Novel protein kinase C phosphorylated kinase inhibitor-matrine suppresses replication of hepatitis B virus via modulating the mitogen-activated protein kinase signal.

HBV (hepatitis B virus) infection still threatens human health. Therefore, it is essential to find new effective anti-HBV compounds. Here, we identified matrine as a novel inhibitor of PKC (protein kinase C) phosphorylated kinase by screening a natural compound library. After HepG2.215 cells were treated with matrine, we carried out a phosphorylated proteomics sequence study and analyzed the prediction of related kinase expression level. In the case of HBV infection, it was found that PKC kinase mediates the activation of mitogen-activated protein kinase (MAPK) signaling pathway known as son of sevenless (SOS) activation. It was also found that PKC kinase inhibits the expression of C-X-C Motif Chemokine Ligand 8 (CXCL8) by inhibiting the activity of activating transcription factor 2/ cAMP response element binding protein (ATF2/CREB), and this effect is independent of its activated MAPK signaling pathway. Finally, Western blot was used to detect the expression of MAPK, ATF2, CREB3 phosphorylation and nonphosphorylation in matrine-treated cells and PKC-treated cells. PKC phosphorylated kinase inhibitor-matrine suppresses the replication of HBV via modulating the MAPK/ATF2 signal. Matrine is a good clinical drug to enhance the autoimmunity in the adjuvant treatment of chronic HBV infection.

Vasorelaxing cell permeant phosphopeptide mimetics for subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) due to rupture of an intracranial aneurysm leads to vasospasm resulting in delayed cerebral ischemia. Therapeutic options are currently limited to hemodynamic optimization and nimodipine, which have marginal clinical efficacy. Nitric oxide (NO) modulates cerebral blood flow through activation of the cGMP-Protein Kinase G (PKG) pathway. Our hypothesis is that SAH results in downregulation of signaling components in the NO-PKG pathway which could explain why treatments for vasospasm targeting this pathway lack efficacy and that treatment with a cell permeant phosphopeptide mimetic of downstream effector prevents delayed vasospasm after SAH. Using a rat endovascular perforation model, reduced levels of NO-PKG pathway molecules were confirmed. Additionally, it was determined that expression and phosphorylation of a PKG substrate: Vasodilator-stimulated phosphoprotein (VASP) was downregulated. A family of cell permeant phosphomimetic of VASP (VP) was wasdesigned and shown to have vasorelaxing property that is synergistic with nimodipine in intact vascular tissuesex vivo. Hence, treatment targeting the downstream effector of the NO signaling pathway, VASP, may bypass receptors and signaling elements leading to vasorelaxation and that treatment with VP can be explored as a therapeutic strategy for SAH induced vasospasm and ameliorate neurological deficits.

Mimosine increases the expressions of tyrosine phosphorylated protein in mouse seminal vesicles / La mimosina aumenta la expresión de la proteína tirosina fosforilada en las vesículas seminales del ratón

Acute effect of purified mimosine (MiMo) extracted from Leucaena leucocephala on testicular histopathology has been documented with seminal vesicle (SV) atrophy. Since protein phosphorylation and seminal secretions play important roles in sperm physiology, this study aimed to study the alteration of substances including tyrosine phosphorylated (TyrPho) proteins in seminal vesicle treated with MiMo. Male mice were divided into a control and experimental groups treated with purified MiMo at 3 doses of 15, 30, and 60 mg/KgBW, respectively for 35 consecutive days. The morphology and weights of SV were compared among groups. The levels of magnesium and fructosamine in SV fluid were assayed. The profiles of equally SV total proteins were compared using SDS-PAGE. The expression of seminal TyrPho proteins was detected by western blotting. Recent results showed the decreased weights of SV in MiMo treated mice compared to control. However MiMo in all doses did not affect the levels of magnesium and fructosamine in SV fluid. The SV protein expression of 130 and 55 kDas was obviously decreased in a high dose MiMo. In dose-dependent response, the expressions of 72 and 55 kDas TyrPho proteins of SV were increased. In conclusion, MiMo could affect SV morphological size and protein secretions especially TyrPho proteins.

El efecto agudo de la mimosina purificada (MiMo) extraída de Leucaena leucocephala en la histopatología testicular se ha documentado con atrofia de vesícula seminal (VS). Debido a que la fosforilación de proteínas y las secreciones seminales tienen un papel importante en la fisiología de los espermatozoides, este estudio tuvo como objetivo estudiar la alteración de sustancias como la proteína tirosina fosforilada (TyrPho) en vesículas seminales tratadas con MiMo. Los ratones se dividieron en un grupo control y un grupo experimental y se trataron con MiMo purificado en 3 dosis de 15, 30 y 60 mg / KgBW, respectivamente, durante 35 días seguidos. La morfología y los pesos de VS se compararon entre los grupos. Fueron analizados los niveles de magnesio y fructosamina en el fluido VS. Los perfiles de las proteínas totales de VS se compararon utilizando SDS-PAGE. La expresión de la proteína TyrPho en las vesículas seminales se detectó mediante transferencia de Western blot. Los resultados recientes muestran la disminución del peso de las VS en ratones tratados con MiMo, en comparación con el grupo control. Sin embargo, en ninguna de las dosis se vieron afectados por mimosina purificada los niveles de magnesio y fructosamina en el líquido de las VS. La expresión de la proteína en VS de 130 y 55 kDas disminuyó notablemente en una dosis alta de MiMo. En la respuesta dependiente de la dosis, aumentaron las expresiones de 72 y 55 kDas de las proteínas TyrPho en las VS. En conclusión, la mimosina purificada podría afectar el tamaño morfológico de las VS y la expresión de proteínas, especialmente las proteínas TyrPho.

Polyphenol extracts from dried sugarcane inhibit inflammatory mediators in an in vitro colon cancer model.

Sugarcane is an important crop grown in tropical regions for sugar, and for ethanol production. Sugarcane is also a source of phytochemicals but its nutraceutical potential has been under-explored. We show that ethanol extracts of whole dried sugarcane (WDS) recovers a rich content of polyphenols, flavonoids and antioxidant activity that act on inflammatory mediator proteins. To investigate the mechanisms of this activity, we stimulated SW480 colon cancer cells with lipopolysaccharide, exposed cells to WDS and quantitated changes to the proteome and phosphoproteome using label-free mass spectrometry. The grape-derived anti-inflammatory polyphenol, resveratrol (RSV) was used as a control. Using SWATH-MS we quantitated ~3000 proteins showing that WDS significantly altered the expression of the oxidative stress regulator SELH. WDS induced changes in protein expression predicted the involvement of NFκB pathway members. Reduced NFκB phosphorylation and IL-8 secretion confirmed this effect. In contrast, RSV was predicted to act primarily through modulation of the PI3K/AKT pathway. Phosphoproteomics studies indicate that WDS interfered in the phosphorylation of cell stress regulators c-Jun, EGFR, PKA, PKCß and SIRT1. Confirmed through pharmacological inhibition, kinase enrichment analysis presented C-Raf to modulate WDS activity. These results demonstrate the anti-inflammatory utility of WSD and define aspects of its mechanisms of action. SIGNIFICANCE: Despite the increasing interest of nutraceuticals in health promotion, scientific evidence proving the molecular mechanisms involved is still lacking. This study investigated some of the mechanistic aspects of in vitro use of whole dried sugarcane extracts in the context of regulating cellular inflammation by using proteomics and phosphoproteomics strategies. We determined that WDS extracts regulate key inflammatory pathways including NFκB, while kinase enrichment analysis from phosphoproteomics demonstrated a role for C-Raf in controlling this mechanism. We demonstrated that the mechanism of WDS extracts on controlling inflammation differs from that of the polyphenol, resveratrol. The results presented herein contribute towards unravelling the activity of nutraceuticals extracted from sugarcane.

Fourth-ventricle leptin infusions dose-dependently activate hypothalamic signal transducer and activator of transcription 3.

Previous studies have shown that very low-dose infusions of leptin into the third or the fourth ventricle alone have little effect on energy balance, but simultaneous low-dose infusions cause rapid weight loss and increased phosphorylation of STAT3 (p-STAT3) in hypothalamic sites that express leptin receptors. Other studies show that injecting high doses of leptin into the fourth ventricle inhibits food intake and weight gain. Therefore, we tested whether fourth-ventricle leptin infusions that cause weight loss are associated with increased leptin signaling in the hypothalamus. In a dose response study 14-day infusions of increasing doses of leptin showed significant hypophagia, weight loss, and increased hypothalamic p-STAT3 in rats receiving at least 0.9 µg leptin/day. In a second study 0.6 µg leptin/day transiently inhibited food intake and reduced carcass fat, but had no significant effect on energy expenditure. In a final study, we identified the localization of STAT3 activation in the hypothalamus of rats receiving 0, 0.3, or 1.2 µg leptin/day. The high dose of leptin, which caused weight loss in the first experiment, increased p-STAT3 in the ventromedial, dorsomedial, and arcuate nuclei of the hypothalamus. The low dose that increased brown fat UCP1 but did not affect body composition in the first experiment had little effect on hypothalamic p-STAT3. We propose that hindbrain leptin increases the precision of control of energy balance by lowering the threshold for leptin signaling in the forebrain. Further studies are needed to directly test this hypothesis.

Effect of Alkaloids from Nelumbinis Plumula against Insulin Resistance of High-Fat Diet-Induced Nonalcoholic Fatty Liver Disease in Mice.

This study aimed to investigate the effects of total alkaloids from Nelumbinis Plumula (NPA) on insulin resistance (IR) of high-fat diet- (HFD-) induced nonalcoholic fatty liver disease (NAFLD). Rats were fed with HFD for 8 weeks to induce NAFLD. Then, the effect of NPA on ameliorating IR in HFD-induced NAFLD was evaluated. Fasting serum insulin was determined using an enzyme-linked immunosorbent assay (ELISA) kit for insulin following the manufacturer's protocol. Some inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) were determined using ELISA kits to assess the inflammatory burden in rats. The results showed that HFD could induce a significant increase in blood glucose and IR in rats. However, rats treated with NPA (400 or 600 mg/kg) showed improved IR and reduction in serum inflammatory cytokines TNF-α and IL-6. Further investigation indicated that NPA could inhibit IR by restoring the insulin receptor substrate-1 (IRS-1) and suppressing the expression of c-Jun N-terminal kinase (JNK) phosphorylation. The present results supported the view that the pathogenesis of NAFLD was complex with inflammation, together with increasing serum glucose and IR. Also, JNK and IRS phosphorylation were suggested for their involvement in the modulating of IR during NAFLD progression. Therefore, NPA may serve as a potential natural remedy against IR in NAFLD.

Scutellarin attenuates endothelium-dependent aasodilation impairment induced by hypoxia reoxygenation, through regulating the PKG signaling pathway in rat coronary artery.

Scutellarin (SCU), a flavonoid from a traditional Chinese medicinal plant. Our previous study has demonstrated that SCU relaxes mouse aortic arteries mainly in an endothelium-depend-ent manner. In the present study, we investigated the vasoprotective effects of SCU against HR-induced endothelial dysfunction (ED) in isolated rat CA and the possible mechanisms involving cyclic guanosine monophosphate (cGMP) dependent protein kinase (PKG). The isolated endothelium-intact and endothelium-denuded rat CA rings were treated with HR injury. Evaluation of endothelium-dependent and -independent vasodilation relaxation of the CA rings were performed using wire myography and the protein expressions were assayed by Western blotting. SCU (10-1 000 µmol·L(-1)) could relax the endothelium-intact CA rings but not endothelium-denuded ones. In the intact CA rings, the PKG inhibitor, Rp-8-Br-cGMPS (PKGI-rp, 4 µmol·L(-1)), significantly blocked SCU (10-1 000 µmol·L(-1))-induced relaxation. The NO synthase (NOS) inhibitor, NO-nitro-L-arginine methylester (L-NAME, 100 µmol·L(-1)), did not significantly change the effects of SCU (10-1 000 µmol·L(-1)). HR treatment significantly impaired ACh-induced relaxation, which was reversed by pre-incubation with SCU (500 µmol·L(-1)), while HR treatment did not altered NTG-induced vasodilation. PKGI-rp (4 µmol·L(-1)) blocked the protective effects of SCU in HR-treated CA rings. Additionally, HR treatment reduced phosphorylated vasodilator-stimulated phosphoprotein (p-VASP, phosphorylated product of PKG), which was reversed by SCU pre-incubation, suggesting that SCU activated PKG phosphorylation against HR injury. SCU induces CA vasodilation in an endothelium-dependent manner to and repairs HR-induced impairment via activation of PKG signaling pathway.

Resveratrol appears to protect against oxidative stress and steroidogenesis collapse in mice fed high-calorie and high-cholesterol diet.

The detrimental effects on Leydig cells steroidogenesis in mice on high-calorie and high-cholesterol diet (HCD) were determined, and the possible protection conferred by resveratrol supplementation was investigated. Male C57BL/6J mice were fed high-calorie and alone (HCD group) or with resveratrol supplementation (HCD + Res group) for 18 weeks. Male C57BL/6J mice fed standard diet without or with the same dose of resveratrol served as controls. At the end of the experiment, there were significant declines of serum testosterone and luteinising hormone (LH) in HCD group as compared to controls. In line with the hormone alterations, the expressions of StAR and steroidogenic enzymes in testicular tissues were significantly down-regulated in HCD group. Resveratrol supplementation could significantly improve expressions of StAR and steroidogenic enzymes, and increase serum testosterone and LH concentrations in HCD + Res group. Mice in HCD group also showed a statistically significant down-regulation in the mRNA expressions of MnSOD and GPx4. Resveratrol supplementation improved testicular MnSOD and GPx4 expression in comparison with HCD group. We propose that resveratrol may attenuate detrimental effects on Leydig cells steroidogenesis in HCD-fed mice, and its upregulations of antioxidant defence mechanisms and LH level may play a role in its protection. Our data suggest resveratrol appears to have the potential for therapeutic approaches targeting male obesity-associated secondary hypogonadism.

Effects of a triple antibiotic solution on pulpal dynamics after intentionally delayed tooth replantation in mice.

INTRODUCTION: This study analyzed the detailed biological events underlying pulpal dynamics evoked by 3Mix (the mixture of ciprofloxacin, metronidazole, and minocycline) solution after intentionally delayed tooth replantation because 3Mix improves pulpal healing after tooth injuries. METHODS: The maxillary first molars of 3-week-old mice were extracted and immersed in 3Mix solution for 30 minutes in comparison with phosphate buffered saline (PBS) alone. Cell proliferation, apoptosis, and differentiation were assessed in extracted/replanted teeth during days 0-14 using immunohistochemistry, apoptosis assay, and reverse-transcriptase polymerase chain reaction. RESULTS: 3Mix solution accelerated odontoblast differentiation in the coronal pulp on day 7 and tertiary dentin formation on day 14, whereas the regenerative process was delayed in the PBS group. Cell proliferation and apoptosis occurred in the pulp of the 3Mix group during days 5-7 and subsequently decreased from days 7-14. On day 5, dentin sialophosphoprotein and nestin were first recovered in the 3Mix group, whereas expression levels for alkaline phosphatase, osteopontin, and osteocalcin increased in the PBS group. The expression levels for octamer-binding factor 3/4A and 3/4B reached the maximum level on day 1 and were sharply decreased on day 3 in both groups. High expression levels of Cd11c were first observed in the 3Mix group on day 1 and later at days 5 and 7. CONCLUSIONS: The results suggest that the application of 3Mix may suppress osteoblast differentiation by the migration of dendritic cells to the injury site and via the activation of stem/progenitor cells, resulting in the acceleration of odontoblastlike cell differentiation.

Regenerative capacity of human dental pulp and apical papilla cells after treatment with a 3-antibiotic mixture.

INTRODUCTION: A 3-antibiotic combination (3Mix) has been widely used in regenerative endodontics. Recent studies recommend that a safe concentration of 3Mix is in the range of 0.39 µg/mL and 1 mg/mL because higher concentrations may limit tissue regeneration. The aim of this study was to determine the regenerative capacity of isolated human dental pulp cells (DPCs) and apical papilla cells (APCs) after a 7-day treatment with selected doses of 3Mix. METHODS: Primary human DPCs/APCs from the third passage were divided into control and experimental groups. In the control group, cells were cultured in regular complete media. In the experimental group, cells were cultured in complete media containing 0.39 µg/mL or 1 mg/mL of 3Mix for 7 days. After the treatment period, the media were changed, and the cells were further tested for proliferation and differentiation potential. For cell proliferation, a colorimetric qualification of 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide was used on days 1, 3, 5, and 7. For differentiation potential, a dentinogenic differentiation medium was added into treated cells and cultured for 7, 14, and 21 days. Results were analyzed using quantitative alizarin red S staining and real-time reverse-transcription polymerase chain reaction. RESULTS: After 7 days of treatment, 100% cell death was discovered in the 1-mg/mL 3Mix group. The proliferative capacity of 0.39 µg/mL 3Mix-treated DPCs and APCs was significantly lower than that of untreated cells at all time points (P < .05). Mineralized nodule formation was found both in the 3Mix-treated and control groups, but it was significantly less in the 3Mix-treated groups at 7, 14, and 21 days (P < .01). Quantitative reverse-transcription polymerase chain reaction showed no statistically significant difference (95% confidence interval) in bone sialoprotein, alkaline phosphatase, and dentin matrix protein 1 gene expression in either 3Mix-treated DPCs or APCs compared with control groups. CONCLUSIONS: One milligram per milliliter of 3Mix had strong toxicity to DPCs/APCs when applied for 7 days, whereas 0.39 µg/mL 3Mix showed no toxicity but still affected cell proliferation and mineralization potential. However, no differences in dentinogenic gene expressions were observed between the 3Mix-treated and untreated groups.

Cyclopia maculata (honeybush tea) stimulates lipolysis in 3T3-L1 adipocytes.

We have previously, for the first time, demonstrated that hot water extracts of Cyclopia maculata and Cyclopia subternata, endemic South African plants that are consumed as herbal teas, inhibit adipogenesis in 3T3-L1 adipocytes. The aim of this study was to extend the anti-obesity investigations of these plants by quantifying lipolysis in mature 3T3-L1 adipocytes. Glycerol concentration in culture supernatants was used as a marker of adipocyte lipolysis. Isoproterenol, a ß-adrenergic agonist and a known lipolytic agent, was used as a positive control in our assays. Lipolysis was stimulated by all extracts, although statistical significance was noted for fermented (oxidised) C. maculata only. A concentration of 80µg/ml of C. maculata extract induced maximal lipolysis (1.8-fold, p<0.001). The increased lipolysis was accompanied by an increase in the expression of hormone sensitive lipase (1.6-fold, p<0.05) and perilipin (1.6-fold, p<0.05). The plant extracts, at the concentration range assayed (0-100µg/ml), were not cytotoxic in terms of mitochondrial dehydrogenase and adenosine-5'-triphosphate activity. These results showed that C. maculata stimulates lipolysis in mature 3T3-L1 adipocytes, providing further support for the anti-obesity effects of Cyclopia spp.

Effect of methotrexate and leucovorin on female reproductive tract of albino rats.

Methotrexate (MTX) an antifolate drug and leucovorin its antidote, are used in the treatment of both neoplastic and non-neoplastic diseases in young women. We hypothesize that MTX treatment might comprise a deleterious effect on fast proliferating reproductive cells, an unavoidable and unwanted side effect. MTX given dose dependently to rats for 20 days prevented vaginal cyclicity and caused a reduction in serum progesterone and estradiol. External morphology of reproductive tract displayed thinning of organs and reduction in their weights. To reveal mechanism of MTX action, we examined the histology of ovary, oviduct, uterus, cervix and vagina. Results suggested that in a dose-dependent fashion MTX restrained preantral and antral follicular growth in ovary. Epithelium and stroma of oviduct, uterus, cervix and vagina were disrupted and lost their normal structures. Such alterations in ovarian function raised serum follicle stimulating hormone, luteinizing hormonal profiles. Expression of steroidogenic acute regulatory protein and P450 cholesterol side chain cleavage gene, which are both essential for steroidogenesis, markedly decreased in ovary upon MTX treatment. Total RNA, DNA and protein concentrations, glucose 6 phosphate dehydrogenase, lactate dehydrogenase and alkaline phosphatase enzyme activities in ovary were distinctly altered. Leucovorin supplementation and withdrawal of the treatment, improved MTX caused effects partially. These results for the first time indicate that the malfunction of female reproductive organs by MTX treatment in young women is not only correlated to the disrupted circulating levels of hormones and histoarchitecture of tissues but also discrepancies in steroidogenic genes and hormone regulated enzyme activities in ovary.

In vitro cytocompatibility of N-acetylcysteine-supplemented dentin bonding agents.

INTRODUCTION: Cytotoxic resin components of dentin bonding agents are known to cause oxidative damage and suppress odontogenic differentiation of dental pulp cells. Because antioxidants were found to protect cells from cytotoxicity of resin monomers in previous studies, we investigated the effect of N-acetylcysteine (NAC) on cytotoxicity and anti-differentiation activity of bonding agents. METHODS: Human dental pulp cells were treated with the extracts of dentin bonding agents (Prime & Bond NT, Adper Single Bond, and Dentin Cement), and then cell viability, alkaline phosphatase (ALP) activity, and matrix mineralization were observed. To assess the effects of NAC, NAC was directly added into culture media or mixed with bonding agents before extraction. Release of NAC from bonding agents was also observed by high-performance liquid chromatography. RESULTS: NAC enhanced ALP activity and mRNA expression of dentin sialophosphoprotein gene, whereas extracts of dentin bonding agents inhibited the induction of ALP activity. When the cells were treated with extracts of the bonding agents, the NAC in the culture media reduced the cytotoxicity of the bonding agents. When NAC was incorporated into bonding agents, a protective effect was only seen for Prime & Bond NT containing more than 1% NAC. The disruption of ALP activity and matrix mineralization in pulp cells was partially reversed by NAC only in Prime & Bond NT-treated cells. High-performance liquid chromatography analysis of NAC showed that the amount of NAC effluxed from Prime & Bond NT was not greater than that effluxed from Adper Single Bond. CONCLUSIONS: NAC was useful for reversing cytotoxicity and anti-differentiation effects of Prime & Bond NT on human dental pulp cells.

1,2-vinyldithiin from garlic inhibits differentiation and inflammation of human preadipocytes.

Obesity is a state of chronic low-grade inflammation. Limiting white adipose tissue (WAT) expansion and therefore reducing inflammation could be effective in preventing the progression of obesity and the development of associated complications. We investigated the effects of 1,2-vinyldithiin (1,2-DT), a garlic-derived organosulfur, on the differentiation and inflammatory state of human preadipocytes. Preadipocytes were prepared from subcutaneous adipose tissue of nonobese young women and differentiated in the presence of 1,2-DT. Inflammatory preadipocytes were obtained following treatment with human macrophage-secreted factors. 1,2-DT (100 micromol/L) significantly reduced gene expression of PPARgamma2 (-40%), CCAAT/enhancer binding protein-alpha (-25%), lipoprotein lipase (-22%), leptin (-30%), and adiponectin (-15%). Lipid accumulation was also significantly diminished in preadipocytes differentiated in the presence of 100 micromol/L 1,2-DT (-37%) compared with controls. Furthermore, 100 micromol/L 1,2-DT treatment for 10 d significantly reduced PPARgamma activity (-27%). The protein expression of perilipin and the secretion levels for 2 adipokines, leptin and adiponectin, were significantly diminished in 1,2-DT-cultured preadipocytes (-37, -51, and -43%, respectively). Moreover, the secretion of inflammatory molecules (interleukin-6 and monocyte chemoattractant protein-1) induced by macrophage-secreted factors was partially abolished in 100 micromol/L 1,2-DT-treated preadipocytes (-28 and -25%, respectively). In conclusion, we demonstrated that 1,2-DT, a garlic-derived organosulfur, has antiadipogenic and antiinflammatory actions on human preadipocytes and may be a novel, antiobesity nutraceutical.

Structure-based drug design: from nucleic acid to membrane protein targets.

The in silico methods for drug discovery are becoming increasingly powerful and useful. That, in combination with increasing computer processor power, in our case using a novel distributed computing grid, has enabled us to greatly enhance our virtual screening efforts. Herein we review some of these efforts using both receptor and ligand-based virtual screening, with the goal of finding new anti-cancer agents. In particular, nucleic acids are a neglected set of targets, especially the different morphologies of duplex, triplex, and quadruplex DNA, many of which have increasing biological relevance. We also review examples of molecular modeling to understand receptors and using virtual screening against G-protein coupled receptor membrane proteins.

Involvement of oxidative pathways in cytokine-induced secretory phospholipase A2-IIA in astrocytes.

Recent studies have suggested the involvement of secretory phospholipase A2-IIA (sPLA2-IIA) in neuroinflammatory diseases. Although sPLA2-IIA is transcriptionally induced through the NF-kappaB pathway by pro-inflammatory cytokines, whether this induction pathway is affected by other intracellular signaling pathways has not been investigated in detail. In this study, we demonstrated the induction of sPLA2-IIA mRNA and protein expression in astrocytes by cytokines and detected the protein in the culture medium after stimulation. We further investigated the effects of oxidative pathways and botanical antioxidants on the induction pathway and observed that IL-1beta-induced sPLA2-IIA mRNA expression in astrocytes is dependent on ERK1/2 and PI-3 kinase, but not p38 MAPK. In addition to apocynin, a known NADPH oxidase inhibitor, botanical antioxidants, such as resveratrol and epigallocatechin gallate, also inhibited IL-1beta-induced sPLA2-IIA mRNA expression. These compounds also suppressed IL-1beta-induced ERK1/2 activation and translocation of the NADPH oxidase subunit p67 phox from cytosol to membrane fraction. Taken together, these results support the involvement of reactive oxygen species from NADPH oxidase in cytokine induction of sPLA2-IIA in astrocytes and promote the use of botanical antioxidants as protective agents for inhibition of inflammatory responses in these cells.

Evodiamine and rutaecarpine inhibit migration by LIGHT via suppression of NADPH oxidase activation.

LIGHT acted as a new player in the atherogenesis. The dried, unripe fruit of Evodia Fructus (EF) has long been used as a traditional Chinese herbal medicine, and is currently widely used for the treatment of headache, abdominal pain, vomiting, colds and reduced blood circulation. Evodiamine and rutaecarpine are active components of EF. In this study, we investigated the inhibitory effect of evodiamine and rutaecarpine on LIGHT-induced migration in human monocytes. Evodiamine and rutaecarpine decreased the LIGHT-induced production of ROS, IL-8, monocyte chemoattractant protein-1 (MCP-1), TNF-alpha, and IL-6, as well as the expression of chemokine receptor (CCR) 1, CCR2 and ICAM-1 and the phosphorylation of the ERK 1/2 and p38 MAPK. Furthermore, NADPH oxidase assembly inhibitor, AEBSF, blocked LIGHT-induced migration and activation of CCR1, CCR2, ICAM-1, and MAPK such as ERK and p38 in a manner similar to evodiamine and rutaecarpine. These findings indicate that the inhibitory effects of evodiamine and rutaecarpine on LIGHT-induced migration and the activation of CCR1, CCR2, ICAM-1, ERK, and p38 MAPK occurs via decreased ROS production and NADPH oxidase activation. Taken together, these results indicate that evodiamine and rutaecarpine have the potential for use as an anti-atherosclerosis agent.

N-acetyl cysteine (NAC)-assisted detoxification of PMMA resin.

Despite its proven cytotoxicity, poly-methyl methacrylate (PMMA) resin is one of the most frequently and extensively used materials in dental practice. This study hypothesized that an anti-oxidant amino acid, N-acetyl cysteine (NAC), has the potential to detoxify this material. Ten percent of the rat dental pulp cells were viable when cultured on the PMMA resin for 24 hours, while over 70% of the cells were viable on the NAC-added resin. Nearly all suppressed alkaline phosphatase activity, matrix mineralizing capability, and odontoblastic gene expression, such as dentin sialoprotein, on the untreated control resin was recovered by NAC in a concentration-dependent manner. A Ca/P ratio of 1.65 was found in the extracellular matrix of cultures on NAC-added resin, while that in the untreated resin culture was 0.70. The addition of NAC to PMMA resin significantly ameliorated its cytotoxicity to the dental pulp cells and restored their odontoblast-like cell phenotype to a biologically significant degree.

Altered protein synthesis is a trigger for long-term memory formation.

There is ongoing debate concerning whether new protein synthesis is necessary for, or even contributes to, memory formation and storage. This review summarizes a contemporary model proposing a role for altered protein synthesis in memory formation and its subsequent stabilization. One defining aspect of the model is that altered protein synthesis serves as a trigger for memory consolidation. Thus, we propose that specific alterations in the pattern of neuronal protein translation serve as an initial event in long-term memory formation. These specific alterations in protein readout result in the formation of a protein complex that then serves as a nidus for subsequent perpetuating reinforcement by a positive feedback mechanism. The model proposes this scenario as a minimal but requisite component for long-term memory formation. Our description specifies three aspects of prevailing scenarios for the role of altered protein synthesis in memory that we feel will help clarify what, precisely, is typically proposed as the role for protein translation in memory formation. First, that a relatively short initial time window exists wherein specific alterations in the pattern of proteins translated (not overall protein synthesis) is involved in initializing the engram. Second, that a self-perpetuating positive feedback mechanism maintains the altered pattern of protein expression (synthesis or recruitment) locally. Third, that other than the formation and subsequent perpetuation of the unique initializing proteins, ongoing constitutive protein synthesis is all that is minimally necessary for formation and maintenance of the engram. We feel that a clear delineation of these three principles will assist in interpreting the available experimental data, and propose that the available data are consistent with a role for protein synthesis in memory.

Oestrogen synthesis in the hippocampus: role in axon outgrowth.

Ovarian oestrogens have been postulated to be neuroprotective. It has also been shown that considerable amounts of oestrogens are synthesised in hippocampal neurones. In the present study, we focused on a potential role of hippocampus-derived oestradiol compared to gonad-derived oestradiol on axon outgrowth of hippocampal neurones. To address the role of hippocampus-derived oestradiol, we inhibited oestrogen synthesis by treatment of neonatal hippocampal cell cultures with letrozole, a specific aromatase inhibitor. As an alternative, we used siRNA against steroidogenic acute regulatory protein (StAR). Axon outgrowth and GAP-43 expression were significantly down-regulated in response to letrozole and in siRNA-StAR transfected cells. The effects after inhibition of oestrogen synthesis in response to letrozole and in siRNA-StAR transfected cells were reversed by oestrogen supplementation. No difference was found between ovariectomised animals, cycling animals at pro-oestrus and ovariectomised and subsequently oestradiol-treated animals. However, high pharmacological doses of oestradiol promoted axon outgrowth, which was possible to abolish by the oestrogen receptor antagonist ICI 182,780. Our results show that oestradiol-induced neurite outgrowth is very likely mediated by genomic oestrogen receptors and requires higher doses of oestradiol than physiological serum concentrations derived from the gonads.