Phytoremediation of Uranium-Contaminated Soil by Perennial Ryegrass (Lolium Perenne L.) Enhanced with Citric Acid Application

Perennial ryegrass (Lolium perenne L.) was planted in uranium-contaminated soil mixture (river sand and vermiculite mixed with equal volume ratio) supplemented with different amounts of citric acid (0, 1, 5, and 10 mmol/kg) and divided into 4 treatments (Con+0, Con+1, Con+5, and Con+10) to investigate the effects of citric acid concentrations on the remediation efficiency and enhanced mechanism of perennial ryegrass in the lab. The uranium content in the plant tissues showed that the roots were the predominant tissue for uranium accumulation, and the subcellular distributions of uranium in the root cells was in the order: cell wall fraction > cytosol fraction > organelle fraction. However, in the shoot cells the order was cell wall fraction > organelle fraction > cytosol fraction. In this study, the optimal concentration of citric acid added was 5 mmol/kg, and the removal efficiency of U in the shoots and roots increased by 47.37% and 30.10% respectively. The treatment with 5 mmol/kg citric acid had the highest contents of photosynthetic pigment and soluble protein, the highest activity of antioxidant enzymes, and the lowest content of MDA (malondialdehyde) and relative electrical conductivity. Moreover, the damage to the cell ultrastructure of perennial ryegrass observed by TEM (transmission electron microscope) was significantly alleviated when 5 mmol/kg citric acid was added. All results indicate that perennial ryegrass can accumulate uranium with elevated uranium tolerance and enrichment ability when 5 mmol/kg citric acid is added under uranium stress. These results suggest that citric acid has significant effects on improving the uranium phytoremediation potential of perennial ryegrass.

Dysregulation of protein kinase C in adult depression and suicide: evidence from postmortem brain studies

Background Several lines of evidence suggest the abnormalities of protein kinase C (PKC) signaling system in mood disorders and suicide based primarily on the studies of PKC and its isozymes in the platelets and postmortem brain of depressed and suicidal subjects. In this study we examined the role of PKC isozymes in depression and suicide. Methods We determined the protein and mRNA expression of various PKC isozymes in the prefrontal cortical region [Brodmann area 9 (BA9)] in 24 normal control (NC) subjects, 24 depressed suicide (DS) subjects and 12 depressed non-suicide (DNS) subjects. The levels of mRNA in the prefrontal cortex (PFC) were determined by qRT-PCR and the protein expression was determined by Western blotting. Results We observed a significant decrease in mRNA expression of PKCα, PKCβI, PKCδ and PKCε and decreased protein expression either in the membrane or the cytosol fraction of PKC isozymes - PKCα, PKCβI, PKCβII and PKCδ in DS and DNS subjects compared with NC subjects. Conclusions The current study provides detailed evidence of specific dysregulation of certain PKC isozymes in the postmortem brain of DS and DNS subjects and further supports earlier evidence for the role of PKC in the platelets and brain of adult and teenage depressed and suicidal population. This comprehensive study may lead to further knowledge of the involvement of PKC in the pathophysiology of depression and suicide.

Activation of Astroglial Connexin Is Involved in Concentration-Dependent Double-Edged Sword Clinical Action of Clozapine

Clozapine (CLZ) is a gold-standard antipsychotic against treatment-refractory schizophrenia, but is one of the most toxic antipsychotic agents. Pharmacological mechanisms of the double-edged sword clinical action of CLZ remain to be clarified. To explore the mechanisms of CLZ, the present study determined the astroglial transmission associated with connexin43 (Cx43), which is the most principal expression in astrocytes and myocardial cells, and expression of Cx43 in primary cultured astrocytes. Both acute and subchronic administrations of CLZ concentration-dependently increased Cx43-associated astroglial release of l-glutamate and d-serine, whereas therapeutic-relevant concentration of CLZ acutely did not affect but subchronically increased astroglial release. In contrast, after the subchronic administration of therapeutic-relevant concentration of valproate (VPA), acute administration of therapeutic-relevant concentration of CLZ drastically increased Cx43-associated astroglial releases. VPA increased Cx43 expression in cytosol fraction without affecting plasma membrane fraction, whereas CLZ increased Cx43 expression in both fractions. Acute administration of therapeutic-relevant concentration of CLZ drastically increased Cx43 expression in the plasma membrane fraction of astrocytes subchronically treated with VPA. The present findings suggest that CLZ-induced the activation of Cx43-associated channel activity and transported Cx43 to plasma membrane, probably contribute to the double-edged sword clinical action of CLZ, such as improvement of cognitive dysfunction and CLZ-induced myocarditis.

Abstract TP283: Phosphorylation of IRF5/4 by IRAK4/MyD88 Regulates Microglial Inflammatory Responses to Ischemia

Introduction: Interferon Regulatory Factors 5 and 4 (IRF5/4) have been suggested to play a determinant role in regulating pro-inflammatory (M1) or anti-inflammatory (M2) microglia/macrophage response. However, how IRF5/4 signaling are activated in these phagocytes has not been well understood. Our previous study has shown an increased level of phosphorylated-IRF4 in both the cytosolic and nuclear fraction of cultured microglial homogenates after ischemia, suggesting phosphorylation is important for IRF4 activation. Interleukin-1 receptor associated kinase 4 (IRAK4) and the adaptor protein MyD88 are the upstream signals of IRF5. In the present study we hypothesized that IRAK4/MyD88 phosphorylate IRF5/4 in ischemic microglia and trigger the translocation of IRF5/4 from the cytoplasm to the nucleus, to promote expression of inflammatory mediators. Methods: Spontaneously Immortalized (SIM)-A9 or primary microglia from C57BL/6 mice were cultured and exposed to oxygen-glucose deprivation (OGD) for 4 hours; after OGD the cells were reperfused overnight with glucose-containing medium in room air. Phosphorylation of IRF5/4 by IRAK4 was determined with IRAK4 inhibitor (ND-2158). The interaction between IRAK4/MyD88 with IRF5/4 and the nuclear translocation of phosphorylated-IRF5/4 were examined by immunocytochemistry (ICC) and co-immunoprecipitation (Co-IP)/Western Blots (WB). Production of inflammatory cytokines was measured by ELISA in the culture medium. Results: WB showed more robust nuclear p-IRF5 signals; ELISA found increased level of TNFα, IL-1β, and IL6 in OGD vs. normoxic microglia. A similar pattern was observed for p-IRF4 nuclear translocation but with moderately increased anti-inflammatory cytokine (IL-4 and IL-10) levels with OGD. Co-IP revealed a higher ratio of p-IRF5/4 over total IRF5/4 in the nuclear vs. cytosol fraction, and interactions of IRAK4/MyD88 with both IRFs. ND-2158 treatment led to decreased phosphorylation of IRF5/4 after OGD compared to the vehicle group. Conclusion: IRAK4/MyD88 complex phosphorylates microglial IRF5/4 which subsequently mediates the production of inflammatory cytokines after ischemia. Manipulation of IRF5/4 signaling potentially represents a novel therapeutic strategy for stroke.

Fecal Calprotectin in Preterm Infants Sepsis with and without Necrotizing Enterocolitis Symptoms

Necrotizing enterocolitis (NEC) is one of the severe gastrointestinal disorder that predominantly affects preterm infants with high morbidity and mortality. The initial clinical manifestations of NEC are non-specific and indistinguishable from sepsis which making delay in diagnosis. Delayed diagnosis might require surgery and even cause death. Calprotectin is a calcium-binding protein, abundantly present in cytosol fraction of neutrophils, also found in feces, and has been found to increase significantly in gastrointestinal inflammation. This study purpose to compare fecal calprotectin in sepsis preterm infants with symptoms of NEC to sepsis preterm infants without symptoms of NEC. The study was a comparative cross-sectional analytic study performed at the Neonatology ward of Dr. Hasan Sadikin General Hospital Bandung, from October 2013 to January 2014 on 40 sepsis preterm infants aged <28 days. Fecal calprotectin was analyzed using enzyme-linked immunoassay (ELISA) kit. Mann-Whitney U test was used to compare the difference of fecal calprotectin concentration in both groups. There were 20 sepsis preterm infants with symptoms of NEC compared to 20 sepsis infants without abdominal symptoms. The concentration of fecal calprotectin was significantly higher in preterm sepsis infants with symptoms of NEC (790.67 μg/g) than preterm sepsis infants without symptoms of NEC (247.93 μg/g, p=0.019). The increasing of fecal calprotectin might provide relevant clinical information to pediatricians for early warning signs of NEC in preterm sepsis infants. In conclusion, fecal calprotectin in preterm sepsis infants with symptoms of NEC is higher compared to those without abdominal symptoms. CALPROTECTIN FESES PADA BAYI KURANG BULAN SEPSIS DENGAN DAN TANPA GEJALA ENTEROKOLITIS NEKROTIKANSEnterokolitis nekrotikans (EKN) merupakan salah satu gangguan gastrointestinal yang serius terutama pada bayi kurang bulan dengan angka kesakitan dan kematian yang tinggi. Gejala klinis awal EKN yang tidak spesifik dan sulit dibedakan dengan sepsis menyebabkan keterlambatan diagnosis. Keterlambatan diagnosis dapat menyebabkan diperlukan tindakan pembedahan bahkan kematian. Calprotectin merupakan protein yang berikatan dengan kalsium banyak terdapat dalam sitosol neutrofil, dapat ditemukan dalam feses, dan diketahui meningkat signifikan pada keadaan inflamasi gastrointestinal. Penelitian ini bertujuan membandingkan kadar calprotectin feses pada bayi kurang bulan (BKB) sepsis dengan BKB sepsis tanpa gejala EKN. Penelitian ini merupakan penelitian observasional analitik dengan rancangan kasus kontrol yang dilakukan di ruang rawat Neonatologi RSUP Dr. Hasan Sadikin Bandung dari Oktober 2013 sampai Januari 2014 terhadap 40 BKB sepsis berusia <28 hari. Kadar calprotectin feses dianalisis menggunakan kit enzim-linked immunoassay (ELISA). Analisis data menggunakan Mann-Whitney U test untuk membandingkan kadar calprotectin feses antara kedua kelompok. Terdapat 20 BKB sepsis dengan gejala EKN yang dibanding dengan 20 BKB sepsis tanpa gejala EKN. Konsentrasi calprotectin feses pada kelompok BKB sepsis dengan gejala EKN lebih tinggi (790,67 μg/g) secara bermakna dibanding dengan kelompok BKB sepsis tanpa EKN (247,93 μg/g, p=0,019). Peningkatan kadar calprotectin pada feses dapat memberikan informasi klinis bagi dokter sebagai tanda awal EKN pada BKB sepsis. Simpulan, kadar calprotectin feses pada BKB sepsis dengan gejala EKN lebih tinggi dibanding dengan BKB sepsis tanpa gejala EKN.

Polar constituents of Ligustrum vulgare L. and their effect on lipoxygenase activity

The present work summarizes results of isolation and identification of polar constituents of the methanolic extract of Ligustrum vulgare L. leaves and of the evaluation of inhibiting activity of selected isolates on rat lung cytosol fraction lipoxygenase. Six different compounds were isolated from the ethylacetate and butanol portions of the methanolic extract (hydroxytyrosol and its glucoside, ligustroflavon, oleuropein, acteoside, echinacoside). The inhibitory activity of oleuropein, echinacoside and the water infusion of Ligustrum vulgare leaves tested on LOX was expressed as IC50. Kinetic parameters (K M, V max) and type of inhibition were determined. As the most effective in competitive inhibition of LOX, oleuropein was proved.

Occurrence of myo-inositol-1-phosphate phosphatase in pteridophytes: characteristics of the enzyme from the reproductive pinnules of Dryopteris filix-mas (L.) Schott

Evident myo-inositol-1-phosphate phosphatase (MIPP) activity has been detected both in the vegetative as well as in the spore-bearing organs of some selected pteridophytes having wide phylogenetic diversity. The basic characterization of this enzyme was carried out using the cosmopolitan fern Dryopteris filix-mas. The enzyme was partially purified from the cytosol fraction obtained from the reproductive pinnules of the plant to about 41-fold over the initial homogenate following low-speed centrifugation, streptomycin sulfate precipitation, 25-70% ammonium sulfate fractionation, CM Sephadex C-50 chromatography and finally gel-filtration on Ultrogel AcA 34. The apparent molecular weight of the native MIPP was estimated to be 94 kDa. The enzyme activity increased linearly with respect to protein concentration to about 150 µg and with respect to time up to 75 min. The temperature optimum was found at 40ºC. However, the enzyme showed good activity over the temperature range of 30-50ºC. This enzyme used D/L-myo-inositol-1-phosphate as its principal substrate (95-100%), however, about 16% activity was recorded when D-myo-inositol-3-phosphate substituted as substrate. Furthermore, weak (3%) activity of this MIPP was observed with 2-glycerophosphate as substrate. The apparent Km for pteridophytic MIPP was 0.083 mM. The enzyme was functional in a narrow pH range of 7.5 to 8.5. The activity of this MIPP enzyme was remarkably inhibited by the presence of a monovalent cation, lithium, and even moderately so at a low concentration such as 1 mM. On the other hand, magnesium, a divalent cation, enhanced activity at least up to 10 mM. Calcium diminished MIPP activity at concentrations over 4 mM.

Estrogen-induced modification of uterine RNA polymerase activity depends on localization of the estrogen receptor

The aim of this study was to examine the effects of estradiol (E2) on activity of RNA polymerase I and RNA polymerase II in uterine nuclei of ovariectomized (OVX) female rats. The obtained results show that estrogen-receptor (E-R) complexes in 30 min induced an increase of polymerase II activity. A second increase of polymerase II activity was observed after 3 h-incubation of nuclei with the E-R complex formed in the cytosol fraction. However, activity of polymerase I was increased 2 h after the start of incubation, with highest activity detected at 3 h in nuclei incubated with E-R complexes. On the contrary, no stimulatory effect on either polymerase I or polymerase II activity was observed in nuclei incubated with E2 alone. These results indicate that E2 stimulates the cytosolic estrogen receptor (ER), which in turn causes uterotrophic responses in OVX rats. In addition, they suggest that in order to provoke uterotrophic responses E-R complexes formed in the cytosol need to be retained in the nucleus for a longer period of time. .

Estren Behaves as a Weak Estrogen Rather than a Nongenomic Selective Activator in the Mouse Uterus

A proposed membrane-mediated mechanism of rapid nongenomic response to estrogen has been the intense focus of recent research. Estren, a synthetic steroid, is reported to act selectively through a rapid membrane-mediated pathway, rather than through the classical nuclear estrogen receptor (ER)-mediated pathway, to maintain bone density in ovariectomized mice without uterotropic effects. To evaluate the mechanism and physiological effects of estren, we studied responses in adult ovariectomized mice. In a 3-d uterine bioassay, we found that 300 μg estren significantly increased uterine weight; in comparison, a more maximal response was seen with 1 μg estradiol (E2). The estren response was partly ERα independent, because ERα knockout (αERKO) uteri also exhibited a more moderate weight increase. Estren induced epithelial cell proliferation in wild-type, but not αERKO, mice, indicating ERα dependence of the epithelial growth response. Examination of estren-regulated uterine genes by microarray indicated that early (2 h) changes in gene expression are similar to the early responses to E2. These gene responses are ERα dependent, because they are not seen in αERKO mice. Later estren-induced changes in gene expression (24 h) are blunted compared with those seen 24 h after E2. In contrast to early genes, these later estren responses are independent of ERα, because the αERKO shows a similar response to estren at 24 h. We found that E2 or estren treatments lead to depletion of ERα in the uterine cytosol fraction and accumulation in the nuclear fraction within 30–60 min, consistent with the ability of estren to regulate genes through a nuclear ERα rather than a nongenomic mechanism. Interestingly, estren, but not E2, induces accumulation of androgen receptor (AR) in the nuclear fraction of both wild-type and αERKO samples, suggesting that AR might be involved in the later ERα-independent genomic responses to estren. In conclusion, our studies suggest that estren is weakly estrogenic in the mouse uterus and might induce nuclear ERα- and AR-mediated responses. Given its activity in our uterine model, the use of estren as a bone-selective clinical compound needs to be reconsidered.

Ischemic preconditioning, insulin, and morphine all cause hexokinase redistribution

Association of hexokinase (HK) with mitochondria preserves mitochondrial integrity and is an important mechanism by which cancer cells are protected against hypoxic conditions. Maintenance of mitochondrial integrity also figures prominently as a major characteristic of many cardioprotective manipulations. In this study, we provide evidence that cardioprotective interventions may promote HK redistribution from the cytosol to the mitochondria in the heart. Isolated Langendorff-perfused rat hearts ( n = 6/group) were subjected to normoxic perfusion (control, Con), three 5-min ischemia-reperfusion periods (ischemic preconditioning, IPC), 1 U/l insulin (Ins), or 1 μM morphine (Mor). Hearts were immediately homogenized and centrifuged to obtain whole cell, cytosolic, and mitochondrial fractions. HK, lactate dehydrogenase (LDH), and citrate synthase (CS) enzyme activities were determined. No change in LDH or CS present in the cytosol fraction relative to whole cell activity was observed with any of the cardioprotective interventions. By contrast, HK present in the cytosol fraction relative to whole cell activity decreased significantly ( P < 0.05) with all cardioprotective interventions, from 0.58 ± 0.03 (Con) to 0.46 ± 0.04 (IPC), 0.41 ± 0.01 (Ins), and 0.45 ± 0.02 (Mor). In addition, HK relative to CS activity in the mitochondrial fraction increased significantly with cardioprotection, from 0.15 ± 0.001 (Con) to 0.21 ± 0.002 (IPC), 0.18 ± 0.003 (Ins), and 0.21 ± 0.005 (Mor). Our novel data suggest that well-known cardioprotective interventions share a common end-effector mechanism of cytosolic HK translocation. Association of HK with mitochondria may promote inhibition of the mitochondrial permeability transition pore and thereby reduce cell death and apoptosis.