Role of p53 isoforms in the DNA damage response during Drosophila oogenesis.

The tumor suppressor p53 is involved in the DNA damage response and induces cell cycle arrest or apoptosis upon DNA damage. Drosophila p53 encodes two isoforms, p53A and p53B, that induce apoptosis in somatic cells. To investigate the roles of Drosophila p53 isoforms in female germline cells, the DNA damage response was analyzed in the adult ovary. Early oogenesis was sensitive to irradiation and lok-, p53-, and hid-dependent cell death occurred rapidly after both low- and high-dose irradiation. Both p53 isoforms were responsible for this cell death. On the other hand, delayed cell death in mid-oogenesis was induced at a low level only after high-dose irradiation in a p53-independent manner. The daily egg production, which did not change after low-dose irradiation, was severely reduced after high-dose irradiation in p53 mutant females due to the loss of germline stem cells. When the p53A or p53B isoform was expressed in the germline cells in the p53 mutant females at levels that do not affect normal oogenesis, p53A, but not p53B, restored the fertility of the irradiated female. In summary, moderate expression of p53A is critical to maintain the function of germline stem cells during normal oogenesis as well as after high-dose irradiation.

The caspase-8/Bid/cytochrome c axis links signals from death receptors to mitochondrial reactive oxygen species production.

Ligation of the death receptors for TNF-α, FasL, and TRAIL triggers two common pathways, caspase-dependent intrinsic apoptosis and intracellular reactive oxygen species (ROS) generation. The apoptotic pathway is well characterized; however, a signaling linker between the death receptor and ROS production has not been clearly elucidated. Here, we found that death receptor-induced ROS generation was strongly inhibited by mitochondrial complex I and II inhibitors, but not by inhibitors of NADPH oxidase, lipoxygenase, cyclooxygenase or xanthine oxidase, indicating that ROS are mostly generated by the impairment of the mitochondrial respiratory chain. ROS generation was accompanied by caspase-8 activation, Bid cleavage, and cytochrome c release; it was blocked in FADD- and caspase-8-deficient cells, as well as by caspase-8 knockdown and inhibitor. Moreover, Bid knockdown abrogated TNF-α- or TRAIL-induced ROS generation, whereas overexpression of truncated Bid (tBid) or knockdown of cytochrome c spontaneously elevated ROS production. In addition, p53-overexpressing cells accumulated intracellular ROS via cytochrome c release mediated by the BH3-only protein Noxa induction. In a cell-free reconstitution system, caspase-8-mediated Bid cleavage and recombinant tBid induced mitochondrial cytochrome c release and ROS generation, which were blocked by Bcl-xL and antioxidant enzymes. These data suggest that anti-apoptotic Bcl-2 proteins play an important role in mitochondrial ROS generation by preventing cytochrome c release. These data provide evidence that the FADD/caspase-8/Bid/cytochrome c axis is a crucial linker between death receptors and mitochondria, where they play a role in ROS generation and apoptosis.

Carbon Monoxide Potentiation of L-Type Ca2+ Channel Activity Increases HIF-1α-Independent VEGF Expression via an AMPKα/SIRT1-Mediated PGC-1α/ERRα Axis.

AIMS: The heme oxygenase-1 (HO-1)/carbon monoxide (CO) pathway induced in astrocytes after ischemic brain injury promotes vascular endothelial growth factor (VEGF) expression to maintain and repair neurovascular function. Although HO-1-derived CO has been shown to induce hypoxia-inducible factor-1α (HIF-1α)-dependent VEGF expression, the underlying mechanism independent of HIF-1α remains to be elucidated. RESULTS: HO-1 and VEGF were coexpressed in astrocytes of ischemic mouse brain tissues. Experiments with specific siRNAs and pharmacological activators/inhibitors of various target genes demonstrated that astrocytes pre-exposed to the CO-releasing compound, CORM-2, or transfected with HO-1 increased HIF-1α-independent VEGF expression via sequential activation of the following signal cascades; Ca2+/calmodulin-dependent protein kinase kinase ß-mediated AMP-activated protein kinase (AMPK)α activation, AMPKα-induced increases in nicotinamide phosphoribosyltransferase (NAMPT) expression and cellular NAD+ level, sirtuin 1 (SIRT1)-dependent peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) stabilization and activation, and PGC-1α/estrogen-related receptor (ERR)α-mediated VEGF expression. All of these sequential events were blocked by an L-type voltage-gated Ca2+ channel inhibitor and Ca2+ chelators, but not by other Ca2+ channel inhibitors. INNOVATION: HO-1-derived CO elicits Ca2+ influx by activating L-type Ca2+ channels, which is a key player in HIF-1α-independent VEGF expression by activating the AMPKα-NAMPT-SIRT1-PGC-1α/ERRα pathway. CONCLUSION: Our results provide new mechanistic insight into the possible role for L-type Ca2+ channels in HO-1/CO-induced angiogenesis. Antioxid. Redox Signal. 27, 21-36.

p-Hydroxybenzyl alcohol-containing biodegradable nanoparticle improves functional blood flow through angiogenesis in a mouse model of hindlimb ischemia.

Therapeutic angiogenesis has achieved promising results for ischemic diseases or peripheral artery disease in preclinical and early-phase clinical studies. We examined the therapeutic angiogenic effects of HPOX, which is biodegradable polymer composing the antioxidant p-hydroxybenzyl alcohol (HBA), in a mouse model of hindlimb ischemia. HPOX effectively stimulated blood flow recovery, compared with its degraded compounds HBA and 1,4-cyclohexendimethanol, via promotion of capillary vessel density in the ischemic hindlimb. These effects were highly correlated with levels of angiogenic inducers, vascular endothelial cell growth factor (VEGF), heme oxygenase-1 (HO-1), and Akt/AMPK/endothelial nitric oxide synthase (eNOS) in ischemic mouse hindlimb muscle. Blood perfusion and neovascularization induced by HPOX were reduced in eNOS(-/-) and HO-1(+/-) mice. HPOX also elevated the endothelial cell markers VEGF receptor-2, CD31, and eNOS mRNAs in the ischemic hindlimb, indicating that HPOX increases endothelial cell population and angiogenesis in the ischemic muscle. However, this nanoparticle suppressed expression levels of several inflammatory genes in ischemic tissues. These results suggest that HPOX significantly promotes angiogenesis and blood flow perfusion in the ischemic mouse hindlimb via increased angiogenic inducers, along with suppression of inflammatory gene expression. Thus, HPOX can be used potentially as a noninvasive drug intervention to facilitate therapeutic angiogenesis.

Novel shortcut estimation method for regeneration energy of amine solvents in an absorption-based carbon capture process.

Among various CO2 capture processes, the aqueous amine-based absorption process is considered the most promising for near-term deployment. However, the performance evaluation of newly developed solvents still requires complex and time-consuming procedures, such as pilot plant tests or the development of a rigorous simulator. Absence of accurate and simple calculation methods for the energy performance at an early stage of process development has lengthened and increased expense of the development of economically feasible CO2 capture processes. In this paper, a novel but simple method to reliably calculate the regeneration energy in a standard amine-based carbon capture process is proposed. Careful examination of stripper behaviors and exploitation of energy balance equations around the stripper allowed for calculation of the regeneration energy using only vapor-liquid equilibrium and caloric data. Reliability of the proposed method was confirmed by comparing to rigorous simulations for two well-known solvents, monoethanolamine (MEA) and piperazine (PZ). The proposed method can predict the regeneration energy at various operating conditions with greater simplicity, greater speed, and higher accuracy than those proposed in previous studies. This enables faster and more precise screening of various solvents and faster optimization of process variables and can eventually accelerate the development of economically deployable CO2 capture processes.

Promoting alkali and alkaline-earth metals on MgO for enhancing CO2 capture by first-principles calculations.

Developing next-generation solid sorbents to improve the economy of pre- and post-combustion carbon capture processes has been challenging for many researchers. Magnesium oxide (MgO) is a promising sorbent because of its moderate sorption-desorption temperature and low heat of sorption. However, its low sorption capacity and thermal instability need to be improved. Various metal-promoted MgO sorbents have been experimentally developed to enhance the CO2 sorption capacities. Nevertheless, rigorous computational studies to screen an optimal metal promoter have been limited to date. We conducted first-principles calculations to select metal promoters of MgO sorbents. Five alkali (Li-, Na-, K-, Rb-, and Cs-) and 4 alkaline earth metals (Be-, Ca-, Sr-, and Ba-) were chosen as a set of promoters. Compared with the CO2 adsorption energy on pure MgO, the adsorption energy on the metal-promoted MgO sorbents is higher, except for the Na-promoter, which indicates that metal promotion on MgO is an efficient approach to enhance the sorption capacities. Based on the stabilized binding of promoters on the MgO surface and the regenerability of sorbents, Li, Ca, and Sr were identified as adequate promoters among the 9 metals on the basis of PW91/GGA augmented with DFT+D2. The adsorption energies of CO2 on metal-promoted MgO sorbents for Li, Ca, and Sr atoms are -1.13, -1.68, and -1.48 eV, respectively.

Hypoxia-responsive microRNA-101 promotes angiogenesis via heme oxygenase-1/vascular endothelial growth factor axis by targeting cullin 3.

AIMS: Hypoxia induces expression of various genes and microRNAs (miRs) that regulate angiogenesis and vascular function. In this study, we investigated a new functional role of new hypoxia-responsive miR-101 in angiogenesis and its underlying mechanism for regulating heme oxygenase-1 (HO-1) and vascular endothelial growth factor (VEGF) expression. RESULTS: We found that hypoxia induced miR-101, which binds to the 3'untranslated region of cullin 3 (Cul3) and stabilizes nuclear factor erythroid-derived 2-related factor 2 (Nrf2) via inhibition of the proteasomal degradation pathway. miR-101 overexpression promoted Nrf2 nuclear accumulation, which was accompanied with increases in HO-1 induction, VEGF expression, and endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) production. The elevated NO-induced S-nitrosylation of Kelch-like ECH-associated protein 1 and subsequent induction of Nrf2-dependent HO-1 lead to further elevation of VEGF production via a positive feedback loop between the Nrf2/HO-1 and VEGF/eNOS axes. Moreover, miR-101 promoted angiogenic signals and angiogenesis both in vitro and in vivo, and these events were attenuated by inhibiting the biological activity of HO-1, VEGF, or eNOS. Moreover, these effects were also observed in aortic rings from HO-1(+/-) and eNOS(-/-) mice. Local overexpression of miR-101 improved therapeutic angiogenesis and perfusion recovery in the ischemic mouse hindlimb, whereas antagomiR-101 diminished regional blood flow. INNOVATION: Hypoxia-responsive miR-101 stimulates angiogenesis by activating the HO-1/VEGF/eNOS axis via Cul3 targeting. Thus, miR-101 is a novel angiomir. CONCLUSION: Our results provide new mechanistic insights into a functional role of miR-101 as a potential therapeutic target in angiogenesis and vascular remodeling.

Functional role of NF-κB in expression of human endothelial nitric oxide synthase.

The transcription factor NF-κB has an essential role in inflammation in endothelial cells. Endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) prevents vascular inflammation. However, the molecular mechanism underlying NF-κB-mediated regulation of eNOS expression has not been clearly elucidated. We here found that NF-κB-activating stimuli, such as lipopolysaccharide, tumor necrosis factor-α (TNF-α), and interleukin-1ß, suppressed eNOS mRNA and protein levels by decreasing mRNA stability, without affecting promoter activity. TNF-α-mediated suppression of eNOS expression, mRNA stability, and 3'-untranslated region (3'UTR) activity were inhibited by NF-κB inhibitors and Dicer knockdown, but not by p38 MAPK and MEK inhibitors, suggesting the involvement of NF-κB-responsive miRNAs in eNOS expression. Moreover, TNF-α increased MIR155HG expression and promoter activity as well as miR-155 biogenesis, and these increases were blocked by NF-κB inhibitors. Transfection with antagomiR-155 blocked TNF-α-mediated suppression of eNOS 3'UTR activity, eNOS mRNA and protein levels, and NO and cGMP production. These data provide evidence that NF-κB is a negative regulator of eNOS expression via upregulation of miR-155 under inflammatory conditions. These results suggest that NF-κB is a potential therapeutic target for preventing vascular inflammation and endothelial dysfunction induced by suppression of miR-155-mediated eNOS expression.

TRAIL negatively regulates VEGF-induced angiogenesis via caspase-8-mediated enzymatic and non-enzymatic functions.

Solid tumors supply oxygen and nutrients required for angiogenesis by producing vascular endothelial growth factor (VEGF). Thus, inhibitors of VEGF signaling abrogate tumor angiogenesis, resulting in the suppression of tumor growth and metastasis. We here investigated the effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on VEGF-induced angiogenesis. TRAIL inhibited VEGF-induced in vitro angiogenesis of human umbilical vein endothelial cells (HUVECs) and in vivo neovascularization in chicken embryos and mice. TRAIL blocked VEGF-induced angiogenic signaling by inhibiting ERK, Src, FAK, paxillin, Akt, and eNOS. Further, TRAIL blocked intracellular Ca(2+) elevation and actin reorganization in HUVECs stimulated with VEGF, without inhibiting VEGF receptor-2 tyrosine phosphorylation. TRAIL increased caspase-8 activity, without inducing caspase-9/-3 activation and apoptosis. Moreover, TRAIL resulted in cleavage of FAK into FAK-related non-kinase-like fragments in VEGF-stimulated HUVECs, which was blocked by a caspase-8 inhibitor and cellular caspase-8-like inhibitory protein. Biochemical and pharmacological inhibition of caspase-8 and FAK blocked the inhibitory effects of TRAIL on VEGF-stimulated anti-angiogenic signaling and events. In addition, caspase-8 knockdown also suppressed VEGF-mediated signaling and angiogenesis, suggesting that procaspase-8 plays a role of a non-apoptotic modulator in VEGF-induced angiogenic signaling. These results suggest that TRAIL inhibits VEGF-induced angiogenesis by increasing caspase-8 activity and subsequently decreasing non-apoptotic signaling functions of procaspase-8, without inducing caspase-3 activation and endothelial cell cytotoxicity. These data indicate that caspase-8 may be used as an anti-angiogenic drug for solid tumors resistant to TRAIL and anti-tumor drugs.

Aqueous extract of unripe Rubus coreanus fruit attenuates atherosclerosis by improving blood lipid profile and inhibiting NF-κB activation via phase II gene expression.

ETHNOPHARMACOLOGICAL RELEVANCE: The fruit of Rubus coreanus has been used as a traditional herbal medicine for alleviation of inflammatory and vascular diseases in Asian countries. AIM OF THE STUDY: The anti-atherogenic effect of unripe Rubus coreanus fruit extract (URFE) and its underlying mechanism were analyzed in mice fed a high-fat diet (HFD) and in cell culture system. MATERIALS AND METHODS: Mouse was freely given HFD alone or supplemented with URFE for 14 weeks, followed by analysis of atherosclerotic lesions and serum lipid levels. For in vitro assay, macrophages were pretreated with URFE, followed by stimulation with lipopolysaccharide (LPS). Expression levels of inflammatory genes (TNF-α, IL-1ß, and iNOS) and phase II genes (heme oxygenase-1, glutamate cysteine lygase, and peroxiredoxine-1) as well as intracellular reactive oxygen species (ROS) level and NF-κB activation pathway were analyzed in cultured macrophages as well as mouse sera and aortic tissues. RESULTS: URFE supplementation reduced HFD-induced atherosclerotic lesion formation which was correlated with decreased levels of lipids, lipid peroxides, and inflammatory mediators (TNF-α, IL-1ß, and nitric oxide) in sera as well as suppression of inflammatory gene in aortic tissues. In addition, pre-treatment of macrophages with URFE also suppressed LPS-induced NF-κB activation, ROS production, and inflammatory and phase II gene expressions. Inhibition of phase II enzyme and protein activities attenuated the suppressive effects URFE on ROS production, NF-κB activation, and inflammatory gene expression. CONCLUSION: These results suggest that URFE attenuates atherosclerosis by improving blood lipid profile and inhibiting NF-κB activation via phase II antioxidant gene expression.

Kurarinone promotes TRAIL-induced apoptosis by inhibiting NF-κB-dependent cFLIP expression in HeLa cells.

This study was designed to investigate the effects of the prenylated flavonoid kurarinone on TNF-related apoptosis inducing ligand (TRAIL)-induced apoptosis and its underlying mechanism. A low dose of kurarinone had no significant effect on apoptosis, but this compound markedly promoted tumor cell death through elevation of Bid cleavage, cytochrome c release release and caspase activation in HeLa cells treated with TRAIL. Caspase inhibitors inhibited kurarinone-mediated cell death, which indicates that the cytotoxic effect of this compound is mediated by caspase-dependent apoptosis. The cytotoxic effect of kurarinone was not associated with expression levels of Bcl-2 and IAP family proteins, such as Bcl-2, Bcl-xL, Bid, Bad, Bax, XIAP, cIAP-1 and cIAP-2. In addition, this compound did not regulate the death-inducing receptors DR4 and DR5. On the other hand, kurarinone significantly inhibited TRAIL-induced IKK activation, IκB degradation and nuclear translocation of NF-κB, as well as effectively suppressed cellular FLICE-inhibitory protein long form (cFLIPL) expression. The synergistic effects of kurarinone on TRAIL-induced apoptosis were mimicked when kurarinone was replaced by the NF-κB inhibitor withaferin A or following siRNA-mediated knockdown of cFLIPL. Moreover, cFLIP overexpression effectively antagonized kurarinone-mediated TRAIL sensitization. These data suggest that kurarinone sensitizes TRAIL-induced tumor cell apoptosis via suppression of NF-κB-dependent cFLIP expression, indicating that this compound can be used as an anti-tumor agent in combination with TRAIL.

Functional dissection of Nrf2-dependent phase II genes in vascular inflammation and endotoxic injury using Keap1 siRNA.

Keap1 is a cytoplasmic repressor of the transcription factor Nrf2, and its degradation induces Nrf2 activation, leading to upregulation of antioxidant phase II genes. We investigated the roles of phase II genes in vascular inflammation and septic injury using Keap1 siRNA and elucidated its underlying mechanism. Selective knockdown of Keap1 with siRNA promoted Nrf2-dependent expression of phase II genes in endothelial cells, such as heme oxygenase-1 (HO-1), glutamate-cysteine ligase (GCL), and peroxiredoxin-1 (Prx1), resulting in the elevation of cellular glutathione levels and suppression of tumor necrosis factor (TNF)-α-induced intracellular H(2)O(2) accumulation. Keap1 knockdown inhibited TNF-α-induced expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) by suppressing NF-κB activation via inhibition of its upstream modulators, Akt, NIK, and IKK, resulting in the elevation of monocyte adhesion to endothelial cells. Importantly, these events were reversed by HO-1 and GCL inhibitors and Prx1-specific siRNA. Keap1 knockdown also inhibited endotoxin-induced expression of inducible nitric oxide synthase (iNOS) and TNF-α by upregulating HO-1, GCL, and Prx1 expression in macrophages. Moreover, in vivo Keap1 knockdown increased the expression of phase II genes and suppressed the expression of ICAM-1, VCAM-1, iNOS, and TNF-α in an endotoxemic mouse model, resulting in significant protection against liver and lung injuries and lethality. Our results indicate that Keap1 knockdown prevents NF-κB-mediated vascular inflammation and endotoxic shock by suppressing NF-κB-mediated inflammatory gene expression via upregulation of Nrf2-mediated antioxidant genes. Thus, siRNA targeting Keap1 may provide a new therapeutic approach for inflammation-associated vascular diseases and sepsis.

Bi-level optimizing control of a simulated moving bed process with nonlinear adsorption isotherms.

A bi-level optimizing control scheme originally proposed for a simulated moving bed (SMB) with linear isotherms has been extended to an SMB with nonlinear isotherms. Cyclic steady state optimization is performed in the upper level to determine the optimum switching period and time-varying feed/desorbent flow rates, and repetitive model predictive control is run in the lower level for purity regulation, taking the decision variables from the upper level as feed-forward information. Experimental as well as numerical study for an SMB process separating a high-concentration mixture of aqueous L-ribose and L-arabinose solutions showed that the proposed scheme performs satisfactorily against various disturbances. In contrast, an alternative scheme based on an SMB model with linear isotherms showed a limitation in the control performance; this scheme was apt to fail in purity regulation.

Prx1 enhances androgen receptor function in prostate cancer cells by increasing receptor affinity to dihydrotestosterone.

Androgen receptor (AR) signaling plays a critical role in the development and progression of prostate cancer. It has been reported previously that peroxiredoxin-1 (Prx1), a member of a novel family of peroxidases, interacts physically with AR to enhance AR transactivation of target genes. In the present study, we evaluated the biological significance of Prx1 in modulating dihydrotestosterone (DHT)-stimulated growth and AR target gene expression of prostate cancer cells. We also investigated the mechanism by which Prx1 might potentiate AR signaling. The contribution of Prx1 was assessed mainly by using the approach of stable Prx1 knockdown. The major observations are as follows: (a) A low level of Prx1 desensitizes cells to growth stimulation and AR target gene induction by DHT, such that exposure to a higher level of DHT is required to reach the same magnitude of response when Prx1 is depressed; (b) Prx1 increases the affinity of AR to DHT and decreases the rate of DHT dissociation from the occupied receptor; (c) Prx1 enhances the NH2 terminus and COOH terminus interaction of AR; a stronger N-C interaction is consistent with a more robust AR activation signal by keeping DHT tight in the ligand-binding pocket; (d) the stimulatory effects of Prx1 on AR ligand binding affinity and AR N-C interaction are manifested regardless of a wild-type or mutant AR. The above findings led us to believe that Prx1 may be a therapeutic target in blocking the transition of prostate cancer from an androgen-dependent to an androgen-refractory phenotype.

Spin dimer analysis of the magnetic structures of Ba3Cr2O8, Ba3Mn2O8, Na4FeO4, and Ba2CoO4 with a three-dimensional network of isolated MO4 (M = Cr, Mn, Fe, Co) tetrahedra.

The spin exchange interactions of the magnetic oxides Ba3Cr2O8, Ba3Mn2O8, Na4FeO4, and Ba2CoO4 with a three-dimensional network of isolated MO4 (M = Cr, Mn, Fe, Co) tetrahedra were examined by performing spin dimer analysis on the basis of tight-binding electronic structure calculations. Although the shortest O...O distances between adjacent MO4 tetrahedra are longer than the van der Waals distance, our analysis shows that the super-superexchange interactions between adjacent MO4 tetrahedra are substantial and determine the magnetic structures of these oxides. In agreement with experiment, our analysis predicts a weakly interacting isolated AFM dimer model for both Ba3Cr2O8 and Ba3Mn2O8, the (0.0, 0.5, 0.0) magnetic superstructure for Na4FeO4, the (0.5, 0.0, 0.5) magnetic superstructure for Ba2CoO4, and the presence of magnetic frustration in Ba2CoO4. The comparison of the intra- and interdimer spin exchange interactions of Ba3Cr2O8 and Ba3Mn2O8 indicates that orbital ordering should be present in Ba3Cr2O8.

Amiloride potentiates TRAIL-induced tumor cell apoptosis by intracellular acidification-dependent Akt inactivation.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor gene family, is considered as one of the most promising cancer therapeutic agents due to its ability to selectively induce tumor cell apoptosis. In this study, we investigated whether the Na(+)/H(+) exchanger inhibitor, amiloride, promotes TRAIL-induced apoptotic death both in sensitive and resistant tumor cells, HeLa and LNCaP cells, respectively, and its underlying molecular mechanism. Amiloride enhanced TRAIL-induced apoptosis and activation of caspase-3 and -8 in both cells. This compound increased TRAIL-induced mitochondrial cytochrome c release and poly(ADP-ribose) polymerase cleavage. Moreover, amiloride-induced intracellular acidification, and inhibited the phosphorylated activation of the serine/threonine kinase Akt, which is known to promote cell survival, in both tumor cells. These data suggest that amiloride sensitizes both tumor cells to TRAIL-induced apoptosis by promoting Akt dephosphorylation and caspase-8 activation via the intracellular acidification and that Na(+)/H(+) exchanger inhibitors may play an important role in the anti-cancer activity of TRAIL, especially, in TRAIL-resistant tumors with highly active and expressed Akt.

Complestatin prevents apoptotic cell death: inhibition of a mitochondrial caspase pathway through AKT/PKB activation.

Complestatin, a bicyclo hexapeptide from Streptomyces, was isolated as a possible regulator of neuronal cell death. In this study, we report an anti-apoptotic activity of complestatin and its underlying molecular mechanism. Complestatin blocked TRAIL (TNF-related apoptosis-inducing ligand)-induced apoptosis and activation of caspase-3 and -8 at micromolar concentration levels without inhibiting the catalytic activities of these caspases. Complestatin potently induced a rapid and sustained AKT/PKB activation and Bad phosphorylation, resulting in inhibition of mitochondrial cytochrome c release. These anti-apoptotic activities of complestatin were significantly abrogated in cells expressing dominant negative AKT/PKB. Taken together, our results suggest that complestatin prevents apoptotic cell death via AKT/PKB-dependent inhibition of the mitochondrial apoptosis signal pathway. The novel property of complestatin may be valuable for developing new pharmaceutical means that will control unwanted cell death.

Astaxanthin inhibits nitric oxide production and inflammatory gene expression by suppressing I(kappa)B kinase-dependent NF-kappaB activation.

Astaxanthin, a carotenoid without vitamin A activity, has shown anti-oxidant and anti-inflammatory activities; however, its molecular action and mechanism have not been elucidated. We examined in vitro and in vivo regulatory function of astaxanthin on production of nitric oxide (NO) and prostaglandin E2 (PGE2) as well as expression of inducible NO synthase (iNOS), cyclooxygenase-2, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta). Astaxanthin inhibited the expression or formation production of these proinflammatory mediators and cytokines in both lipopolysaccharide (LPS)-stimulated RAW264.7 cells and primary macrophages. Astaxanthin also suppressed the serum levels of NO, PGE2, TNF-alpha, and IL-1beta in LPS-administrated mice, and inhibited NF-kappaB activation as well as iNOS promoter activity in RAW264.7 cells stimulated with LPS. This compound directly inhibited the intracellular accumulation of reactive oxygen species in LPS-stimulated RAW264.7 cells as well as H2O2-induced NF-kappaB activation and iNOS expression. Moreover, astaxanthin blocked nuclear translocation of NF-kappaB p65 subunit and I(kappa)B(alpha) degradation, which correlated with its inhibitory effect on I(kappa)B kinase (IKK) activity. These results suggest that astaxanthin, probably due to its antioxidant activity, inhibits the production of inflammatory mediators by blocking NF-kappaB activation and as a consequent suppression of IKK activity and I(kappa)B-alpha degradation.

Nitric oxide (NO) pretreatment increases cytokine-induced NO production in cultured rat hepatocytes by suppressing GTP cyclohydrolase I feedback inhibitory protein level and promoting inducible NO synthase dimerization.

Nitric oxide (NO) regulates the biological activity of many enzymes and other functional proteins as well as gene expression. In this study, we tested whether pretreatment with NO regulates NO production in response to cytokines in cultured rat hepatocytes. Hepatocytes were recovered in fresh medium for 24 h following pretreatment with the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) and stimulated to express the inducible NO synthase (iNOS) with interleukin-1beta and interferon-gamma or transfected with the human iNOS gene. NO pretreatment resulted in a significant increase in NO production without changing iNOS expression for both conditions. This effect, which did not occur in macrophages and smooth muscle cells, was inhibited when NO was scavenged using red blood cells. Pretreatment with oxidized SNAP, 8-Br-cGMP, NO(2)(-), or NO(3)(-) did not increase the cytokine-induced NO production. SNAP pretreatment increased cytosolic iNOS activity measured only in the absence of exogenous tetrahydrobiopterin (BH(4)). SNAP pretreatment suppressed the level of GTP cyclohydrolase I (GTPCHI) feedback regulatory protein (GFRP) and increased GTPCHI activity without changing GTPCHI protein level. SNAP pretreatment also increased total cellular levels of biopterin and active iNOS dimer. These results suggest that SNAP pretreatment increased NO production from iNOS by elevating cellular BH(4) levels and promoting iNOS subunit dimerization through the suppression of GFRP levels and subsequent activation of GTPCHI.