-weighting factors and different possible irradiation geometries of the source cosmic-radiation field. A computational analysis with Monte Carlo N-Particle eXtended Code was further used to estimate additional aircrew exposure that may result from sporadic solar energetic particle events considering real-time monitoring by the Geosynchronous Operational Environmental Satellite. These predictions were compared with the ambient dose equivalent rates measured on-board an aircraft and to count rate data observed at various ground-level neutron monitors.

Levels of retinyl palmitate and retinol in the skin of SKH-1 mice topically treated with retinyl palmitate and concomitant exposure to simulated solar light for thirteen weeks.

Retinyl esters account for more than 70% of the endogenous vitamin A found in human skin, and retinyl palmitate is one of the retinyl esters in this pool. Human skin is also exposed to retinyl palmitate exogenously through the topical application of cosmetic and skin care products that contain retinyl palmitate. To date, there is limited information on the penetration and distribution of retinyl palmitate and vitamin A within in the skin. In this study, the accumulation of retinyl palmitate and generation of retinol in the skin of male and female SKH-1 mice that received repeated topical applications of creams containing 0.0%, 0.1%, 0.5%, 1.0%, 5.0%, 10%, or 13% of retinyl palmitate 5 days a week for a period of 13 weeks were studied. Because products containing retinyl palmitate are frequently applied to sun-exposed skin, and because it is well established that exposure to sunlight and UV light can alter cutaneous levels of retinoids, mice in this study were additionally exposed 5 days a week to simulated solar light. The results showed that retinyl palmitate diffused into the skin and was partially hydrolyzed to retinol. The levels of retinyl palmitate in the skin of mice that were administered retinyl palmitate cream were higher than control values, and levels of both retinyl palmitate and retinol increased with the application of higher concentrations of retinyl palmitate in the cream. Our results indicate that topically applied retinyl palmitate may alter the normal physiological levels of retinyl palmitate and retinol in the skin of SKH-1 mice and may have a significant impact on vitamin A homeostasis in the skin.

Dietary exposure to 2-aminoanthracene induces morphological and immunocytochemical changes in pancreatic tissues of Fisher-344 rats.

Toxic chemicals ingested as the result of environmental exposures or other risk factors such as cigarette smoking may increase the risk of developing cancer and other diseases such as diabetes. 2-Aminoanthracene (2-AA) was investigated to determine toxic effects of chronic dietary exposure upon major organ systems including the pancreas. Fisher-344 rats were fed 2-AA (50-100 mg/kg of diet) and euthanized at 14, 30, 63, and 80 days. Growth, tissue histological, immunocytochemical, and clinical pathological end points were examined at each time point. Significantly elevated plasma glucose and glycated hemoglobins and reduced serum protein levels were recognized after 80 days of feeding (100 mg/kg of diet 2-AA group). Similar results were observed in rats exposed to 75 mg/kg of diet but appeared to be absent in the 50-mg/kg group. An unexpected pattern of responses suggestive of diabetic sequelae was observed in a glucose tolerance test conducted during the seventh week. After 63 and 80 days, large cytoplasmic vacuoles in islet cells were observed by light microscopy. In addition, the immunocytochemical study demonstrated beta cell insulin insufficiency at 63 and 80 days. No inflammatory infiltration of the islets was observed. These findings suggest that depletion of secretory granules occurred in the beta cells. Necrotic changes occurred in the acinar cells of the pancreas with increasing duration and dose of 2-AA. The cytological, immunocytochemical, and histological results demonstrate that chronic dietary exposure to 2-amino anthracene alters the endocrine and exocrine pancreas cellular morphology and induces diabetic-like symptoms in the Fisher-344 rat.

Activity in ventral and dorsal premotor cortex in response to predictable force-pulse perturbations in a precision grip task.

This study compared the responses of ventral and dorsal premotor cortex (PMv and PMd) neurons to predictable force-pulse perturbations applied during a precision grip. Three monkeys were trained to grasp an unseen instrumented object between the thumb and index finger and to lift and hold it stationary within a position window for 2-2.5 s. The grip and load forces and the object displacement were measured on each trial. Single-unit activity was recorded from the hand regions in the PMv and PMd. In some conditions a predictable perturbation was applied to the object after 1,500 ms of static holding, whereas in other conditions different random combinations of perturbed and unperturbed trials were given. In the perturbed conditions, some were randomly and intermittently presented with a warning flash, whereas some were unsignaled. The activities of 198 cells were modulated during the task performance. Of these cells, 151 were located in the PMv, and 47 were located in the PMd. Although both PMv and PMd neurons had similar discharge patterns, more PMd neurons (84 vs. 43%) showed early pregrip activity. Forty of 106 PMv and 10/30 PMd cells responded to the perturbation with reflexlike triggered reactions. The latency of this response was always <100 ms with a mean of about 55 ms in both the PMv and the PMd. In contrast, 106 PMv and 30 PMd cells tested with the perturbations, only 9 and 10%, respectively, showed significant but nonspecific adaptations to the perturbation. The warning stimulus did not increase the occurrence of specific responses to the perturbation even though 21 of 42 cells related to the grip task also responded to moving visual stimuli. The responses were retinal and frequently involved limited portions of both foveal and peripheral visual fields. When tested with a 75 x 5.5-cm dark bar on a light background, these cells were sensitive to the direction of movement. In summary, the periarcuate premotor area activity to related to predictable force-pulse perturbations seems to reflect a general increase in excitability in contrast to a more specific anticipatory activity such as recorded in the cerebellum. In spite of the strong cerebello-thalamo-cortical projections, the results of the present study suggest that the cortical premotor areas are not involved in the elaboration of adaptive internal models of hand-object dynamics.

Activity in rostral motor cortex in response to predictable force-pulse perturbations in a precision grip task.

The purpose of this investigation was to characterize the discharge of neurons in the rostral area 4 motor cortex (MI) during performance of a precision grip task. Three monkeys were trained to grasp an object between the thumb and index finger and to lift and hold it stationary for 2-2.5 s within a narrow position window. The grip and load forces and the vertical displacement of the object were recorded on each trial. On some trials a downward force-pulse perturbation generating a shear force and slip on the skin was applied to the object after 1.5 s of static holding. In total, 72 neurons were recorded near the rostral limit of the hand area of the motor cortex, located close to the premotor areas. Of these, 30 neurons were examined for receptive fields, and all 30 were found to receive proprioceptive inputs from finger muscles. Intracortical microstimulation applied to 38 recording sites evoked brief hand movements, most frequently involving the thumb and index finger with an average threshold of 12 microA. Slightly more than one-half of the neurons (38/72) demonstrated significant increases in firing rate that on average began 284 +/- 186 ms before grip onset. Of 54 neurons tested with predictable force-pulse perturbations, 29 (53.7%) responded with a reflexlike reaction at a mean latency of 54.2 +/- 16.8 ms. This latency was 16 ms longer than the mean latency of reflexlike activity evoked in neurons with proprioceptive receptive fields in the more caudal motor cortex. No neurons exhibited anticipatory activity that preceded the perturbation even when the perturbations were delivered randomly and signaled by a warning stimulus. The results indicate the presence of a strong proprioceptive input to the rostral motor cortex, but raise the possibility that the afferent pathway or intracortical processing may be different because of the slightly longer latency.

Suppression of arylamine toxicity in the Fischer-344 rat following ingestion of a complex mixture.

The toxic effects of a mixture of 2-aminoanthracene (2-AA), benzanthracene (BA), and dinitropyrene isomers (DNP), and the toxic effects of these compounds individually, were investigated in the Fischer-344 rat following dietary exposure via a powdered basal diet. Animals were sacrificed at 14-, 30-, and 80-days of dietary exposure. Exposure to dietary 2-AA alone induced anorexia, cachexia, variable mortality, and altered serum chemistry profiles in the F-344 rat. Reduced lymphocyte counts were also shown in rats exposed to 2-AA. A temporal pattern of effect of 2-AA dietary exposure was observed in the progression of hepatic lesions in exposed animals. Dietary exposure to either DNP isomers or BA at a 10-fold higher concentration in the diet, relative to 2-AA, did not induce detectable toxic responses. However, exposure of rats to a mixture of 2-AA, BA, and DNP isomers (100 mg/kg, 1.0 g/kg, and 1.0 g/kg of diet, respectively) resulted in the attenuation of toxic effects when compared to exposure of F-344 rats to 2-AA alone. These results indicate that the toxic effects of 2-AA are suppressed by co-administration of DNP and BA and suggest that compound interactions need to be considered when predicting the toxic potential of specific environmental pollutants.

Suppression of tumor cell growth both in nude mice and in culture by n-3 polyunsaturated fatty acids: mediation through cyclooxygenase-independent pathways.

Dietary n-3 polyunsaturated fatty acids (PUFAs), as compared with n-6 PUFAs, suppress cellular production of prostaglandins and tumor cell growth both in vitro and in vivo. However, the mechanism by which n-3 PUFAs suppress tumor growth is not understood. We investigated whether the suppression of tumor cell growth by dietary n-3 PUFAs is mediated through inhibition of cyclooxygenase (COX). A colon tumor cell line, HCT-116, that does not express COX was stably transfected with the constitutively expressed COX-1 or the inducible COX-2 cDNA using a retroviral transfection and infection system. Athymic nude mice transplanted with the cells expressing enzymatically active COX were fed isocaloric diets containing either safflower oil or fish oil for 2 weeks before the start of the experiment and for an additional 21 days after transplantation. Both tumor volume and tumor burden (tumor volume/body weight) were significantly reduced in mice fed the fish oil diet as compared with safflower oil-fed mice. This reduction occurred even in control mice that received injections with cells infected with the retroviral vector without COX-1 or COX-2 cDNA. The growth of tumor cells expressing COX was not different from the growth of those transfected with the vector alone in the nude mice and in soft agar. N-3 PUFAs, as compared with linoleic acid, also inhibited the growth of these cells in culture. This growth inhibition by n-3 PUFAs was not affected by COX-1 or COX-2 overexpression. Contrary to general belief, these results indicate that the suppression of tumor growth by dietary n-3 PUFAs is mediated through COX-independent pathways.

Two opposing effects of non-steroidal anti-inflammatory drugs on the expression of the inducible cyclooxygenase. Mediation through different signaling pathways.

The efficacy of non-steroidal anti-inflammatory drugs (NSAIDs) is considered to be a result of their inhibitory effect on cyclooxygenase (COX) activity. Here, we report that flufenamic acid shows two opposing effects on COX-2 expression; it induces COX-2 expression in the colon cancer cell line (HT-29) and macrophage cell line (RAW 264.7); conversely, it inhibits tumor necrosis factor alpha (TNFalpha)- or lipopolysaccharide (LPS)-induced COX-2 expression. This inhibition correlates with the suppression of TNFalpha- or LPS-induced NFkappaB activation by flufenamic acid. The inhibitor of extracellular signal-regulated protein kinase, p38, or NFkappaB does not affect the NSAID-induced COX-2 expression. These results suggest that the NSAID-induced COX-2 expression is not mediated through activation of NFkappaB and mitogen-activated protein kinases. An activator of peroxisome proliferator-activated receptor gamma, 15-deoxy-Delta(12,14)-prostaglandin J(2), also induces COX-2 expression and inhibits TNFalpha-induced NFkappaB activation and COX-2 expression. Flufenamic acid and 15-deoxy-Delta(12,14)-prostaglandin J(2) also inhibit LPS-induced expression of inducible form of nitric-oxide synthase and interleukin-1alpha in RAW 264.7 cells. Together, these results indicate that the NSAIDs inhibit mitogen-induced COX-2 expression while they induce COX-2 expression. Furthermore, the results suggest that the anti-inflammatory effects of flufenamic acid and some other NSAIDs are due to their inhibitory action on the mitogen-induced expression of COX-2 and downstream markers of inflammation in addition to their inhibitory effect on COX enzyme activity.

Application of in situ ellipsometry in the fabrication of thin-film optical coatings on semiconductors.

Thin-film interference filters, suitable for use on GaAs- and InP-based lasers, have been fabricated by use of the electron-cyclotron resonance plasma-enhanced chemical vapor deposition technique. Multilayer film structures composed of silicon oxynitride material have been deposited at low temperatures with an in situ rotating compensator ellipsometer for monitoring the index of refraction and thickness of the deposited layers. Individual layers with an index of refraction from 3.3 to 1.46 at 633 nm have been produced with a run-to-run reproducibility of 0.005 and a thickness control of 10 A. Several filter designs have been implemented, including high-reflection filters, one- and two-layer anitreflection filters, and narrow-band high-reflection filters. It is shown that an accurate measurement of the filter optical properties during deposition is possible and that controlled reflectance spectra can be obtained.

Activation and inactivation of cyclo-oxygenase in rat alveolar macrophages by aqueous cigarette tar extracts.

Cyclo-oxygenase (COX) activity and its level of expression, the release of arachidonic acid (AA), and the accumulation of prostaglandins (PGs) were determined in isolated rat pulmonary alveolar macrophages (PAM) exposed to aqueous cigarette tar (ACT) extracts. COX activity increased 3-fold above the initial activity within 2 h of incubation with ACT extracts and gradually decreased below the initial activity after 8 h of incubation. The increased COX activity after 2 h of incubation did not lead to increased accumulation of PGE2. Accumulated levels of PGE2 increased dramatically after 12 h of incubation despite decreased COX activity in cells incubated with ACT extracts. This increased accumulation of PGE2 was greater in cells derived from vitamin E deficient rats compared with control rats. Release of AA from cells was dramatically increased in cells incubated with ACT extracts in parallel to PG accumulation. Thus increased accumulation of PGE2 despite decreased COX activity after 12 h of incubation is likely the result of increased substrate availability. These results suggest that, contrary to earlier reports, cigarette smoke stimulates the formation of PGs in alveolar macrophages. Increased PG production may lead to suppressed immune response and enhanced risk of tumorigenesis in smokers' lungs.

The effects of muscimol inactivation of small regions of motor and somatosensory cortex on independent finger movements and force control in the precision grip.

This study investigated the effects of inactivating small regions of the primary somatosensory (SI) and motor (MI) cortex on the control of finger forces in a precision grip. A monkey was trained to grasp and lift a computer-controlled object between the thumb and index finger and to hold it stationary within a narrow position window for 2 s. The grip force applied perpendicular to the object surface, the lifting or load force applied tangentially in the vertical direction, and the vertical displacement were sampled at 100 Hz. Also, the ability of the monkey to extract small pieces of food from narrow wells of a Klüver board was analyzed from video-tape. Preliminary single-unit recordings and microstimulation studies were used to map the extent of the thumb and index-finger representation within SI and MI. Two local injections of 1 microl each (5 microg/microl) of the GABA(A)-agonist muscimol were used to inactivate the thumb and index region of either the pre- or post-central gyrus. The precision grip was differently affected by muscimol injection into either SI or MI. MI injections produced a deficit in the monkey's ability to perform independent finger movements and a general weakness in the finger muscles. Whole-hand grasping movements were inappropriately performed in an attempt to grasp either the instrumented object or morsels of food. Although the effect seemed strongest on intrinsic hand muscles, a clear deficit in digit extension was also noted. As a result, the monkey was unable to lift and maintain the object within the position window for the required 2 s, and, over time, the grip force decreased progressively until the animal stopped working. Following SI injections, the most obvious effect was a loss of finger coordination. In grasping, the placement of the fingers on the object was often abnormal and the monkey seemed unable to control the application of prehensile and lifting forces. However, the detailed analysis of forces revealed that a substantial increase in the grip force occurred well before any deficit in the coordination of finger movements was noted. This observation suggests that cutaneous feedback to SI is essential for the fine control of grip forces.

Depolarization and neurotrophins converge on the phosphatidylinositol 3-kinase-Akt pathway to synergistically regulate neuronal survival.

In this report, we have examined the mechanisms whereby neurotrophins and neural activity coordinately regulate neuronal survival, focussing on sympathetic neurons, which require target-derived NGF and neural activity for survival during development. When sympathetic neurons were maintained in suboptimal concentrations of NGF, coincident depolarization with concentrations of KCl that on their own had no survival effect, synergistically enhanced survival. Biochemical analysis revealed that depolarization was sufficient to activate a Ras-phosphatidylinositol 3-kinase-Akt pathway (Ras-PI3-kinase-Akt), and function-blocking experiments using recombinant adenovirus indicated that this pathway was essential for approximately 50% of depolarization-mediated neuronal survival. At concentrations of NGF and KCl that promoted synergistic survival, these two stimuli converged to promote increased PI3-kinase-dependent Akt phosphorylation. This convergent PI3-kinase-Akt pathway was essential for synergistic survival. In contrast, inhibition of calcium/calmodulin-dependent protein kinase II revealed that, while this molecule was essential for depolarization-induced survival, it had no role in KCl- induced Akt phosphorylation, nor was it important for synergistic survival by NGF and KCl. Thus, NGF and depolarization together mediate survival of sympathetic neurons via intracellular convergence on a Ras-PI3-kinase-Akt pathway. This convergent regulation of Akt may provide a general mechanism for coordinating the effects of growth factors and neural activity on neuronal survival throughout the nervous system.

Expression of gamma-IFN responsive genes in scavenger receptor over-expressing monocytes is associated with xanthomatosis.

We have recently described an inherited over-expression of the macrophage scavenger receptor (SR) in blood monocytes from members of a kindred, only two of whom displayed extensive xanthomatosis. Using mRNA differential display we demonstrated abnormally high expression of the signal transducer and activator of transcription (STAT1alpha) in monocytes from the proband II-2. Expression of gamma-interferon inducible protein 10 (IP-10), a STAT1alpha-responsive gene and mediator of inflammatory response, was also abnormally expressed in the monocytes from II-2. Over-expression of both genes was restricted to monocytes from II-2 and was not observed in monocytes from the clinically unaffected family members, unlike that of SR. Gel retardation assays with THP-1 cell extracts identified gamma-IFN inducible DNA binding activity to three potential STATI DNA binding elements in the human IP-10 promoter region from nucleotides - 245 to - 188. Taken together these results suggest that gamma-interferon mediated cell activation is responsible for STAT1alpha-induced transcription of the IP-10 gene in THP-1 macrophages as well as in monocytes from II-2. Analysis of monocytes from familial hypercholesterolemic (FH) subjects, who frequently develop xanthomatosis, revealed a significant number of subjects with elevated STAT1alpha and IP-10 expression. Our data suggest that the inflammatory effects of gamma-IFN signaling could play a role in foam cell formation and xanthomatosis.

Effect of Strawberry Density on the Spread of Anthracnose Caused by Colletotrichum acutatum.

ABSTRACT Spread of strawberry anthracnose, resulting from the rain splash dispersal of Colletotrichum acutatum conidia, was determined in field plots by assessing fruit disease incidence at a range of distances from an introduced point source of infected fruit with sporulating lesions. Four within-row plant densities were established in replicated plots in each of 2 years. A generalized linear model with a logit link function and binomial distribution for incidence was used to quantify the effects of distance and side of the row relative to the inoculum source, plant density treatment, and their interactions on disease incidence. At all assessment times, there was a significant (P

Does vegetable oil attenuate the beneficial effects of fish oil in reducing risk factors for cardiovascular disease?

Contradictory reports on the protective effect of fish consumption on cardiovascular disease (CVD) risk could be due to variations in the intake of n-3 and n-6 polyunsaturated fatty acids (PUFAs). Metabolic competition between n-3 and n-6 PUFAs suggests that n-6 PUFAs in vegetable oils could attenuate the efficacy of n-3 PUFAs in fish oil to favorably alter endpoints relevant to CVD risk. We determined the effects of varying dietary amounts of fish oil on lipid and thrombotic endpoints relevant to risk factors for CVD and whether these effects were attenuated by vegetable oils. Two randomized, double-blind, placebo-controlled, parallel studies were conducted in human subjects fed varying amounts of n-3 and n-6 PUFAs; n-3 PUFA intake was varied by using fish or placebo oil capsules, and n-6 PUFA intake was modified by incorporating varying amounts of safflower oil into the diet. Endpoints included changes in membrane fatty acid composition, blood lipids, and thrombotic profile. The results indicated that absolute amounts of fish oil, and not the relative amounts of fish and vegetable oil (ratios of n-3 to n-6 PUFAs), determined the magnitude of the reduction of arachidonic acid and increase in eicosapentaenoic acid in phospholipids of plasma and platelets. The suppression of plasma triacylglycerols by fish oil was not affected by varying amounts of dietary n-6 PUFAs. Fibrinogen concentrations decreased with 15 g but not with 9 g fish oil/d fed at the same ratio of n-3 to n-6 PUFAs. The efficacy of fish oil in favorably modifying certain risk factors for CVD was not attenuated by vegetable oil.

Expression of mitogen-inducible cyclooxygenase induced by lipopolysaccharide: mediation through both mitogen-activated protein kinase and NF-kappaB signaling pathways in macrophages.

The mitogen-inducible cyclooxygenase (COX-2) is selectively expressed in lipopolysaccharide (LPS)-stimulated macrophages. However, the signaling pathways that lead to the expression of COX-2 in LPS-stimulated macrophages are not well understood. LPS activates members of mitogen-activated protein kinases (MAPKs) and NF-kappaB transcription factor in macrophages. We have shown that protein tyrosine kinase (PTK) inhibitors suppress the LPS-induced expression of COX-2 in macrophages (Chanmugam et al., J Biol Chem 270: 5418-5426, 1995). These PTK inhibitors also inhibit LPS-induced activation of MAPKs. Thus, in the present study, we determined whether the activation of MAPKs and NF-kappaB is necessary for the signaling pathway for the LPS-induced expression of COX-2 in the murine macrophage cell line RAW 264.7. The findings demonstrated that inhibition of extracellular signal-regulated protein kinases 1 and 2 (ERK-1 and -2) by the selective inhibitor PD98059 or inhibition of P38 by the specific inhibitor SB203580 results in partial suppression of COX-2 expression. However, activation of MAPKs by phorbol 12-myristate 13-acetate, H2O2, sorbitol, sodium vanadate, or a combination of these agents failed to induce the expression of COX-2. Inhibitors of NF-kappaB suppressed COX-2 expression without affecting tyrosine phosphorylation of MAPKs. The PTK inhibitors that suppressed the activation of MAPKs and COX-2 expression also inhibited the degradation of IkappaB-alpha. Together, these results indicate that the activation of NF-kappaB is required to induce the expression of COX-2 in LPS-stimulated RAW 264.7 cells. Inhibition of ERK-1 and 2 or P38 results in partial suppression of COX-2 expression. However, the activation of MAPKs alone is not sufficient to induce the expression of COX-2 in these cells.

Characterization of a binding site for angiotensin IV on bovine aortic endothelial cells.

We have characterized a specific binding site for angiotensin IV on bovine aortic endothelial cell membranes. Pseudo-equilibrium studies at 37 degrees C for 2 h have shown that this binding site recognizes angiotensin IV with a high affinity (Kd = 0.71; average of two experiments that yielded values of 0.71 and 0.72 nM). The binding site is saturable and relatively abundant with a maximal binding capacity of 0.59 pmol/mg protein (average of two experiments that yielded values of 0.39 and 0.78 pmol/mg of protein). Non-equilibrium kinetic analyses at 37 degree C revealed a calculated Kd of 59 pM (average of two experiments that yielded values of 67 and 50 pM). The binding site displays a high affinity for angiotensin receptors AT1 or AT2. An analysis of specificity showed that the binding site displays a high affinity for angiotensin IV, low affinities for angiotensin II, [Sar1, Val5, Ala8]angiotensin II and does not recognize L-158,809 (5,7-dimethyl-2-ethyl-3-[(2'-(1 H-tetrazole-5-yl)[1,1'-biphenyl]-4-yl)methyl]-3H-imidazo[4, 5-beta]pyridine H2O) and PD 123319 (1-[4-dimethylamino)3-methylphenyl]methyl-5-(diphenylacetyl) 4,5,6,7-tetrahydro-1 H-imidazo[4,5-c]pyridine-6-carboxylic acid). A few unrelated hormones (bradykinin, [Arg8] vasopressin, endothelin-1, atrial natriuretic factor, isoproterenol and adrenocorticotropic hormone) were unable to inhibit any 125I-angiotensin IV binding. The affinities of different structural analogues of angiotensin IV revealed that the N-terminal position is critical for receptor recognition and the C-terminal proline is also important. GTP gamma S and polyvinyl sulfate did not affect the binding, suggesting that the receptor is not coupled to a G-protein. The divalent cations Mg2+ and Ca2+ were shown to diminish the binding of 125I-angiotensin IV. Cross-linking of 125I-angiotensin IV to bovine aortic endothelial cell membranes in the presence of disuccinimidyl suberate, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a major band of 186 +/- 12 kDa. The presence in high concentration of this angiotensin binding site on aortic endothelial cells suggest the existence of a novel mechanism involved in the control of vascular tone or vascular permeability.

Safety and feasibility of a self-monitored, home-based phase II exercise program for high risk patients after cardiac surgery.

OBJECTIVE: To determine the safety and effectiveness of a self-monitored, home-based phase II exercise program for high risk patients after cardiac surgery. METHODS: High risk patients were defined as those presenting with severe left ventricular dysfunction with an ejection fraction less than 35%, severe ventricular arrhythmias, incomplete revascularization, abnormal response to a standard walking test or significant (grade 3/4) valvular regurgitation persisting postoperatively. Eighty patients (mean age 58.5 +/- 8.9 years) were randomly assigned to two groups. The experimental group (n = 37) received a home program of aerobic training with an intensity gradually increasing from 1.5 to 4.0 multiples of resting oxygen consumption (METs). This program was started at discharge from the hospital and lasted eight weeks. The control group (n = 43) received general guidelines for progressive increase of their activity level. Functional capacity was measured at discharge by the 6 min walking test and between the sixth and eighth week following discharge by a symptom-limited exercise test, according to the Naughton protocol. RESULTS: No cardiovascular complications occurred during the training program. At the final evaluation, there was no significant difference between the experimental and control groups regarding aerobic capacity (5.1 +/- 1.8 versus 4.9 +/- 1.6 METs respectively, P = 0.61), nor peak rate-pressure product (22.8 +/- 4.9 versus 23.6 +/- 5.2 beats/min x mmHg x 10(3) respectively, P = 0.54).