Effect of a Caregiver's Education Program on Stroke Rehabilitation.

OBJECTIVE: To evaluate effects of caregiver's education program on their satisfaction, as well as patient functional recovery, performed in addition to daily conventional rehabilitation treatment. METHODS: Three hundred eleven subjects diagnosed with first-onset stroke and transferred to the Department of Physical Medicine and Rehabilitation of Inha University Hospital were surveyed. In 2015, caregivers attended an education program for acute and subacute stroke patients. Patients who received an additional rehabilitation therapy were assigned to the experimental group (n=81), whereas the control group (n=100) consisted of transfer cases in 2014 with only conventional treatment. The experimental group was classified by severity using the Korean version of the National Institutes of Health Stroke Scale (K-NIHSS), which was administered to all 181 subjects, in addition to, the Korean version of the Mini Mental Status Examination (K-MMSE), a Modified Barthel Index (K-MBI), and the Berg Balance Scale (K-BBS). Caregiver satisfaction and burden before and after education programs were assessed using the Canadian Occupational Performance Measure (COPM), as well as family burden and caregiver burnout scales. RESULTS: No significant intergroup difference was observed between initial K-NIHSS, K-MMSE, K-BBS, K-MBI scores, and times from admission to transfer. Those with moderate or severe strokes under the experimental condition showed a more significant improvement than the control group as determined by the K-NIHSS and K-BBS, as well as tendential K-MMSE and K-MBI score increases. Satisfaction was significantly greater for family members and formal caregivers of patients with strokes of moderate severity in the experimental group. CONCLUSION: The caregiver's education program for stroke subjects had a positive outcome on patients' functional improvement and caregiver satisfaction. The authors believe that the additional rehabilitation therapy with the education program aids patients to achieve functional improvements for an optimal return to social life.

Mechanism of anti-platelet activity of Oligoporus tephroleucus oligoporin A: involvement of extracellular signal-regulated kinase phosphorylation and cyclic nucleotide elevation.

This study investigated the inhibitory effects of oligoporin A on platelet aggregation and the mechanism of its action on downstream signaling molecules. Oligoporin A was isolated from the fruiting bodies of Oligoporus tephroleucus (Polyporaceae). The anti-platelet activities of oligoporin A were studied using rat platelets. The effects of oligoporin A on intracellular Ca(2+) mobilization, ATP release, production of the cyclic nucleotides cAMP and cGMP, extracellular signal-regulated kinase (ERK) 2 phosphorylation, and fibrinogen binding to active integrin α(II)(b)ß(3) were assessed. Oligoporin A, but not oligoporins B and C, inhibited collagen-induced platelet aggregation in a concentration-dependent manner. Interestingly, oligoporin A did not affect ADP- and thrombin-induced platelet aggregations, which act on different types of membrane receptors. Granule secretion analysis demonstrated that oligoporin A significantly and dose-dependently reduced collagen-induced ATP release and intracellular Ca(2+) mobilization. Additionally, oligoporin A induced the dynamic increase in cAMP and cGMP. Increased cGMP production was further confirmed by the simultaneous production of nitric oxide. Pretreatment with oligoporin A significantly blocked collagen-induced ERK2 phosphorylation. Finally, oligoporin A vaguely diminished the binding of fibrinogen to its cognate receptor, integrin α(II)(b)ß(3). The results indicate that oligoporin A inhibits only collagen-induced platelet aggregation mediated through the modulation of downstream signaling molecules. Oligoporin A may be beneficial against cardiovascular disease provoked by aberrant platelet activation.

Phellinus baumii ethyl acetate extract inhibits lipopolysaccharide-induced iNOS, COX-2, and proinflammatory cytokine expression in RAW264.7 cells.

Mushrooms are valuable sources of biologically active compounds possessing anticancer, antiplatelet, and anti-inflammatory properties. Phellinus baumii is a mushroom used in folk medicine for a variety of human diseases. However, its potential anti-inflammatory effect has remained unclear. Therefore, we studied the effect of P. baumii ethyl acetate extract (PBEAE) on inflammatory mediator and proinflammatory cytokine protein and/or mRNA expression levels using the nitric oxide (NO) assay, enzyme immunoassay (EIA), western blot, and reverse transcription polymerase chain reaction (RT-PCR) in lipopolysaccharide (LPS)-stimulated macrophage like RAW264.7 cells. PBEAE markedly inhibited NO generation and prostaglandin E(2) (PGE(2)) synthesis in a concentration-dependent pattern without any cytotoxic effect at the concentration range used. PBEAE also suppressed inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression. In addition, LPS-induced iNOS and COX-2 mRNA expression levels were dose-dependently inhibited by PBEAE pretreatment. Furthermore, PBEAE attenuated the mRNA expression levels of proinflammatory cytokines, specifically interleukin (IL)-1ß, IL-6, and granulocyte macrophage colony-stimulating factor (GM-CSF), in a concentration-dependent fashion. Our study suggests that P. baumii might exhibit anti-inflammatory properties by downregulating proinflammatory mediators. Thus, further study on compounds isolated from PBEAE is warranted to investigate the associated molecular mechanisms and identify the potential therapeutic targets.

Korean Red Ginseng Saponin Fraction Downregulates Proinflammatory Mediators in LPS Stimulated RAW264.7 Cells and Protects Mice against Endotoxic Shock.

Korean red ginseng has shown therapeutic effects for a number of disease conditions. However, little is known about the antiinflammatory effect of Korean red ginseng saponin fraction (RGSF) in vitro and in vivo. Therefore, in this study, we showed that RGSF containing 20(S)-protopanaxadiol type saponins inhibited nitric oxide production and attenuated the release of tumor necrotic factor (TNF)-α, interleukin (IL)-6, granulocyte monocyte colony stimulating factor (GMCSF), and macrophage chemo-attractant protein-1 in lipopolysaccharide (LPS) stimulated murine macrophage RAW264.7 cells. Moreover, RGSF down-regulated the mRNA expressions of inducible nitric oxide synthase, cyclooxyginase-2, IL-1ß, TNF-α, GMCSF, and IL-6. Furthermore, RGSF reduced the level of TNF-α in the serum and protected mice against LPS mediated endotoxic shock. In conclusion, these results indicated that ginsenosides from RGSF and their metabolites could be potential sources of therapeutic agents against inflammation.

Hepatitis B virus X protein regulates hepatic glucose homeostasis via activation of inducible nitric oxide synthase.

Dysregulation of liver functions leads to insulin resistance causing type 2 diabetes mellitus and is often found in chronic liver diseases. However, the mechanisms of hepatic dysfunction leading to hepatic metabolic disorder are still poorly understood in chronic liver diseases. The current work investigated the role of hepatitis B virus X protein (HBx) in regulating glucose metabolism. We studied HBx-overexpressing (HBxTg) mice and HBxTg mice lacking inducible nitric oxide synthase (iNOS). Here we show that gene expressions of the key gluconeogenic enzymes were significantly increased in HepG2 cells expressing HBx (HepG2-HBx) and in non-tumor liver tissues of hepatitis B virus patients with high levels of HBx expression. In the liver of HBxTg mice, the expressions of gluconeogenic genes were also elevated, leading to hyperglycemia by increasing hepatic glucose production. However, this effect was insufficient to cause systemic insulin resistance. Importantly, the actions of HBx on hepatic glucose metabolism are thought to be mediated via iNOS signaling, as evidenced by the fact that deficiency of iNOS restored HBx-induced hyperglycemia by suppressing the gene expression of gluconeogenic enzymes. Treatment of HepG2-HBx cells with nitric oxide (NO) caused a significant increase in the expression of gluconeogenic genes, but JNK1 inhibition was completely normalized. Furthermore, hyperactivation of JNK1 in the liver of HBxTg mice was also suppressed in the absence of iNOS, indicating the critical role for JNK in the mutual regulation of HBx- and iNOS-mediated glucose metabolism. These findings establish a novel mechanism of HBx-driven hepatic metabolic disorder that is modulated by iNOS-mediated activation of JNK.

Antiplatelet activity of Phellinus baummii methanol extract is mediated by cyclic AMP elevation and inhibition of collagen-activated integrin-α(IIb) ß3 and MAP kinase.

Phellinus baumii is a mushroom that has been used as folk medicine against various diseases and is reported to have antidiabetic, anticancer, antioxidant, antiinflammatory and antihypertensive activities. However, information on the effects of P. baumii extract in platelet function is limited. Therefore, the aim of this study was to examine the impact of a P. baumii methanol extract (PBME) on platelet activation and to investigate the mechanism behind its antiplatelet activity. PBME effects on agonist-induced platelet aggregation, granule secretion, [Ca²âº](i) mobilization, α(IIb) ß3 activation, cyclic AMP release and mitogen-activated protein kinase (MAPK) phosphorylations were studied using rat platelets. PBME dose-dependently inhibited collagen, thrombin and ADP-induced platelet aggregation with an IC50 of 51.0 ± 2.4, 54.0 ± 2.1 and 53.0 ± 4.3 µg/mL, respectively. Likewise, thrombin-induced [Ca²âº](i) and collagen-activated ATP secretions were suppressed in PBME treated platelets. Aggregation and ATP secretion were also markedly attenuated by PBME alone or in combination with PP2 (Src inhibitor) and U-73122 (PLC inhibitor) in collagen-stimulated platelets. Besides, PBME treatment elevated basal cyclic AMP levels and inhibited collagen-induced integrin-α(IIb) ß3 activation. Moreover, PBME attenuated extracellular-signal-regulated protein kinase 2 (ERK2) and c-Jun N-terminal kinase 1 (JNK1) phosphorylations. Further PD98059 (ERK inhibitor) and SP60025 (JNK inhibitor) reduced collagen-induced platelet aggregation and ATP secretion. In conclusion, the observed PBME antiplatelet activity may be mediated by activation of cyclic AMP and inhibition of ERK2 and JNK1 phosphorylations. Finally, these data suggest that PBME may have therapeutic potential for the treatment of cardiovascular diseases that involve aberrant platelet function.

Microglial peroxiredoxin V acts as an inducible anti-inflammatory antioxidant through cooperation with redox signaling cascades.

Reactive oxygen species (ROS) actively participate in microglia-mediated pathogenesis as pro-inflammatory molecules. However, little is known about the involvement of specific antioxidants in maintaining the microglial oxidative balance. We demonstrate that microglial peroxiredoxin (Prx) 5 expression is up-regulated by lipopolysaccharide (LPS) through activation of the ROS-sensitive signaling pathway and is involved in attenuation of both microglial activation and nitric oxide (NO) generation. Unlike in stimulation of oxidative insults with paraquat and hydrogen peroxide, Prx V expression is highly sensitive to LPS-stimulation in microglia. Reduction of ROS level by treatment with either NADPH oxidase inhibitor or antioxidant ablates LPS-mediated Prx V up-regulation in BV-2 microglial cells and is closely associated with the activation of the c-jun N-terminal kinase (JNK) signaling pathway. This suggests the involvement of ROS/JNK signaling in LPS-mediated Prx V induction. Furthermore, NO induces Prx V up-regulation that is ablated by the addition of inducible nitric oxide synthase inhibitor or deleted mutation of inducible nitric oxide synthase in LPS-stimulated microglia. Therefore, these results suggest that Prx V is induced by cooperative action among the ROS, RNS, and JNK signaling cascades. Interestingly, knockdown of Prx V expression causes the acceleration of microglia activation, including augmented ROS generation and JNK-dependent NO production. In summary, we demonstrate that Prx V plays a key role in the microglial activation process through modulation of the balance between ROS/NO generation and the corresponding JNK cascade activation.

A noble function of BAY 11-7082: Inhibition of platelet aggregation mediated by an elevated cAMP-induced VASP, and decreased ERK2/JNK1 phosphorylations.

Platelets, though anucleated, possess several transcription factors, including NF-kappaB, that exert non-genomic functions regulating platelet activation. Since platelets have not only been recognized as central players of homeostasis, but also participated in pathological conditions such as thrombosis, atherosclerosis, and inflammation, we examined rat platelet NF-kappaB expression and evaluated the effects of anti-inflammatory drug BAY 11-7082, an inhibitor of NF-kappaB activation, in platelet physiology. Western blotting revealed that rat platelets express NF-kappaB. BAY 11-7082, dose dependently, inhibited collagen- or thrombin-induced-platelet aggregation. ATP release, TXB(2) formation, P-selectin expression, and intercellular Ca(2+) concentration activated by collagen were reduced in BAY 11-7082-treated platelets. BAY 11-7082 elevated intracellular levels of cAMP, but not cGMP, and its co-incubation with cAMP-activating agent (forskolin) or its hydrolyzing enzyme inhibitor (3-isobutyl-1-methylxanthine, IBMX), synergistically inhibited collagen-induced-platelet aggregation. In addition, vasodilator-stimulated-phosphoprotein (VASP) phosphorylation was enhanced in BAY 11-7082-treated platelets, which was partially inhibited by a protein kinase A (PKA) inhibitor, H-89. Moreover, while p38 mitogen-activated protein kinase (MAPK) was not affected, BAY 11-7082 attenuated c-Jun N-terminal kinase 1 (JNK1) and extracellular-signal-regulated protein kinase 2 (ERK2) phosphorylations. In conclusion, BAY 11-7082 inhibits platelet activation, granule secretion, and aggregation, and that this effect is mediated by inhibition of JNK1 and ERK2 phosphorylations, and partially by stimulation of cAMP-dependent PKA VASP phosphorylation. The ability of BAY 11-7082 to inhibit platelet function might be relevant in cases involving aberrant platelet activation where the drug is considered as anti-atherothrombosis, and anti-inflammatory therapy.

Automated control of blood glucose in the OR and surgical ICU.

A device which integrates existing intravenous continuous glucose monitors and infusion pumps into a central hub for automated intravenous intensive insulin therapy, targeting non-diabetic critically-ill patients is presented. Additionally, a fuzzy logic based controller that is capable of automatically making closed-loop decisions to achieve tight glycemic control between a euglycemic range of 90 to 120 mg/dl is presented. Initial bench top testing shows a significant improvement in glycemic control with fuzzy logic control when compared to manual infusion protocols currently used in hospitals; future animal testing will be performed to verify these results in vivo.

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent activation of phosphoinositide 3-kinase and p38 mitogen-activated protein kinase signal pathways is required for lipopolysaccharide-induced microglial phagocytosis.

The importance of microglial reactive oxygen species (ROS) signaling in neuroinflammatory processes has been well demonstrated; however, relatively little is known regarding the related mechanisms underlying these processes. Here, we show that ROS-dependent signal pathways that govern microglial phagocytosis are highly dependent upon nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) activation. Specifically, phagocytosis was greatly reduced by both antioxidant and Nox inhibitor treatments in lipopolysaccharide (LPS)-stimulated BV-2 microglia. Additionally, there was a marked reduction in intracellular ROS content. These results suggest that Nox is the main ROS source for LPS-induced microglial phagocytosis. More decisive evidence for the involvement of ROS in phagocytosis was obtained from an examination of phosphatidyl inositol 3-kinase (PI3-K) and p38 mitogen-activated protein kinase (MAPK) signal pathway activation under reduced ROS levels. These two kinases were activated by LPS treatment and inhibited by ROS neutralization and Nox inhibition. We conclude that microglial phagocytosis requires ROS-dependent PI3-K and p38 MAPK activation and that Nox-derived ROS functions as an upstream regulator of both PI3-K and p38 MAPK. These findings will provide a fundamental basis for a therapeutic modality in inflammation-mediated neurodiseases.

Evaluation of antioxidant, antinociceptive, and anti-inflammatory activities of ethanol extracts from Aloe saponaria Haw.

Aloe species are traditionally prescribed for hypertension, burning, and rheumatoid arthritis. To elucidate the mechanism of the antihypertensive and anti-inflammatory activities of this herb, the ethanol fraction from A. saponaria Haw. was evaluated for antioxidative activity using xanthine-xanthine oxidase (XO) assay, 2,2-Diphenyl-lpicrylhydrazyl radical (DPPH) assay, lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 cell, and antinociceptive activity using a tail-flick assay and hind paw pressure assay in cisplatin-treated hyperalgesic rats. The ethanol fraction displayed potent antioxidative activities in XO assay. In addition, ethanol fractions showed potent scavenging effects in DPPH assay. We next examined whether ethanol fractions showed anti-inflammatory activities. Ethanol fractions significantly suppressed NO production from LPS-activated RAW264.7 cells. As expected, ethanol fractions dose-dependently inhibited the messenger RNA expression of inducible NO synthase (iNOS). Moreover, ethanol fractions potently suppressed the expression of cycloxygenase (COX)-2 and granulocyte-macrophage colony-stimulating factor (GM-CSF), which are stimulated by LPS in RAW264.7 cells. In addition, ethanol fractions significantly blocked cisplatin-induced hyperalgesia using tail-flick assay and hind paw pressure test in rats. Taken altogether, ethanol extracts of aloe may be useful as a functional food or as a drug against reactive oxygen species (ROS) mediated diseases.

Peroxiredoxin I is an indicator of microglia activation and protects against hydrogen peroxide-mediated microglial death.

Imbalance between oxidative stress and antioxidative defence system is generally known as one of mechanisms causing an oxidative stress-medieated neuropathogenesis. Peroxiredoxins (Prxs), a family of antioxidative enzymes neutralizing cellular hydroperoxides, was characterized recently, but their distributions and roles have not been resolved clearly or controversial in the central nervous system, Therefore, the present study was carried out to determine the specific cell types that express Prx I in the mouse brain and primary neural cells, and to examine its antioxidative role in the preferential cell types. Immunohistochemical reactivity for Prx I was detected dominantly in oligodendrocytes and rarely in microglia, whereas strong and specific immunoreactivity for Prx I was observed exclusively in microglia of primary neural cell culture. Further evidences for Prx I specificity were its relatively high expression in BV-2 microglial cells and its upregulated expression in microglia after lipopolysaccharide (LPS) stimulation. These results imply that Prx I can be used as an indicator of microglial activation. Inhibition of p38 MAPK ablated LPS-mediated Prx I upregulation and sensitized the microglia to H(2)O(2)-mediated cell death. These findings indicate that Prx I function as a scavenger for H(2)O(2) generated during microglial activation. The results of this study will help in unraveling the neuropathologic roles of the six Prx isoforms in neural function.

The mechanism of anti-platelet activity of davallialactone: involvement of intracellular calcium ions, extracellular signal-regulated kinase 2 and p38 mitogen-activated protein kinase.

This study was designed to investigate the effect of davallialactone, which was isolated from the mushroom Inonotus xeranticus, on platelet aggregation induced by collagen, thrombin and ADP. We found that davallialactone dose-dependently inhibited platelet aggregation that was stimulated either by collagen (2.5 microg/ml), a potent ligand of integrin alpha2beta1 and glycoprotein VI, or by thrombin (0.1U/ml), a potent agonist of the protease-activated receptors (PARs) PAR1 and PAR3. In addition, davallialactone inhibited platelet aggregation induced by ADP, an agonist of P2Y receptor. To understand the mechanism of anti-platelet activity, we determined whether davallialactone affected the downstream signaling in collagen-activated platelets. Using the fura-2/AM fluorometric assay, we found that davallialactone dose-dependently inhibited intracellular calcium concentration levels ([Ca2+]i). Moreover, davallialactone inhibited the phosphorylation of extracellular signal-regulated protein kinase (ERK)-2 and p38 mitogen-activated protein kinase (MAPK), in a dose-dependent manner. The tyrosine phosphorylation of 60 and 85kDa proteins, which were activated by collagen, were differentially inhibited by davallialactone. Taken together, these data suggest that davallialactone may have potential anti-platelet aggregation activity via suppression of intracellular downstream signaling pathways.

In vitro anti-oxidative and anti-inflammatory effects of solvent-extracted fractions from Suaeda asparagoides.

Suaeda asparagoides Miq. (Chenopodiaceae: S. asparagoides) is a salt-marsh plant that has long been prescribed in traditional Oriental medicine for the treatment of hypertension and hepatitis. In order to elucidate the pharmacological mechanisms of the herb, we conducted an examination of the anti-oxidative and anti-inflammatory properties of solvent-extracts of S. asparagoides. All of the solvent fractions showed potent anti-oxidative effects, as assessed using a radical generation assay system (xanthine oxidase assay) and an electron-donating activity system (DPPH [2,2-diphenyl-l-picrylhydrazyl radical] assay), with IC50 values ranging from 9 to 42 microg/ml. In agreement with this pattern, the total phenolic contents were widely distributed in the various solvent fractions, and ranged from 36.5 to 50.3 mg/g of dry weight. All of the solvent fractions significantly suppressed NO production in RAW264.7 cells induced by lipopolysaccharide (LPS, 0.1 microg/ml) and of the fractions, only the chloroform (CHC) fraction completely blocked the expression of inducible NO synthase (iNOS). Additionally, the hexane (HEX) and CHC fractions suppressed the mRNA expression of granulocyte/macrophage colony-stimulating factor (GM-CSF) and monocyte chemoattractant protein 1 (MCP-1), respectively, in the LPS-stimulated RAW264.7 cells. Therefore, these results suggest that the pharmacological action of S. asparagoides is due to its potent anti-oxidative effects and anti-inflammatory effects, and that therefore it can be applied to other diseases caused by oxidative stress and inflammation, such as cardiovascular diseases.

The expression patterns of RGS transcripts in platelets.

Regulators of G protein signalling (RGS) are involved in the negative regulation of cell activation processes and are involved in the pathophysiology of cardiovascular diseases. To get some further evidence for a role of RGS proteins in platelets, we determined the expression profile of RGS-specific mRNA in rat platelets using reverse transcription-polymerase chain reaction (RT-PCR) with a poly dT18 primer and transcript-specific primers. We found that RGS2, RGS3, RGS5, RGS6, RGS10, RGS14, RGS16 and RGS18, Leukemia-associated Rho-GEF factor (LARG), and Galpha interacting protein (GAIP) were differentially expressed in platelets. The highest expression rate was found for RGS18 (about 1.3 fold when compared to GAPDH), followed by LARG, RGS6, RGS10 and RGS16 (0.7 to 0.95), whereas expression rates for RGS2, RGS3, RGS5, RGS14, and GAIP were in a range of 0.1 to 0.3. Our results suggest that G-protein-coupled receptor-mediated signalling in platelet may be regulated mainly by RGS 18, 16, 10, 6, and LARG.

Mechanism of isoproterenol-induced RGS2 up-regulation in astrocytes.

Regulators of G protein signaling (RGSs) are inducibly expressed in response to various stimuli and the up-regulation of RGSs leads to significant decreases in GPCR responsiveness. Isoproterenol, an adrenergic receptor agonist, stimulated RGS2 mRNA in C6 rat astrocytoma cells. The up-regulation of RGS2 mRNA was abrogated by genistein, a protein tyrosine kinase inhibitor (PTK), and by broad-spectrum protein kinase C (PKC) inhibitors (staurosporine and GF109203X). alpha-Adrenergic antagonist (prazocin), beta-adrenergic antagonist (prazocin), and pertussis toxin only partially blocked the RGS2 up-regulation, suggesting that the RGS2 up-regulation is concomitantly mediated by Galphai, Galphas, and Galphaq. It is interesting to note that SB203580, a potent p38 mitogen-activated protein kinase (MAPK) inhibitor, completely inhibited the isoproterenol-mediated RGS2 expression. In addition, isoproterenol also markedly stimulated RGS2 mRNA in rat primary astrocytes, which were sensitive to SB203580 and staurosporine. Therefore, our data suggest that adrenergic receptor-mediated signaling (induced by isoproterenol) may be involved in the regulation of RGS2 expression in astrocytes via activating PTK, PKC, and p38 MAPK.

Surfactin C inhibits platelet aggregation.

This study was designed to investigate the effect of surfactin C, which is derived from Bacillus subtilis, on platelet aggregation and homotypic leucocyte aggregation. Surfactin C strongly and dose-dependently inhibited platelet aggregation, which was stimulated both by thrombin (0.1 U mL(-1)), a potent agonist that activates the G protein-coupled protease receptor, and by collagen (5 microg mL(-1)), a potent ligand that activates alpha(IIb)beta(3) with IC50 values (concentration inhibiting platelet aggregation by 50%) of 10.9 and 17.0 microM, respectively. Moreover, surfactin C significantly suppressed the intracellular Ca(2+) mobilization in thrombin-activated platelets. Surfactin C, however, did not affect various integrin-mediated U937 cell aggregation, implying that the anti-platelet activity of surfactin C was not due to its detergent effect but by its action on the downstream signalling pathway. Therefore, the results suggest that surfactin C may have a beneficial therapeutic effect on aberrant platelet aggregation-mediated cardiovascular diseases.

BRCA1 modulates xenobiotic stress-inducible gene expression by interacting with ARNT in human breast cancer cells.

Previously, we have reported that BRCA1 regulates the expression of various classes of genes, including genes involved in xenobiotic stress responses (Bae, I., Fan, S., Meng, Q., Rih, J. K., Kim, H. J., Kang, H. J., Xu, J., Goldberg, I. D., Jaiswal, A. K., and Rosen, E. M. (2004) Cancer Res. 64, 7893-7909). In the present study, we have investigated the effects of BRCA1 on xenobiotic stress-inducible gene expression. In response to aryl hydrocarbon receptor (AhR) ligands, cytoplasmic AhR becomes activated and then translocates to the nucleus where it forms a complex with the aryl hydrocarbon receptor nuclear translocator (ARNT). Subsequently, the AhR.ARNT complex binds to the enhancer or promoter of genes containing a xenobiotic stress-responsive element and regulates the expression of multiple target genes including cytochrome P450 subfamily polypeptide 1 (CYP1A1). In this study, we have found that endogenous and overexpressed exogenous wild-type BRCA1 affect xenobiotic stress-induced CYP1A1 gene expression. Using a standard chromatin immunoprecipitation assay, we have demonstrated that BRCA1 is recruited to the promoter regions of CYP1A1 and CYP1B1 along with ARNT and/or AhR following xenobiotic exposure. Our findings suggest that BRCA1 may be physiologically important for mounting a normal response to xenobiotic insults and that it may function as a coactivator for ARNT activity. Using immunoprecipitation, Western blotting, and glutathione S-transferase capture assays, a xenobiotic-independent interaction between BRCA1 and ARNT has been identified, although it is not yet known whether this is a direct or indirect interaction. We have also found that the inducibility of CYP1A1 and CYP1B1 transcripts following xenobiotic stress was significantly attenuated in BRCA1 knockdown cells. This reduced inducibility is associated with an altered stability of ARNT and was almost completely reversed in cells transfected with an ARNT expression vector. Finally, we have found that xenobiotic (TCDD) treatments of breast cancer cells containing reduced levels of BRCA1 cause the transcription factor ARNT to become unstable.

Radical scavenging and anti-inflammatory activity of extracts from Opuntia humifusa Raf.

Opuntia humifusa Raf. (O. humifusa Raf.) is a member of the Cactaceae family. To determine the antioxidative and anti-inflammatory effects of this herb, various solvent fractions (methanol, hexane, chloroform, ethyl acetate, butanol, and water) prepared from the leaves of cacti were tested using DPPH (2,2-diphenyl-l-picrylhydrazyl radical) and xanthine oxidase assays, and nitric oxide (NO)-producing macrophage cells. We found that O. humifusa Raf. displayed potent antioxidative and anti-inflammatory activity. Thus, all solvent fractions, except for the water layer, showed potent scavenging effects. The scavenging effect of the ethyl acetate fraction was higher than that of the other fractions, with IC50 values of 3.6 and 48.2 microg mL(-1). According to activity-guided fractionation, one of the active radical scavenging principles in the ethyl acetate fraction was found to be quercetin. In contrast, only two fractions (chloroform and ethyl acetate) significantly suppressed nitric oxide production from the lipopolysaccharide (LPS)-activated RAW264.7 cells. In addition, chloroform and ethyl acetate fractions significantly blocked the expression of inducible nitric oxide synthetase (iNOS) and interleukin-6 (IL-6) from the RAW264.7 cells stimulated by LPS. Moreover, ethyl acetate fractions significantly blocked the expression of IL-1beta from the RAW264.7 cells stimulated by LPS. Therefore, the results suggested that O. humifusa Raf. may modulate radical-induced toxicity via both direct scavenging activity and the inhibition of reactive species generation, and the modulation of the expression of inflammatory cytokines. Finally, O. humifusa Raf. may be useful as a functional food or drug against reactive species-mediated disease.

Histone deacetylase 1 contributes to cell cycle and apoptosis.

Histone deacetylases (HDACs) are generally thought to play important roles in human disease. However, little information is available concerning the specific functions of individual HDACs. We previously reported on transgenic mice that expressed human HDAC1 and experienced steatosis and nuclear pleomorphism in their hepatic tissues. To find out if the over-expression of HDAC1 contributes to the expression of genes related to the cell cycle, apoptosis, and lipid metabolism that eventually contribute to the pathological changes in the livers of the transgenic mice, the expression profiles of the related genes in liver tissues were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. The activated human HDAC1 significantly induced the expression levels of mRNA for p53, PPAR-gamma and Bak and reduced the p21 expression level compared with the levels in control littermates. However, the protein levels of p53 and PPAR-gamma were significantly decreased. In conclusion, our results indicate that HDAC1 can regulate gene expression at the mRNA and protein levels independently and that this may be a potential cytopathic factor for hepatic tissue in transgenic mice that over-express HDAC1.