Lysophosphatidic Acid Disrupts Junctional Integrity and Epithelial Cohesion in Ovarian Cancer Cells

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Yueying Liu

2012-01-01

Full Text Available Ovarian cancer metastasizes via exfoliation of free-floating cells and multicellular aggregates from the primary tumor to the peritoneal cavity. A key event in EOC metastasis is disruption of cell-cell contacts via modulation of intercellular junctional components including cadherins. Ascites is rich in lysophosphatidic acid (LPA, a bioactive lipid that may promote early events in ovarian cancer dissemination. The objective of this paper was to assess the effect of LPA on E-cadherin junctional integrity. We report a loss of junctional E-cadherin in OVCAR3, OVCA429, and OVCA433 cells exposed to LPA. LPA-induced loss of E-cadherin was concentration and time dependent. LPA increased MMP-9 expression and promoted MMP-9-catalyzed E-cadherin ectodomain shedding. Blocking LPA receptor signaling inhibited MMP-9 expression and restored junctional E-cadherin staining. LPA-treated cells demonstrated a significant decrease in epithelial cohesion. Together these data support a model wherein LPA induces MMP-9 expression and MMP-9-catalyzed E-cadherin ectodomain shedding, resulting in loss of E-cadherin junctional integrity and epithelial cohesion, facilitating metastatic dissemination of ovarian cancer cells.

Toluene diisocyanate: Induction of the autotaxin-lysophosphatidic acid axis and its association with airways symptoms

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Broström, Julia M. [Division of Occupational and Environmental Medicine, Lund University, SE 221 85 Lund (Sweden); Ye, Zhi-wei [Institute of Environmental Medicine, Karolinska Institutet, Box 210, SE 171 77 Stockholm (Sweden); Axmon, Anna; Littorin, Margareta; Tinnerberg, Håkan; Lindh, Christian H. [Division of Occupational and Environmental Medicine, Lund University, SE 221 85 Lund (Sweden); Zheng, Huiyuan; Ghalali, Aram; Stenius, Ulla [Institute of Environmental Medicine, Karolinska Institutet, Box 210, SE 171 77 Stockholm (Sweden); Jönsson, Bo A.G. [Division of Occupational and Environmental Medicine, Lund University, SE 221 85 Lund (Sweden); Högberg, Johan, E-mail: johan.hogberg@ki.se [Institute of Environmental Medicine, Karolinska Institutet, Box 210, SE 171 77 Stockholm (Sweden)

2015-09-15

Diisocyanates are industrial chemicals which have a wide range of applications in developed and developing countries. They are notorious lung toxicants and respiratory sensitizers. However, the mechanisms behind their adverse effects are not adequately characterized. Autotaxin (ATX) is an enzyme producing lysophosphatidic acid (LPA), and the ATX-LPA axis has been implicated in lung related inflammatory conditions and diseases, including allergic asthma, but not to toxicity of environmental low-molecular-weight chemicals. We investigated effects of toluene diisocyanate (TDI) on ATX induction in human lung epithelial cell models, and we correlated LPA-levels in plasma to biomarkers of TDI exposure in urine collected from workers exposed to < 5 ppb (parts per billion). Information on workers' symptoms was collected through interviews. One nanomolar TDI robustly induced ATX release within 10 min in vitro. A P2X7- and P2X4-dependent microvesicle formation was implicated in a rapid ATX release and a subsequent protein synthesis. Co-localization between purinergic receptors and ATX was documented by immunofluorescence and confocal microscopy. The release was modulated by monocyte chemoattractant protein-1 (MCP-1) and by extracellular ATP. In workers, we found a dose–response relationship between TDI exposure biomarkers in urine and LPA levels in plasma. Among symptomatic workers reporting “sneezing”, the LPA levels were higher than among non-symptomatic workers. This is the first report indicating induction of the ATX-LPA axis by an environmental low-molecular-weight chemical, and our data suggest a role for the ATX-LPA axis in TDI toxicity. - Highlights: • Human epithelial cells release autotaxin in response to 1 nM toluene diisocyanate (TDI). • The release involves P2X4 and P2X7 receptors and is modulated by ATP and MCP-1. • Lysophosphatidic acid (LPA) was measured in workers exposed to < 5 ppb TDI. • LPA in plasma correlated to TDI exposure

1-Oleoyl lysophosphatidic acid: a new mediator of emotional behavior in rats.

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Estela Castilla-Ortega

Full Text Available The role of lysophosphatidic acid (LPA in the control of emotional behavior remains to be determined. We analyzed the effects of the central administration of 1-oleoyl-LPA (LPA 18∶1 in rats tested for food consumption and anxiety-like and depression-like behaviors. For this purpose, the elevated plus-maze, open field, Y maze, forced swimming and food intake tests were performed. In addition, c-Fos expression in the dorsal periaqueductal gray matter (DPAG was also determined. The results revealed that the administration of LPA 18∶1 reduced the time in the open arms of the elevated plus-maze and induced hypolocomotion in the open field, suggesting an anxiogenic-like phenotype. Interestingly, these effects were present following LPA 18∶1 infusion under conditions of novelty but not under habituation conditions. In the forced swimming test, the administration of LPA 18∶1 dose-dependently increased depression-like behavior, as evaluated according to immobility time. LPA treatment induced no effects on feeding. However, the immunohistochemical analysis revealed that LPA 18∶1 increased c-Fos expression in the DPAG. The abundant expression of the LPA1 receptor, one of the main targets for LPA 18∶1, was detected in this brain area, which participates in the control of emotional behavior, using immunocytochemistry. These findings indicate that LPA is a relevant transmitter potentially involved in normal and pathological emotional responses, including anxiety and depression.

Ginseng pharmacology: a new paradigm based on gintonin-lysophosphatidic acid receptor interactions

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Seung-Yeol eNah

2015-10-01

Full Text Available Ginseng, the root of Panax ginseng, is used as a traditional medicine. Despite the long history of the use of ginseng, there is no specific scientific or clinical rationale for ginseng pharmacology besides its application as a general tonic. The ambiguous description of ginseng pharmacology might be due to the absence of a predominant active ingredient that represents ginseng pharmacology. Recent studies show that ginseng abundantly contains lysophosphatidic acids (LPAs, which are phospholipid-derived growth factor with diverse biological functions including those claimed to be exhibited by ginseng. LPAs in ginseng form a complex with ginseng proteins, which can bind and deliver LPA to its cognate receptors with a high affinity. As a first messenger, gintonin produces second messenger Ca2+ via G protein-coupled LPA receptors. Ca2+ is an intracellular mediator of gintonin and initiates a cascade of amplifications for further intercellular communications by activation of Ca2+-dependent kinases, receptors, gliotransmitter and neurotransmitter release. Ginsenosides, which have been regarded as primary ingredients of ginseng, cannot elicit intracellular [Ca2+]i transients, since they lack specific cell surface receptor. However, ginsenosides exhibit non-specific ion channel and receptor regulations. This is the key characteristic that distinguishes gintonin from ginsenosides. Although the current discourse on ginseng pharmacology is focused on ginsenosides, gintonin can definitely provide a mode of action for ginseng pharmacology that ginsenosides cannot. This review article introduces a novel concept of ginseng ligand-LPA receptor interaction and proposes to establish a paradigm that shifts the focus from ginsenosides to gintonin as a major ingredient representing ginseng pharmacology.

Lysophosphatidic acid stimulates thrombomodulin lectin-like domain shedding in human endothelial cells

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Wu Hualin; Lin ChiIou; Huang Yuanli; Chen, Pin-Shern; Kuo, Cheng-Hsiang; Chen, Mei-Shing; Wu, G.C.-C.; Shi, G.-Y.; Yang, H.-Y.; Lee Hsinyu

2008-01-01

Thrombomodulin (TM) is an anticoagulant glycoprotein highly expressed on endothelial cell surfaces. Increased levels of soluble TM in circulation have been widely accepted as an indicator of endothelial damage or dysfunction. Previous studies indicated that various proinflammatory factors stimulate TM shedding in various cell types such as smooth muscle cells and epithelial cells. Lysophosphatidic acid (LPA) is a bioactive lipid mediator present in biological fluids during endothelial damage or injury. In the present study, we first observed that LPA triggered TM shedding in human umbilical vein endothelial cells (HUVECs). By Cyflow analysis, we showed that the LPA-induced accessibility of antibodies to the endothelial growth factor (EGF)-like domain of TM is independent of matrix metalloproteinases (MMPs), while LPA-induced TM lectin-like domain shedding is MMP-dependent. Furthermore, a stable cell line expressing TM without its lectin-like domain exhibited a higher cell proliferation rate than a stable cell line expressing full-length TM. These results imply that LPA induces TM lectin-like domain shedding, which might contribute to the exposure of its EGF-like domain for EGF receptor (EGFR) binding, thereby stimulating subsequent cell proliferation. Based on our findings, we propose a novel mechanism for the exposure of TM EGF-like domain, which possibly mediates LPA-induced EGFR transactivation

Lysophosphatidic acid mediates pleiotropic responses in skeletal muscle cells

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Jean-Baptiste, Gael; Yang Zhao; Khoury, Chamel; Greenwood, Michael T.

2005-01-01

Lysophosphatidic acid (LPA) is a potent modulator of growth, cell survival, and apoptosis. Although all four LPA receptors are expressed in skeletal muscle, very little is known regarding the role they play in this tissue. We used RT-PCR to demonstrate that cultured skeletal muscle C2C12 cells endogenously express multiple LPA receptor subtypes. The demonstration that LPA mediates the activation of ERK1/2 MAP kinase and Akt/PKB in C2C12 cells is consistent with the widely observed mitogenic properties of LPA. In spite of these observations, LPA did not induce proliferation in C2C12 cells. Paradoxically, we found that prolonged treatment of C2C12 cells with LPA led to caspase 3 and PARP cleavage as well as the activation of stress-associated MAP kinases JNK and p38. In spite of these typically pro-apoptotic responses, LPA did not induce cell death. Blocking ERK1/2 and Akt/PKB activation with specific pharmacological inhibitors, nevertheless, stimulated LPA-mediated apoptosis. Taken together, these results suggest that both mitogenic and apoptotic responses serve to counterbalance the effects of LPA in cultured C2C12 cells

Involvement of the lysophosphatidic acid-generating enzyme autotaxin in lymphocyte-endothelial cell interactions.

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Nakasaki, Tae; Tanaka, Toshiyuki; Okudaira, Shinichi; Hirosawa, Michi; Umemoto, Eiji; Otani, Kazuhiro; Jin, Soojung; Bai, Zhongbin; Hayasaka, Haruko; Fukui, Yoshinori; Aozasa, Katsuyuki; Fujita, Naoya; Tsuruo, Takashi; Ozono, Keiichi; Aoki, Junken; Miyasaka, Masayuki

2008-11-01

Autotaxin (ATX) is a secreted protein with lysophospholipase D activity that generates lysophosphatidic acid (LPA) from lysophosphatidylcholine. Here we report that functional ATX is selectively expressed in high endothelial venules (HEVs) of both lymph nodes and Peyer's patches. ATX expression was developmentally regulated and coincided with lymphocyte recruitment to the lymph nodes. In adults, ATX expression was independent of HEV-expressed chemokines such as CCL21 and CXCL13, innate immunity signals including those via TLR4 or MyD88, and of the extent of lymphocyte trafficking across the HEVs. ATX expression was induced in venules at sites of chronic inflammation. Receptors for the ATX enzyme product LPA were constitutively expressed in HEV endothelial cells (ECs). In vitro, LPA induced strong morphological changes in HEV ECs. Forced ATX expression caused cultured ECs to respond to lysophosphatidylcholine, up-regulating lymphocyte binding to the ECs in a LPA receptor-dependent manner under both static and flow conditions. Although in vivo depletion of circulating ATX did not affect lymphocyte trafficking into the lymph nodes, we surmise, based on the above data, that ATX expressed by HEVs acts on HEVs in situ to facilitate lymphocyte binding to ECs and that ATX in the general circulation does not play a major role in this process. Tissue-specific inactivation of ATX will verify this hypothesis in future studies of its mechanism of action.

Autotaxin-lysophosphatidic acid axis is a novel molecular target for lowering intraocular pressure.

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Padma Iyer

Full Text Available Primary open-angle glaucoma is the second leading cause of blindness in the United States and is commonly associated with elevated intraocular pressure (IOP resulting from diminished aqueous humor (AH drainage through the trabecular pathway. Developing effective therapies for increased IOP in glaucoma patients requires identification and characterization of molecular mechanisms that regulate IOP and AH outflow. This study describes the identification and role of autotaxin (ATX, a secretory protein and a major source for extracellular lysophosphatidic acid (LPA, in regulation of IOP in a rabbit model. Quantitative proteomics analysis identified ATX as an abundant protein in both human AH derived from non-glaucoma subjects and in AH from different animal species. The lysophospholipase D (LysoPLD activity of ATX was found to be significantly elevated (by ∼1.8 fold; n=20 in AH derived from human primary open angle glaucoma patients as compared to AH derived from age-matched cataract control patients. Immunoblotting analysis of conditioned media derived from primary cultures of human trabecular meshwork (HTM cells has confirmed secretion of ATX and the ability of cyclic mechanical stretch of TM cells to increase the levels of secreted ATX. Topical application of a small molecular chemical inhibitor of ATX (S32826, which inhibited AH LysoPLD activity in vitro (by >90%, led to a dose-dependent and significant decrease of IOP in Dutch-Belted rabbits. Single intracameral injection of S32826 (∼2 µM led to significant reduction of IOP in rabbits, with the ocular hypotensive response lasting for more than 48 hrs. Suppression of ATX expression in HTM cells using small-interfering RNA (siRNA caused a decrease in actin stress fibers and myosin light chain phosphorylation. Collectively, these observations indicate that the ATX-LPA axis represents a potential therapeutic target for lowering IOP in glaucoma patients.

Lysophosphatidic Acid Upregulates Laminin-332 Expression during A431 Cell Colony Dispersal

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Hironobu Yamashita

2010-01-01

Full Text Available Lysophosphatidic acid (LPA is a bioactive phospholipid that affects various biological functions, such as cell proliferation, migration, survival, wound healing, and tumor invasion through LPA receptors. Previously, we reported that LPA induces A431 colony dispersal, accompanied by disruption of cell-cell contacts and cell migration. However, it remains unclear how LPA affects cell migration and gene expression during A431 colony dispersal. In this paper, we performed cDNA microarray analysis to investigate this question by comparing gene expression between untreated and LPA-treated A431 cells. Interestingly, these results revealed that LPA treatment upregulates several TGF-β1 target genes, including laminin-332 (Ln-332 components (α3, β3, and γ2 chains. Western blot analysis also showed that LPA increased phosphorylation of Smad2, an event that is carried out by TGF-β1 interactions. Among the genes upregulated, we further addressed the role of Ln-332. Real-time PCR analysis confirmed the transcriptional upregulation of all α3, β3, and γ2 chains of Ln-332 by LPA, corresponding to the protein level increases revealed by western blot. Further, the addition of anti-Ln-332 antibody prevented LPA-treated A431 colonies from dispersing. Taken together, our results suggest that LPA-induced Ln-332 plays a significant role in migration of individual cells from A431 colonies.

Mechanism of sphingosine 1-phosphate- and lysophosphatidic Acid-induced up-regulation of adhesion molecules and eosinophil chemoattractant in nerve cells.

LENUS (Irish Health Repository)

Costello, Richard W

2012-02-01

The lysophospholipids sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) act via G-protein coupled receptors S1P(1-5) and LPA(1-3) respectively, and are implicated in allergy. Eosinophils accumulate at innervating cholinergic nerves in asthma and adhere to nerve cells via intercellular adhesion molecule-1 (ICAM-1). IMR-32 neuroblastoma cells were used as an in vitro cholinergic nerve cell model. The G(i) coupled receptors S1P(1), S1P(3), LPA(1), LPA(2) and LPA(3) were expressed on IMR-32 cells. Both S1P and LPA induced ERK phosphorylation and ERK- and G(i)-dependent up-regulation of ICAM-1 expression, with differing time courses. LPA also induced ERK- and G(i)-dependent up-regulation of the eosinophil chemoattractant, CCL-26. The eosinophil granule protein eosinophil peroxidase (EPO) induced ERK-dependent up-regulation of transcription of S1P(1), LPA(1), LPA(2) and LPA(3), providing the situation whereby eosinophil granule proteins may enhance S1P- and\\/or LPA- induced eosinophil accumulation at nerve cells in allergic conditions.

Mechanism of sphingosine 1-phosphate- and lysophosphatidic Acid-induced up-regulation of adhesion molecules and eosinophil chemoattractant in nerve cells.

LENUS (Irish Health Repository)

Costello, Richard W

2011-05-01

The lysophospholipids sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) act via G-protein coupled receptors S1P(1-5) and LPA(1-3) respectively, and are implicated in allergy. Eosinophils accumulate at innervating cholinergic nerves in asthma and adhere to nerve cells via intercellular adhesion molecule-1 (ICAM-1). IMR-32 neuroblastoma cells were used as an in vitro cholinergic nerve cell model. The G(i) coupled receptors S1P(1), S1P(3), LPA(1), LPA(2) and LPA(3) were expressed on IMR-32 cells. Both S1P and LPA induced ERK phosphorylation and ERK- and G(i)-dependent up-regulation of ICAM-1 expression, with differing time courses. LPA also induced ERK- and G(i)-dependent up-regulation of the eosinophil chemoattractant, CCL-26. The eosinophil granule protein eosinophil peroxidase (EPO) induced ERK-dependent up-regulation of transcription of S1P(1), LPA(1), LPA(2) and LPA(3), providing the situation whereby eosinophil granule proteins may enhance S1P- and\\/or LPA- induced eosinophil accumulation at nerve cells in allergic conditions.

Lysophosphatidic acid-functionalised titanium as a superior surface for supporting human osteoblast (MG63 maturation

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JP Mansell

2012-05-01

Full Text Available Covalent modifications of titanium with small molecules known to promote human osteoblast maturation are especially attractive in developing superior biomaterials. An important step in securing competent bone formation at implant sites is promoting the formation of mature osteoblasts, either from committed pre-osteoblasts or from their mesenchymal progenitors. To this end our research has focussed on identifying molecules that enhance human osteoblast formation and maturation and to develop ways of covalently attaching these molecules to implant surfaces so that they are more likely to withstand the rigors of the implantation process whilst still retaining their bioactivity. Herein we report the novel production of lipid-functionalised titanium using lysophosphatidic acid or a related compound, (3S 1-fluoro-3-hydroxy-4-butyl-1-phosphonate. Both lipids were especially effective at co-operating with calcitriol to promote human osteoblast maturation at these modified Ti surfaces in vitro. The novel findings presented offer enticing new developments towards the fabrication of next-generation implant devices with the potential to significantly enhance the osseointegration process and with it improvements in future prosthesis performance and longevity.

Bovine ovarian follicular growth and development correlate with lysophosphatidic acid expression.

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Sinderewicz, Emilia; Grycmacher, Katarzyna; Boruszewska, Dorota; Kowalczyk-Zięba, Ilona; Staszkiewicz, Joanna; Ślężak, Tomasz; Woclawek-Potocka, Izabela

2018-01-15

The basis of successful reproduction is proper ovarian follicular growth and development. In addition to prostaglandins and vascular endothelial growth factor, a number of novel factors are suggested as important regulators of follicular growth and development: PGES, TFG, CD36, RABGAP1, DBI and BTC. This study focuses on examining the expression of these factors in granulosa and thecal cells that originate from different ovarian follicle types and their link with the expression of lysophosphatidic acid (LPA), known local regulator of reproductive functions in the cow. Ovarian follicles were divided into healthy, transitional, and atretic categories. The mRNA expression levels for PGES, TFG, CD36, RABGAP1, DBI and BTC in granulosa and thecal cells in different follicle types were measured by real-time PCR. The correlations among expression of enzymes synthesizing LPA (autotaxin, phospholipase A2), receptors for LPA and examined factors were measured. Immunolocalization of PGES, TFG, CD36, RABGAP1, DBI and BTC was examined by immunohistochemistry. We investigated follicle-type dependent mRNA expression of factors potentially involved in ovarian follicular growth and development, both in granulosa and thecal cells of bovine ovarian follicles. Strong correlations among receptors for LPA, enzymes synthesizing LPA, and the examined factors in healthy and transitional follicles were observed, with its strongest interconnection with TFG, DBI and RABGAP1 in granulosa cells, and TFG in thecal cells; whereas no correlations in atretic follicles were detected. A greater number of correlations were found in thecal cells than in granulosa cells as well as in healthy follicles than in transitional follicles. These data indicate the role of LPA in the growth, development and physiology of the bovine ovarian follicle. Copyright © 2017 Elsevier Inc. All rights reserved.

Lysophosphatidic acid induces chemotaxis in MC3T3-E1 osteoblastic cells

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Masiello, Lisa M.; Fotos, Joseph S.; Galileo, Deni S.; Karin, Norm J.

2006-07-01

Lysophosphatidic acid (LPA) is a bioactive lipid that has pleiotropic effects on a variety of cell types and enhances the migration of endothelial and cancer cells, but it is not known if this lipid can alter osteoblast motility. We performed transwell migration assays using MC3T3-E1 osteoblastic cells and found LPA to be a potent chemotactic agent. Quantitative time-lapse video analysis of osteoblast migration after wounds were introduced into cell monolayers indicated that LPA stimulated both migration velocity and the average migration distance per cell. LPA also elicited substantial changes in cell shape and actin cytoskeletal structure; lipid-treated cells contained fewer stress fibers and displayed long membrane processes that were enriched in F-actin. Quantitative RT-PCR analysis showed that MC3T3-E1 cells express all four known LPA-specific G protein-coupled receptors (LPA1-LPA4) with a relative mRNA abundance of LPA1 > LPA4 > LPA2 >> LPA3. LPA-induced changes in osteoblast motility and morphology were antagonized by both pertussis toxin and Ki16425, a subtype-specific blocker of LPA1 and LPA3 receptor function. Cell migration in many cell types is linked to changes in intracellular Ca2+. Ki16425 also inhibited LPA-induced Ca2+ signaling in a dose-dependent manner, suggesting a link between LPA-induced Ca2+ transients and osteoblast chemotaxis. Our data show that LPA stimulates MC3T3-E1 osteoblast motility via a mechanism that is linked primarily to the G protein-coupled receptor LPA1.

Biofunctionalized Lysophosphatidic Acid/Silk Fibroin Film for Cornea Endothelial Cell Regeneration

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Jeon, Hayan; Oliveira, Joaquim Miguel; Reis, Rui Luis; Khang, Gilson

2018-01-01

Cornea endothelial cells (CEnCs) tissue engineering is a great challenge to repair diseased or damaged CEnCs and require an appropriate biomaterial to support cell proliferation and differentiation. Biomaterials for CEnCs tissue engineering require biocompatibility, tunable biodegradability, transparency, and suitable mechanical properties. Silk fibroin-based film (SF) is known to meet these factors, but construction of functionalized graft for bioengineering of cornea is still a challenge. Herein, lysophosphatidic acid (LPA) is used to maintain and increase the specific function of CEnCs. The LPA and SF composite film (LPA/SF) was fabricated in this study. Mechanical properties and in vitro studies were performed using a rabbit model to demonstrate the characters of LPA/SF. ATR-FTIR was characterized to identify chemical composition of the films. The morphological and physical properties were performed by SEM, AFM, transparency, and contact angle. Initial cell density and MTT were performed for adhesion and cell viability in the SF and LPA/SF film. Reverse transcription polymerase chain reactions (RT-PCR) and immunofluorescence were performed to examine gene and protein expression. The results showed that films were designed appropriately for CEnCs delivery. Compared to pristine SF, LPA/SF showed higher biocompatibility, cell viability, and expression of CEnCs specific genes and proteins. These indicate that LPA/SF, a new biomaterial, offers potential benefits for CEnCs tissue engineering for regeneration. PMID:29710848

Lysophosphatidic acid acyltransferase β (LPAATβ promotes the tumor growth of human osteosarcoma.

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Farbod Rastegar

2010-12-01

Full Text Available Osteosarcoma is the most common primary malignancy of bone with poorly characterized molecular pathways important in its pathogenesis. Increasing evidence indicates that elevated lipid biosynthesis is a characteristic feature of cancer. We sought to investigate the role of lysophosphatidic acid acyltransferase β (LPAATβ, aka, AGPAT2 in regulating the proliferation and growth of human osteosarcoma cells. LPAATβ can generate phosphatidic acid, which plays a key role in lipid biosynthesis as well as in cell proliferation and survival. Although elevated expression of LPAATβ has been reported in several types of human tumors, the role of LPAATβ in osteosarcoma progression has yet to be elucidated.Endogenous expression of LPAATβ in osteosarcoma cell lines is analyzed by using semi-quantitative PCR and immunohistochemical staining. Adenovirus-mediated overexpression of LPAATβ and silencing LPAATβ expression is employed to determine the effect of LPAATβ on osteosarcoma cell proliferation and migration in vitro and osteosarcoma tumor growth in vivo. We have found that expression of LPAATβ is readily detected in 8 of the 10 analyzed human osteosarcoma lines. Exogenous expression of LPAATβ promotes osteosarcoma cell proliferation and migration, while silencing LPAATβ expression inhibits these cellular characteristics. We further demonstrate that exogenous expression of LPAATβ effectively promotes tumor growth, while knockdown of LPAATβ expression inhibits tumor growth in an orthotopic xenograft model of human osteosarcoma.Our results strongly suggest that LPAATβ expression may be associated with the aggressive phenotypes of human osteosarcoma and that LPAATβ may play an important role in regulating osteosarcoma cell proliferation and tumor growth. Thus, targeting LPAATβ may be exploited as a novel therapeutic strategy for the clinical management of osteosarcoma. This is especially attractive given the availability of selective

Lysophosphatidic acid (LPA) effects on endometrial carcinoma in vitro proliferation, invasion, and matrix metalloproteinase activity.

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Wang, Feng-qiang; Ariztia, Edgardo V; Boyd, Leslie R; Horton, Faith R; Smicun, Yoel; Hetherington, Jessica A; Smith, Phillip J; Fishman, David A

2010-04-01

Lysophosphatidic acid (LPA) has potent growth-regulatory effect in many cell types and has been linked to the in vivo tumor growth and metastasis in several malignancies. The goal of this study was to assess the regulation of (EC) microenvironment by LPA through the examination of its effect on cell proliferation, migration, invasion, uPA activity, and matrix metalloproteinase (MMP) secretion/activation. All experiments were performed in vitro using an EC cell line, HEC-1A. Cell proliferation was determined using the Promega MTS proliferation assay following 48 h of exposures to different concentrations of LPA (0.1, 1.0 and 10.0 microM). Cell invasion was assessed using a modified Boyden chamber assay with collagen I coated on the membrane. HEC-1A motility was examined by Boyden chamber migration assay as well as the scratch wound closure assay on type I collagen. MMP secretion/activation in HEC-1A conditioned medium was detected by gelatin zymography. MMP-7 mRNA expression was determined using real-time PCR. uPA activity was measured using a coupled colorimetric assay. LPA, at the concentrations of 0.1 and 1.0 microM, significantly induced the proliferation of HEC-1A cells (p0.05). Gelatin zymogram showed that HEC-1A cells secreted high levels of MMP-7, while MMP-2 and MMP-9 are barely detectable. In addition, LPA significantly enhanced uPA activity in HEC-1A conditioned medium in a concentration-dependent manner. LPA is a potent modulator of cellular proliferation and invasion for EC cells. It also has the capacity to stimulate the secretion/activity of uPA and MMP-7. Those results suggest that LPA is a bioactive modulator of EC microenvironment and may have a distinct regulation mechanism as observed in epithelial ovarian cancer. Copyright 2009. Published by Elsevier Inc.

Mice Deficient in lysophosphatidic acid acyltransferase delta (Lpaatδ)/acylglycerophosphate acyltransferase 4 (Agpat4) Have Impaired Learning and Memory.

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Bradley, Ryan M; Mardian, Emily B; Bloemberg, Darin; Aristizabal Henao, Juan J; Mitchell, Andrew S; Marvyn, Phillip M; Moes, Katherine A; Stark, Ken D; Quadrilatero, Joe; Duncan, Robin E

2017-11-15

We previously characterized LPAATδ/AGPAT4 as a mitochondrial lysophosphatidic acid acyltransferase that regulates brain levels of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI). Here, we report that Lpaat δ -/- mice display impaired spatial learning and memory compared to wild-type littermates in the Morris water maze and our investigation of potential mechanisms associated with brain phospholipid changes. Marker protein immunoblotting suggested that the relative brain content of neurons, glia, and oligodendrocytes was unchanged. Relative abundance of the important brain fatty acid docosahexaenoic acid was also unchanged in phosphatidylserine, phosphatidylglycerol, and cardiolipin, in agreement with prior data on PC, PE and PI. In phosphatidic acid, it was increased. Specific decreases in ethanolamine-containing phospholipids were detected in mitochondrial lipids, but the function of brain mitochondria in Lpaat δ -/- mice was unchanged. Importantly, we found that Lpaat δ -/- mice have a significantly and drastically lower brain content of the N -methyl-d-asparate (NMDA) receptor subunits NR1, NR2A, and NR2B, as well as the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluR1, compared to wild-type mice. However, general dysregulation of PI-mediated signaling is not likely responsible, since phospho-AKT and phospho-mTOR pathway regulation was unaffected. Our findings indicate that Lpaat δ deficiency causes deficits in learning and memory associated with reduced NMDA and AMPA receptors. Copyright © 2017 American Society for Microbiology.

Lysophosphatidic acid rescues bone mesenchymal stem cells from hydrogen peroxide-induced apoptosis.

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Wang, Xian-Yun; Fan, Xue-Song; Cai, Lin; Liu, Si; Cong, Xiang-Feng; Chen, Xi

2015-03-01

The increase of reactive oxygen species in infracted heart significantly reduces the survival of donor mesenchymal stem cells, thereby attenuating the therapeutic efficacy for myocardial infarction. In our previous study, we demonstrated that lysophosphatidic acid (LPA) protects bone marrow-derived mesenchymal stem cells (BMSCs) against hypoxia and serum deprivation-induced apoptosis. However, whether LPA protects BMSCs from H2O2-induced apoptosis was not examined. In this study, we report that H2O2 induces rat BMSC apoptosis whereas LPA pre-treatment effectively protects BMSCs from H2O2-induced apoptosis. LPA protection of BMSC from the induced apoptosis is mediated mostly through LPA3 receptor. Furthermore, we found that membrane G protein Gi2 and Gi3 are involved in LPA-elicited anti-apoptotic effects through activation of ERK1/2- and PI3 K-pathways. Additionally, H2O2 increases levels of type II of light chain 3B (LC3B II), an autophagy marker, and H2O2-induced autophagy thus protected BMSCs from apoptosis. LPA further increases the expression of LC3B II in the presence of H2O2. In contrast, autophagy flux inhibitor bafilomycin A1 has no effect on LPA's protection of BMSC from H2O2-induced apoptosis. Taken together, our data suggest that LPA rescues H2O2-induced apoptosis mainly by interacting with Gi-coupled LPA3, resulting activation of the ERK1/2- and PI3 K/AKT-pathways and inhibition caspase-3 cleavage, and LPA protection of BMSCs against the apoptosis is independent of it induced autophagy.

Clinical significance and correlation of the level change of plasma lysophosphatidic acid in patients before and during radiotherapy

International Nuclear Information System (INIS)

Yu Liang; Li Hong; Zhu Shengjie

2011-01-01

Objective: To investigate the effects of radiotherapy on the level of lysophosphatidic acid (LPA) in different patients. Methods: Three groups of patients (metastatic brain tumor group, non-brain tumor group and primary brain tumor group) were given external irradiation (by linear accelerator). LPA level, white blood cell count and platelet count in the blood plasma were evaluated pre-irradiation and after irradiation with 40 Gy and 60 Gy respectively. Results: The LPA level decreased gradually as irradiation doses increase in metastatic brain tumor group non-brain tumor group (after irradiation with 60 Gy 0.05), and neoplasm volume changed little. White blood cell count and platelet count gradually decreased with the increasing irradiation doses in metastatic brain tumor group and primary brain tumor group, but there was no significant correlation to LPA level. However, there was a negative correlation to LPA level in non-brain tumor group (r=-0.285 and r=-0.237, both P<0.05 ). Conclusions: There is a negative correlation between radiotherapy dose and LPA level in metastatic brain tumor patients and non-brain tumor patients. LPA level could be used as a predictor of the effect of the radiotherapy in tumor treatment. (authors)

Lysophosphatidic acid activates peroxisome proliferator activated receptor-γ in CHO cells that over-express glycerol 3-phosphate acyltransferase-1.

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Cliona M Stapleton

2011-04-01

Full Text Available Lysophosphatidic acid (LPA is an agonist for peroxisome proliferator activated receptor-γ (PPARγ. Although glycerol-3-phosphate acyltransferase-1 (GPAT1 esterifies glycerol-3-phosphate to form LPA, an intermediate in the de novo synthesis of glycerolipids, it has been assumed that LPA synthesized by this route does not have a signaling role. The availability of Chinese Hamster Ovary (CHO cells that stably overexpress GPAT1, allowed us to analyze PPARγ activation in the presence of LPA produced as an intracellular intermediate. LPA levels in CHO-GPAT1 cells were 6-fold higher than in wild-type CHO cells, and the mRNA abundance of CD36, a PPARγ target, was 2-fold higher. Transactivation assays showed that PPARγ activity was higher in the cells that overexpressed GPAT1. PPARγ activity was enhanced further in CHO-GPAT1 cells treated with the PPARγ ligand troglitazone. Extracellular LPA, phosphatidic acid (PA or a membrane-permeable diacylglycerol had no effect, showing that PPARγ had been activated by LPA generated intracellularly. Transient transfection of a vector expressing 1-acylglycerol-3-phosphate acyltransferase-2, which converts endogenous LPA to PA, markedly reduced PPARγ activity, as did over-expressing diacylglycerol kinase, which converts DAG to PA, indicating that PA could be a potent inhibitor of PPARγ. These data suggest that LPA synthesized via the glycerol-3-phosphate pathway can activate PPARγ and that intermediates of de novo glycerolipid synthesis regulate gene expression.

Lysophosphatidic acid induces expression of genes in human oral keratinocytes involved in wound healing.

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Thorlakson, Hong Huynh; Engen, Stian Andre; Schreurs, Olav; Schenck, Karl; Blix, Inger Johanne Schytte

2017-08-01

Epithelial cells participate in wound healing by covering wounds, but also as important mediators of wound healing processes. Topical application of the phospholipid growth factor lysophosphatidic acid (LPA) accelerates dermal wound healing and we hypothesized that LPA can play a role in human oral wound healing through its effects on human oral keratinocytes (HOK). HOK were isolated from gingival biopsies and exposed to LPA. The LPA receptor profile, signal transduction pathways, gene expression and secretion of selected cytokines were analyzed. HOK expressed the receptors LPA 1 , LPA 5 and LPA 6 and LPA activated the ERK1/2, JNK and p38 intracellular pathways, substantiated by secretion of IL-6 and IL-8. The early (2h) and intermediate (6h) gene expression profiles of HOK after LPA treatment showed a wide array of regulated genes. The majority of the strongest upregulated genes were related to chemotaxis and inflammation, and became downregulated after 6h. At 6h, genes coding for factors involved in extracellular matrix remodeling and re-epithelialization became highly expressed. IL-36γ, not earlier known to be regulated by LPA, was strongly transcribed and translated but not secreted. After stimulation with LPA, HOK responded by regulating factors and genes that are essential in wound healing processes. As LPA is found in saliva and is released by activated cells after wounding, our results indicate that LPA has a favorable physiological role in oral wound healing. This may further point towards a beneficial role for application of LPA on oral surgical or chronic wounds. Copyright © 2017 Elsevier Ltd. All rights reserved.

CD14 is a key mediator of both lysophosphatidic acid and lipopolysaccharide induction of foam cell formation.

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An, Dong; Hao, Feng; Zhang, Fuqiang; Kong, Wei; Chun, Jerold; Xu, Xuemin; Cui, Mei-Zhen

2017-09-01

Macrophage uptake of oxidized low-density lipoprotein (oxLDL) plays an important role in foam cell formation and the pathogenesis of atherosclerosis. We report here that lysophosphatidic acid (LPA) enhances lipopolysaccharide (LPS)-induced oxLDL uptake in macrophages. Our data revealed that both LPA and LPS highly induce the CD14 expression at messenger RNA and protein levels in macrophages. The role of CD14, one component of the LPS receptor cluster, in LPA-induced biological functions has been unknown. We took several steps to examine the role of CD14 in LPA signaling pathways. Knockdown of CD14 expression nearly completely blocked LPA/LPS-induced oxLDL uptake in macrophages, demonstrating for the first time that CD14 is a key mediator responsible for both LPA- and LPS-induced oxLDL uptake/foam cell formation. To determine the molecular mechanism mediating CD14 function, we demonstrated that both LPA and LPS significantly induce the expression of scavenger receptor class A type I (SR-AI), which has been implicated in lipid uptake process, and depletion of CD14 levels blocked LPA/LPS-induced SR-AI expression. We further showed that the SR-AI-specific antibody, which quenches SR-AI function, blocked LPA- and LPS-induced foam cell formation. Thus, SR-AI is the downstream mediator of CD14 in regulating LPA-, LPS-, and LPA/LPS-induced foam cell formation. Taken together, our results provide the first experimental evidence that CD14 is a novel connecting molecule linking both LPA and LPS pathways and is a key mediator responsible for LPA/LPS-induced foam cell formation. The LPA/LPS-CD14-SR-AI nexus might be the new convergent pathway, contributing to the worsening of atherosclerosis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Lysophosphatidic acid-induced RhoA signaling and prolonged macrophage infiltration worsens fibrosis and fatty infiltration following rotator cuff tears.

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Davies, Michael R; Lee, Lawrence; Feeley, Brian T; Kim, Hubert T; Liu, Xuhui

2017-07-01

Previous studies have suggested that macrophage-mediated chronic inflammation is involved in the development of rotator cuff muscle atrophy and degeneration following massive tendon tears. Increased RhoA signaling has been reported in chronic muscle degeneration, such as muscular dystrophy. However, the role of RhoA signaling in macrophage infiltration and rotator muscle degeneration remains unknown. Using a previously established rat model of massive rotator cuff tears, we found RhoA signaling is upregulated in rotator cuff muscle following a massive tendon-nerve injury. This increase in RhoA expression is greatly potentiated by the administration of a potent RhoA activator, lysophosphatidic acid (LPA), and is accompanied by increased TNFα and TGF-β1 expression in rotator cuff muscle. Boosting RhoA signaling with LPA significantly worsened rotator cuff muscle atrophy, fibrosis, and fatty infiltration, accompanied with massive monocytic infiltration of rotator cuff muscles. Co-staining of RhoA and the tissue macrophage marker CD68 showed that CD68+ tissue macrophages are the dominant cell source of increased RhoA signaling in rotator cuff muscles after tendon tears. Taken together, our findings suggest that LPA-mediated RhoA signaling in injured muscle worsens the outcomes of atrophy, fibrosis, and fatty infiltration by increasing macrophage infiltraion in rotator cuff muscle. Clinically, inhibiting RhoA signaling may represent a future direction for developing new treatments to improve muscle quality following massive rotator cuff tears. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1539-1547, 2017. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Endogenous lysophosphatidic acid participates in vascularisation and decidualisation at the maternal-fetal interface in the rat.

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Sordelli, Micaela S; Beltrame, Jimena S; Zotta, Elsa; Gomez, Natalia; Dmytrenko, Ganna; Sales, María Elena; Blois, Sandra M; Davio, Carlos; Martinez, Silvina Perez; Franchi, Ana M; Ribeiro, María L

2017-10-01

Lysophosphatidic acid (LPA) affects several female reproductive functions through G-protein-coupled receptors. LPA contributes to embryo implantation via the lysophospholipid LPA 3 receptor. In the present study we investigated the participation of endogenous LPA signalling through the LPA 3 receptor in vascularisation and decidualisation, two crucial events at the maternal-fetal interface. Pregnant rats were treated with diacylglycerol pyrophosphate (DGPP), a highly selective antagonist of LPA 3 receptors, on Day 5 of gestation. Pregnant rats received intrauterine (i.u.) injections of single doses of DGPP (0.1mgkg -1 ) in a total volume of 2μL in the left horn (treated horn) in the morning of GD5. DGPP treatment produced aberrant embryo spacing and increased embryo resorption. The LPA 3 receptor antagonist decreased the cross-sectional length of the uterine and arcuate arteries and induced histological anomalies in the decidua and placentas. Marked haemorrhagic processes, infiltration of immune cells and tissue disorganisation were observed in decidual and placental tissues from sites of resorption. The mRNA expression of three vascularisation markers, namely interleukin 10 (Il10), vascular endothelial growth factor (Vegfa) and vascular endothelial growth factor receptor 1 (Vegfr1), was reduced at sites of resorption from Day 8. The results show that the disruption of endogenous LPA signalling by blocking the LPA 3 receptor modified the development of uterine vessels with consequences in the formation of the decidua and placenta and in the growth of embryos.

Clinical significance of plasma lysophosphatidic acid levels in the differential diagnosis of ovarian cancer

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Yun-Jie Zhang

2015-01-01

Full Text Available Objective: To investigate the value of lysophosphatidic acid (LPA in the diagnosis of ovarian cancer. Materials and Methods: We first performed a hospital-based, case-control study involving 123 ovarian cancer patients and 101 benign ovarian tumor patients, and then conducted a meta-analysis with 19 case-control studies to assess the correlation between ovarian cancer and plasma LPA levels. Results: The case-control study results demonstrated that ovarian cancer patients have increased LPA and cancer antigen (CA-125 levels compared to patients with benign ovarian tumor (LPA: Ovarian cancer vs benign ovarian tumor: 5.28 ± 1.52 vs 1.82 ± 0.77 μmol/L; CA-125: Ovarian cancer vs benign ovarian tumor: 87.17 ± 45.81 vs. 14.03 ± 10.14 U/mL, which showed statistically significant differences (both P < 0.05. LPA with advanced sensitivity, specificity, positive predictive value, negative predictive value, and accuracy rate of diagnosis excelled CA-125 in the diagnosis of ovarian cancer (both P < 0.05. The areas under the receiver operating characteristic (ROC curve in the diagnosis of ovarian cancer (LPA: 0.983; CA-125: 0.910 were statistically significant compared with the reference (both P < 0.001 and the difference of the areas of ROC curve between LPA and CA-125 in the diagnosis of ovarian cancer showed statistically significant difference (P < 0.05. The meta-analysis results suggested that plasma LPA levels were higher in ovarian cancer tissues than in benign tissues (standardized mean difference (SMD =2.36, 95% confidence interval (CI: 1.61-3.11, P < 0.001 and normal tissues (SMD = 2.32, 95% CI: 1.77-2.87, P < 0.001. Conclusion: LPA shows greater value in the diagnosis of ovarian cancer compared to CA-125 and may be employed as a biological index to diagnose ovarian cancer.

Lysophosphatidic acid directly induces macrophage-derived foam cell formation by blocking the expression of SRBI.

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Chen, Linmu; Zhang, Jun; Deng, Xiao; Liu, Yan; Yang, Xi; Wu, Qiong; Yu, Chao

2017-09-23

The leading cause of morbidity and mortality is the result of cardiovascular disease, mainly atherosclerosis. The formation of macrophage foam cells by ingesting ox-LDL and focal retention in the subendothelial space are the hallmarks of the early atherosclerotic lesion. Lysophosphatidic acid (LPA), which is a low-molecular weight lysophospholipid enriched in oxidized LDL, exerts a range of effects on the cardiovascular system. Previous reports show that LPA increases the uptake of ox-LDL to promote the formation of foam cells. However, as the most active component of ox-LDL, there is no report showing whether LPA directly affects foam cell formation. The aim of this study was to investigate the effects of LPA on foam cell formation, as well as to elucidate the underlying mechanism. Oil red O staining and a Cholesterol/cholesteryl ester quantitation assay were used to evaluate foam cell formation in Raw264.7 macrophage cells. We utilized a Western blot and RT-PCR to investigate the relationship between LPA receptors and lipid transport related proteins. We found that LPA promoted foam cell formation, using 200 μM for 24 h. Meanwhile, the expression of the Scavenger receptor BI (SRBI), which promotes the efflux of free cholesterol, was decreased. Furthermore, the LPA 1/3 receptor antagonist Ki16425 significantly abolished the LPA effects, indicating that LPA 1/3 was involved in the foam cell formation and SRBI expression induced by LPA. Additionally, the LPA-induced foam cell formation was blocked with an AKT inhibitor. Our results suggest that LPA-enhanced foam cell formation is mediated by LPA 1/3 -AKT activation and subsequent SRBI expression. Copyright © 2017. Published by Elsevier Inc.

Aromatic hydrocarbon receptor inhibits lysophosphatidic acid-induced vascular endothelial growth factor-A expression in PC-3 prostate cancer cells

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Wu, Pei-Yi; Lin, Yueh-Chien; Lan, Shun-Yan; Huang, Yuan-Li; Lee, Hsinyu

2013-01-01

Highlights: •LPA-induced VEGF-A expression was regulated by HIF-1α and ARNT. •PI3K mediated LPA-induced VEGF-A expression. •AHR signaling inhibited LPA-induced VEGF-A expression in PC-3 cells. -- Abstract: Lysophosphatidic acid (LPA) is a lipid growth factor with multiple biological functions and has been shown to stimulate cancer cell secretion of vascular endothelial growth factor-A (VEGF-A) and trigger angiogenesis. Hypoxia-inducible factor-1 (HIF-1), a heterodimer consisting of HIF-1α and HIF-1β (also known as aromatic hydrocarbon receptor nuclear translocator (ARNT)) subunits, is an important regulator of angiogenesis in prostate cancer (PC) through the enhancement of VEGF-A expression. In this study, we first confirmed the ability of LPA to induce VEGF-A expression in PC-3 cells and then validated that LPA-induced VEGF-A expression was regulated by HIF-1α and ARNT through phosphatidylinositol 3-kinase activation. Aromatic hydrocarbon receptor (AHR), a receptor for dioxin-like compounds, functions as a transcription factor through dimerization with ARNT and was found to inhibit prostate carcinogenesis and vanadate-induced VEGF-A production. Since ARNT is a common dimerization partner of AHR and HIF-1α, we hypothesized that AHR might suppress LPA-induced VEGF-A expression in PC-3 cells by competing with HIF-1α for ARNT. Here we demonstrated that overexpression and ligand activation of AHR inhibited HIF-1-mediated VEGF-A induction by LPA treatment of PC-3 cells. In conclusion, our results suggested that AHR activation may inhibit LPA-induced VEGF-A expression in PC-3 cells by attenuating HIF-1α signaling, and subsequently, suppressing angiogenesis and metastasis of PC. These results suggested that AHR presents a potential therapeutic target for the prevention of PC metastasis

Spinal cord compression injury in lysophosphatidic acid 1 receptor-null mice promotes maladaptive pronociceptive descending control.

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Suardíaz, M; Galan-Arriero, I; Avila-Martin, G; Estivill-Torrús, G; de Fonseca, F R; Chun, J; Gómez-Soriano, J; Bravo-Esteban, E; Taylor, J

2016-02-01

Although activation of the lysophosphatidic acid receptor 1 (LPA1) is known to mediate pronociceptive effects in peripheral pain models, the role of this receptor in the modulation of spinal nociception following spinal cord injury (SCI) is unknown. In this study, LPA1 regulation of spinal excitability mediated by supraspinal descending antinociceptive control systems was assessed following SCI in both wild-type (WT) and maLPA1-null receptor mice. The effect of a T8 spinal compression in WT and maLPA1-null mice was assessed up to 1 month after SCI using histological, immunohistochemical and behavioural techniques analysis including electrophysiological recording of noxious toes-Tibialis Anterior (TA) stimulus-response reflex activity. The effect of a T3 paraspinal transcutaneous electrical conditioning stimulus on TA noxious reflex temporal summation was also assessed. Histological analysis demonstrated greater dorsolateral funiculus damage after SCI in maLPA1-null mice, without a change in the stimulus-response function of the TA noxious reflex when compared to WT mice. While T3 conditioning stimulation in the WT group inhibited noxious TA reflex temporal summation after SCI, this stimulus strongly excited TA reflex temporal summation in maLPA1-null mice. The functional switch from descending inhibition to maladaptive facilitation of central excitability of spinal nociception demonstrated in maLPA1-null mice after SCI was unrelated to a general change in reflex activity. These data suggest that the LPA1 receptor is necessary for inhibition of temporal summation of noxious reflex activity, partly mediated via long-tract descending modulatory systems acting at the spinal level. © 2015 European Pain Federation - EFIC®

Enhancement of Human Endothelial Cell Adhesion to Type I Collagen by Lysophosphatidic Acid (LPA and Sphingosine-1-Phosphate (S1P

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Hsinyu Lee

2004-06-01

Full Text Available The diverse cellular effects of lysophosphatidic acid (LPA and sphingosine-1-phosphate (S1P are transduced by two structurally homologous subfamilies of G protein-coupled receptors, which are encoded by endothelial differentiation genes (Edg Rs. Human umbilical cord vein endothelial cells (HUVECs express Edg Rs for LPA (Edg2 and S1P (Edg1 and 3, which transduce signals for migration of HUVECs through micropore filters coated with type I collagen. Since activation of integrins is essential for optimal migration of endothelial cells, we now examine the capacity of LPA and S1P to augment integrin mediation of endothelial cell binding to type I collagen. Lysophospholipid enhancement of HUVEC adhesion to type I collagen is detectable within 20 minutes. Enhancement of adhesion by both LPA and S1P is significant at 50 nM and optimal at 5µM. Pertussis toxin (PTx, a specific inhibitor of Gi, and C3 exotoxin, a specific inhibitor of Rho, both suppress LPA and S1P enhancement of HUVEC adhesion. In contrast, PD98059, which blocks MAP kinase kinase (MEK, and wortmannin, which inhibits phosphatidylinositol 3-kinase (PI3K, had no effect on LPA- or S1P-enhancement of HUVEC adhesion. Neutralizing monoclonal antibodies specific for α2 and β1 integrin chains, concomitantly decrease LPA and S1P enhancement of HUVEC adhesion to type I collagen. LPA and S1P thus promote type I collagen-dependent adhesion and migration of HUVECs by recruiting α2 and β1 integrin through both Gi and Rho pathways. Integrin α2/β1 therefore appears to be critical on the effects of LPA and S1P on endothelial cell physiology.

Lysophosphatidic acid induces reactive oxygen species generation by activating protein kinase C in PC-3 human prostate cancer cells

International Nuclear Information System (INIS)

Lin, Chu-Cheng; Lin, Chuan-En; Lin, Yueh-Chien; Ju, Tsai-Kai; Huang, Yuan-Li; Lee, Ming-Shyue; Chen, Jiun-Hong; Lee, Hsinyu

2013-01-01

Highlights: •LPA induces ROS generation through LPA 1 and LPA 3 . •LPA induces ROS generation by activating PLC. •PKCζ mediates LPA-induced ROS generation. -- Abstract: Prostate cancer is one of the most frequently diagnosed cancers in males, and PC-3 is a cell model popularly used for investigating the behavior of late stage prostate cancer. Lysophosphatidic acid (LPA) is a lysophospholipid that mediates multiple behaviors in cancer cells, such as proliferation, migration and adhesion. We have previously demonstrated that LPA enhances vascular endothelial growth factor (VEGF)-C expression in PC-3 cells by activating the generation of reactive oxygen species (ROS), which is known to be an important mediator in cancer progression. Using flow cytometry, we showed that LPA triggers ROS generation within 10 min and that the generated ROS can be suppressed by pretreatment with the NADPH oxidase (Nox) inhibitor diphenylene iodonium. In addition, transfection with LPA 1 and LPA 3 siRNA efficiently blocked LPA-induced ROS production, suggesting that both receptors are involved in this pathway. Using specific inhibitors and siRNA, phospholipase C (PLC) and protein kinase C (PKC) were also suggested to participate in LPA-induced ROS generation. Overall, we demonstrated that LPA induces ROS generation in PC-3 prostate cancer cells and this is mediated through the PLC/PKC/Nox pathway

Lysophosphatidic acid induces reactive oxygen species generation by activating protein kinase C in PC-3 human prostate cancer cells

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Lin, Chu-Cheng; Lin, Chuan-En; Lin, Yueh-Chien [Institute of Zoology, College of Life Science, National Taiwan University, Taipei, Taiwan, ROC (China); Ju, Tsai-Kai [Instrumentation Center, National Taiwan University, Taipei, Taiwan, ROC (China); Technology Commons, College of Life Science, National Taiwan University, Taipei, Taiwan, ROC (China); Huang, Yuan-Li [Department of Biotechnology, Asia University, Taichung, Taiwan, ROC (China); Lee, Ming-Shyue [Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan, ROC (China); Chen, Jiun-Hong [Institute of Zoology, College of Life Science, National Taiwan University, Taipei, Taiwan, ROC (China); Department of Life Science, College of Life Science, National Taiwan University, Taipei, Taiwan, ROC (China); Lee, Hsinyu, E-mail: hsinyu@ntu.edu.tw [Institute of Zoology, College of Life Science, National Taiwan University, Taipei, Taiwan, ROC (China); Department of Life Science, College of Life Science, National Taiwan University, Taipei, Taiwan, ROC (China); Center for Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Research Center for Developmental Biology and Regenerative Medicine, National Taiwan University, Taipei, Taiwan, ROC (China)

2013-11-01

Highlights: •LPA induces ROS generation through LPA{sub 1} and LPA{sub 3}. •LPA induces ROS generation by activating PLC. •PKCζ mediates LPA-induced ROS generation. -- Abstract: Prostate cancer is one of the most frequently diagnosed cancers in males, and PC-3 is a cell model popularly used for investigating the behavior of late stage prostate cancer. Lysophosphatidic acid (LPA) is a lysophospholipid that mediates multiple behaviors in cancer cells, such as proliferation, migration and adhesion. We have previously demonstrated that LPA enhances vascular endothelial growth factor (VEGF)-C expression in PC-3 cells by activating the generation of reactive oxygen species (ROS), which is known to be an important mediator in cancer progression. Using flow cytometry, we showed that LPA triggers ROS generation within 10 min and that the generated ROS can be suppressed by pretreatment with the NADPH oxidase (Nox) inhibitor diphenylene iodonium. In addition, transfection with LPA{sub 1} and LPA{sub 3} siRNA efficiently blocked LPA-induced ROS production, suggesting that both receptors are involved in this pathway. Using specific inhibitors and siRNA, phospholipase C (PLC) and protein kinase C (PKC) were also suggested to participate in LPA-induced ROS generation. Overall, we demonstrated that LPA induces ROS generation in PC-3 prostate cancer cells and this is mediated through the PLC/PKC/Nox pathway.

Lysophosphatidic Acid Level and the Incidence of Silent Brain Infarction in Patients with Nonvalvular Atrial Fibrillation

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Li Zhou

2010-10-01

Full Text Available Lysophosphatidic acid (LPA, which is proposed to play an important role in normal physiological situations such as wound healing, vascular tone, vascular integrity and reproduction, may be involved in the etiology of some diseases such as atherosclerosis, cancer, obesity or myocardial infarction. Abnormal findings, including silent brain infarction (SBI, are frequently observed by magnetic resonance imaging (MRI in patients with nonvalvular atrial fibrillation (NVAF. However, whether there is a relationship between LPA level and the prevalence of SBI has not been extensively studied. In the present study, the association between them was investigated. 235 patients with NVAF, 116 cases of SBI without NVAF and 120 cases of healthy volunteers (control group, who did not receive any antithrombotic therapy, were enrolled in this study. Plasma LPA levels in the NVAF with SBI group were significantly higher than that in the control group (p < 0.01, NVAF without SBI group (p < 0.01 and SBI without NVAF group (p < 0.01. The LPA levels are lower in the control group than in the NVAF without SBI and SBI without NVAF groups (p < 0.01, however, the latter two groups did not significantly differ from each other for LPA levels (p > 0.05 There were significant differences in the positive rate of platelet activation between each of the groups (p < 0.01. The positive rate of platelet activation was significantly higher in the NVAF with SBI group. We suggest that LPA might be a novel marker for estimation of the status of platelet activation and the risk factor for SBI onset in NVAF patients. We expected that plasma LPA levels could predict the occurrence of SBI in NVAF patients.

Role of lysophosphatidic acid receptor LPA2 in the development of allergic airway inflammation in a murine model of asthma

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Chun Jerold

2009-11-01

Full Text Available Abstract Background Lysophosphatidic acid (LPA plays a critical role in airway inflammation through G protein-coupled LPA receptors (LPA1-3. We have demonstrated that LPA induced cytokine and lipid mediator release in human bronchial epithelial cells. Here we provide evidence for the role of LPA and LPA receptors in Th2-dominant airway inflammation. Methods Wild type, LPA1 heterozygous knockout mice (LPA1+/-, and LPA2 heterozygous knockout mice (LPA2+/- were sensitized with inactivated Schistosoma mansoni eggs and local antigenic challenge with Schistosoma mansoni soluble egg Ag (SEA in the lungs. Bronchoalveolar larvage (BAL fluids and lung tissues were collected for analysis of inflammatory responses. Further, tracheal epithelial cells were isolated and challenged with LPA. Results BAL fluids from Schistosoma mansoni egg-sensitized and challenged wild type mice (4 days of challenge showed increase of LPA level (~2.8 fold, compared to control mice. LPA2+/- mice, but not LPA1+/- mice, exposed to Schistosoma mansoni egg revealed significantly reduced cell numbers and eosinophils in BAL fluids, compared to challenged wild type mice. Both LPA2+/- and LPA1+/- mice showed decreases in bronchial goblet cells. LPA2+/- mice, but not LPA1+/- mice showed the decreases in prostaglandin E2 (PGE2 and LPA levels in BAL fluids after SEA challenge. The PGE2 production by LPA was reduced in isolated tracheal epithelial cells from LPA2+/- mice. These results suggest that LPA and LPA receptors are involved in Schistosoma mansoni egg-mediated inflammation and further studies are proposed to understand the role of LPA and LPA receptors in the inflammatory process.

Lysophosphatidic acid receptor activation affects the C13NJ microglia cell line proteome leading to alterations in glycolysis, motility, and cytoskeletal architecture

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Bernhart, Eva; Kollroser, Manfred; Rechberger, Gerald; Reicher, Helga; Heinemann, Akos; Schratl, Petra; Hallström, Seth; Wintersperger, Andrea; Nusshold, Christoph; DeVaney, Trevor; Zorn-Pauly, Klaus; Malli, Roland; Graier, Wolfgang; Malle, Ernst; Sattler, Wolfgang

2014-01-01

Microglia, the immunocompetent cells of the CNS, are rapidly activated in response to injury and microglia migration towards and homing at damaged tissue plays a key role in CNS regeneration. Lysophosphatidic acid (LPA) is involved in signaling events evoking microglia responses through cognate G protein-coupled receptors. Here we show that human immortalized C13NJ microglia express LPA receptor subtypes LPA1, LPA2, and LPA3 on mRNA and protein level. LPA activation of C13NJ cells induced Rho and extracellular signal-regulated kinase activation and enhanced cellular ATP production. In addition, LPA induced process retraction, cell spreading, led to pronounced changes of the actin cytoskeleton and reduced cell motility, which could be reversed by inhibition of Rho activity. To get an indication about LPA-induced global alterations in protein expression patterns a 2-D DIGE/LC-ESI-MS proteomic approach was applied. On the proteome level the most prominent changes in response to LPA were observed for glycolytic enzymes and proteins regulating cell motility and/or cytoskeletal dynamics. The present findings suggest that naturally occurring LPA is a potent regulator of microglia biology. This might be of particular relevance in the pathophysiological context of neurodegenerative disorders where LPA concentrations can be significantly elevated in the CNS. PMID:19899077

SLC1 and SLC4 encode partially redundant acyl-coenzyme A 1-acylglycerol-3-phosphate O-acyltransferases of budding yeast

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Benghezal, Mohammed; Roubaty, Carole; Veepuri, Vijayanath

2007-01-01

Phosphatidic acid is the intermediate, from which all glycerophospholipids are synthesized. In yeast, it is generated from lysophosphatidic acid, which is acylated by Slc1p, an sn-2-specific, acyl-coenzyme A-dependent 1-acylglycerol-3-phosphate O-acyltransferase. Deletion of SLC1 is not lethal...

Lysophosphatidic acid modulates the association of PTP1B with N-cadherin/catenin complex in SKOV3 ovarian cancer cells.

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Huang, Ruby Yun-Ju; Wen, Chen-Chen; Liao, Chih-Kai; Wang, Shu-Huei; Chou, Liang-Yin; Wu, Jiahn-Chun

2012-09-01

LPA (lysophosphatidic acid) is a natural phospholipid that plays important roles in promoting cancer cell proliferation, invasion and metastases. We previously reported that LPA induces ovarian cancer cell dispersal and disruption of AJ (adherens junction) through the activation of SFK (Src family kinases). In this study, we have investigated the regulatory mechanisms during the early phase of LPA-induced cell dispersal. An in vitro model of the ovarian cancer cell line SKOV3 for cell dispersal was used. LPA induces rapid AJ disruption by increasing the internalization of N-cadherin-β-catenin. By using immunoprecipitations, LPA was shown to induce increased tyrosine phosphorylation of β-catenin and alter the balance of β-catenin-bound SFK and PTP1B (phosphotyrosine phosphatase 1B). The altered balance of tyrosine kinase/phosphatase correlated with a concomitant disintegration of the β-catenin-α-catenin, but not the β-catenin-N-cadherin complex. This disintegration of β-catenin from α-catenin and the cell dispersal caused by LPA can be rescued by blocking SFK activity with the chemical inhibitor, PP2. More importantly, PP2 also restores the level of PTP1B bound to β-catenin. We propose that LPA signalling alters AJ stability by changing the dynamics of tyrosine kinase/phosphatase bound to AJ proteins. This work provides further understanding of the early signalling events regulating ovarian cancer cell dispersal and AJ disruption induced by LPA. © The Author(s) Journal compilation © 2012 International Federation for Cell Biology.

Enhanced root growth in phosphate-starved Arabidopsis by stimulating de novo phospholipid biosynthesis through the overexpression of LYSOPHOSPHATIDIC ACID ACYLTRANSFERASE 2 (LPAT2).

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Angkawijaya, Artik Elisa; Nguyen, Van Cam; Nakamura, Yuki

2017-09-01

Upon phosphate starvation, plants retard shoot growth but promote root development presumably to enhance phosphate assimilation from the ground. Membrane lipid remodelling is a metabolic adaptation that replaces membrane phospholipids by non-phosphorous galactolipids, thereby allowing plants to obtain scarce phosphate yet maintain the membrane structure. However, stoichiometry of this phospholipid-to-galactolipid conversion may not account for the massive demand of membrane lipids that enables active growth of roots under phosphate starvation, thereby suggesting the involvement of de novo phospholipid biosynthesis, which is not represented in the current model. We overexpressed an endoplasmic reticulum-localized lysophosphatidic acid acyltransferase, LPAT2, a key enzyme that catalyses the last step of de novo phospholipid biosynthesis. Two independent LPAT2 overexpression lines showed no visible phenotype under normal conditions but showed increased root length under phosphate starvation, with no effect on phosphate starvation response including marker gene expression, root hair development and anthocyanin accumulation. Accompanying membrane glycerolipid profiling of LPAT2-overexpressing plants revealed an increased content of major phospholipid classes and distinct responses to phosphate starvation between shoot and root. The findings propose a revised model of membrane lipid remodelling, in which de novo phospholipid biosynthesis mediated by LPAT2 contributes significantly to root development under phosphate starvation. © 2017 John Wiley & Sons Ltd.

Involvement of aberrant DNA methylation on reduced expression of lysophosphatidic acid receptor-1 gene in rat tumor cell lines

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Tsujiuchi, Toshifumi; Shimizu, Kyoko; Onishi, Mariko; Sugata, Eriko; Fujii, Hiromasa; Mori, Toshio; Honoki, Kanya; Fukushima, Nobuyuki

2006-01-01

Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation, migration, and protects cells from apoptosis. It interacts with specific G protein-coupled transmembrane receptors. Recently, it has been reported that alterations of LPA receptor expression might be important in the malignant transformation of tumor cells. Therefore, to assess an involvement of DNA methylation in reduced expression of the LPA receptor-1 (lpa1) gene, we investigated the expression of the lpa1 gene and its DNA methylation patterns in rat tumor cell lines. Both rat brain-derived neuroblastoma B103 and liver-derived hepatoma RH7777 cells used in this study indicated no expression of lpa1. For the analysis of methylation status, bisulfite sequencing was performed with B103 and RH7777 cells, comparing with other lpa1 expressed cells and normal tissues of brain and liver. The lpa1 expressed cells and tissues were all unmethylated in this region of lpa1. In contrast, both B103 and RH7777 cells were highly methylated, correlating with reduced expression of the lpa1. Treatment with 5-aza 2'-deoxycytidine induced expression of lpa1 gene in B103 and RH7777 cells after 24 h. In RH7777 cells treated with 5-aza 2'-deoxycytidine, stress fiber formation was also observed in response to LPA in RH7777 cells, but not in untreated RH7777 cells. These results suggest that aberrant DNA methylation of the lpa1 gene may be involved in its reduced expression in rat tumor cells

Lysophosphatidic acid receptor-5 negatively regulates cellular responses in mouse fibroblast 3T3 cells

Energy Technology Data Exchange (ETDEWEB)

Dong, Yan; Hirane, Miku; Araki, Mutsumi [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Fukushima, Nobuyuki [Division of Molecular Neurobiology, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Tsujiuchi, Toshifumi, E-mail: ttujiuch@life.kindai.ac.jp [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan)

2014-04-04

Highlights: • LPA{sub 5} inhibits the cell growth and motile activities of 3T3 cells. • LPA{sub 5} suppresses the cell motile activities stimulated by hydrogen peroxide in 3T3 cells. • Enhancement of LPA{sub 5} on the cell motile activities inhibited by LPA{sub 1} in 3T3 cells. • The expression and activation of Mmp-9 were inhibited by LPA{sub 5} in 3T3 cells. • LPA signaling via LPA{sub 5} acts as a negative regulator of cellular responses in 3T3 cells. - Abstract: Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA{sub 1}–LPA{sub 6}) mediates a variety of biological functions, including cell migration. Recently, we have reported that LPA{sub 1} inhibited the cell motile activities of mouse fibroblast 3T3 cells. In the present study, to evaluate a role of LPA{sub 5} in cellular responses, Lpar5 knockdown (3T3-L5) cells were generated from 3T3 cells. In cell proliferation assays, LPA markedly stimulated the cell proliferation activities of 3T3-L5 cells, compared with control cells. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3-L5 cells were significantly higher than those of control cells. The activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. 3T3-L5 cells stimulated the activation of Mmp-2, correlating with the expression levels of Mmp-2 gene. Moreover, to assess the co-effects of LPA{sub 1} and LPA{sub 5} on cell motile activities, Lpar5 knockdown (3T3a1-L5) cells were also established from Lpar1 over-expressing (3T3a1) cells. 3T3a1-L5 cells increased the cell motile activities of 3T3a1 cells, while the cell motile activities of 3T3a1 cells were significantly lower than those of control cells. These results suggest that LPA{sub 5} may act as a negative regulator of cellular responses in mouse fibroblast 3T3 cells, similar to the case for LPA{sub 1}.

Lysophosphatidic acid receptor-5 negatively regulates cellular responses in mouse fibroblast 3T3 cells

International Nuclear Information System (INIS)

Dong, Yan; Hirane, Miku; Araki, Mutsumi; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

2014-01-01

Highlights: • LPA 5 inhibits the cell growth and motile activities of 3T3 cells. • LPA 5 suppresses the cell motile activities stimulated by hydrogen peroxide in 3T3 cells. • Enhancement of LPA 5 on the cell motile activities inhibited by LPA 1 in 3T3 cells. • The expression and activation of Mmp-9 were inhibited by LPA 5 in 3T3 cells. • LPA signaling via LPA 5 acts as a negative regulator of cellular responses in 3T3 cells. - Abstract: Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA 1 –LPA 6 ) mediates a variety of biological functions, including cell migration. Recently, we have reported that LPA 1 inhibited the cell motile activities of mouse fibroblast 3T3 cells. In the present study, to evaluate a role of LPA 5 in cellular responses, Lpar5 knockdown (3T3-L5) cells were generated from 3T3 cells. In cell proliferation assays, LPA markedly stimulated the cell proliferation activities of 3T3-L5 cells, compared with control cells. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3-L5 cells were significantly higher than those of control cells. The activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. 3T3-L5 cells stimulated the activation of Mmp-2, correlating with the expression levels of Mmp-2 gene. Moreover, to assess the co-effects of LPA 1 and LPA 5 on cell motile activities, Lpar5 knockdown (3T3a1-L5) cells were also established from Lpar1 over-expressing (3T3a1) cells. 3T3a1-L5 cells increased the cell motile activities of 3T3a1 cells, while the cell motile activities of 3T3a1 cells were significantly lower than those of control cells. These results suggest that LPA 5 may act as a negative regulator of cellular responses in mouse fibroblast 3T3 cells, similar to the case for LPA 1

Lysophosphatidic acid signaling through its receptor initiates profibrotic epithelial cell fibroblast communication mediated by epithelial cell derived connective tissue growth factor.

Science.gov (United States)

Sakai, Norihiko; Chun, Jerold; Duffield, Jeremy S; Lagares, David; Wada, Takashi; Luster, Andrew D; Tager, Andrew M

2017-03-01

The expansion of the fibroblast pool is a critical step in organ fibrosis, but the mechanisms driving expansion remain to be fully clarified. We previously showed that lysophosphatidic acid (LPA) signaling through its receptor LPA 1 expressed on fibroblasts directly induces the recruitment of these cells. Here we tested whether LPA-LPA 1 signaling drives fibroblast proliferation and activation during the development of renal fibrosis. LPA 1 -deficient (LPA 1 -/- ) or -sufficient (LPA 1 +/+ ) mice were crossed to mice with green fluorescent protein expression (GFP) driven by the type I procollagen promoter (Col-GFP) to identify fibroblasts. Unilateral ureteral obstruction-induced increases in renal collagen were significantly, though not completely, attenuated in LPA 1 -/- Col-GFP mice, as were the accumulations of both fibroblasts and myofibroblasts. Connective tissue growth factor was detected mainly in tubular epithelial cells, and its levels were suppressed in LPA 1 -/- Col-GFP mice. LPA-LPA 1 signaling directly induced connective tissue growth factor expression in primary proximal tubular epithelial cells, through a myocardin-related transcription factor-serum response factor pathway. Proximal tubular epithelial cell-derived connective tissue growth factor mediated renal fibroblast proliferation and myofibroblast differentiation. Administration of an inhibitor of myocardin-related transcription factor/serum response factor suppressed obstruction-induced renal fibrosis. Thus, targeting LPA-LPA 1 signaling and/or myocardin-related transcription factor/serum response factor-induced transcription could be promising therapeutic strategies for renal fibrosis. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Lysophosphatidic acid signaling via LPA_1 and LPA_3 regulates cellular functions during tumor progression in pancreatic cancer cells

International Nuclear Information System (INIS)

Fukushima, Kaori; Takahashi, Kaede; Yamasaki, Eri; Onishi, Yuka; Fukushima, Nobuyuki; Honoki, Kanya; Tsujiuchi, Toshifumi

2017-01-01

Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors exhibits a variety of biological effects, such as cell proliferation, motility and differentiation. The aim of this study was to evaluate the roles of LPA_1 and LPA_3 in cellular functions during tumor progression in pancreatic cancer cells. LPA_1 and LPA_3 knockdown cells were generated from PANC-1 cells. The cell motile and invasive activities of PANC-1 cells were inhibited by LPA_1 and LPA_3 knockdown. In gelatin zymography, LPA_1 and LPA_3 knockdown cells indicated the low activation of matrix metalloproteinase-2 (MMP-2) in the presence of LPA. Next, to assess whether LPA_1 and LPA_3 regulate cellular functions induced by anticancer drug, PANC-1 cells were treated with cisplatin (CDDP) for approximately 6 months. The cell motile and invasive activities of long-term CDDP treated cells were markedly higher than those of PANC-1 cells, correlating with the expression levels of LPAR1 and LPAR3 genes. In soft agar assay, the long-term CDDP treated cells formed markedly large sized colonies. In addition, the cell motile and invasive activities enhanced by CDDP were significantly suppressed by LPA_1 and LPA_3 knockdown as well as colony formation. These results suggest that LPA signaling via LPA_1 and LPA_3 play an important role in the regulation of cellular functions during tumor progression in PANC-1 cells. - Highlights: • The cell motile and invasive activities of PANC-1 cells were stimulated by LPA_1 and LPA_3. • LPA_1 and LPA_3 enhanced MMP-2 activation in PANC-1 cells. • The expressions of LPAR1 and LPAR3 genes were elevated in PANC-1 cells treated with cisplatin. • The cell motile and invasive activities of PANC-1 cells treated with cisplatin were suppressed by LPA_1 and LPA_3 knockdown. • LPA_1 and LPA_3 are involved in the regulation of cellular functions during tumor progression in PANC-1 cells.

Expression of lysophosphatidic acid receptor 1 and relation with cell proliferation, apoptosis, and angiogenesis on preneoplastic changes induced by cadmium chloride in the rat ventral prostate.

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Riánsares Arriazu

Full Text Available BACKGROUND: Lysophosphatidic acid (LPA is a phospholipid growth factor involved in cell proliferation, differentiation, migration, inflammation, angiogenesis, wound healing, cancer invasion, and survival. This study was directed to evaluate the immunoexpression of LPA-1, cell proliferation, apoptosis, and angiogenesis markers in preneoplastic lesions induced with cadmium chloride in rat prostate. METHODS: The following parameters were calculated in ventral prostate of normal rats and rats that received Cd in drinking water during 24 months: percentages of cells immunoreactive to LPA-1 (LILPA1, PCNA (LIPCNA, MCM7 (LIMCM7, ubiquitin (LIUBI, apoptotic cells (LIAPO, and p53 (LIp53; volume fraction of Bcl-2 (VFBcl-2; and length of microvessels per unit of volume (LVMV/mm3. Data were analyzed using Student's t-test and Pearson correlation test. RESULTS: The LILPA1 in dysplastic lesions and normal epithelium of Cd-treated rats was significantly higher than those in the control group. Markers of proliferation were significantly increased in dysplastic lesions, whereas some apoptotic markers were significantly decreased. No significant differences between groups were found in VFBcl-2. Dysplastic lesions showed a significant increase of LIp53. The length of microvessels per unit of volume was elevated in dysplastic acini. Statistically significant correlations were found only between LILPA1 and LIUBI. CONCLUSIONS: Our results suggest that LPA-1 might be implicated in dysplastic lesions induced by cadmium chloride development. More studies are needed to confirm its potential contribution to the disease.

Derivatives of Dictyostelium differentiation-inducing factors inhibit lysophosphatidic acid–stimulated migration of murine osteosarcoma LM8 cells

Energy Technology Data Exchange (ETDEWEB)

Kubohara, Yuzuru, E-mail: ykuboha@juntendo.ac.jp [Department of Molecular and Cellular Biology, Institute for Molecular and Cellular Regulation (IMCR), Gunma University, Maebashi 371-8512 (Japan); Department of Health Science, Juntendo University Graduate School of Health and Sports Science, Inzai 270-1695 (Japan); Komachi, Mayumi [Department of Molecular and Cellular Biology, Institute for Molecular and Cellular Regulation (IMCR), Gunma University, Maebashi 371-8512 (Japan); Department of Radiation Oncology, Gunma University Graduate School of Medicine, Maebashi 371-8511 (Japan); Homma, Yoshimi [Department of Biomolecular Science, Institute of Biomedical Sciences, Fukushima Medical University School of Medicine, Fukushima 960-1295 (Japan); Kikuchi, Haruhisa; Oshima, Yoshiteru [Laboratory of Natural Product Chemistry, Tohoku University Graduate School of Pharmaceutical Sciences, Aoba-yama, Aoba-ku, Sendai 980-8578 (Japan)

2015-08-07

Osteosarcoma is a common metastatic bone cancer that predominantly develops in children and adolescents. Metastatic osteosarcoma remains associated with a poor prognosis; therefore, more effective anti-metastatic drugs are needed. Differentiation-inducing factor-1 (DIF-1), −2, and −3 are novel lead anti-tumor agents that were originally isolated from the cellular slime mold Dictyostelium discoideum. Here we investigated the effects of a panel of DIF derivatives on lysophosphatidic acid (LPA)-induced migration of mouse osteosarcoma LM8 cells by using a Boyden chamber assay. Some DIF derivatives such as Br-DIF-1, DIF-3(+2), and Bu-DIF-3 (5–20 μM) dose-dependently suppressed LPA-induced cell migration with associated IC{sub 50} values of 5.5, 4.6, and 4.2 μM, respectively. On the other hand, the IC{sub 50} values of Br-DIF-1, DIF-3(+2), and Bu-DIF-3 versus cell proliferation were 18.5, 7.2, and 2.0 μM, respectively, in LM8 cells, and >20, 14.8, and 4.3 μM, respectively, in mouse 3T3-L1 fibroblasts (non-transformed). Together, our results demonstrate that Br-DIF-1 in particular may be a valuable tool for the analysis of cancer cell migration, and that DIF derivatives such as DIF-3(+2) and Bu-DIF-3 are promising lead anti-tumor agents for the development of therapies that suppress osteosarcoma cell proliferation, migration, and metastasis. - Highlights: • LPA induces cell migration (invasion) in murine osteosarcoma LM8 cells. • DIFs are novel lead anti-tumor agents found in Dictyostelium discoideum. • We examined the effects of DIF derivatives on LPA-induced LM8 cell migration in vitro. • Some of the DIF derivatives inhibited LPA-induced LM8 cell migration.

PSI1 is responsible for the stearic acid enrichment that is characteristic of phosphatidylinositol in yeast

DEFF Research Database (Denmark)

Le Guédard, Marina; Bessoule, Jean-Jacques; Boyer, Valérie

2009-01-01

complete disappearance of stearic (but not of palmitic acid) at the sn-1 position of this phospholipid. Moreover, it was found that, whereas glycerol 3-phosphate, lysophosphatidic acid and 1-acyl lysophosphatidylinositol acyltransferase activities were similar in microsomal membranes isolated from wild......-acyl-1-lysolysophosphatidylinositol acyltransferase activity was recovered, and was accompanied by a strong increase in the stearic acid content of lysophosphatidylinositol. As previously suggested for phosphatidylinositol from animal cells (which contains almost exclusively stearic acid...... as the saturated fatty acid), the results obtained in the present study demonstrate that the existence of phosphatidylinositol species containing stearic acid in yeast results from a remodeling of neo-synthesized molecules of phosphatidylinositol....

Genetic and Functional Evidence Supports LPAR1 as a Susceptibility Gene for Hypertension.

Science.gov (United States)

Xu, Ke; Ma, Lu; Li, Yang; Wang, Fang; Zheng, Gu-Yan; Sun, Zhijun; Jiang, Feng; Chen, Yundai; Liu, Huirong; Dang, Aimin; Chen, Xi; Chun, Jerold; Tian, Xiao-Li

2015-09-01

Essential hypertension is a complex disease affected by genetic and environmental factors and serves as a major risk factor for cardiovascular diseases. Serum lysophosphatidic acid correlates with an elevated blood pressure in rats, and lysophosphatidic acid interacts with 6 subtypes of receptors. In this study, we assessed the genetic association of lysophosphatidic acid receptors with essential hypertension by genotyping 28 single-nucleotide polymorphisms from genes encoding for lysophosphatidic acid receptors, LPAR1, LPAR2, LPAR3, LPAR4, LPAR5, and LPAR6 and their flanking sequences, in 3 Han Chinese cohorts consisting of 2630 patients and 3171 controls in total. We identified a single-nucleotide polymorphism, rs531003 in the 3'-flanking genomic region of LPAR1, associated with hypertension (the Bonferroni corrected P=1.09×10(-5), odds ratio [95% confidence interval]=1.23 [1.13-1.33]). The risk allele C of rs531003 is associated with the increased expression of LPAR1 and the susceptibility of hypertension, particularly in those with a shortage of sleep (P=4.73×10(-5), odds ratio [95% confidence interval]=1.75 [1.34-2.28]). We further demonstrated that blood pressure elevation caused by sleep deprivation and phenylephrine-induced vasoconstriction was both diminished in LPAR1-deficient mice. Together, we show that LPAR1 is a novel susceptibility gene for human essential hypertension and that stress, such as shortage of sleep, increases the susceptibility of patients with risk allele to essential hypertension. © 2015 American Heart Association, Inc.

Lysophosphatidic acid signaling via LPA{sub 1} and LPA{sub 3} regulates cellular functions during tumor progression in pancreatic cancer cells

Energy Technology Data Exchange (ETDEWEB)

Fukushima, Kaori; Takahashi, Kaede; Yamasaki, Eri; Onishi, Yuka [Division of Molecular Oncology, Department of Life Science, Faculty of Science and Engineering, Kindai University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Fukushima, Nobuyuki [Division of Molecular Neurobiology, Department of Life Science, Faculty of Science and Engineering, Kindai University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Honoki, Kanya [Department of Orthopedic Surgery, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Tsujiuchi, Toshifumi, E-mail: ttujiuch@life.kindai.ac.jp [Division of Molecular Oncology, Department of Life Science, Faculty of Science and Engineering, Kindai University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan)

2017-03-01

Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors exhibits a variety of biological effects, such as cell proliferation, motility and differentiation. The aim of this study was to evaluate the roles of LPA{sub 1} and LPA{sub 3} in cellular functions during tumor progression in pancreatic cancer cells. LPA{sub 1} and LPA{sub 3} knockdown cells were generated from PANC-1 cells. The cell motile and invasive activities of PANC-1 cells were inhibited by LPA{sub 1} and LPA{sub 3} knockdown. In gelatin zymography, LPA{sub 1} and LPA{sub 3} knockdown cells indicated the low activation of matrix metalloproteinase-2 (MMP-2) in the presence of LPA. Next, to assess whether LPA{sub 1} and LPA{sub 3} regulate cellular functions induced by anticancer drug, PANC-1 cells were treated with cisplatin (CDDP) for approximately 6 months. The cell motile and invasive activities of long-term CDDP treated cells were markedly higher than those of PANC-1 cells, correlating with the expression levels of LPAR1 and LPAR3 genes. In soft agar assay, the long-term CDDP treated cells formed markedly large sized colonies. In addition, the cell motile and invasive activities enhanced by CDDP were significantly suppressed by LPA{sub 1} and LPA{sub 3} knockdown as well as colony formation. These results suggest that LPA signaling via LPA{sub 1} and LPA{sub 3} play an important role in the regulation of cellular functions during tumor progression in PANC-1 cells. - Highlights: • The cell motile and invasive activities of PANC-1 cells were stimulated by LPA{sub 1} and LPA{sub 3}. • LPA{sub 1} and LPA{sub 3} enhanced MMP-2 activation in PANC-1 cells. • The expressions of LPAR1 and LPAR3 genes were elevated in PANC-1 cells treated with cisplatin. • The cell motile and invasive activities of PANC-1 cells treated with cisplatin were suppressed by LPA{sub 1} and LPA{sub 3} knockdown. • LPA{sub 1} and LPA{sub 3} are involved in the regulation of cellular functions during tumor

Dual Action of Lysophosphatidate-Functionalised Titanium: Interactions with Human (MG63 Osteoblasts and Methicillin Resistant Staphylococcus aureus.

Directory of Open Access Journals (Sweden)

Mette Elena Skindersoe

Full Text Available Titanium (Ti is a widely used material for surgical implants; total joint replacements (TJRs, screws and plates for fixing bones and dental implants are forged from Ti. Whilst Ti integrates well into host tissue approximately 10% of TJRs will fail in the lifetime of the patient through a process known as aseptic loosening. These failures necessitate revision arthroplasties which are more complicated and costly than the initial procedure. Finding ways of enhancing early (osseointegration of TJRs is therefore highly desirable and continues to represent a research priority in current biomaterial design. One way of realising improvements in implant quality is to coat the Ti surface with small biological agents known to support human osteoblast formation and maturation at Ti surfaces. Lysophosphatidic acid (LPA and certain LPA analogues offer potential solutions as Ti coatings in reducing aseptic loosening. Herein we present evidence for the successful bio-functionalisation of Ti using LPA. This modified Ti surface heightened the maturation of human osteoblasts, as supported by increased expression of alkaline phosphatase. These functionalised surfaces also deterred the attachment and growth of Staphylococcus aureus, a bacterium often associated with implant failures through sepsis. Collectively we provide evidence for the fabrication of a dual-action Ti surface finish, a highly desirable feature towards the development of next-generation implantable devices.

Identification of Farnesyl Pyrophosphate and N-Arachidonylglycine as Endogenous Ligands for GPR92*S⃞

OpenAIRE

Oh, Da Young; Yoon, Jung Min; Moon, Mi Jin; Hwang, Jong-Ik; Choe, Han; Lee, Ju Yeon; Kim, Jae Il; Kim, Sunoh; Rhim, Hyewhon; O'Dell, David K.; Walker, J. Michael; Na, Heung Sik; Lee, Min Goo; Kwon, Hyuk Bang; Kim, Kyungjin

2008-01-01

A series of small compounds acting at the orphan G protein-coupled receptor GPR92 were screened using a signaling pathway-specific reporter assay system. Lipid-derived molecules including farnesyl pyrophosphate (FPP), N-arachidonylglycine (NAG), and lysophosphatidic acid were found to activate GPR92. FPP and lysophosphatidic acid were able to activate both Gq/11- and Gs-mediated signaling pathways, whereas NAG activated only the Gq/11-mediated signaling pathway. Co...

Co-ordinate regulation of growth factor receptors and lipid phosphate phosphatase-1 controls cell activation by exogenous lysophosphatidate.

Science.gov (United States)

Pilquil, C; Ling, Z C; Singh, I; Buri, K; Zhang, Q X; Brindley, D N

2001-11-01

The serum-derived lipid growth factors, lysophosphatidate (LPA) and sphingosine 1-phosphate (S1P), activate cells selectively through different members of a family of endothelial differentiation gene (EDG) receptors. Activation of EDG receptors by LPA and S1P provides a variety of signalling cascades depending upon the G-protein coupling of the different EDG receptors. This leads to chemotactic and mitogenic responses, which are important in wound healing. For example, LPA stimulates fibroblast division and S1P stimulates the chemotaxis and division of endothelial cells leading to angiogenesis. Counteracting these effects of LPA and S1P, are the actions of lipid phosphate phosphatases (LPP, or phosphatidate phosphohydrolases, Type 2). The isoform LPP-1 is expressed in the plasma membrane with its active site outside the cell. This enzyme is responsible for 'ecto-phosphatase' activity leading to the degradation of exogenous lipid phosphate mediators, particularly LPA. Expression of LPP-1 decreases cell activation by exogenous LPA. The mechanism for this is controversial and several mechanisms have been proposed. Evidence will be presented that the LPPs cross-talk with EDG and other growth factor receptors, thus, regulating the responses of the cells to lipid phosphate mediators of signal transduction.

Association of fatty acids and lipids metabolism in placenta with early spontaneous pregnancy loss in Chinese women.

Science.gov (United States)

Li, Kelei; Zhang, Xiaotian; Chen, Gong; Pei, Lijun; Xiao, Hailong; Jiang, Jiajing; Li, Jiaomei; Zheng, Xiaoying; Li, Duo

2018-02-21

The aim of the present study was to evaluate the association of fatty acids and lipids metabolism in placenta with early spontaneous pregnancy loss (ESPL) in Chinese women. Seventy women with ESPL and 29 healthy pregnant women who asked for legal induced abortion were included in the case and control groups, respectively. The gestational age of the subject foetuses in both the case and control groups ranged from 4 to 10 weeks. The total fatty acids composition in the decidual and villous tissues was detected by gas-liquid chromatography using a standard method. Metabonomics analysis of the decidual and villous tissues was conducted by ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-QTOFMS). The total C18:3n-3 in the decidual and villous tissues, total n-3 polyunsaturated fatty acid (n-3 PUFA) in the decidual tissue, and total C18:2n-6 in the villous tissue were all significantly lower in the case group than in the control group. The ratio of C20:4n-6/C20:5n-3 in villous tissue was significantly higher, but prostaglandin I 2 as well as hydroxyeicosapentaenoic acid, leukotriene B 5 and thromboxane B 3 in the villous tissue were significantly lower in the case group than in the control group. In addition, the low content of lysophosphatide in the decidual and villous tissues and the low content of diacylglycerol in the villous tissue were also associated with the occurance of ESPL. In conclusion, the lack of essential fatty acids, high ratio of C20:4n-6/C20:5n-3, abnormal eicosanoids metabolism and low content of lysophosphatide and diacylglycerol in the placenta were all potential risk factors for ESPL in Chinese.

Helicase-dependent amplification of nucleic acids.

Science.gov (United States)

Cao, Yun; Kim, Hyun-Jin; Li, Ying; Kong, Huimin; Lemieux, Bertrand

2013-10-11

Helicase-dependent amplification (HDA) is a novel method for the isothermal in vitro amplification of nucleic acids. The HDA reaction selectively amplifies a target sequence by extension of two oligonucleotide primers. Unlike the polymerase chain reaction (PCR), HDA uses a helicase enzyme to separate the deoxyribonucleic acid (DNA) strands, rather than heat denaturation. This allows DNA amplification without the need for thermal cycling. The helicase used in HDA is a helicase super family II protein obtained from a thermophilic organism, Thermoanaerobacter tengcongensis (TteUvrD). This thermostable helicase is capable of unwinding blunt-end nucleic acid substrates at elevated temperatures (60° to 65°C). The HDA reaction can also be coupled with reverse transcription for ribonucleic acid (RNA) amplification. The products of this reaction can be detected during the reaction using fluorescent probes when incubations are conducted in a fluorimeter. Alternatively, products can be detected after amplification using a disposable amplicon containment device that contains an embedded lateral flow strip. Copyright © 2013 John Wiley & Sons, Inc.

Na--dependent transport of basic, zwitterionic, and bicyclic amino acids by a broad-scope system in mouse blastocysts

International Nuclear Information System (INIS)

Van Winkle, L.J.; Christensen, H.N.; Campione, A.L.

1985-01-01

Mouse blastocysts which had been activated from diapause in utero appeared to take up amino acids via a Na - -dependent transport system with novel characteristics. In contrast to other cell types, uptake of 3-aminoendobicyclo [3,2,1]octane-3-carboxylic acid (BCO) by blastocysts was largely Na - dependent. Moreover, L-alanine and BCO met standard criteria for mutual competitive inhibition of the Na - -dependent transport of each other. The Ki for each of these amino acids as an inhibitor of transport of the other had a value similar to the value of its Km for transport. In addition, both 2-aminoendobicyclo [2,2,1]heptane-2-carboxylic acid and L-valine appeared to inhibit Na - -dependent transport of alanine and BCO competitively. Finally, alanine and L-lysine appeared to compete for the same Na+-dependent transport sites in blastocysts. For these reasons, the authors conclude that lysine, alanine, and BCO are transported by a common Na+-dependent system in blastocysts. In addition, the apparent interaction of the system with other basic amino acids, such as 1-dimethylpiperidine-4-amino-4-carboxylic acid, which has a nondissociable positive charge on its side chain, and L-arginine and L-homoarginine, whose cationic forms are highly predominant at neutral pH, suggests that the cationic forms of basic amino acids are transported by the wide-scope system

Arginine-dependent acid resistance in Salmonella enterica serovar Typhimurium

NARCIS (Netherlands)

Kieboom, J.; Abee, T.

2006-01-01

Salmonella enterica serovar Typhimurium does not survive a pH 2.5 acid challenge under conditions similar to those used for Escherichia coli (J. W. Foster, Nat. Rev. Microbiol. 2:898-907, 2004). Here, we provide evidence that S. enterica serovar Typhimurium can display arginine-dependent acid

EGFR-dependent signalling reduced and p38 dependent apoptosis required by Gallic acid in Malignant Mesothelioma cells.

Science.gov (United States)

Demiroglu-Zergeroglu, Asuman; Candemir, Gulsife; Turhanlar, Ebru; Sagir, Fatma; Ayvali, Nurettin

2016-12-01

The unrestrained EGFR signalling contributes to malignant phenotype in a number of cancers including Malignant Mesotheliomas. Present study was designed to evaluate EGFR-dependent anti-proliferative and apoptotic effects of Gallic acid in transformed Mesothelial (MeT-5A) and Malignant Mesothelioma (SPC212) cells. Gallic acid reduced the viability of Malignant Mesothelioma cells in a concentration and time-dependent manner. However, viability of mesothelial cells reduced only at high concentration and longer time periods. Gallic acid restrained the activation of EGFR, ERK1/2 and AKT proteins and down regulated expression of Cyclin D and Bcl-2 genes, but upregulated the expression of p21 gene in EGF-induced SPC212 cells. GA-induced transitory G1 arrest and triggered mitochondrial and death receptor mediated apoptosis, which requires p38MAPK activation. The data provided here indicate that GA is able to inhibit EGFR dependent proliferation and survival signals and induces p38 pathway dependent apoptosis in Malignant Mesothelioma cells. On the basis of these experimental findings it is worthwhile to investigate further the biological activity of Gallic acid on other Mesothelioma cell lines harbouring aberrant EGFR signals. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Poly(aspartic acid) with adjustable pH-dependent solubility.

Science.gov (United States)

Németh, Csaba; Gyarmati, Benjámin; Abdullin, Timur; László, Krisztina; Szilágyi, András

2017-02-01

Poly(aspartic acid) (PASP) derivatives with adjustable pH-dependent solubility were synthesized and characterized to establish the relationship between their structure and solubility in order to predict their applicability as a basic material for enteric coatings. Polysuccinimide, the precursor of PASP, was modified with short chain alkylamines, and the residual succinimide rings were subsequently opened to prepare the corresponding PASP derivatives. Study of the effect of the type and concentration of the side groups on the pH-dependent solubility of PASP showed that solubility can be adjusted by proper selection of the chemical structure. The Henderson-Hasselbalch (HH) and the extended HH equations were used to describe the pH-dependent solubility of the polymers quantitatively. The estimate provided by the HH equation is poor, but an accurate description of the pH-dependent solubility can be found with the extended HH equation. The dissolution rate of a polymer film prepared from a selected PASP derivative was determined by fluorescence marking. The film dissolved rapidly when the pH was increased above its pK a . Cellular viability tests show that PASP derivatives are non-toxic to a human cell line. These polymers are thus of great interest as starting materials for enteric coatings. Poly(amino acid) type biocompatible polymers were synthesized for future use as pharmaceutical film coatings. To this end, we tailored the pH-dependent solubility of poly(aspartic acid) (PASP). It was found that both the solubility and the pK a values of the modified PASP depended strongly on composition. Fluorescent marking was used to characterize the dissolution of a chosen PASP derivative. In acidic media only a negligible amount of the polymer dissolved, but dissolution was very fast and complete at the pH values that prevail in the small intestine. As a consequence, enteric coatings based on such PASP derivatives may be used for drug delivery in the gastrointestinal tract

E1A FUNCTIONS AS A COACTIVATOR OF RETINOIC ACID-DEPENDENT RETINOIC ACID RECEPTOR-BETA-2 PROMOTER ACTIVATION

NARCIS (Netherlands)

KRUYT, FAE; FOLKERS, GE; WALHOUT, AJM; VANDERLEEDE, BM; VANDERSAAG, PT; Kruyt, Frank

The retinoic acid (RA) receptor (RAR) beta2 promoter is strongly activated by RA in embryonal carcinoma (EC) cells. We examined this activation in the P19 EC-derived END-2 cell line and in E1A-expressing counterparts and found strong RA-dependent RARbeta2 promoter activation in the E1A-expressing

NblA1/A2-Dependent Homeostasis of Amino Acid Pools during Nitrogen Starvation in Synechocystis sp. PCC 6803.

Science.gov (United States)

Kiyota, Hiroshi; Hirai, Masami Yokota; Ikeuchi, Masahiko

2014-06-30

Nutrient balance is important for photosynthetic growth and biomass production in microalgae. Here, we investigated and compared metabolic responses of amino acid pools to nitrogen and sulfur starvation in a unicellular model cyanobacterium, Synechocystis sp. PCC 6803, and its mutant nblA1/A2. It is known that NblA1/A2-dependent and -independent breakdown of abundant photosynthetic phycobiliproteins and other cellular proteins supply nutrients to the organism. However, the contribution of the NblA1/A2-dependent nutrient supply to amino acid pool homeostasis has not been studied. Our study demonstrates that changes in the pool size of many amino acids during nitrogen starvation can be categorized as NblA1/A2-dependent (Gln, Glu, glutathione, Gly, Ile, Leu, Met, Phe, Pro, Ser, Thr, Tyr and Val) and NblA1/A2-independent (Ala, Asn, Lys, and Trp). We also report unique changes in amino acid pool sizes during sulfur starvation in wild type and the mutant and found a generally marked increase in the Lys pool in cyanobacteria during nutrient starvation. In conclusion, the NblA1/A2-dependent protein turnover contributes to the maintenance of many amino acid pools during nitrogen starvation.

NblA1/A2-Dependent Homeostasis of Amino Acid Pools during Nitrogen Starvation in Synechocystis sp. PCC 6803

Directory of Open Access Journals (Sweden)

Hiroshi Kiyota

2014-06-01

Full Text Available Nutrient balance is important for photosynthetic growth and biomass production in microalgae. Here, we investigated and compared metabolic responses of amino acid pools to nitrogen and sulfur starvation in a unicellular model cyanobacterium, Synechocystis sp. PCC 6803, and its mutant nblA1/A2. It is known that NblA1/A2-dependent and -independent breakdown of abundant photosynthetic phycobiliproteins and other cellular proteins supply nutrients to the organism. However, the contribution of the NblA1/A2-dependent nutrient supply to amino acid pool homeostasis has not been studied. Our study demonstrates that changes in the pool size of many amino acids during nitrogen starvation can be categorized as NblA1/A2-dependent (Gln, Glu, glutathione, Gly, Ile, Leu, Met, Phe, Pro, Ser, Thr, Tyr and Val and NblA1/A2-independent (Ala, Asn, Lys, and Trp. We also report unique changes in amino acid pool sizes during sulfur starvation in wild type and the mutant and found a generally marked increase in the Lys pool in cyanobacteria during nutrient starvation. In conclusion, the NblA1/A2-dependent protein turnover contributes to the maintenance of many amino acid pools during nitrogen starvation.

Perception of low red:far-red ratio comprises both salicylic acid- and jasmonic acid-dependent pathogen defences in Arabidopsis

NARCIS (Netherlands)

De Wit, M. de; Spoel, S.H.; Sanchez-Perez, G.F.; Gommers, C.M.M.; Pieterse, C.M.J.; Voesenek, L.A.C.J.; Pierik, R.

2013-01-01

In dense stands of plants, such as agricultural monocultures, plants are exposed simultaneously to competition for light and other stresses such as pathogen infection. Here, we show that both salicylic acid (SA)-dependent and jasmonic acid (JA)-dependent disease resistance is inhibited by a

Autotaxin: A protein with two faces

Energy Technology Data Exchange (ETDEWEB)

Tania, Mousumi; Khan, Md. Asaduzzaman; Zhang, Huaiyuan; Li, Jinhua [Department of Biochemistry, School of Biological Science and Technology, Central South University, Changsha, Hunan 410013 (China); Song, Yuanda, E-mail: yuanda_song@hotmail.com [Department of Biochemistry, School of Biological Science and Technology, Central South University, Changsha, Hunan 410013 (China)

2010-10-29

Research highlights: {yields} Autotaxin (ATX) has lysophospholipase D activity. {yields} ATX catalyzes the formation of lysophosphatidic acid (LPA). {yields} LPA is a mitogen, and thus is responsible for cancer. {yields} ATX also catalyzes the formation of anti-cancerous cyclic phosphatidic acid. {yields} Autotaxin is a novel target of cancer therapy research. -- Abstract: Autotaxin (ATX) is a catalytic protein, which possesses lysophospholipase D activity, and thus involved in cellular membrane lipid metabolism and remodeling. Primarily, ATX was thought as a culprit protein for cancer, which potently stimulates cancer cell proliferation and tumor cell motility, augments the tumorigenicity and induces angiogenic responses. The product of ATX catalyzed reaction, lysophosphatidic acid (LPA) is a potent mitogen, which facilitates cell proliferation and migration, neurite retraction, platelet aggregation, smooth muscle contraction, actin stress formation and cytokine and chemokine secretion. In addition to LPA formation, later ATX has been found to catalyze the formation of cyclic phosphatidic acid (cPA), which have antitumor role by antimitogenic regulation of cell cycle, inhibition of cancer invasion and metastasis. Furthermore, the very attractive information to the scientists is that the LPA/cPA formation can be altered at different physiological conditions. Thus the dual role of ATX with the scope of product manipulation has made ATX a novel target for cancer treatment.

Time-dependent inhibition of CYP3A4 by gallic acid in human liver microsomes and recombinant systems.

Science.gov (United States)

Pu, Qiang-Hong; Shi, Liang; Yu, Chao

2015-03-01

1.Gallic acid is a main polyphenol in various fruits and plants. Inhibitory characteristics of gallic acid on CYP3A4 were still unclear. The objective of this work is hence to investigate inhibitory characteristics of gallic acid on CYP3A4 using testosterone as the probe substrate in human liver microsomes (HLMs) and recombinant CYP3A4 (rCYP3A4) systems. 2.Gallic acid caused concentration-dependent loss of CYP3A4 activity with IC50 values of 615.2 μM and 669.5 μM in HLM and rCYP3A4 systems, respectively. IC50-shift experiments showed that pre-incubation with gallic acid in the absence of NADPH contributed to 12- or 14-fold reduction of IC50 in HLM and rCYP3A4 systems, respectively, supporting a time-dependent inhibition. In HLM, time-dependent inactivation variables KI and Kinact were 485.8 μM and 0.05 min(-1), respectively. 3.Compared with the presence of NADPH, pre-incubation of gallic acid in the absence of NADPH markedly increased its inhibitory effects in HLM and rCYP3A4 systems. Those results indicate that CYP3A4 inactivation by gallic acid was independent on NADPH and was mainly mediated its oxidative products. 4.In conclusion, we showed that gallic acid weakly and time-dependently inactivated CYP3A4 via its oxidative products.

Association of connexin43 with a receptor protein tyrosine phosphatase

NARCIS (Netherlands)

Giepmans, Ben N G; Feiken, Elles; Gebbink, Martijn F B G; Moolenaar, Wouter H

2003-01-01

Connexin-43(Cx43)-based gap junctional communication is transiently inhibited by certain G protein-coupled receptor agonists, including lysophosphatidic acid, endothelin and thrombin. Our previous studies have implicated the c-Src protein tyrosine kinase in mediating closure of Cx43 based gap

Lysophosphatidic acid inhibition of the accumulation of Pseudomonas aeruginosa PAO1 alginate, pyoverdin, elastase and LasA

DEFF Research Database (Denmark)

Laux, D.C.; Corson, J.M.; Givskov, Michael Christian

2002-01-01

. In the present study, a lysophospholipid, 1-paimitoyl-2-hydroxy-sn-glycero-3-phosphate [also called monopalmitoylphosphatidic acid (MPPA)], which accumulates in inflammatory exudates, was shown to inhibit the extracellular accumulation of P. aeruginosa PAO1 alginate, elastase, LasA protease and the siderophore...

Zinc-ion-dependent acid phosphatase exhibits magnesium-ion-dependent myo-inositol-1-phosphatase activity.

Science.gov (United States)

Fujimoto, S; Okano, I; Tanaka, Y; Sumida, Y; Tsuda, J; Kawakami, N; Shimohama, S

1996-06-01

We have purified bovine brain Zn(2+)-dependent acid phosphatase (Zn(2+)-APase), which requires Zn2+ ions to hydrolyze the substrate p-nitrophenyl phosphate (pNPP) in an acidic environment. The substrate specificity and metal requirement of Zn(2+)-APase at a physiological pH was also studied. The enzyme exhibited hydrolytic activity on myo-inositol-1- and -2-monophosphates, 2'-adenosine monophosphate, 2'-guanosine monophosphate, and the alpha- and beta-glycerophosphates, glucose-1-phosphate, and fructose-6-phosphate in 50 mM Tris-HCl buffer (pH 7.4) in the presence of Mg2+ ions, but not on pNPP and phosphotyrosine. Zn2+, Mn2+ and Co2+ ions were less effective for activation. Among the above substrates, myo-inositol-1-phosphate was the most susceptible to hydrolysis by the enzyme in the presence of 3 mM Mg2+ ions. The enzyme exhibited an optimum pH at around 8 for myo-inositol-1-phosphate in the presence of 3 mM Mg2+ ions. The Mg(2+)-dependent myo-inositol-1-phosphatase activity of the enzyme was significantly inhibited by Li+ ions. The Zn(2+)-dependent p-nitrophenyl phosphatase activity and Mg(2+)-dependent myo-inositol-1-phosphatase activity of the purified enzyme fraction exhibited similar behavior on Sephadex G-100 and Mono Q colomns. These findings suggest that Zn(2+)-APase also exhibits Mg(2+)-dependent myo-inositol-1-phosphatase activity under physiological conditions.

Glucagon Couples Hepatic Amino Acid Catabolism to mTOR-Dependent Regulation of α-Cell Mass

Directory of Open Access Journals (Sweden)

Mark J. Solloway

2015-07-01

Full Text Available Understanding the regulation of islet cell mass has important implications for the discovery of regenerative therapies for diabetes. The liver plays a central role in metabolism and the regulation of endocrine cell number, but liver-derived factors that regulate α-cell and β-cell mass remain unidentified. We propose a nutrient-sensing circuit between liver and pancreas in which glucagon-dependent control of hepatic amino acid metabolism regulates α-cell mass. We found that glucagon receptor inhibition reduced hepatic amino acid catabolism, increased serum amino acids, and induced α-cell proliferation in an mTOR-dependent manner. In addition, mTOR inhibition blocked amino-acid-dependent α-cell replication ex vivo and enabled conversion of α-cells into β-like cells in vivo. Serum amino acids and α-cell proliferation were increased in neonatal mice but fell throughout postnatal development in a glucagon-dependent manner. These data reveal that amino acids act as sensors of glucagon signaling and can function as growth factors that increase α-cell proliferation.

Involvement of PlsX and the acyl-phosphate dependent sn-glycerol-3-phosphate acyltransferase PlsY in the initial stage of glycerolipid synthesis in Bacillus subtilis.

Science.gov (United States)

Hara, Yoshinori; Seki, Masahide; Matsuoka, Satoshi; Hara, Hiroshi; Yamashita, Atsushi; Matsumoto, Kouji

2008-12-01

The gene responsible for the first acylation of sn-glycerol-3-phosphate (G3P) in Bacillus subtilis has not yet been determined with certainty. The product of this first acylation, lysophosphatidic acid (LPA), is subsequently acylated again to form phosphatidic acid (PA), the primary precursor to membrane glycerolipids. A novel G3P acyltransferase (GPAT), the gene product of plsY, which uses acyl-phosphate formed by the plsX gene product, has recently been found to synthesize LPA in Streptococcus pneumoniae. We found that in B. subtilis growth arrests after repression of either a plsY homologue or a plsX homologue were overcome by expression of E. coli plsB, which encodes an acyl-acylcarrier protein (acyl-ACP)-dependent GPAT, although in the case of plsX repression a high level of plsB expression was required. B. subtilis has, therefore, a capability to use the acyl-ACP dependent GPAT of PlsB. Simultaneous expression of plsY and plsX suppressed the glycerol requirement of a strict glycerol auxotrophic derivative of the E. coli plsB26 mutant, although either one alone did not. Membrane fractions from B. subtilis cells catalyzed palmitoylphosphate-dependent acylation of [14C]-labeled G3P to synthesize [14C]-labeled LPA, whereas those from DeltaplsY cells did not. The results indicate unequivocally that PlsY is an acyl-phosphate dependent GPAT. Expression of plsX corrected the glycerol auxotrophy of a DeltaygiH (the deleted allele of an E. coli homologue of plsY) derivative of BB26-36 (plsB26 plsX50), suggesting an essential role of plsX other than substrate supply for acyl-phosphate dependent LPA synthesis. Two-hybrid examinations suggested that PlsY is associated with PlsX and that each may exist in multimeric form.

The Obesity-Associated FTO Gene Encodes a 2-Oxoglutarate–Dependent Nucleic Acid Demethylase

Science.gov (United States)

Gerken, Thomas; Girard, Christophe A.; Tung, Yi-Chun Loraine; Webby, Celia J.; Saudek, Vladimir; Hewitson, Kirsty S.; Yeo, Giles S. H.; McDonough, Michael A.; Cunliffe, Sharon; McNeill, Luke A.; Galvanovskis, Juris; Rorsman, Patrik; Robins, Peter; Prieur, Xavier; Coll, Anthony P.; Ma, Marcella; Jovanovic, Zorica; Farooqi, I. Sadaf; Sedgwick, Barbara; Barroso, Inês; Lindahl, Tomas; Ponting, Chris P.; Ashcroft, Frances M.; O'Rahilly, Stephen; Schofield, Christopher J.

2009-01-01

Variants in the FTO (fat mass and obesity associated) gene are associated with increased body mass index in humans. Here, we show by bioinformatics analysis that FTO shares sequence motifs with Fe(II)- and 2-oxoglutarate–dependent oxygenases. We find that recombinant murine Fto catalyzes the Fe(II)- and 2OG-dependent demethylation of 3-methylthymine in single-stranded DNA, with concomitant production of succinate, formaldehyde, and carbon dioxide. Consistent with a potential role in nucleic acid demethylation, Fto localizes to the nucleus in transfected cells. Studies of wild-type mice indicate that Fto messenger RNA (mRNA) is most abundant in the brain, particularly in hypothalamic nuclei governing energy balance, and that Fto mRNA levels in the arcuate nucleus are regulated by feeding and fasting. Studies can now be directed toward determining the physiologically relevant FTO substrate and how nucleic acid methylation status is linked to increased fat mass. PMID:17991826

Plasma Ascorbic Acid in Insulin and Non-insulin Dependent Diabetes

African Journals Online (AJOL)

Blood glucose, plasma ascorbic acid and haemoglobin levels were estimated in insulin dependent diabetics, non-insulin dependent diabetics and controls matched for number, sex and age. Significantly higher levels of these parameters were found in control group than in the other two groups. Statistically differences were ...

Factor VIIa response to a fat-rich meal does not depend on fatty acid composition: A randomized controlled trial

NARCIS (Netherlands)

Mennen, L.; Maat, M. de; Meijer, G.; Zock, P.; Grobbee, D.; Kok, F.; Kluft, C.; Schouten, E.

1998-01-01

A fat-rich meal increases activated factor VII (FVIIa), but it is not clear whether this increase depends on the fatty acid composition of the meal. Therefore, we studied the FVIIa response to fat-rich meals with different fatty acid composition in a randomized controlled crossover trial and

Dependence of the metabolic fecal amino acids on the amino acid content of the feed. 1

International Nuclear Information System (INIS)

Krawielitzki, K.; Schadereit, R.; Voelker, T.; Reichel, K.

1981-01-01

The amount of metabolic fecal amino acids (MFAA) in dependence on the amino acid intake was determined for graded maize rations in 15 N-labelled rats and the part of labelled endogenous amino acids in feces was calculated by the isotope dilution method. The excretion of amino acids and MFAA in feces are described as functions of the amino acid intake for 17 amino acids and calculated regressively. For all 17 amino acids investigated, there was a more or less steep increase of MFAA according to an increasing amino acid intake. In contrast to N-free feeding, the MFAA increase to the 2- to 4.5-fold value in feeding with pure maize (16.5% crude protein). The thesis of the constancy of the excretion of MFAA can consequently be no longer maintained. The true digestibility according to the conventional method is, on an average of all amino acids, 7.3 units below ascertained according to the 15 N method. The limiting amino acids lysine and threonine revealed the greatest difference. Tryptophane as first limiting amino acid could not be determined. The true digestibility of nearly all amino acids ascertained for maize by the isotope method is above 90%. (author)

Lysophosphatidic acids are new substrates for the phosphatase domain of soluble epoxide hydrolase[S

OpenAIRE

Oguro, Ami; Imaoka, Susumu

2012-01-01

Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has a C-terminus epoxide hydrolase domain and an N-terminus phosphatase domain. The endogenous substrates of epoxide hydrolase are known to be epoxyeicosatrienoic acids, but the endogenous substrates of the phosphatase activity are not well understood. In this study, to explore the substrates of sEH, we investigated the inhibition of the phosphatase activity of sEH toward 4-methylumbelliferyl phosphate by using lecithin and its hyd...

Sulfuric acid nucleation: power dependencies, variation with relative humidity, and effect of bases

Directory of Open Access Journals (Sweden)

J. H. Zollner

2012-05-01

Full Text Available Nucleation of particles composed of sulfuric acid, water, and nitrogen base molecules was studied using a continuous flow reactor. The particles formed from these vapors were detected with an ultrafine condensation particle counter, while vapors of sulfuric acid and nitrogen bases were detected by chemical ionization mass spectrometry. Variation of particle numbers with sulfuric acid concentration yielded a power dependency on sulfuric acid of 5 ± 1 for relative humidities of 14–68% at 296 K; similar experiments with varying water content yielded power dependencies on H₂O of ~7. The critical cluster contains about 5 H₂SO₄ molecules and a new treatment of the power dependency for H₂O suggests about 12 H₂O molecules for these conditions. Addition of 2-to-45 pptv of ammonia or methyl amine resulted in up to millions of times more particles than in the absence of these compounds. Particle detection capabilities, sulfuric acid and nitrogen base detection, wall losses, and the extent of particle growth are discussed. Results are compared to previous laboratory nucleation studies and they are also discussed in terms of atmospheric nucleation scenarios.

Abscisic acid-dependent multisite phosphorylation regulates the activity of a transcription activator AREB1

OpenAIRE

Furihata, Takashi; Maruyama, Kyonoshin; Fujita, Yasunari; Umezawa, Taishi; Yoshida, Riichiro; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

2006-01-01

bZIP-type transcription factors AREBs/ABFs bind an abscisic acid (ABA)-responsive cis-acting element named ABRE and transactivate downstream gene expression in Arabidopsis. Because AREB1 overexpression could not induce downstream gene expression, activation of AREB1 requires ABA-dependent posttranscriptional modification. We confirmed that ABA activated 42-kDa kinase activity, which, in turn, phosphorylated Ser/Thr residues of R-X-X-S/T sites in the conserved regions of AREB1. Amino acid subs...

Stimulation of apical sodium-dependent bile acid transporter expands the bile acid pool and generates bile acids with positive feedback properties.

Science.gov (United States)

Rudling, Mats; Bonde, Ylva

2015-01-01

Bile acid synthesis has been considered a prototype for how a physiological process is controlled by end product feedback inhibition. By this feedback inhibition, bile acid concentrations are kept within safe ranges. However, careful examination of published rodent data strongly suggests that bile acid synthesis is also under potent positive feedback control by hydrophilic bile acids. Current concepts on the regulation of bile acid synthesis are derived from mouse models. Recent data have shown that mice have farnesoid X receptor (FXR) antagonistic bile acids capable of quenching responses elicited by FXR agonistic bile acids. This is important to recognize to understand the regulation of bile acid synthesis in the mouse, and in particular to clarify if mouse model findings are valid also in the human situation. In addition to classic end product feedback inhibition, regulation of bile acid synthesis in the mouse largely appears also to be driven by changes in hepatic levels of murine bile acids such as α- and β-muricholic acids. This has not been previously recognized. Stimulated bile acid synthesis or induction of the apical sodium-dependent bile acid transporter in the intestine, increase the availability of chenodeoxycholic acid in the liver, thereby promoting hepatic conversion of this bile acid into muricholic acids. Recognition of these mechanisms is essential for understanding the regulation of bile acid synthesis in the mouse, and for our awareness of important species differences in the regulation of bile acid synthesis in mice and humans. 2015 S. Karger AG, Basel.

Slc3a2 Mediates Branched-Chain Amino-Acid-Dependent Maintenance of Regulatory T Cells

Directory of Open Access Journals (Sweden)

Kayo Ikeda

2017-11-01

Full Text Available Summary: Foxp3+ regulatory T (Treg cells, which suppress immune responses, are highly proliferative in vivo. However, it remains unclear how the active replication of Treg cells is maintained in vivo. Here, we show that branched-chain amino acids (BCAAs, including isoleucine, are required for maintenance of the proliferative state of Treg cells via the amino acid transporter Slc3a2-dependent metabolic reprogramming. Mice fed BCAA-reduced diets showed decreased numbers of Foxp3+ Treg cells with defective in vivo proliferative capacity. Mice lacking Slc3a2 specifically in Foxp3+ Treg cells showed impaired in vivo replication and decreased numbers of Treg cells. Slc3a2-deficient Treg cells showed impaired isoleucine-induced activation of the mTORC1 pathway and an altered metabolic state. Slc3a2 mutant mice did not show an isoleucine-induced increase of Treg cells in vivo and exhibited multi-organ inflammation. Taken together, these findings demonstrate that BCAA controls Treg cell maintenance via Slc3a2-dependent metabolic regulation. : Treg cells regulate excess immune responses and are highly proliferative in vivo. Ikeda et al. find that branched-chain amino acids (BCAAs are essentially required to maintain expansion and the suppressive capacity of Treg cells via Slc3a2 and mTORC1. Keywords: Treg cells, amino acids, immunometabolism, immune regulation, transporter

Mycophenolic Acid Pharmacokinetics and Relapse in Children with Steroid–Dependent Idiopathic Nephrotic Syndrome

Science.gov (United States)

Tellier, Stéphanie; Dallocchio, Aymeric; Guigonis, Vincent; Saint-Marcoux, Frank; Llanas, Brigitte; Ichay, Lydia; Bandin, Flavio; Godron, Astrid; Morin, Denis; Brochard, Karine; Gandia, Peggy; Bouchet, Stéphane; Marquet, Pierre; Decramer, Stéphane

2016-01-01

Background and objectives Therapeutic drug monitoring of mycophenolic acid can improve clinical outcome in organ transplantation and lupus, but data are scarce in idiopathic nephrotic syndrome. The aim of our study was to investigate whether mycophenolic acid pharmacokinetics are associated with disease control in children receiving mycophenolate mofetil for the treatment of steroid–dependent nephrotic syndrome. Design, setting, participants, & measurements This was a retrospective multicenter study including 95 children with steroid–dependent nephrotic syndrome treated with mycophenolate mofetil with or without steroids. Area under the concentration-time curve of mycophenolic acid was determined in all children on the basis of sampling times at 20, 60, and 180 minutes postdose, using Bayesian estimation. The association between a threshold value of the area under the concentration-time curve of mycophenolic acid and the relapse rate was assessed using a negative binomial model. Results In total, 140 areas under the concentration-time curve of mycophenolic acid were analyzed. The findings indicate individual dose adaptation in 53 patients (38%) to achieve an area under the concentration-time curve target of 30–60 mg·h/L. In a multivariable negative binomial model including sex, age at disease onset, time to start of mycophenolate mofetil, previous immunomodulatory treatment, and concomitant prednisone dose, a level of area under the concentration-time curve of mycophenolic acid >45 mg·h/L was significantly associated with a lower relapse rate (rate ratio, 0.65; 95% confidence interval, 0.46 to 0.89; P=0.01). Conclusions Therapeutic drug monitoring leading to individualized dosing may improve the efficacy of mycophenolate mofetil in steroid–dependent nephrotic syndrome. Additional prospective studies are warranted to determine the optimal target for area under the concentration-time curve of mycophenolic acid in this population. PMID:27445161

Characterisation of a thiamine diphosphate-dependent alpha-keto acid decarboxylase from Proteus mirabilis JN458.

Science.gov (United States)

Wang, Biying; Bai, Yajun; Fan, Taiping; Zheng, Xiaohui; Cai, Yujie

2017-10-01

Alpha-keto acid decarboxylases can convert keto acids to their corresponding aldehydes, which are often volatile aroma compounds. The gene encoding α-keto acid decarboxylase in Proteus mirabilis JN458 was cloned, and the enzyme overexpressed in Escherichia coli BL21 (DE3), purified in high yield, and characterised. The molecular weight is 62.291kDa by MALDI-TOF MS, and optimum activity at pH 6.0 and 40-50°C. The enzyme is a typical decarboxylase, dependent on thiamine diphosphate and Mg 2+ as cofactors. For the decarboxylation reaction, the enzyme displayed a broad substrate range. Kinetic parameters were determined using 4-methyl-2-oxopentanoic acid, phenyl pyruvate and 3-methyl-2-oxopentanoic acid as substrates. K m and k cat values for phenyl pyruvate were 0.62mM and 77.38s -1 , respectively, and the k cat /K m value was 124.81mM -1 s -1 . The enzyme properties suggest it may act effectively under cheese ripening conditions. Copyright © 2017. Published by Elsevier Ltd.

Analysis of hydroxycinnamic acid degradation in Agrobacterium fabrum reveals a coenzyme A-dependent, beta-oxidative deacetylation pathway.

Science.gov (United States)

Campillo, Tony; Renoud, Sébastien; Kerzaon, Isabelle; Vial, Ludovic; Baude, Jessica; Gaillard, Vincent; Bellvert, Floriant; Chamignon, Cécile; Comte, Gilles; Nesme, Xavier; Lavire, Céline; Hommais, Florence

2014-06-01

The soil- and rhizosphere-inhabiting bacterium Agrobacterium fabrum (genomospecies G8 of the Agrobacterium tumefaciens species complex) is known to have species-specific genes involved in ferulic acid degradation. Here, we characterized, by genetic and analytical means, intermediates of degradation as feruloyl coenzyme A (feruloyl-CoA), 4-hydroxy-3-methoxyphenyl-β-hydroxypropionyl-CoA, 4-hydroxy-3-methoxyphenyl-β-ketopropionyl-CoA, vanillic acid, and protocatechuic acid. The genes atu1416, atu1417, and atu1420 have been experimentally shown to be necessary for the degradation of ferulic acid. Moreover, the genes atu1415 and atu1421 have been experimentally demonstrated to be essential for this degradation and are proposed to encode a phenylhydroxypropionyl-CoA dehydrogenase and a 4-hydroxy-3-methoxyphenyl-β-ketopropionic acid (HMPKP)-CoA β-keto-thiolase, respectively. We thus demonstrated that the A. fabrum hydroxycinnamic degradation pathway is an original coenzyme A-dependent β-oxidative deacetylation that could also transform p-coumaric and caffeic acids. Finally, we showed that this pathway enables the metabolism of toxic compounds from plants and their use for growth, likely providing the species an ecological advantage in hydroxycinnamic-rich environments, such as plant roots or decaying plant materials.

Journal of Biosciences | Indian Academy of Sciences

Indian Academy of Sciences (India)

Lysophosphatidyl acyltransferase (LPAT) is the important enzyme responsible for the acylation of lysophosphatidic acid (LPA), leading to the generation of phosphatidic acid (PA) in plant. Its encoding gene is an essential candidate for oil crops to improve oil composition and increase seed oil content through genetic ...

Multiple Mechanisms are Responsible for Transactivation of the Epidermal Growth Factor Receptor in Mammary Epithelial Cells

Energy Technology Data Exchange (ETDEWEB)

Rodland, Karin D.; Bollinger, Nikki; Ippolito, Danielle L.; Opresko, Lee; Coffey, Robert J.; Zangar, Richard C.; Wiley, H. S.

2008-11-14

REVIEW ENTIRE DOCUMENT AT: https://pnlweb.pnl.gov/projects/bsd/ERICA%20Manuscripts%20for%20Review/KD%20Rodland%20D7E80/HMEC_transactivation_ms01_15+Figs.pdf ABSTRACT: Using a single nontransformed strain of human mammary epithelial cells, we found that the ability of multiple growth factors and cytokines to induce ERK phosphorylation was dependent on EGFR activity. These included lysophosphatidic acid (LPA), uridine triphosphate, growth hormone, vascular endothelial growth factor, insulin-like growth factor-1 (IGF-1), and tumor necrosis factoralpha. In contrast, hepatocyte growth factor could stimulate ERK phosphorylation independent of EGFR activity...

Real-time electrochemical monitoring of isothermal helicase-dependent amplification of nucleic acids.

Science.gov (United States)

Kivlehan, Francine; Mavré, François; Talini, Luc; Limoges, Benoît; Marchal, Damien

2011-09-21

We described an electrochemical method to monitor in real-time the isothermal helicase-dependent amplification of nucleic acids. The principle of detection is simple and well-adapted to the development of portable, easy-to-use and inexpensive nucleic acids detection technologies. It consists of monitoring a decrease in the electrochemical current response of a reporter DNA intercalating redox probe during the isothermal DNA amplification. The method offers the possibility to quantitatively analyze target nucleic acids in less than one hour at a single constant temperature, and to perform at the end of the isothermal amplification a DNA melt curve analysis for differentiating between specific and non-specific amplifications. To illustrate the potentialities of this approach for the development of a simple, robust and low-cost instrument with high throughput capability, the method was validated with an electrochemical system capable of monitoring up to 48 real-time isothermal HDA reactions simultaneously in a disposable microplate consisting of 48-electrochemical microwells. Results obtained with this approach are comparable to that obtained with a well-established but more sophisticated and expensive fluorescence-based method. This makes for a promising alternative detection method not only for real-time isothermal helicase-dependent amplification of nucleic acid, but also for other isothermal DNA amplification strategies.

Arabidopsis MAP kinase 4 regulates salicylic acid- and jasmonic acid/ethylene-dependent responses via EDS1 and PAD4

DEFF Research Database (Denmark)

Brodersen, P; Petersen, M; Nielsen, Henrik Bjørn

2006-01-01

Arabidopsis MPK4 has been implicated in plant defense regulation because mpk4 knockout plants exhibit constitutive activation of salicylic acid (SA)-dependent defenses, but fail to induce jasmonic acid (JA) defense marker genes in response to JA. We show here that mpk4 mutants are also defective...

Hydroxyoctadecadienoic acids regulate apoptosis in human THP-1 cells in a PPARγ-dependent manner.

Science.gov (United States)

Vangaveti, Venkat N; Shashidhar, Venkatesh M; Rush, Catherine; Malabu, Usman H; Rasalam, Roy R; Collier, Fiona; Baune, Bernhard T; Kennedy, Richard L

2014-12-01

Macrophage apoptosis, a key process in atherogenesis, is regulated by oxidation products, including hydroxyoctadecadienoic acids (HODEs). These stable oxidation products of linoleic acid (LA) are abundant in atherosclerotic plaque and activate PPARγ and GPR132. We investigated the mechanisms through which HODEs regulate apoptosis. The effect of HODEs on THP-1 monocytes and adherent THP-1 cells were compared with other C18 fatty acids, LA and α-linolenic acid (ALA). The number of cells was reduced within 24 hours following treatment with 9-HODE (p labelling of cells (p blocked by the caspase inhibitor DEVD-CHO. The PPARγ antagonist T0070907 further increased apoptosis, suggestive of the PPARγ-regulated apoptotic effects induced by 9-HODE. The use of siRNA for GPR132 showed no evidence that the effect of HODEs was mediated through this receptor. 9-HODE and 13-HODE are potent--and specific--regulators of apoptosis in THP-1 cells. Their action is PPARγ-dependent and independent of GPR132. Further studies to identify the signalling pathways through which HODEs increase apoptosis in macrophages may reveal novel therapeutic targets for atherosclerosis.

Abscisic acid-dependent multisite phosphorylation regulates the activity of a transcription activator AREB1.

Science.gov (United States)

Furihata, Takashi; Maruyama, Kyonoshin; Fujita, Yasunari; Umezawa, Taishi; Yoshida, Riichiro; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

2006-02-07

bZIP-type transcription factors AREBs/ABFs bind an abscisic acid (ABA)-responsive cis-acting element named ABRE and transactivate downstream gene expression in Arabidopsis. Because AREB1 overexpression could not induce downstream gene expression, activation of AREB1 requires ABA-dependent posttranscriptional modification. We confirmed that ABA activated 42-kDa kinase activity, which, in turn, phosphorylated Ser/Thr residues of R-X-X-S/T sites in the conserved regions of AREB1. Amino acid substitutions of R-X-X-S/T sites to Ala suppressed transactivation activity, and multiple substitution of these sites resulted in almost complete suppression of transactivation activity in transient assays. In contrast, substitution of the Ser/Thr residues to Asp resulted in high transactivation activity without exogenous ABA application. A phosphorylated, transcriptionally active form was achieved by substitution of Ser/Thr in all conserved R-X-X-S/T sites to Asp. Transgenic plants overexpressing the phosphorylated active form of AREB1 expressed many ABA-inducible genes, such as RD29B, without ABA treatment. These results indicate that the ABA-dependent multisite phosphorylation of AREB1 regulates its own activation in plants.

Developmentally dependent and different roles of fatty acids OMEGA-6 and OMEGA-3

DEFF Research Database (Denmark)

Mourek, J; Mourek, J

2011-01-01

The developmentally-dependent differences in the biological significances and effects of PUFA-OMEGA-6 (namely of arachidonic acid) and PUFA-OMEGA-3 (namely of docosahexaenoic acid) are discussed. The clinical results as well as developmental experiences are indicating a hypothesis of the evolution...... that created mutual relationship between those two substances (with immunological basis and following recuperation). The anti-inflammatory actions of PUFA-OMEGA-3 are the most visible (and significant) contrasts as compared with the large affects of namely arachidonic acid and its metabolites....

Bias-dependent amino-acid-induced conductance changes in short semi-metallic carbon nanotubes

International Nuclear Information System (INIS)

Abadir, G B; Walus, K; Pulfrey, D L

2010-01-01

We study the interaction between short semi-metallic carbon nanotubes and different amino acids using molecular dynamics and ab initio (density functional theory/non-equilibrium Green's function) simulations. We identify two different mechanisms of nanotube conductance change upon adsorption of amino acids: one due to the change of the coordinates of the nanotube arising from van der Waals forces of interaction with the adsorbed amino acid; and one due to electrostatic interactions, which appear only in the case of charged amino acids. We also find that the transport mechanism and the changes in the conductance of the tube upon amino acid adsorption are bias dependent.

Identification and Manipulation of a Novel Signaling Mechanism to Improve Articular Cartilage Restoration After Posttraumatic Joint Injury

Science.gov (United States)

2014-10-01

collagen II (COL II). As a result, the newly-formed fibrocartilage has poor biomechanical properties and often evidences poor integration with the... fibrocartilage generation at the site of injury defines a new potential avenue of intervention in the field of cartilage restoration. We defined the...lysophosphatidic acid (LPA)-autotaxin (ATX; encoded by the ENPP2 gene) signaling axis as an important mediator of fibrocartilage formation both in vitro and in

Pruritus in chronic cholestatic liver diseases

Directory of Open Access Journals (Sweden)

E. V. Vinnitskaya

2017-01-01

Full Text Available Pruritus can be a prominent symptom  in patients with chronic liver disorders, especially those  with cholestasis,  and  substantially  affects  quality  of life. Management of pruritus  in cholestatic  liver diseases  remains  a  complicated   medical  problem. The review article deals with pathophysiological mechanisms of pruritus in cholestatic liver diseases, in particular, with the role of bile acids, endogenous opioids, serotonin, and histamine. There is new data on the key pathophysiological elements, such as neuronal activation lysophosphatidic acid and autotaxin, an enzyme that produces lysophosphatidic acid and whose serum activity is associated with the intensity of pruritus. Pathophysiology-based management approaches include administration of anionic exchange resin cholestyramine, ursodeoxycholic acid, rifampicin agonists, an opioid antagonist naltrexone and a  serotonin-reuptake inhibitor sertraline. These agents are recommended for the use as a stepped treatment algorithm. Patients who do not respond to these therapies can become candidates for albumin dialysis, plasmapheresis, ultraviolet B phototherapy, or need some other individualized approaches. New knowledge on the pathophysiology of pruritus may potentially result in the development of new agents for cholestatic pruritus.

Voltage-dependent motion of the catalytic region of voltage-sensing phosphatase monitored by a fluorescent amino acid.

Science.gov (United States)

Sakata, Souhei; Jinno, Yuka; Kawanabe, Akira; Okamura, Yasushi

2016-07-05

The cytoplasmic region of voltage-sensing phosphatase (VSP) derives the voltage dependence of its catalytic activity from coupling to a voltage sensor homologous to that of voltage-gated ion channels. To assess the conformational changes in the cytoplasmic region upon activation of the voltage sensor, we genetically incorporated a fluorescent unnatural amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), into the catalytic region of Ciona intestinalis VSP (Ci-VSP). Measurements of Anap fluorescence under voltage clamp in Xenopus oocytes revealed that the catalytic region assumes distinct conformations dependent on the degree of voltage-sensor activation. FRET analysis showed that the catalytic region remains situated beneath the plasma membrane, irrespective of the voltage level. Moreover, Anap fluorescence from a membrane-facing loop in the C2 domain showed a pattern reflecting substrate turnover. These results indicate that the voltage sensor regulates Ci-VSP catalytic activity by causing conformational changes in the entire catalytic region, without changing their distance from the plasma membrane.

Voltage-dependent motion of the catalytic region of voltage-sensing phosphatase monitored by a fluorescent amino acid

Science.gov (United States)

Sakata, Souhei; Jinno, Yuka; Kawanabe, Akira; Okamura, Yasushi

2016-01-01

The cytoplasmic region of voltage-sensing phosphatase (VSP) derives the voltage dependence of its catalytic activity from coupling to a voltage sensor homologous to that of voltage-gated ion channels. To assess the conformational changes in the cytoplasmic region upon activation of the voltage sensor, we genetically incorporated a fluorescent unnatural amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), into the catalytic region of Ciona intestinalis VSP (Ci-VSP). Measurements of Anap fluorescence under voltage clamp in Xenopus oocytes revealed that the catalytic region assumes distinct conformations dependent on the degree of voltage-sensor activation. FRET analysis showed that the catalytic region remains situated beneath the plasma membrane, irrespective of the voltage level. Moreover, Anap fluorescence from a membrane-facing loop in the C2 domain showed a pattern reflecting substrate turnover. These results indicate that the voltage sensor regulates Ci-VSP catalytic activity by causing conformational changes in the entire catalytic region, without changing their distance from the plasma membrane. PMID:27330112

UDP-glucuronosyltransferase-dependent bioactivation of clofibric acid to a DNA-damaging intermediate in mouse hepatocytes.

Science.gov (United States)

Ghaoui, Roula; Sallustio, Benedetta C; Burcham, Philip C; Fontaine, Frank R

2003-05-06

Glucuronidation of a number of carboxyl-containing drugs generates reactive acyl glucuronide metabolites. These electrophilic species alkylate cell proteins and may be implicated in the pathogenesis of a number of toxic syndromes seen in patients receiving the parent aglycones. Whether acyl glucuronides also attack nuclear DNA is unknown, although the acyl glucuronide formed from clofibric acid was recently found to decrease the transfection efficiency of phage DNA and generate strand breaks in plasmid DNA in vitro. To determine if such a DNA damage occurs within a cellular environment, the comet assay (i.e. single-cell gel electrophoresis) was used to detect DNA lesions in the nuclear genome of isolated mouse hepatocytes cultured with clofibric acid. Overnight exposure to 50 microM and higher concentrations of clofibric acid produced concentration-dependent increases in the comet areas of hepatocyte nuclei, with 1 mM clofibrate producing a 3.6-fold elevation over controls. These effects closely coincided with culture medium concentrations of the glucuronide metabolite formed from clofibric acid, 1-O-beta-clofibryl glucuronide. Consistent with a role for glucuronidation in the DNA damage observed, the glucuronidation inhibitor borneol diminished glucuronide formation from 100 microM clofibrate by 98% and returned comet areas to baseline levels. Collectively, these results suggest that the acyl glucuronide formed from clofibric acid is capable of migrating from its site of formation within the endoplasmic reticulum to generate strand nicks in nuclear DNA.

Nor-ursodeoxycholic acid reverses hepatocyte-specific nemo-dependent steatohepatitis.

Science.gov (United States)

Beraza, Naiara; Ofner-Ziegenfuss, Lisa; Ehedego, Haksier; Boekschoten, Mark; Bischoff, Stephan C; Mueller, Michael; Trauner, Michael; Trautwein, Christian

2011-03-01

Hepatocyte-specific NEMO/NF-κB deleted mice (NEMO(Δhepa)) develop spontaneous non-alcoholic steatohepatitis (NASH). Free fatty acids and bile acids promote DR5 expression. TRAIL/NK cell-mediated activation of TRAIL-R2/DR5 plays an important role during acute injury in NEMO(Δhepa) mice. To inhibit the progression of NASH in the absence of hepatocyte-NEMO/NF-kB signaling. NEMOf/f and NEMO(Δhepa) mice were fed with a low-fat diet, and with two anticholestatic diets; UDCA and NorUDCA. The impact of these treatments on the progression of NASH was evaluated. We show that high expression of DR5 in livers from NEMO(Δhepa) mice is accompanied by an abundant presence of bile acids (BAs), misregulation of BA transporters and significant alteration of lipid metabolism-related genes. Additionally, mice lacking NEMO in hepatocytes spontaneously showed ductular response at young age. Unexpectedly, feeding of NEMO(Δhepa) mice with low-fat diet failed to improve chronic liver injury. Conversely, anti-cholestatic treatment with nor-ursodeoxycholic acid (NorUDCA), but not with ursodeoxycholic acid (UDCA), led to a significant attenuation of liver damage in NEMO(Δhepa) mice. The strong therapeutic effect of NorUDCA relied on a significant downregulation of LXR-dependent lipogenesis and the normalisation of BA metabolism through mechanisms involving cross-talk between Cyp7a1 and SHP. This was associated with the significant improvement of liver histology, NEMO(Δhepa)/NorUDCA-treated mice showed lower apoptosis and reduced CyclinD1 expression, indicating attenuation of the compensatory proliferative response to hepatocellular damage. Finally, fibrosis and ductular reaction markers were significantly reduced in NorUDCA-treated NEMO(Δhepa) mice. Overall, our work demonstrates the contribution of bile acids metabolism to the progression of NASH in the absence of hepatocyte-NF-kB through mechanisms involving DR5-apoptosis, inflammation and fibrosis. Our work suggests a potential

Role of NPR1 dependent and NPR1 independent genes in response to Salicylic acid

Directory of Open Access Journals (Sweden)

Neha Agarwal

2017-10-01

Full Text Available NPR1 (Nonexpressor of pathogenesis-related gene is a transcription coactivator and central regulator of systemic acquired resistance (SAR pathway. It controls wide range of pathogenesis related genes involved in various defense responses, acts by sensing SAR signal molecule, Salicylic acid (SA. Mutation in NPR1 results in increased susceptibility to pathogen infection and less expression of pathogenesis related genes. The present study aimed to identify the role of NPR1 in gene expression after the Salicylic acid induction. For this RNA-seq was performed in Arabidopsis thaliana Col-0 and npr1-1 in response to Salicylic acid. RNA-seq analysis revealed a total of 3811 differentially expressed gene in which 2109 genes are up-regulated and 1702 genes are down-regulated. We have divided these genes in 6 categories SA induced (SI, SA repressed (SR, NPR1 dependent SI (ND-SI, NPR1 dependent SR (ND-SR, NPR1 independent SI (NI-SI, NPR1 independent SR (NI-SR. Further, Gene ontology and MapMan pathway analysis of differentially expressed genes suggested variety of biological processes and metabolic pathways that are enriched during SAR defense pathway. These results contribute to shed light on importance of both NPR1-dependent (ND and NPR1-independent (NI gene acting downstream to Salicylic acid induction in SAR pathway. The present study aimed to identify the role of NPR1 in gene expression after the Salicylic acid induction.

Activation of the Glutamic Acid-Dependent Acid Resistance System in Escherichia coli BL21(DE3) Leads to Increase of the Fatty Acid Biotransformation Activity.

Science.gov (United States)

Woo, Ji-Min; Kim, Ji-Won; Song, Ji-Won; Blank, Lars M; Park, Jin-Byung

The biosynthesis of carboxylic acids including fatty acids from biomass is central in envisaged biorefinery concepts. The productivities are often, however, low due to product toxicity that hamper whole-cell biocatalyst performance. Here, we have investigated factors that influence the tolerance of Escherichia coli to medium chain carboxylic acid (i.e., n-heptanoic acid)-induced stress. The metabolic and genomic responses of E. coli BL21(DE3) and MG1655 grown in the presence of n-heptanoic acid indicated that the GadA/B-based glutamic acid-dependent acid resistance (GDAR) system might be critical for cellular tolerance. The GDAR system, which is responsible for scavenging intracellular protons by catalyzing decarboxylation of glutamic acid, was inactive in E. coli BL21(DE3). Activation of the GDAR system in this strain by overexpressing the rcsB and dsrA genes, of which the gene products are involved in the activation of GadE and RpoS, respectively, resulted in acid tolerance not only to HCl but also to n-heptanoic acid. Furthermore, activation of the GDAR system allowed the recombinant E. coli BL21(DE3) expressing the alcohol dehydrogenase of Micrococcus luteus and the Baeyer-Villiger monooxygenase of Pseudomonas putida to reach 60% greater product concentration in the biotransformation of ricinoleic acid (i.e., 12-hydroxyoctadec-9-enoic acid (1)) into n-heptanoic acid (5) and 11-hydroxyundec-9-enoic acid (4). This study may contribute to engineering E. coli-based biocatalysts for the production of carboxylic acids from renewable biomass.

Activation of the Glutamic Acid-Dependent Acid Resistance System in Escherichia coli BL21(DE3 Leads to Increase of the Fatty Acid Biotransformation Activity.

Directory of Open Access Journals (Sweden)

Ji-Min Woo

Full Text Available The biosynthesis of carboxylic acids including fatty acids from biomass is central in envisaged biorefinery concepts. The productivities are often, however, low due to product toxicity that hamper whole-cell biocatalyst performance. Here, we have investigated factors that influence the tolerance of Escherichia coli to medium chain carboxylic acid (i.e., n-heptanoic acid-induced stress. The metabolic and genomic responses of E. coli BL21(DE3 and MG1655 grown in the presence of n-heptanoic acid indicated that the GadA/B-based glutamic acid-dependent acid resistance (GDAR system might be critical for cellular tolerance. The GDAR system, which is responsible for scavenging intracellular protons by catalyzing decarboxylation of glutamic acid, was inactive in E. coli BL21(DE3. Activation of the GDAR system in this strain by overexpressing the rcsB and dsrA genes, of which the gene products are involved in the activation of GadE and RpoS, respectively, resulted in acid tolerance not only to HCl but also to n-heptanoic acid. Furthermore, activation of the GDAR system allowed the recombinant E. coli BL21(DE3 expressing the alcohol dehydrogenase of Micrococcus luteus and the Baeyer-Villiger monooxygenase of Pseudomonas putida to reach 60% greater product concentration in the biotransformation of ricinoleic acid (i.e., 12-hydroxyoctadec-9-enoic acid (1 into n-heptanoic acid (5 and 11-hydroxyundec-9-enoic acid (4. This study may contribute to engineering E. coli-based biocatalysts for the production of carboxylic acids from renewable biomass.

Molecular cloning and expression analysis of jasmonic acid dependent but salicylic acid independent LeWRKY1.

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Lu, M; Wang, L F; Du, X H; Yu, Y K; Pan, J B; Nan, Z J; Han, J; Wang, W X; Zhang, Q Z; Sun, Q P

2015-11-30

Various plant genes can be activated or inhibited by phytohormones under conditions of biotic and abiotic stress, especially in response to jasmonic acid (JA) and salicylic acid (SA). Interactions between JA and SA may be synergistic or antagonistic, depending on the stress condition. In this study, we cloned a full-length cDNA (LeWRKY1, GenBank accession No. FJ654265) from Lycopersicon esculentum by rapid amplification of cDNA ends. Sequence analysis showed that this gene is a group II WRKY transcription factor. Analysis of LeWRKY1 mRNA expression in various tissues by qRT-PCR showed that the highest and lowest expression occurred in the leaves and stems, respectively. In addition, LeWRKY1 expression was induced by JA and Botrytis cinerea Pers., but not by SA.

pH-, Lactic acid-, and non-lactic acid-dependent activities of probiotic Lactobacilli against Salmonella enterica Serovar Typhimurium.

Science.gov (United States)

Fayol-Messaoudi, Domitille; Berger, Cédric N; Coconnier-Polter, Marie-Hélène; Liévin-Le Moal, Vanessa; Servin, Alain L

2005-10-01

The mechanism(s) underlying the antibacterial activity of probiotic Lactobacillus strains appears to be multifactorial and includes lowering of the pH and the production of lactic acid and of antibacterial compounds, including bacteriocins and nonbacteriocin, non-lactic acid molecules. Addition of Dulbecco's modified Eagle's minimum essential medium to the incubating medium delays the killing activity of lactic acid. We found that the probiotic strains Lactobacillus johnsonii La1, Lactobacillus rhamnosus GG, Lactobacillus casei Shirota YIT9029, L. casei DN-114 001, and L. rhamnosus GR1 induced a dramatic decrease in the viability of Salmonella enterica serovar Typhimurium SL1344 mainly attributable to non-lactic acid molecule(s) present in the cell-free culture supernatant (CFCS). These molecules were more active against serovar Typhimurium SL1344 in the exponential growth phase than in the stationary growth phase. We also showed that the production of the non-lactic acid substance(s) responsible for the killing activity was dependent on growth temperature and that both unstable and stable substances with killing activity were present in the CFCSs. We found that the complete inhibition of serovar Typhimurium SL1344 growth results from a pH-lowering effect.

Isolimonic acid interferes with Escherichia coli O157:H7 biofilm and TTSS in QseBC and QseA dependent fashion

Science.gov (United States)

2012-01-01

Background E. coli O157:H7 (EHEC) is an important human pathogen. The antibiotic treatment of EHEC reportedly results in release of Shiga toxin and is therefore discouraged. Consequently, alternative preventive or therapeutic strategies for EHEC are required. The objective of the current study was to investigate the effect of citrus limonoids on cell-cell signaling, biofilm formation and type III secretion system in EHEC. Results Isolimonic acid and ichangin were the most potent inhibitors of EHEC biofilm (IC25=19.7 and 28.3 μM, respectively) and adhesion to Caco-2 cells. The qPCR analysis revealed that isolimonic acid and ichangin repressed LEE encoded genes by ≈3 to 12 fold. In addition, flhDC was repressed by the two limonoids (≈3 to 7 fold). Further studies suggested that isolimonic acid interferes with AI-3/epinephrine activated cell-cell signaling pathway. Loss of biofilm inhibitory activity of isolimonic acid in ΔqseBC mutant, which could be restored upon complementation, suggested a dependence on functional QseBC. Additionally, overexpression of qseBC in wild type EHEC abated the inhibitory effect of isolimonic acid. Furthermore, the isolimonic acid failed to differentially regulate ler in ΔqseA mutant, while plasmid borne expression of qseA in ΔqseA background restored the repressive effect of isolimonic acid. Conclusions Altogether, results of study seem to suggest that isolimonic acid and ichangin are potent inhibitors of EHEC biofilm and TTSS. Furthermore, isolimonic acid appears to interfere with AI-3/epinephrine pathway in QseBC and QseA dependent fashion. PMID:23153211

Bile acids induce necrosis in pancreatic stellate cells dependent on calcium entry and sodium‐driven bile uptake

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Jakubowska, Monika A.; Gerasimenko, Julia V.; Gerasimenko, Oleg V.; Petersen, Ole H.

2016-01-01

Key points Acute biliary pancreatitis is a sudden and severe condition initiated by bile reflux into the pancreas.Bile acids are known to induce Ca2+ signals and necrosis in isolated pancreatic acinar cells but the effects of bile acids on stellate cells are unexplored.Here we show that cholate and taurocholate elicit more dramatic Ca2+ signals and necrosis in stellate cells compared to the adjacent acinar cells in pancreatic lobules; whereas taurolithocholic acid 3‐sulfate primarily affects acinar cells.Ca2+ signals and necrosis are strongly dependent on extracellular Ca2+ as well as Na+; and Na+‐dependent transport plays an important role in the overall bile acid uptake in pancreatic stellate cells.Bile acid‐mediated pancreatic damage can be further escalated by bradykinin‐induced signals in stellate cells and thus killing of stellate cells by bile acids might have important implications in acute biliary pancreatitis. Abstract Acute biliary pancreatitis, caused by bile reflux into the pancreas, is a serious condition characterised by premature activation of digestive enzymes within acinar cells, followed by necrosis and inflammation. Bile acids are known to induce pathological Ca2+ signals and necrosis in acinar cells. However, bile acid‐elicited signalling events in stellate cells remain unexplored. This is the first study to demonstrate the pathophysiological effects of bile acids on stellate cells in two experimental models: ex vivo (mouse pancreatic lobules) and in vitro (human cells). Sodium cholate and taurocholate induced cytosolic Ca2+ elevations in stellate cells, larger than those elicited simultaneously in the neighbouring acinar cells. In contrast, taurolithocholic acid 3‐sulfate (TLC‐S), known to induce Ca2+ oscillations in acinar cells, had only minor effects on stellate cells in lobules. The dependence of the Ca2+ signals on extracellular Na+ and the presence of sodium–taurocholate cotransporting polypeptide (NTCP) indicate a Na+‐dependent

Pipecolic Acid Orchestrates Plant Systemic Acquired Resistance and Defense Priming via Salicylic Acid-Dependent and -Independent Pathways.

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Bernsdorff, Friederike; Döring, Anne-Christin; Gruner, Katrin; Schuck, Stefan; Bräutigam, Andrea; Zeier, Jürgen

2016-01-01

We investigated the relationships of the two immune-regulatory plant metabolites, salicylic acid (SA) and pipecolic acid (Pip), in the establishment of plant systemic acquired resistance (SAR), SAR-associated defense priming, and basal immunity. Using SA-deficient sid2, Pip-deficient ald1, and sid2 ald1 plants deficient in both SA and Pip, we show that SA and Pip act both independently from each other and synergistically in Arabidopsis thaliana basal immunity to Pseudomonas syringae. Transcriptome analyses reveal that SAR establishment in Arabidopsis is characterized by a strong transcriptional response systemically induced in the foliage that prepares plants for future pathogen attack by preactivating multiple stages of defense signaling and that SA accumulation upon SAR activation leads to the downregulation of photosynthesis and attenuated jasmonate responses systemically within the plant. Whereas systemic Pip elevations are indispensable for SAR and necessary for virtually the whole transcriptional SAR response, a moderate but significant SA-independent component of SAR activation and SAR gene expression is revealed. During SAR, Pip orchestrates SA-dependent and SA-independent priming of pathogen responses in a FLAVIN-DEPENDENT-MONOOXYGENASE1 (FMO1)-dependent manner. We conclude that a Pip/FMO1 signaling module acts as an indispensable switch for the activation of SAR and associated defense priming events and that SA amplifies Pip-triggered responses to different degrees in the distal tissue of SAR-activated plants. © 2016 American Society of Plant Biologists. All rights reserved.

Palmitoleic Acid Improves Metabolic Functions in Fatty Liver by PPARα-Dependent AMPK Activation.

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de Souza, Camila O; Teixeira, Alexandre A S; Biondo, Luana A; Lima Junior, Edson A; Batatinha, Helena A P; Rosa Neto, Jose C

2017-08-01

Palmitoleic acid, since described as lipokine, increases glucose uptake by modulation of 5'AMP-activated protein kinase (AMPK), as well as increasing lipolysis by activation of peroxisome proliferator-activated receptor-α (PPARα), in adipose tissue. However, in liver, the effects of palmitoleic acid on glucose metabolism and the role of PPARα remain unknown. To investigate whether palmitoleic acid improved the hepatic insulin sensitivity of obese mice. C57BL6 and PPARα knockout (KO) mice were fed for 12 weeks with a standard diet (SD) or high-fat diet (HF), and in the last 2 weeks were treated with oleic or palmitoleic acid. Palmitoleic acid promoted a faster uptake of glucose in the body, associated with higher insulin concentration; however, even when stimulated with insulin, palmitoleic acid did not modulate the insulin pathway (AKT, IRS). Palmitoleic acid increased the phosphorylation of AMPK, upregulated glucokinase and downregulated SREBP-1. Regarding AMPK downstream, palmitoleic acid increased the production of FGF-21 and stimulated the expression of PPARα. Palmitoleic acid treatment did not increase AMPK phosphorylation, modulate glucokinase or increase FGF-21 in liver of PPARα KO mice. In mice fed with a high-fat diet, palmitoleic acid supplementation stimulated the uptake of glucose in liver through activation of AMPK and FGF-21, dependent on PPARα. J. Cell. Physiol. 232: 2168-2177, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

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Indian Academy of Sciences (India)

PRAKASH KUMAR

Although originally described as an intracellular second messenger, sphingosine 1-phosphate (S1P) has recently been shown to ... lysophosphatidic acid; LPS, lipopolysaccharides; mDC, mature DC; MHC, major histocompatibility complex; NK, natural killer; PCR, ..... -the enigmatic lipid class: biochemistry, physiology, and.

Temperature dependence of relaxation times in proton components of fatty acids

International Nuclear Information System (INIS)

Kuroda, Kagayaki; Iwabuchi, Taku; Saito, Kensuke; Obara, Makoto; Honda, Masatoshi; Imai, Yutaka

2011-01-01

We examined the temperature dependence of relaxation times in proton components of fatty acids in various samples in vitro at 11 tesla as a standard calibration data for quantitative temperature imaging of fat. The spin-lattice relaxation time, T 1 , of both the methylene (CH 2 ) chain and terminal methyl (CH 3 ) was linearly related to temperature (r>0.98, P 2 signal for calibration and observed the signal with 18% of CH 3 to estimate temperature. These findings suggested that separating the fatty acid components would significantly improve accuracy in quantitative thermometry for fat. Use of the T 1 of CH 2 seems promising in terms of reliability and reproducibility in measuring temperature of fat. (author)

Beta-and gamma-turns in proteins revisited: a new set of amino acid turn-type dependent positional preferences and potentials.

Science.gov (United States)

Guruprasad, K; Rajkumar, S

2000-06-01

The number of beta-turns in a representative set of 426 protein three-dimensional crystal structures selected from the recent Protein Data Bank has nearly doubled and the number of gamma-turns in a representative set of 320 proteins has increased over seven times since the previous analysis. Beta-turns (7153) and gamma-turns (911) extracted from these proteins were used to derive a revised set of type-dependent amino acid positional preferences and potentials. Compared with previous results, the preference for proline, methionine and tryptophan has increased and the preference for glutamine, valine, glutamic acid and alanine has decreased for beta-turns. Certain new amino acid preferences were observed for both turn types and individual amino acids showed turn-type dependent positional preferences. The rationale for new amino acid preferences are discussed in the light of hydrogen bonds and other interactions involving the turns. Where main-chain hydrogen bonds of the type NH(i + 3) --> CO(i) were not observed for some beta-turns, other main-chain hydrogen bonds or solvent interactions were observed that possibly stabilize such beta-turns. A number of unexpected isolated beta-turns with proline at i + 2 position were also observed. The NH(i + 2) --> CO(i) hydrogen bond was observed for almost all gamma-turns. Nearly 20% classic gamma-turns and 43% inverse gamma-turns are isolated turns.

The Cirque du Soleil of Golgi membrane dynamics.

Science.gov (United States)

Bankaitis, Vytas A

2009-07-27

The role of lipid metabolic enzymes in Golgi membrane remodeling is a subject of intense interest. Now, in this issue, Schmidt and Brown (2009. J. Cell Biol. doi:10.1083/jcb.200904147) report that lysophosphatidic acid-specific acyltransferase, LPAAT3, contributes to Golgi membrane dynamics by suppressing tubule formation.

The Cirque du Soleil of Golgi membrane dynamics

OpenAIRE

Bankaitis, Vytas A.

2009-01-01

The role of lipid metabolic enzymes in Golgi membrane remodeling is a subject of intense interest. Now, in this issue, Schmidt and Brown (2009. J. Cell Biol. doi:10.1083/jcb.200904147) report that lysophosphatidic acid?specific acyltransferase, LPAAT3, contributes to Golgi membrane dynamics by suppressing tubule formation.

Perception of low red:far-red ratio compromises both salicylic acid- and jasmonic acid-dependent pathogen defences in Arabidopsis.

Science.gov (United States)

de Wit, Mieke; Spoel, Steven H; Sanchez-Perez, Gabino F; Gommers, Charlotte M M; Pieterse, Corné M J; Voesenek, Laurentius A C J; Pierik, Ronald

2013-07-01

In dense stands of plants, such as agricultural monocultures, plants are exposed simultaneously to competition for light and other stresses such as pathogen infection. Here, we show that both salicylic acid (SA)-dependent and jasmonic acid (JA)-dependent disease resistance is inhibited by a simultaneously reduced red:far-red light ratio (R:FR), the early warning signal for plant competition. Conversely, SA- and JA-dependent induced defences did not affect shade-avoidance responses to low R:FR. Reduced pathogen resistance by low R:FR was accompanied by a strong reduction in the regulation of JA- and SA-responsive genes. The severe inhibition of SA-responsive transcription in low R:FR appeared to be brought about by the repression of SA-inducible kinases. Phosphorylation of the SA-responsive transcription co-activator NPR1, which is required for full induction of SA-responsive transcription, was indeed reduced and may thus play a role in the suppression of SA-mediated defences by low R:FR-mediated phytochrome inactivation. Our results indicate that foraging for light through the shade-avoidance response is prioritised over plant immune responses when plants are simultaneously challenged with competition and pathogen attack. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

Cross-talk between abscisic acid-dependent and abscisic acid-independent pathways during abiotic stress.

Science.gov (United States)

Roychoudhury, Aryadeep; Paul, Saikat; Basu, Supratim

2013-07-01

Salinity, drought and low temperature are the common forms of abiotic stress encountered by land plants. To cope with these adverse environmental factors, plants execute several physiological and metabolic responses. Both osmotic stress (elicited by water deficit or high salt) and cold stress increase the endogenous level of the phytohormone abscisic acid (ABA). ABA-dependent stomatal closure to reduce water loss is associated with small signaling molecules like nitric oxide, reactive oxygen species and cytosolic free calcium, and mediated by rapidly altering ion fluxes in guard cells. ABA also triggers the expression of osmotic stress-responsive (OR) genes, which usually contain single/multiple copies of cis-acting sequence called abscisic acid-responsive element (ABRE) in their upstream regions, mostly recognized by the basic leucine zipper-transcription factors (TFs), namely, ABA-responsive element-binding protein/ABA-binding factor. Another conserved sequence called the dehydration-responsive element (DRE)/C-repeat, responding to cold or osmotic stress, but not to ABA, occurs in some OR promoters, to which the DRE-binding protein/C-repeat-binding factor binds. In contrast, there are genes or TFs containing both DRE/CRT and ABRE, which can integrate input stimuli from salinity, drought, cold and ABA signaling pathways, thereby enabling cross-tolerance to multiple stresses. A strong candidate that mediates such cross-talk is calcium, which serves as a common second messenger for abiotic stress conditions and ABA. The present review highlights the involvement of both ABA-dependent and ABA-independent signaling components and their interaction or convergence in activating the stress genes. We restrict our discussion to salinity, drought and cold stress.

Inhibition of time-dependent enhancement of amino acid transport by leukemic leukocytes: a possible index of the sensitivity of cells to drugs

Energy Technology Data Exchange (ETDEWEB)

Frengley, P A; Peck, W A; Lichtman, M A

1975-01-01

Leukemic leukocytes increase their rates of alpha aminoisobutyric acid (AIB) accumulation when incubated for prolonged periods in amino acid deficient media. The time-dependent increase was prevented by concurrent exposure of cells to cycloheximide or actinomycin D in vitro. In addition, the increase in AIB uptake was not present in leukemic blasts studied in vitro when the cells were obtained from subjects with acute myeloblastic leukemia who had received antileukemic therapy. Cortisol added to cell suspensions in vitro inhibited the development of time-dependent increases in AIB uptake in lymphoid cells, but accentuated the process slightly in myeloblasts. Cortisol administered to a subject with CLL by infusion reduced the time-dependent increase in AIB uptake by CLL cells subsequently studied in vitro. These data indicate that the time-dependent increase in AIB uptake may be a means of testing the sensitivity of leukemic cells to drugs.

The effect of humic acids on biodegradation of polycyclic aromatic hydrocarbons depends on the exposure regime

International Nuclear Information System (INIS)

Tejeda-Agredano, Maria-Carmen; Mayer, Philipp; Ortega-Calvo, Jose-Julio

2014-01-01

Binding of polycyclic aromatic hydrocarbons (PAHs) to dissolved organic matter (DOM) can reduce the freely dissolved concentration, increase apparent solubility or enhance diffusive mass transfer. To study the effects of DOM on biodegradation, we used phenanthrene and pyrene as model PAHs, soil humic acids as model DOM and a soil Mycobacterium strain as a representative degrader organism. Humic acids enhanced the biodegradation of pyrene when present as solid crystals but not when initially dissolved or provided by partitioning from a polymer. Synchronous fluorescence spectrophotometry, scintillation counting and a microscale diffusion technique were applied in order to determine the kinetics of dissolution and diffusive mass transfer of pyrene. We suggest that humic acids can enhance or inhibit biodegradation as a result of the balance of two opposite effects, namely, solubilization of the chemicals on the one hand and inhibition of cell adhesion to the pollutant source on the other. Highlights: • Humic acids can enhance the biodegradation of PAHs. • The enhancement depends on how the bacteria are exposed to PAHs. • Humic acids stimulate if PAHs are provided by dissolution form crystals. • An inhibition occurs if PAHs are provided by partitioning from a silicone. • The balance between enhanced dissolution and decreased adhesion is the cause. -- Humic acids cause opposite effects on biodegradation of PAHs depending on the exposure regime

AT32P-dependent estimation of nanomoles of fatty acids: Its use in the assay of phospholipase A2 activity

International Nuclear Information System (INIS)

Sarafianos, S.G.; Nair, P.P.; Kumar, S.

1990-01-01

A procedure for the assay of free fatty acids which has been adapted for the assay of phospholipase A2 is described. This consists of the conversion of long chain fatty acids to fatty acyl-CoA using the Mg2(+)-dependent fatty acyl-CoA synthetase, [alpha-32P]ATP and coenzyme A. In order to ensure the complete conversion of the acid to its CoA ester pyrophosphatase is also added to the incubation mixture. AM32P formed in stoichiometric amounts is separated from the remaining AT32P by polyethyleneimine-cellulose thin-layer chromatography and the fatty acid content is calculated from the specific radioactivity of AT32P. As little as 1 to 3 nmol of fatty acids hydrolyzed from any phospholipid using nanogram amounts of phospholipase A2 can be estimated with reliability. The real advantage of the method is that it combines the sensitivity of a radiochemical procedure without having to use radiolabeled substrates for the assay of phospholipases

Carbon Source-Dependent Inducible Metabolism of Veratryl Alcohol and Ferulic Acid in Pseudomonas putida CSV86

Science.gov (United States)

Mohan, Karishma

2017-01-01

ABSTRACT Pseudomonas putida CSV86 degrades lignin-derived metabolic intermediates, viz., veratryl alcohol, ferulic acid, vanillin, and vanillic acid, as the sole sources of carbon and energy. Strain CSV86 also degraded lignin sulfonate. Cell respiration, enzyme activity, biotransformation, and high-pressure liquid chromatography (HPLC) analyses suggest that veratryl alcohol and ferulic acid are metabolized to vanillic acid by two distinct carbon source-dependent inducible pathways. Vanillic acid was further metabolized to protocatechuic acid and entered the central carbon pathway via the β-ketoadipate route after ortho ring cleavage. Genes encoding putative enzymes involved in the degradation were found to be present at fer, ver, and van loci. The transcriptional analysis suggests a carbon source-dependent cotranscription of these loci, substantiating the metabolic studies. Biochemical and quantitative real-time (qRT)-PCR studies revealed the presence of two distinct O-demethylases, viz., VerAB and VanAB, involved in the oxidative demethylation of veratric acid and vanillic acid, respectively. This report describes the various steps involved in metabolizing lignin-derived aromatic compounds at the biochemical level and identifies the genes involved in degrading veratric acid and the arrangement of phenylpropanoid metabolic genes as three distinct inducible transcription units/operons. This study provides insight into the bacterial degradation of lignin-derived aromatics and the potential of P. putida CSV86 as a suitable candidate for producing valuable products. IMPORTANCE Pseudomonas putida CSV86 metabolizes lignin and its metabolic intermediates as a carbon source. Strain CSV86 displays a unique property of preferential utilization of aromatics, including for phenylpropanoids over glucose. This report unravels veratryl alcohol metabolism and genes encoding veratric acid O-demethylase, hitherto unknown in pseudomonads, thereby providing new insight into the

Autotaxin: Its Role in Biology of Melanoma Cells and as a Pharmacological Target

Directory of Open Access Journals (Sweden)

Maciej Jankowski

2011-01-01

Full Text Available Autotaxin (ATX is an extracellular lysophospholipase D (lysoPLD released from normal cells and cancer cells. Activity of ATX is detected in various biological fluids. The lysophosphatidic acid (LPA is the main product of ATX. LPA acting through specific G protein-coupled receptors (LPA1-LPA6 affects immunological response, normal development, and malignant tumors' formation and progression. In this review, the impact of autotoxin on biology of melanoma cells and potential treatment is discussed.

Conductivity-Dependent Flow Field-Flow Fractionation of Fulvic and Humic Acid Aggregates

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Martha J. M. Wells

2015-09-01

Full Text Available Fulvic (FAs and humic acids (HAs are chemically fascinating. In water, they have a strong propensity to aggregate, but this research reveals that tendency is regulated by ionic strength. In the environment, conductivity extremes occur naturally—freshwater to seawater—warranting consideration at low and high values. The flow field flow fractionation (flow FFF of FAs and HAs is observed to be concentration dependent in low ionic strength solutions whereas the corresponding flow FFF fractograms in high ionic strength solutions are concentration independent. Dynamic light scattering (DLS also reveals insight into the conductivity-dependent behavior of humic substances (HSs. Four particle size ranges for FAs and humic acid aggregates are examined: (1 <10 nm; (2 10 nm–6 µm; (3 6–100 µm; and (4 >100 µm. Representative components of the different size ranges are observed to dynamically coexist in solution. The character of the various aggregates observed—such as random-extended-coiled macromolecules, hydrogels, supramolecular, and micellar—as influenced by electrolytic conductivity, is discussed. The disaggregation/aggregation of HSs is proposed to be a dynamic equilibrium process for which the rate of aggregate formation is controlled by the electrolytic conductivity of the solution.

Spin-dependent transport properties of oleic acid molecule self-assembled La0.7Sr0.3MnO3 nanoparticles

International Nuclear Information System (INIS)

Xi, L.; Du, J.H.; Ma, J.H.; Wang, Z.; Zuo, Y.L.; Xue, D.S.

2013-01-01

Highlights: ► Spin-dependent transport property of LSMO/oleic acid nanoparticles is investigated. ► Transport properties and MR measured by Cu/nanoparticle assembly/elargol device. ► Non-linear I–V curve indicates a tunneling type transport properties. ► Tunnel barrier height around 1.3 ± 0.15 eV was obtained by fitting I–V curves. ► LFMR of LSMO/oleic acid molecules value reaches −18% with current of 0.1 μA at 10 K. - Abstract: Spin-dependent transport property through molecules is investigated using a monolayer of oleic acid molecule self-assembled half metallic La 0.7 Sr 0.3 MnO 3 (LSMO) nanoparticles, which was synthesized using a coprecipitation method. Fourier transform infrared spectroscopy was used to confirm that one-monolayer oleic acid molecules chemically bond to the LSMO nanoparticles. The transport properties and magnetoresistance (MR) effect of the oleic acid molecule coated LSMO nanoparticles were measured by a direct current four probes method using a Cu/nanoparticle assembly/elargol electrode sandwich device with various temperatures and bias voltages. The non-linear I–V curve indicates a tunneling type transport properties. The tunnel barrier height around 1.3 ± 0.15 eV was obtained by fitting the I–V curve according to the Simmons equation. The magnetoresistance curves can be divided to high-field MR and low-field MR (LFMR) parts. The former is ascribed to the influence of spin disorder or canting within the LSMO nanoparticle surface and the latter one with strong bias dependence is attributed to the spin-dependent tunneling effect through the insulating surface layer of LSMO and oleic acid molecules. The enhanced LFMR effect for oleic acid coated LSMO with respect to the bare LSMO was attributed to the enhanced tunneling transport and weak spin scattering in oleic acid molecule barrier.

Metal cation dependence of interactions with amino acids: bond dissociation energies of Rb(+) and Cs(+) to the acidic amino acids and their amide derivatives.

Science.gov (United States)

Armentrout, P B; Yang, Bo; Rodgers, M T

2014-04-24

Metal cation-amino acid interactions are key components controlling the secondary structure and biological function of proteins, enzymes, and macromolecular complexes comprising these species. Determination of pairwise interactions of alkali metal cations with amino acids provides a thermodynamic vocabulary that begins to quantify these fundamental processes. In the present work, we expand a systematic study of such interactions by examining rubidium and cesium cations binding with the acidic amino acids (AA), aspartic acid (Asp) and glutamic acid (Glu), and their amide derivatives, asparagine (Asn) and glutamine (Gln). These eight complexes are formed using electrospray ionization and their bond dissociation energies (BDEs) are determined experimentally using threshold collision-induced dissociation with xenon in a guided ion beam tandem mass spectrometer. Analyses of the energy-dependent cross sections include consideration of unimolecular decay rates, internal energy of the reactant ions, and multiple ion-neutral collisions. Quantum chemical calculations are conducted at the B3LYP, MP2(full), and M06 levels of theory using def2-TZVPPD basis sets, with results showing reasonable agreement with experiment. At 0 and 298 K, most levels of theory predict that the ground-state conformers for M(+)(Asp) and M(+)(Asn) involve tridentate binding of the metal cation to the backbone carbonyl, amino, and side-chain carbonyl groups, although tridentate binding to the carboxylic acid group and side-chain carbonyl is competitive for M(+)(Asn). For the two longer side-chain amino acids, Glu and Gln, multiple structures are competitive. A comparison of these results to those for the smaller alkali cations, Na(+) and K(+), provides insight into the trends in binding energies associated with the molecular polarizability and dipole moment of the side chain. For all four metal cations, the BDEs are inversely correlated with the size of the metal cation and follow the order Asp < Glu

Gallic acid attenuates calcium calmodulin-dependent kinase II-induced apoptosis in spontaneously hypertensive rats.

Science.gov (United States)

Jin, Li; Piao, Zhe Hao; Liu, Chun Ping; Sun, Simei; Liu, Bin; Kim, Gwi Ran; Choi, Sin Young; Ryu, Yuhee; Kee, Hae Jin; Jeong, Myung Ho

2018-03-01

Hypertension causes cardiac hypertrophy and leads to heart failure. Apoptotic cells are common in hypertensive hearts. Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is associated with apoptosis. We recently demonstrated that gallic acid reduces nitric oxide synthase inhibition-induced hypertension. Gallic acid is a trihydroxybenzoic acid and has been shown to have beneficial effects, such as anti-cancer, anti-calcification and anti-oxidant activity. The purpose of this study was to determine whether gallic acid regulates cardiac hypertrophy and apoptosis in essential hypertension. Gallic acid significantly lowered systolic and diastolic blood pressure in spontaneously hypertensive rats (SHRs). Wheat germ agglutinin (WGA) and H&E staining revealed that gallic acid reduced cardiac enlargement in SHRs. Gallic acid treatment decreased cardiac hypertrophy marker genes, including atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), in SHRs. The four isoforms, α, β, δ and γ, of CaMKII were increased in SHRs and were significantly reduced by gallic acid administration. Gallic acid reduced cleaved caspase-3 protein as well as bax, p53 and p300 mRNA levels in SHRs. CaMKII δ overexpression induced bax and p53 expression, which was attenuated by gallic acid treatment in H9c2 cells. Gallic acid treatment reduced DNA fragmentation and the TUNEL positive cells induced by angiotensin II. Taken together, gallic acid could be a novel therapeutic for the treatment of hypertension through suppression of CaMKII δ-induced apoptosis. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Amino acid-dependent signaling via S6K1 and MYC is essential for regulation of rDNA transcription

Science.gov (United States)

Kang, Jian; Kusnadi, Eric P.; Ogden, Allison J.; Hicks, Rodney J.; Bammert, Lukas; Kutay, Ulrike; Hung, Sandy; Sanij, Elaine; Hannan, Ross D.; Hannan, Katherine M.; Pearson, Richard B.

2016-01-01

Dysregulation of RNA polymerase I (Pol I)-dependent ribosomal DNA (rDNA) transcription is a consistent feature of malignant transformation that can be targeted to treat cancer. Understanding how rDNA transcription is coupled to the availability of growth factors and nutrients will provide insight into how ribosome biogenesis is maintained in a tumour environment characterised by limiting nutrients. We demonstrate that modulation of rDNA transcription initiation, elongation and rRNA processing is an immediate, co-regulated response to altered amino acid abundance, dependent on both mTORC1 activation of S6K1 and MYC activity. Growth factors regulate rDNA transcription initiation while amino acids modulate growth factor-dependent rDNA transcription by primarily regulating S6K1-dependent rDNA transcription elongation and processing. Thus, we show for the first time amino acids regulate rRNA synthesis by a distinct, post-initiation mechanism, providing a novel model for integrated control of ribosome biogenesis that has implications for understanding how this process is dysregulated in cancer. PMID:27385002

Lysophosphatidic Acid Regulation and Roles in Human Prostate Cancer

Science.gov (United States)

2006-08-01

IGF-II ( insulin -like growth factor-II), and hormones have been implicated in the growth and survival of prostate cancer cells [1]. A recent addition to...sphingomyelin. In the synthesis of sphingomyelin, a phosphocholine group is transferred from phosphatidylcholine to ceramide. Sphin- gomyelin synthesis...cytotoxicity of TNF-α [78]. A phosphatidylcholine -specific phospholipase C activity was also required for aSMase activa- tion [79,80]. It is assumed that

Engineering an ATP-dependent D-Ala:D-Ala ligase for synthesizing amino acid amides from amino acids.

Science.gov (United States)

Miki, Yuta; Okazaki, Seiji; Asano, Yasuhisa

2017-05-01

We successfully engineered a new enzyme that catalyzes the formation of D-Ala amide (D-AlaNH 2 ) from D-Ala by modifying ATP-dependent D-Ala:D-Ala ligase (EC 6.3.2.4) from Thermus thermophilus, which catalyzes the formation of D-Ala-D-Ala from two molecules of D-Ala. The new enzyme was created by the replacement of the Ser293 residue with acidic amino acids, as it was speculated to bind to the second D-Ala of D-Ala-D-Ala. In addition, a replacement of the position with Glu performed better than that with Asp with regards to specificity for D-AlaNH 2 production. The S293E variant, which was selected as the best enzyme for D-AlaNH 2 production, exhibited an optimal activity at pH 9.0 and 40 °C for D-AlaNH 2 production. The apparent K m values of this variant for D-Ala and NH 3 were 7.35 mM and 1.58 M, respectively. The S293E variant could catalyze the synthesis of 9.3 and 35.7 mM of D-AlaNH 2 from 10 and 50 mM D-Ala and 3 M NH 4 Cl with conversion yields of 93 and 71.4 %, respectively. This is the first report showing the enzymatic formation of amino acid amides from amino acids.

Tributyltin and triphenyltin inhibit osteoclast differentiation through a retinoic acid receptor-dependent signaling pathway

International Nuclear Information System (INIS)

Yonezawa, Takayuki; Hasegawa, Shin-ichi; Ahn, Jae-Yong; Cha, Byung-Yoon; Teruya, Toshiaki; Hagiwara, Hiromi; Nagai, Kazuo; Woo, Je-Tae

2007-01-01

Organotin compounds, such as tributyltin (TBT) and triphenyltin (TPT), have been widely used in agriculture and industry. Although these compounds are known to have many toxic effects, including endocrine-disrupting effects, their effects on bone resorption are unknown. In this study, we investigated the effects of organotin compounds, such as monobutyltin (MBT), dibutyltin (DBT), TBT, and TPT, on osteoclast differentiation using mouse monocytic RAW264.7 cells. MBT and DBT had no effects, whereas TBT and TPT dose-dependently inhibited osteoclast differentiation at concentrations of 3-30 nM. Treatment with a retinoic acid receptor (RAR)-specific antagonist, Ro41-5253, restored the inhibition of osteoclastogenesis by TBT and TPT. TBT and TPT reduced receptor activator of nuclear factor-κB ligand (RANKL) induced nuclear factor of activated T cells (NFAT) c1 expression, and the reduction in NFATc1 expression was recovered by Ro41-5253. Our results suggest that TBT and TPT suppress osteoclastogenesis by inhibiting RANKL-induced NFATc1 expression via an RAR-dependent signaling pathway

Prebiotic Amino Acid Thioester Synthesis: Thiol-Dependent Amino Acid Synthesis from Formose substrates (Formaldehyde and Glycolaldehyde) and Ammonia

Science.gov (United States)

Weber, Arthur L.

1998-01-01

Formaldehyde and glycolaldehyde (substrates of the formose autocatalytic cycle) were shown to react with ammonia yielding alanine and homoserine under mild aqueous conditions in the presence of thiol catalysts. Since similar reactions carried out without ammonia yielded alpha-hydroxy acid thioesters, the thiol-dependent synthesis of alanine and homoserine is presumed to occur via amino acid thioesters-intermediates capable of forming peptides. A pH 5.2 solution of 20 mM formaldehyde, 20 mM glycolaldehyde, 20 mM ammonium chloride, 23 mM 3-mercaptopropionic acid, and 23 mM acetic acid that reacted for 35 days at 40 C yielded (based on initial formaldehyde) 1.8% alanine and 0.08% homoserine. In the absence of thiol catalyst, the synthesis of alanine and homoserine was negligible. Alanine synthesis required both formaldehyde and glycolaldehyde, but homoserine synthesis required only glycolaldehyde. At 25 days the efficiency of alanine synthesis calculated from the ratio of alanine synthesized to formaldehyde reacted was 2.1%, and the yield (based on initial formaldehyde) of triose and tetrose intermediates involved in alanine and homoserine synthesis was 0.3 and 2.1%, respectively. Alanine synthesis was also seen in similar reactions containing only 10 mM each of aldehyde substrates, ammonia, and thiol. The prebiotic significance of these reactions that use the formose reaction to generate sugar intermediates that are converted to reactive amino acid thioesters is discussed.

Pro-lipogenic Action of Lysophosphatidic Acid in Ovarian Cancer

Science.gov (United States)

2014-04-01

Function. IUBMB Life. 2008. 60: 347-354. 11. Tse C, Shoemaker AR, Adickes J, Anderson MG, Chen J, Jin S, Johnson EF, Marsh KC , Mitten MJ, Nimmer P...Ann. Oncol. 9, 437–442 14. Choi, J. W., Herr, D. R., Noguchi, K., Yung, Y. C., Lee, C. W., Mutoh, T., Lin, M. E., Teo , S. T., Park, K. E., Mosley, A

A pH dependent Raman and surface enhanced Raman spectroscopic studies of citrazinic acid aided by theoretical calculations

Science.gov (United States)

Sarkar, Sougata; Chowdhury, Joydeep; Dutta, Soumen; Pal, Tarasankar

2016-12-01

A pH dependent normal Raman scattering (NRS) and surface enhanced Raman scattering (SERS) spectral patterns of citrazinic acid (CZA), a biologically important molecule, have been investigated. The acid, with different pKa values ( 4 and 11) for the two different functional groups (-COOH and -OH groups), shows interesting range of color changes (yellow at pH 14 and brown at pH 2) with the variation in solution pH. Thus, depending upon the pH of the medium, CZA molecule can exist in various protonated and/or deprotonated forms. Here we have prescribed the existence different possible forms of CZA at different pH (Forms ;C;, ;H; and ;Dprot; at pH 14 and Forms ;A;, ;D;, and ;P; at pH 2 respectively). The NRS spectra of these solutions and their respective SERS spectra over gold nanoparticles were recorded. The spectra clearly differ in their spectral profiles. For example the SERS spectra recorded with the CZA solution at pH 2 shows blue shift for different bands compared to its NRS window e.g. 406 to 450 cm- 1, 616 to 632 cm- 1, 1332 to 1343 cm- 1 etc. Again, the most enhanced peak at 1548 cm- 1 in NRS while in the SERS window this appears at 1580 cm- 1. Similar observation was also made for CZA at pH 14. For example, the 423 cm- 1 band in the NRS profile experience a blue shift and appears at 447 cm- 1 in the SERS spectrum as well as other bands at 850, 1067 and 1214 cm- 1 in the SERS window are markedly enhanced. It is also worth noting that the SERS spectra at the different pH also differ from each other. These spectral differences indicate the existence of various adsorptive forms of the CZA molecule depending upon the pH of the solution. Therefore based on the experimental findings we propose different possible molecular forms of CZA at different pH (acidic and alkaline) conditions. For example forms 'A', 'D' and 'P' existing in acidic pH (pH 2) and three other deprotonated forms 'C', 'H' and 'Dprot' in alkaline pH (pH 14). The DFT calculations for these

TrkB-T1 regulates the RhoA signaling and actin cytoskeleton in glioma cells

International Nuclear Information System (INIS)

Ohira, Koji; Homma, Koichi J.; Hirai, Hirohisa; Nakamura, Shun; Hayashi, Motoharu

2006-01-01

Recently, the truncated TrkB receptor, T1, has been reported to be involved in the control of cell morphology via the regulation of Rho proteins, through which T1 binds Rho guanine nucleotide dissociation inhibitor (Rho GDI) 1 and dissociates it in a brain-derived neurotrophic factor (BDNF)-dependent manner. However, it is unclear whether T1 signaling regulates the downstream of Rho signaling and the actin cytoskeleton. In this study, we investigated this question using C6 rat glioma cells, which express T1 endogenously. Rho GDI1 was dissociated from T1 in a BDNF-dependent manner, which also causes decreases in the activities of Rho-signaling molecules such as RhoA, Rho-associated kinase, p21-activated kinase, and extracellular-signal regulated kinase1/2. Moreover, BDNF treatment resulted in the disappearance of stress fibers in the cells treated with lysophosphatidic acid, an activator of RhoA, and in morphological changes in cells. Furthermore, a competitive assay with cyan fluorescent protein fusion proteins of T1-specific sequences reduced the effects of BDNF. These results suggest that T1 regulates the Rho-signaling pathways and the actin cytoskeleton

Large scale purification and characterization of recombinant human autotaxin/lysophospholipase D from mammalian cells

OpenAIRE

Song, Yuanda; Dilger, Emily; Bell, Jessica; Barton, William A; Fang, Xianjun

2010-01-01

We utilized a mammalian expression system to purify and characterize autotaxin (ATX)/lysophospholipase D, an enzyme present in the blood responsible for biosynthesis of lysophosphatidic acid. The human ATX cDNA encoding amino acids 29–915 was cloned downstream of a secretion signal of CD5. At the carboxyl terminus was a thrombin cleavage site followed by the constant domain (Fc) of IgG to facilitate protein purification. The ATX-Fc fusion protein was expressed in HEK293 cells and isolated fro...

Nucleosome acidic patch promotes RNF168- and RING1B/BMI1-dependent H2AX and H2A ubiquitination and DNA damage signaling.

Directory of Open Access Journals (Sweden)

Justin W Leung

2014-03-01

Full Text Available Histone ubiquitinations are critical for the activation of the DNA damage response (DDR. In particular, RNF168 and RING1B/BMI1 function in the DDR by ubiquitinating H2A/H2AX on Lys-13/15 and Lys-118/119, respectively. However, it remains to be defined how the ubiquitin pathway engages chromatin to provide regulation of ubiquitin targeting of specific histone residues. Here we identify the nucleosome acid patch as a critical chromatin mediator of H2A/H2AX ubiquitination (ub. The acidic patch is required for RNF168- and RING1B/BMI1-dependent H2A/H2AXub in vivo. The acidic patch functions within the nucleosome as nucleosomes containing a mutated acidic patch exhibit defective H2A/H2AXub by RNF168 and RING1B/BMI1 in vitro. Furthermore, direct perturbation of the nucleosome acidic patch in vivo by the expression of an engineered acidic patch interacting viral peptide, LANA, results in defective H2AXub and RNF168-dependent DNA damage responses including 53BP1 and BRCA1 recruitment to DNA damage. The acidic patch therefore is a critical nucleosome feature that may serve as a scaffold to integrate multiple ubiquitin signals on chromatin to compose selective ubiquitinations on histones for DNA damage signaling.

Surfactin Protects Wheat against Zymoseptoria tritici and Activates Both Salicylic Acid- and Jasmonic Acid-Dependent Defense Responses

Directory of Open Access Journals (Sweden)

Geraldine Le Mire

2018-01-01

Full Text Available Natural elicitors induce plant resistance against a broad spectrum of diseases, and are currently among the most promising biocontrol tools. The present study focuses on the elicitor properties of the cyclic lipopeptide surfactin on wheat, in order to stimulate the defenses of this major crop against the challenging fungal pathogen Zymoseptoria tritici. The protection efficacy of surfactin extracted from the strain Bacillus amyloliquefaciens S499 was investigated through greenhouse trials. Surfactin protected wheat by 70% against Z. tritici, similarly to the chemical reference elicitor Bion®50WG. In vitro biocidal assays revealed no antifungal activities of surfactin towards the pathogen. A biomolecular RT-qPCR based low-density microarray tool was used to study the relative expression of 23 wheat defense genes. Surfactin significantly induced wheat natural defenses by stimulating both salicylic acid- and jasmonic acid-dependent signaling pathways. Surfactin was successfully tested as an elicitor on the pathosystem wheat–Z. tritici. These results promote further sustainable agricultural practices and the reduction of chemical inputs.

Antimicrobial medium- and long-chain free fatty acids prevent PrfA-dependent activation of virulence genes in Listeria monocytogenes

DEFF Research Database (Denmark)

Sternkopf Lillebæk, Eva Maria; Lambert Nielsen, Stine; Scheel Thomasen, Rikke

2017-01-01

of virulence factors required for bacterial entry, intracellular replication and cell-to-cell spread. PrfA-dependent activation of virulence genes occurs primarily in the blood and during intracellular infection. In contrast, PrfA does not play a significant role in regulation of virulence gene expression...... antimicrobial free fatty acids act to downregulate transcription of PrfA-activated virulence genes. Interestingly, the inhibitory effect is also evident in cells encoding a constitutively active variant of PrfA. Collectively, our data suggest that antimicrobial medium- and long-chain free fatty acids may act...... as signals to prevent PrfA-mediated activation of virulence genes in environments where PrfA activation is not required, such as in food and the gastrointestinal tract....

A single amino acid substitution controls DAF-dependent phenotype of echovirus 11 in rhabdomyosarcoma cells.

Science.gov (United States)

Novoselov, Alexey V; Rezaykin, Alexey V; Sergeev, Alexander G; Fadeyev, Fedor A; Grigoryeva, Julia V; Sokolova, Zoya I

2012-06-01

Decay accelerating factor (DAF, CD55) is used by DAF-dependent (Daf+) variants of echovirus 11 (EV11) as a primary cellular receptor. The interaction of EV11 with DAF is completely reversible, therefore DAF-dependent variants require an unidentified coreceptor to initiate uncoating. Daf- variants of EV11, which do not interact with DAF, use an alternative primary cellular receptor. The aim of this study was to test the hypothesis whether the coreceptor, which is necessary for the uncoating of DAF-dependent variants, may act as an alternative primary receptor for the Daf- variants of EV11. By using the model of the two closely related daf+ and daf- clones of EV11 in rhabdomyosarcoma (RD) cell line, it was shown that a single amino acid substitution in the capsid protein VP2 could control the expression of the DAF-dependent phenotype. Anti-DAF monoclonal antibody has blocked the infection of RD cells by the DAF-dependent daf+ clone, but not by the daf- clone of EV11. Since the structural proteins of the two clones differed only in the receptor binding site for DAF, the unidentified non-DAF primary receptor for the daf- clone might have the same conformation as the uncoating coreceptor required for the daf+ clone. Despite the difference in primary receptors, both daf+ and daf- clones were equally inhibited by a monoclonal antibody to beta2-microglobulin. The monoclonal antibody B9.12.1 to class I human leukocyte antigen molecules showed no inhibitory effect in regards to either clone. The hypothesis of convergent intracellular traffic of Daf+ and Daf- variants of EV11 is discussed. Copyright © 2012 Elsevier B.V. All rights reserved.

Uric acid inhibition of dipeptidyl peptidase IV in vitro is dependent on the intracellular formation of triuret.

Science.gov (United States)

Mohandas, Rajesh; Sautina, Laura; Beem, Elaine; Schuler, Anna; Chan, Wai-Yan; Domsic, John; McKenna, Robert; Johnson, Richard J; Segal, Mark S

2014-08-01

Uric acid affects endothelial and adipose cell function and has been linked to diseases such as hypertension, metabolic syndrome, and cardiovascular disease. Interestingly uric acid has been shown to increase endothelial progenitor cell (EPC) mobilization, a potential mechanism to repair endothelial injury. Since EPC mobilization is dependent on activity of the enzyme CD26/dipeptidyl peptidase (DPP)IV, we examined the effect uric acid will have on CD26/DPPIV activity. Uric acid inhibited the CD26/DPPIV associated with human umbilical vein endothelial cells but not human recombinant (hr) CD26/DPPIV. However, triuret, a product of uric acid and peroxynitrite, could inhibit cell associated and hrCD26/DPPIV. Increasing or decreasing intracellular peroxynitrite levels enhanced or decreased the ability of uric acid to inhibit cell associated CD26/DPPIV, respectively. Finally, protein modeling demonstrates how triuret can act as a small molecule inhibitor of CD26/DPPIV activity. This is the first time that uric acid or a uric acid reaction product has been shown to affect enzymatic activity and suggests a novel avenue of research in the role of uric acid in the development of clinically important diseases. Published by Elsevier Inc.

Morphology-dependent activity of Pt nanocatalysts for ethanol oxidation in acidic media: Nanowires versus nanoparticles

International Nuclear Information System (INIS)

Zhou Weiping; Li Meng; Koenigsmann, Christopher; Ma Chao; Wong, Stanislaus S.; Adzic, Radoslav R.

2011-01-01

Highlights: → We demonstrate the morphology effect of Pt catalysts in electrooxidation of ethanol and CO in an acidic solution. → Pt nanowires and nanoparticles were used as catalysts. → Pt nanowires display a higher catalytic activity by a factor of at least two relative to those nanoparticles for ethanol oxidation. → The rate for CO monolayer oxidation exhibits similar morphology-dependent behavior with a markedly enhanced rate on the Pt nanowires. - Abstract: The morphology of nanostructured Pt catalysts is known to affect significantly the kinetics of various reactions. Herein, we report on a pronounced morphology effect in the electrooxidation of ethanol and carbon monoxide (CO) on Pt nanowires and nanoparticles in an acidic solution. The high resolution transmission electron microscopy analysis showed the inherent morphology difference between these two nanostructured catalysts. Voltammetric and chronoamperometric studies of the ethanol electrooxidation revealed that these nanowires had a higher catalytic activity by a factor of two relative to these nanoparticles. The rate for CO monolayer oxidation exhibits similar morphology-dependent behavior with a markedly enhanced rate on the Pt nanowires. In situ infrared reflection-absorption spectroscopy measurements revealed a different trend for chemisorbed CO formation and CO 2 -to-acetic acid reaction product ratios on these two nanostructures. The morphology-induced change in catalytic activity and selectivity in ethanol electrocatalysis is discussed in detail.

Morphology-dependent activity of Pt nanocatalysts for ethanol oxidation in acidic media: Nanowires versus nanoparticles

Energy Technology Data Exchange (ETDEWEB)

Zhou Weiping, E-mail: wpzhou@bnl.gov [Chemistry Department, Brookhaven National Laboratory, Upton, NY 11973 (United States); Li Meng [Chemistry Department, Brookhaven National Laboratory, Upton, NY 11973 (United States); Koenigsmann, Christopher [Department of Chemistry, State University of New York at Stony Brook, Stony Brook, NY 11794 (United States); Ma Chao [Condensed Matter Physics and Materials Sciences Department, Brookhaven National Laboratory, Building 480, Upton, NY 11973 (United States); Wong, Stanislaus S. [Department of Chemistry, State University of New York at Stony Brook, Stony Brook, NY 11794 (United States); Condensed Matter Physics and Materials Sciences Department, Brookhaven National Laboratory, Building 480, Upton, NY 11973 (United States); Adzic, Radoslav R. [Chemistry Department, Brookhaven National Laboratory, Upton, NY 11973 (United States)

2011-11-30

Highlights: > We demonstrate the morphology effect of Pt catalysts in electrooxidation of ethanol and CO in an acidic solution. > Pt nanowires and nanoparticles were used as catalysts. > Pt nanowires display a higher catalytic activity by a factor of at least two relative to those nanoparticles for ethanol oxidation. > The rate for CO monolayer oxidation exhibits similar morphology-dependent behavior with a markedly enhanced rate on the Pt nanowires. - Abstract: The morphology of nanostructured Pt catalysts is known to affect significantly the kinetics of various reactions. Herein, we report on a pronounced morphology effect in the electrooxidation of ethanol and carbon monoxide (CO) on Pt nanowires and nanoparticles in an acidic solution. The high resolution transmission electron microscopy analysis showed the inherent morphology difference between these two nanostructured catalysts. Voltammetric and chronoamperometric studies of the ethanol electrooxidation revealed that these nanowires had a higher catalytic activity by a factor of two relative to these nanoparticles. The rate for CO monolayer oxidation exhibits similar morphology-dependent behavior with a markedly enhanced rate on the Pt nanowires. In situ infrared reflection-absorption spectroscopy measurements revealed a different trend for chemisorbed CO formation and CO{sub 2}-to-acetic acid reaction product ratios on these two nanostructures. The morphology-induced change in catalytic activity and selectivity in ethanol electrocatalysis is discussed in detail.

The transcription factor ABI4 Is required for the ascorbic acid-dependent regulation of growth and regulation of jasmonate-dependent defense signaling pathways in Arabidopsis.

Science.gov (United States)

Kerchev, Pavel I; Pellny, Till K; Vivancos, Pedro Diaz; Kiddle, Guy; Hedden, Peter; Driscoll, Simon; Vanacker, Hélène; Verrier, Paul; Hancock, Robert D; Foyer, Christine H

2011-09-01

Cellular redox homeostasis is a hub for signal integration. Interactions between redox metabolism and the ABSCISIC ACID-INSENSITIVE-4 (ABI4) transcription factor were characterized in the Arabidopsis thaliana vitamin c defective1 (vtc1) and vtc2 mutants, which are defective in ascorbic acid synthesis and show a slow growth phenotype together with enhanced abscisic acid (ABA) levels relative to the wild type (Columbia-0). The 75% decrease in the leaf ascorbate pool in the vtc2 mutants was not sufficient to adversely affect GA metabolism. The transcriptome signatures of the abi4, vtc1, and vtc2 mutants showed significant overlap, with a large number of transcription factors or signaling components similarly repressed or induced. Moreover, lincomycin-dependent changes in LIGHT HARVESTING CHLOROPHYLL A/B BINDING PROTEIN 1.1 expression were comparable in these mutants, suggesting overlapping participation in chloroplast to nucleus signaling. The slow growth phenotype of vtc2 was absent in the abi4 vtc2 double mutant, as was the sugar-insensitive phenotype of the abi4 mutant. Octadecanoid derivative-responsive AP2/ERF-domain transcription factor 47 (ORA47) and AP3 (an ABI5 binding factor) transcripts were enhanced in vtc2 but repressed in abi4 vtc2, suggesting that ABI4 and ascorbate modulate growth and defense gene expression through jasmonate signaling. We conclude that low ascorbate triggers ABA- and jasmonate-dependent signaling pathways that together regulate growth through ABI4. Moreover, cellular redox homeostasis exerts a strong influence on sugar-dependent growth regulation.

Gibberellic acid and cGMP-dependent transcriptional regulation in arabidopsis thaliana

KAUST Repository

Bastian, René

2010-03-01

An ever increasing amount of transcriptomic data and analysis tools provide novel insight into complex responses of biological systems. Given these resources we have undertaken to review aspects of transcriptional regulation in response to the plant hormone gibberellic acid (GA) and its second messenger guanosine 3\\',5\\'-cyclic monophosphate (cGMP) in Arabidopsis thaliana, both wild type and selected mutants. Evidence suggests enrichment of GA-responsive (GARE) elements in promoters of genes that are transcriptionally upregulated in response to cGMP but downregulated in a GA insensitive mutant (ga1-3). In contrast, in the genes upregulated in the mutant, no enrichment in the GARE is observed suggesting that GARE motifs are diagnostic for GA-induced and cGMP-dependent transcriptional upregulation. Further, we review how expression studies of GA-dependent transcription factors and transcriptional networks based on common promoter signatures derived from ab initio analyses can contribute to our understanding of plant responses at the systems level. © 2010 Landes Bioscience.

Weak Organic Acids Decrease Borrelia burgdorferi Cytoplasmic pH, Eliciting an Acid Stress Response and Impacting RpoN- and RpoS-Dependent Gene Expression

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Daniel P. Dulebohn

2017-09-01

Full Text Available The spirochete Borrelia burgdorferi survives in its tick vector, Ixodes scapularis, or within various hosts. To transition between and survive in these distinct niches, B. burgdorferi changes its gene expression in response to environmental cues, both biochemical and physiological. Exposure of B. burgdorferi to weak monocarboxylic organic acids, including those detected in the blood meal of fed ticks, decreased the cytoplasmic pH of B. burgdorferi in vitro. A decrease in the cytoplasmic pH induced the expression of genes encoding enzymes that have been shown to restore pH homeostasis in other bacteria. These include putative coupled proton/cation exchangers, a putative Na+/H+ antiporter, a neutralizing buffer transporter, an amino acid deaminase and a proton exporting vacuolar-type VoV1 ATPase. Data presented in this report suggested that the acid stress response triggered the expression of RpoN- and RpoS-dependent genes including important virulence factors such as outer surface protein C (OspC, BBA66, and some BosR (Borreliaoxidative stress regulator-dependent genes. Because the expression of virulence factors, like OspC, are so tightly connected by RpoS to general cellular stress responses and cell physiology, it is difficult to separate transmission-promoting conditions in what is clearly a multifactorial and complex regulatory web.

Adaptive Immune Responses Regulate the Pathophysiology of Lymphedema

Science.gov (United States)

2012-09-01

A, Hennig B (2000) Effect of complex decongestive physiotherapy on gene expression for the inflammatory response in peripheral lymphedema. Lymphology...pharmacologic antagonism of lysophosphatidic acid receptor 1 in a mouse model of scleroderma. Arthritis Rheum 63: 1405-1415. 51. Romani L, Mencacci A, Grohmann...severe disease in collagen-induced arthritis . Arthritis Rheum 48: 1452-1460. 29. Nakamura K, Radhakrishnan K, Wong YM, Rockson SG (2009) Anti

3-Nitropropionic acid neurotoxicity in organotypic striatal and corticostriatal slice cultures is dependent on glucose and glutamate

DEFF Research Database (Denmark)

Storgaard, J; Kornblit, B T; Zimmer, J

2000-01-01

of lactate dehydrogenase in the medium and glutamic acid decarboxylase in tissue homogenates. 3-NPA toxicity (25-100 microM in 5 mM glucose, 24-48 h) appeared to be highly dependent on culture medium glucose levels. 3-NPA treatment caused also a dose-dependent lactate increase, reaching a maximum......Mitochondrial inhibition by 3-nitropropionic acid (3-NPA) causes striatal degeneration reminiscent of Huntington's disease. We studied 3-NPA neurotoxicity and possible indirect excitotoxicity in organotypic striatal and corticostriatal slice cultures. Neurotoxicity was quantified by assay...... of threefold increase above control at 100 microM. Both a high dose of glutamate (5 mM) and glutamate uptake blockade by dl-threo-beta-hydroxyaspartate potentiated 3-NPA neurotoxicity in corticostriatal slice cultures. Furthermore, striatum from corticostriatal cocultures was more sensitive to 3-NPA than...

The reported human NADsyn2 is ammonia-dependent NAD synthetase from a pseudomonad.

Science.gov (United States)

Bieganowski, Pawel; Brenner, Charles

2003-08-29

Nicotinamide-adenine dinucleotide (NAD+) synthetases catalyze the last step in NAD+ metabolism in the de novo, import, and salvage pathways that originate from tryptophan (or aspartic acid), nicotinic acid, and nicotinamide, respectively, and converge on nicotinic acid mononucleotide. NAD+ synthetase converts nicotinic acid adenine dinucleotide to NAD+ via an adenylylated intermediate. All of the known eukaryotic NAD+ synthetases are glutamine-dependent, hydrolyzing glutamine to glutamic acid to provide the attacking ammonia. In the prokaryotic world, some NAD+ synthetases are glutamine-dependent, whereas others can only use ammonia. Earlier, we noted a perfect correlation between presence of a domain related to nitrilase and glutamine dependence and then proved in the accompanying paper (Bieganowski, P., Pace, H. C., and Brenner, C. (2003) J. Biol. Chem. 278, 33049-33055) that the nitrilase-related domain is an essential, obligate intramolecular, thiol-dependent glutamine amidotransferase in the yeast NAD+ synthetase, Qns1. Independently, human NAD+ synthetase was cloned and shown to depend on Cys-175 for glutamine-dependent but not ammonia-dependent NAD+ synthetase activity. Additionally, it was claimed that a 275 amino acid open reading frame putatively amplified from human glioma cell line LN229 encodes a human ammonia-dependent NAD+ synthetase and this was speculated largely to mediate NAD+ synthesis in human muscle tissues. Here we establish that the so-called NADsyn2 is simply ammonia-dependent NAD+ synthetase from Pseudomonas, which is encoded on an operon with nicotinic acid phosphoribosyltransferase and, in some Pseudomonads, with nicotinamidase.

The Glutaminase-Dependent System Confers Extreme Acid Resistance to New Species and Atypical Strains of Brucella

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Luca Freddi

2017-11-01

Full Text Available Neutralophilic bacteria have developed specific mechanisms to cope with the acid stress encountered in environments such as soil, fermented foods, and host compartments. In Escherichia coli, the glutamate decarboxylase (Gad-dependent system is extremely efficient: it requires the concerted action of glutamate decarboxylase (GadA/GadB and of the glutamate (Glu/γ-aminobutyrate antiporter, GadC. Notably, this system is operative also in new strains/species of Brucella, among which Brucella microti, but not in the “classical” species, with the exception of marine mammals strains. Recently, the glutaminase-dependent system (named AR2_Q, relying on the deamination of glutamine (Gln into Glu and on GadC activity, was described in E. coli. In Brucella genomes, a putative glutaminase (glsA-coding gene is located downstream of the gadBC genes. We found that in B. microti these genes are expressed as a polycistronic transcript. Moreover, using a panel of Brucella genus-representative strains, we show that the AR2_Q system protects from extreme acid stress (pH ≤2.5, in the sole presence of Gln, only the Brucella species/strains predicted to have functional glsA and gadC. Indeed, mutagenesis approaches confirmed the involvement of glsA and gadC of B. microti in AR2_Q and that the acid-sensitive phenotype of B. abortus can be ascribed to a Ser248Leu substitution in GlsA, leading to loss of glutaminase activity. Furthermore, we found that the gene BMI_II339, of unknown function and downstream of the gadBC–glsA operon, positively affects Gad- and GlsA-dependent AR. Thus, we identified novel determinants that allow newly discovered and marine mammals Brucella strains to be better adapted to face hostile acidic environments. As for significance, this work may contribute to the understanding of the host preferences of Brucella species and opens the way to alternative diagnostic targets in epidemiological surveillance of brucellosis.

Temperature-dependent dynamical transitions of different classes of amino acid residue in a globular protein.

Science.gov (United States)

Miao, Yinglong; Yi, Zheng; Glass, Dennis C; Hong, Liang; Tyagi, Madhusudan; Baudry, Jerome; Jain, Nitin; Smith, Jeremy C

2012-12-05

The temperature dependences of the nanosecond dynamics of different chemical classes of amino acid residue have been analyzed by combining elastic incoherent neutron scattering experiments with molecular dynamics simulations on cytochrome P450cam. At T = 100-160 K, anharmonic motion in hydrophobic and aromatic residues is activated, whereas hydrophilic residue motions are suppressed because of hydrogen-bonding interactions. In contrast, at T = 180-220 K, water-activated jumps of hydrophilic side chains, which are strongly coupled to the relaxation rates of the hydrogen bonds they form with hydration water, become apparent. Thus, with increasing temperature, first the hydrophobic core awakens, followed by the hydrophilic surface.

Tauroursodeoxycholate Protects Rat Hepatocytes from Bile Acid-Induced Apoptosis via β1-Integrin- and Protein Kinase A-Dependent Mechanisms

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Annika Sommerfeld

2015-05-01

Full Text Available Background/Aims: Ursodeoxycholic acid, which in vivo is rapidly converted into its taurine conjugate, is frequently used for the treatment of cholestatic liver disease. Apart from its choleretic effects, tauroursodeoxycholate (TUDC can protect hepatocytes from bile acid-induced apoptosis, but the mechanisms underlying its anti-apoptotic effects are poorly understood. Methods: These mechanisms were investigated in perfused rat liver and isolated rat hepatocytes. Results: It was found that TUDC inhibited the glycochenodeoxycholate (GCDC-induced activation of the CD95 death receptor at the level of association between CD95 and the epidermal growth factor receptor. This was due to a rapid TUDC-induced β1-integrin-dependent cyclic AMP (cAMP signal with induction of the dual specificity mitogen-activated protein (MAP kinase phosphatase 1 (MKP-1, which prevented GCDC-induced phosphorylation of mitogen-activated protein kinase kinase 4 (MKK4 and c-jun-NH2-terminal kinase (JNK activation. Furthermore, TUDC induced a protein kinase A (PKA-mediated serine/threonine phosphorylation of the CD95, which was recently identified as an internalization signal for CD95. Furthermore, TUDC inhibited GCDC-induced CD95 targeting to the plasma membrane in a β1-integrin-and PKA-dependent manner. In line with this, the β1-integrin siRNA knockdown in sodium taurocholate cotransporting polypeptide (Ntcp-transfected HepG2 cells abolished the protective effect of TUDC against GCDC-induced apoptosis. Conclusion: TUDC exerts its anti-apoptotic effect via a β1-integrin-mediated formation of cAMP, which prevents CD95 activation by hydrophobic bile acids at the levels of JNK activation and CD95 serine/threonine phosphorylation.

Insulin-like Growth Factor 1 (IGF-1) Stabilizes Nascent Blood Vessels*

Science.gov (United States)

Jacobo, Sarah Melissa P.; Kazlauskas, Andrius

2015-01-01

Here we report that VEGF-A and IGF-1 differ in their ability to stabilize newly formed blood vessels and endothelial cell tubes. Although VEGF-A failed to support an enduring vascular response, IGF-1 stabilized neovessels generated from primary endothelial cells derived from various vascular beds and mouse retinal explants. In these experimental systems, destabilization/regression was driven by lysophosphatidic acid (LPA). Because previous studies have established that Erk antagonizes LPA-mediated regression, we considered whether Erk was an essential component of IGF-dependent stabilization. Indeed, IGF-1 lost its ability to stabilize neovessels when the Erk pathway was inhibited pharmacologically. Furthermore, stabilization was associated with prolonged Erk activity. In the presence of IGF-1, Erk activity persisted longer than in the presence of VEGF or LPA alone. These studies reveal that VEGF and IGF-1 can have distinct inputs in the angiogenic process. In contrast to VEGF, IGF-1 stabilizes neovessels, which is dependent on Erk activity and associated with prolonged activation. PMID:25564613

ω-3 and ω-6 Fatty Acids Modulate Conventional and Atypical Protein Kinase C Activities in a Brain Fatty Acid Binding Protein Dependent Manner in Glioblastoma Multiforme

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Marwa E. Elsherbiny

2018-04-01

Full Text Available Glioblastoma multiforme (GBM is a highly infiltrative brain cancer with a dismal prognosis. High levels of brain fatty acid binding protein (B-FABP are associated with increased migration/infiltration in GBM cells, with a high ratio of arachidonic acid (AA to docosahexaenoic acid (DHA driving B-FABP-mediated migration. Since several protein kinase Cs (PKCs are overexpressed in GBM and linked to migration, we explored a possible relationship between B-FABP and levels/activity of different PKCs, as a function of AA and DHA supplementation. We report that ectopic expression of B-FABP in U87 cells alters the levels of several PKCs, particularly PKCζ. Upon analysis of PKCζ RNA levels in a panel of GBM cell lines and patient-derived GBM neurospheres, we observed a trend towards moderate positive correlation (r = 0.624, p = 0.054 between B-FABP and PKCζ RNA levels. Analysis of PKC activity in U87 GBM cells revealed decreased typical PKC activity (23.4% in B-FABP-expressing cells compared with nonexpressing cells, with no difference in novel and atypical PKC activities. AA and DHA modulated both conventional and atypical PKC activities in a B-FABP-dependent manner, but had no effect on novel PKC activity. These results suggest that conventional and atypical PKCs are potential downstream effectors of B-FABP/fatty acid-mediated alterations in GBM growth properties.

Cross-Linked Dependency of Boronic Acid-Conjugated Chitosan Nanoparticles by Diols for Sustained Insulin Release

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Nabil A. Siddiqui

2016-10-01

Full Text Available Boronic acids have been widely investigated for their potential use as glucose sensors in glucose responsive polymeric insulin delivery systems. Interactions between cyclic diols and boronic acids, anchored to polymeric delivery systems, may result in swelling of the delivery system, releasing the drug. In this study, 4-formylphenylboronic acid conjugated chitosan was formulated into insulin containing nanoparticles via polyelectrolyte complexation. The nanoparticles had an average diameter of 140 ± 12.8 nm, polydispersity index of 0.17 ± 0.1, zeta potential of +19.1 ± 0.69 mV, encapsulation efficiency of 81% ± 1.2%, and an insulin loading capacity of 46% ± 1.8% w/w. Changes in size of the nanoparticles and release of insulin were type of sugar- and concentration-dependent. High concentration of diols resulted in a sustained release of insulin due to crosslink formation with boronic acid moieties within the nanoparticles. The formulation has potential to be developed into a self-regulated insulin delivery system for the treatment of diabetes.

Constituents of Propolis: Chrysin, Caffeic Acid, p-Coumaric Acid, and Ferulic Acid Induce PRODH/POX-Dependent Apoptosis in Human Tongue Squamous Cell Carcinoma Cell (CAL-27).

Science.gov (United States)

Celińska-Janowicz, Katarzyna; Zaręba, Ilona; Lazarek, Urszula; Teul, Joanna; Tomczyk, Michał; Pałka, Jerzy; Miltyk, Wojciech

2018-01-01

Propolis evokes several therapeutic properties, including anticancer activity. These activities are attributed to the action of polyphenols. Previously it has been demonstrated, that one of the most abundant polyphenolic compounds in ethanolic extracts of propolis are chrysin, caffeic acid, p -coumaric acid, and ferulic acid. Although their pro-apoptotic activity on human tongue squamous cell carcinoma cells (CAL-27) was established previously, the detailed mechanism of this process remains unclear. Considering the crucial role of proline metabolism and proline dehydrogenase/proline oxidase (PRODH/POX) in the regulation of cancer cell survival/apoptosis, we studied these processes in polyphenol-treated CAL-27 cells. All studied polyphenols evoked anti-proliferative activity, accompanied by increased PRODH/POX, P53, active caspases-3 and -9 expressions and decreased collagen biosynthesis, prolidase activity and proline concentration in CAL-27 cells. These data suggest that polyphenols of propolis induce PRODH/POX-dependent apoptosis through up-regulation of mitochondrial proline degradation and down-regulation of proline utilization for collagen biosynthesis.

Acidosis counteracts itch tachyphylaxis to consecutive pruritogen exposure dependent on acid-sensing ion channel 3.

Science.gov (United States)

Jiang, Yi-Ming; Huang, Chen; Peng, Zhong; Han, Shao-Ling; Li, Wei-Guang; Zhu, Michael Xi; Xu, Tian-Le

2017-01-01

Tachyphylaxis of itch refers to a markedly reduced scratching response to consecutive exposures of a pruritogen, a process thought to protect against tissue damage by incessant scratching and to become disrupted in chronic itch. Here, we report that a strong stimulation of the Mas-related G-protein-coupled receptor C11 by its agonist, Ser-Leu-Ile-Gly-Arg-Leu-NH 2 (SL-NH 2 ) or bovine adrenal medulla 8-22 peptide, via subcutaneous injection in mice induces tachyphylaxis to the subsequent application of SL-NH 2 to the same site. Notably, co-application of acid and SL-NH 2 following the initial injection of the pruritogen alone counteracted itch tachyphylaxis by augmenting the scratching behaviors in wild-type but not in acid-sensing ion channel 3-null, animals. Using an activity-dependent silencing strategy, we identified that acid-sensing ion channel 3-mediated itch enhancement mainly occurred via the Mas-related G-protein-coupled receptor C11-responsive sensory neurons. Together, our results indicate that acid-sensing ion channel 3, activated by concomitant acid and certain pruritogens, constitute a novel signaling pathway that counteracts itch tachyphylaxis to successive pruritogenic stimulation, which likely contributes to chronic itch associated with tissue acidosis.

Fatty acid compositions of triglycerides and free fatty acids in sebum depend on amount of triglycerides, and do not differ in presence or absence of acne vulgaris.

Science.gov (United States)

Akaza, Narifumi; Akamatsu, Hirohiko; Numata, Shigeki; Matsusue, Miyuki; Mashima, Yasuo; Miyawaki, Masaaki; Yamada, Shunji; Yagami, Akiko; Nakata, Satoru; Matsunaga, Kayoko

2014-12-01

To clarify the influence of the fatty acid composition of sebum in acne vulgaris, we investigated the amounts and fatty acid compositions of triglycerides (TG) and free fatty acids (FFA), and the amounts of cutaneous superficial Propionibacterium acnes in acne patients and healthy subjects. The foreheads of 18 female patients, 10 male patients, 10 healthy females and 10 healthy males were studied in a Japanese population. There were significant differences in the amounts of sebum, TG and cutaneous superficial P. acnes, as well as the fatty acid compositions of TG and FFA between acne patients and healthy subjects in females. Their fatty acid compositions were correlated with the amount of TG with or without acne. It was clarified that the fatty acid compositions of TG and FFA depended on the amount of TG, and there were no differences in the fatty acid composition in the presence and absence of acne. © 2014 Japanese Dermatological Association.

12(R)-hydroxyicosatetraenoic acid: a cytochrome P450-dependent arachidonate metabolite that inhibits Na+, K+-ATPase in the cornea

International Nuclear Information System (INIS)

Schwartzman, M.L.; Balazy, M.; Masferrer, J.; Abraham, N.G.; McGiff, J.C.; Murphy, R.C.

1987-01-01

When corneal microsomes were incubated with arachidonic acid in the presence of an NADPH-generating system, four polar metabolites (compounds A-D) were formed. Synthesis of these metabolites could be inhibited by carbon monoxide, SKF 525A, and anti-cytochrome c reductase antibodies. One of the metabolites, compound C, was found to inhibit partially purified Na + , K + -ATPase from the corneal epithelium in a dose-dependent manner. After compound C was purified by TLC and HPLC, it was found to have a UV absorption spectrum with a maximum absorbance at 236 nm suggesting the presence of a conjugated diene. Mass spectrometric analysis using positive- and negative-ionization modes was carried out on derivatized compound C. Abundant fragment ions were consistent with compound C being a monooxygenated derivative of arachidonic acid with a hydroxyl substituent at carbon-12 of the icosanoid backbone; all deuterium atoms from [ 2 H 8 ]arachidonate were retained in the structure. Compound C was characterized as a 12-hydroxyicosatetraenoic acid. However, only 12(R) isomer was found to be an inhibitor of the Na + , K + -ATPase from the corneal epithelium, suggesting that the biologically active compound C was 12(R)-hydroxyy-5,8,10,14-icosatetraenoic acid. Such an inhibitor of Na + , K + -ATPase synthesized in the cornea may have an important role in regulating ocular transparency and aqueous human secretion

Probe-Dependent Negative Allosteric Modulators of the Long-Chain Free Fatty Acid Receptor FFA4

DEFF Research Database (Denmark)

Watterson, Kenneth R; Hansen, Steffen V F; Hudson, Brian D

2017-01-01

High-affinity and selective antagonists that are able to block the actions of both endogenous and synthetic agonists of G protein-coupled receptors are integral to analysis of receptor function and to support suggestions of therapeutic potential. Although there is great interest in the potential...... of endogenous and synthetic agonists, clear agonist probe dependence in the nature of allosteric modulation was apparent. Although AH-7614 did not antagonize the second long-chain free fatty acid receptor, free fatty acid receptor 1, the simple chemical structure of AH-7614 containing features found in many...

Crystallization and preliminary X-ray diffraction analysis of a class II phospholipase D from Loxosceles intermedia venom

International Nuclear Information System (INIS)

Ullah, Anwar; Giuseppe, Priscila Oliveira de; Murakami, Mario Tyago; Trevisan-Silva, Dilza; Wille, Ana Carolina Martins; Chaves-Moreira, Daniele; Gremski, Luiza Helena; Silveira, Rafael Bertoni da; Sennf-Ribeiro, Andrea; Chaim, Olga Meiri; Veiga, Silvio Sanches; Arni, Raghuvir Krishnaswamy

2011-01-01

Wild-type and H12A-mutant class II phospholipase D from L. intermedia venom were crystallized; the crystals diffracted to maximum resolutions of 1.95 and 1.60 Å, respectively. Phospholipases D are the major dermonecrotic component of Loxosceles venom and catalyze the hydrolysis of phospholipids, resulting in the formation of lipid mediators such as ceramide-1-phosphate and lysophosphatidic acid which can induce pathological and biological responses. Phospholipases D can be classified into two classes depending on their catalytic efficiency and the presence of an additional disulfide bridge. In this work, both wild-type and H12A-mutant forms of the class II phospholipase D from L. intermedia venom were crystallized. Wild-type and H12A-mutant crystals were grown under very similar conditions using PEG 200 as a precipitant and belonged to space group P12 1 1, with unit-cell parameters a = 50.1, b = 49.5, c = 56.5 Å, β = 105.9°. Wild-type and H12A-mutant crystals diffracted to maximum resolutions of 1.95 and 1.60 Å, respectively

Phenibut (β-Phenyl-γ-aminobutyric Acid) Dependence and Management of Withdrawal: Emerging Nootropics of Abuse.

Science.gov (United States)

Ahuja, Tania; Mgbako, Ofole; Katzman, Caroline; Grossman, Allison

2018-01-01

This case report describes the development of withdrawal from phenibut, a gamma-aminobutyric acid-receptor type B agonist. Although phenibut is not an FDA-approved medication, it is available through online retailers as a nootropic supplement. There are reports of dependence in patients that misuse phenibut. We report a case in which a patient experienced withdrawal symptoms from phenibut and was successfully treated with a baclofen taper. This case report highlights the development of phenibut use disorder with coingestion of alcohol and potential management for phenibut withdrawal. We believe clinicians must be aware of how phenibut dependence may present and how to manage the withdrawal syndrome.

Phenibut (β-Phenyl-γ-aminobutyric Acid Dependence and Management of Withdrawal: Emerging Nootropics of Abuse

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Tania Ahuja

2018-01-01

Full Text Available This case report describes the development of withdrawal from phenibut, a gamma-aminobutyric acid-receptor type B agonist. Although phenibut is not an FDA-approved medication, it is available through online retailers as a nootropic supplement. There are reports of dependence in patients that misuse phenibut. We report a case in which a patient experienced withdrawal symptoms from phenibut and was successfully treated with a baclofen taper. This case report highlights the development of phenibut use disorder with coingestion of alcohol and potential management for phenibut withdrawal. We believe clinicians must be aware of how phenibut dependence may present and how to manage the withdrawal syndrome.

The fatty acid profile in different wheat cultivars depending on the level of contamination with microscopic fungi.

Science.gov (United States)

Stuper-Szablewska, Kinga; Buśko, Maciej; Góral, Tomasz; Perkowski, Juliusz

2014-06-15

Analyses were conducted on 30 winter wheat samples growing under controlled conditions and following inoculation with fungi Fusarium culmorum. In inoculated samples the mean concentration of 30 analysed fatty acids was significantly higher in relation to the control and amounted to 1,396 mg/kg vs. 1,046 mg/kg in the control kernels. Recorded concentrations for individual cultivars were significantly correlated with the concentration of fungal biomass. Higher concentration in the control was recorded only for the acid trans C18:2n-6. It was also found that the acid profiles are characteristic of individual cultivars. Statistical analysis showed that trans C18:2n-6, C18:1, C18:3n-3 and C18:3n-6 were the acids with the greatest discriminatory power in distinguishing inoculated samples from the control. Discriminatory analysis separated individual cultivars into quality classes of winter wheat cultivars depending on the presence of a specific fatty acid profile. Copyright © 2014 Elsevier Ltd. All rights reserved.

d(− Lactic Acid-Induced Adhesion of Bovine Neutrophils onto Endothelial Cells Is Dependent on Neutrophils Extracellular Traps Formation and CD11b Expression

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Pablo Alarcón

2017-08-01

Full Text Available Bovine ruminal acidosis is of economic importance as it contributes to reduced milk and meat production. This phenomenon is mainly attributed to an overload of highly fermentable carbohydrate, resulting in increased d(− lactic acid levels in serum and plasma. Ruminal acidosis correlates with elevated acute phase proteins in blood, along with neutrophil activation and infiltration into various tissues leading to laminitis and aseptic polysynovitis. Previous studies in bovine neutrophils indicated that d(− lactic acid decreased expression of L-selectin and increased expression of CD11b to concentrations higher than 6 mM, suggesting a potential role in neutrophil adhesion onto endothelia. The two aims of this study were to evaluate whether d(− lactic acid influenced neutrophil and endothelial adhesion and to trigger neutrophil extracellular trap (NET production (NETosis in exposed neutrophils. Exposure of bovine neutrophils to 5 mM d(− lactic acid elevated NET release compared to unstimulated neutrophil negative controls. Moreover, this NET contains CD11b and histone H4 citrullinated, the latter was dependent on PAD4 activation, a critical enzyme in DNA decondensation and NETosis. Furthermore, NET formation was dependent on d(− lactic acid plasma membrane transport through monocarboxylate transporter 1 (MCT1. d(− lactic acid enhanced neutrophil adhesion onto endothelial sheets as demonstrated by in vitro neutrophil adhesion assays under continuous physiological flow conditions, indicating that cell adhesion was a NET- and a CD11b/ICAM-1-dependent process. Finally, d(− lactic acid was demonstrated for the first time to trigger NETosis in a PAD4- and MCT1-dependent manner. Thus, d(− lactic acid-mediated neutrophil activation may contribute to neutrophil-derived pro-inflammatory processes, such as aseptic laminitis and/or polysynovitis in animals suffering acute ruminal acidosis.

Glutamate decarboxylase-dependent acid resistance in Brucella spp.: distribution and contribution to fitness under extremely acidic conditions.

Science.gov (United States)

Damiano, Maria Alessandra; Bastianelli, Daniela; Al Dahouk, Sascha; Köhler, Stephan; Cloeckaert, Axel; De Biase, Daniela; Occhialini, Alessandra

2015-01-01

Brucella is an expanding genus of major zoonotic pathogens, including at least 10 genetically very close species occupying a wide range of niches from soil to wildlife, livestock, and humans. Recently, we have shown that in the new species Brucella microti, the glutamate decarboxylase (Gad)-dependent system (GAD system) contributes to survival at a pH of 2.5 and also to infection in mice by the oral route. In order to study the functionality of the GAD system in the genus Brucella, 47 isolates, representative of all known species and strains of this genus, and 16 strains of the closest neighbor genus, Ochrobactrum, were studied using microbiological, biochemical, and genetic approaches. In agreement with the genome sequences, the GAD system of classical species was not functional, unlike that of most strains of Brucella ceti, Brucella pinnipedialis, and newly described species (B. microti, Brucella inopinata BO1, B. inopinata-like BO2, and Brucella sp. isolated from bullfrogs). In the presence of glutamate, these species were more acid resistant in vitro than classical terrestrial brucellae. Expression in trans of the gad locus from representative Brucella species in the Escherichia coli MG1655 mutant strain lacking the GAD system restored the acid-resistant phenotype. The highly conserved GAD system of the newly described or atypical Brucella species may play an important role in their adaptation to acidic external and host environments. Furthermore, the GAD phenotype was shown to be a useful diagnostic tool to distinguish these latter Brucella strains from Ochrobactrum and from classical terrestrial pathogenic Brucella species, which are GAD negative. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Lysosomal metabolomics reveals V-ATPase- and mTOR-dependent regulation of amino acid efflux from lysosomes.

Science.gov (United States)

Abu-Remaileh, Monther; Wyant, Gregory A; Kim, Choah; Laqtom, Nouf N; Abbasi, Maria; Chan, Sze Ham; Freinkman, Elizaveta; Sabatini, David M

2017-11-10

The lysosome degrades and recycles macromolecules, signals to the cytosol and nucleus, and is implicated in many diseases. Here, we describe a method for the rapid isolation of mammalian lysosomes and use it to quantitatively profile lysosomal metabolites under various cell states. Under nutrient-replete conditions, many lysosomal amino acids are in rapid exchange with those in the cytosol. Loss of lysosomal acidification through inhibition of the vacuolar H + -adenosine triphosphatase (V-ATPase) increased the luminal concentrations of most metabolites but had no effect on those of the majority of essential amino acids. Instead, nutrient starvation regulates the lysosomal concentrations of these amino acids, an effect we traced to regulation of the mechanistic target of rapamycin (mTOR) pathway. Inhibition of mTOR strongly reduced the lysosomal efflux of most essential amino acids, converting the lysosome into a cellular depot for them. These results reveal the dynamic nature of lysosomal metabolites and that V-ATPase- and mTOR-dependent mechanisms exist for controlling lysosomal amino acid efflux. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Temperature-dependent deliquescence relative humidities and water activities using humidity controlled thermogravimetric analysis with application to malonic acid.

Science.gov (United States)

Beyer, Keith D; Schroeder, Jason R; Kissinger, Jared A

2014-04-03

We utilize a new experimental technique, humidity-controlled thermogravimetric analysis (HTGA), to determine temperature-dependent deliquescence relative humidities (DRH) and to determine the equilibrium concentration of a solution at a given temperature and relative humidity. To that end, we have investigated the malonic acid/water system determining the DRH and concentration/RH relationship in the temperature range 303-278 K. Excellent agreement is found with literature values for the DRH of malonic acid as a function of temperature and for the concentration/RH relationship at several temperatures. Thus, we extend the DRH and concentration/RH relationship to a broader temperature range and are using the HTGA experiments to investigate other organic acids.

Dependence of α-helical and β-sheet amino acid propensities on the overall protein fold type

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Fujiwara Kazuo

2012-08-01

Full Text Available Abstract Background A large number of studies have been carried out to obtain amino acid propensities for α-helices and β-sheets. The obtained propensities for α-helices are consistent with each other, and the pair-wise correlation coefficient is frequently high. On the other hand, the β-sheet propensities obtained by several studies differed significantly, indicating that the context significantly affects β-sheet propensity. Results We calculated amino acid propensities for α-helices and β-sheets for 39 and 24 protein folds, respectively, and addressed whether they correlate with the fold. The propensities were also calculated for exposed and buried sites, respectively. Results showed that α-helix propensities do not differ significantly by fold, but β-sheet propensities are diverse and depend on the fold. The propensities calculated for exposed sites and buried sites are similar for α-helix, but such is not the case for the β-sheet propensities. We also found some fold dependence on amino acid frequency in β-strands. Folds with a high Ser, Thr and Asn content at exposed sites in β-strands tend to have a low Leu, Ile, Glu, Lys and Arg content (correlation coefficient = −0.90 and to have flat β-sheets. At buried sites in β-strands, the content of Tyr, Trp, Gln and Ser correlates negatively with the content of Val, Ile and Leu (correlation coefficient = −0.93. "All-β" proteins tend to have a higher content of Tyr, Trp, Gln and Ser, whereas "α/β" proteins tend to have a higher content of Val, Ile and Leu. Conclusions The α-helix propensities are similar for all folds and for exposed and buried residues. However, β-sheet propensities calculated for exposed residues differ from those for buried residues, indicating that the exposed-residue fraction is one of the major factors governing amino acid composition in β-strands. Furthermore, the correlations we detected suggest that amino acid composition is related to folding

Recombinant cells and organisms having persistent nonstandard amino acid dependence and methods of making them

Science.gov (United States)

Church, George M.; Mandell, Daniel J.; Lajoie, Marc J.

2017-12-05

Recombinant cells and recombinant organisms persistently expressing nonstandard amino acids (NSAAs) are provided. Methods of making recombinant cells and recombinant organisms dependent on persistently expressing NSAAs for survival are also provided. These methods may be used to make safe recombinant cells and recombinant organisms and/or to provide a selective pressure to maintain one or more reassigned codon functions in recombinant cells and recombinant organisms.

NMR determination of solvent dependent behavior and XRD structural properties of 4-carboxy phenylboronic acid: A DFT supported study

Science.gov (United States)

Dikmen, Gökhan; Alver, Özgür; Parlak, Cemal

2018-04-01

Solvent dependent structural properties of 4-carboxy phenylboronic acid (4-cpba) were investigated by X-ray diffraction (XRD) and nuclear magnetic resonance (NMR) spectroscopic methods. The molecular structure and geometric parameters were determined by some computational methods such as B3LYP/6-31 + G(3df,p), HF/aug-cc-pvtz and MP2/6-31G(d). Detailed elucidation of the structural and spectroscopic properties of 4-cpba was carried out with 1H, HETCOR and DOSY NMR experiments. Solvent effects on the structural properties were monitored on the changes of 1H NMR spectra by using various solvents and it was observed that 4-cpba shows serious structural preferences depending on the solvent used.

Identification of a novel amino acid racemase from a hyperthermophilic archaeon Pyrococcus horikoshii OT-3 induced by D-amino acids.

Science.gov (United States)

Kawakami, Ryushi; Ohmori, Taketo; Sakuraba, Haruhiko; Ohshima, Toshihisa

2015-08-01

To date, there have been few reports analyzing the amino acid requirement for growth of hyperthermophilic archaea. We here found that the hyperthermophilic archaeon Pyrococcus horikoshii OT-3 requires Thr, Leu, Val, Phe, Tyr, Trp, His and Arg in the medium for growth, and shows slow growth in medium lacking Met or Ile. This largely corresponds to the presence, or absence, of genes related to amino acid biosynthesis in its genome, though there are exceptions. The amino acid requirements were dramatically lost by addition of D-isomers of Met, Leu, Val, allo-Ile, Phe, Tyr, Trp and Arg. Tracer analysis using (14)C-labeled D-Trp showed that D-Trp in the medium was used as a protein component in the cells, suggesting the presence of D-amino acid metabolic enzymes. Pyridoxal 5'-phosphate (PLP)-dependent racemase activity toward Met, Leu and Phe was detected in crude extract of P. horikoshii and was enhanced in cells grown in the medium supplemented with D-amino acids, especially D-allo-Ile. The gene encoding the racemase was narrowed down to one open reading frame on the basis of enzyme purification from P. horikoshii cells, and the recombinant enzyme exhibited PLP-dependent racemase activity toward several amino acids, including Met, Leu and Phe, but not Pro, Asp or Glu. This is the first report showing the presence in a hyperthermophilic archaeon of a PLP-dependent amino acid racemase with broad substrate specificity that is likely responsible for utilization of D-amino acids for growth.

The Peanut (Arachis hypogaea L. Gene AhLPAT2 Increases the Lipid Content of Transgenic Arabidopsis Seeds.

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Silong Chen

Full Text Available Lysophosphatidic acid acyltransferase (LPAT, which converts lysophosphatidic acid (LPA to phosphatidic acid (PA, catalyzes the addition of fatty acyl moieties to the sn-2 position of the LPA glycerol backbone in triacylglycerol (TAG biosynthesis. We recently reported the cloning and temporal-spatial expression of a peanut (Arachis hypogaea AhLPAT2gene, showing that an increase in AhLPAT2 transcript levels was closely correlated with an increase in seed oil levels. However, the function of the enzyme encoded by the AhLPAT2 gene remains unclear. Here, we report that AhLPAT2 transcript levels were consistently higher in the seeds of a high-oil cultivar than in those of a low-oil cultivar across different seed developmental stages. Seed-specific overexpression of AhLPAT2 in Arabidopsis results in a higher percentage of oil in the seeds and greater-than-average seed weight in the transgenic plants compared with the wild-type plants, leading to a significant increase in total oil yield per plant. The total fatty acid (FA content and the proportion of unsaturated FAs also increased. In the developing siliques of AhLPAT2-overexpressing plants, the expression levels of genes encoding crucial enzymes involved in de novo FA synthesis, acetyl-CoA subunit (AtBCCP2 and acyl carrier protein 1 (AtACP1 were elevated. AhLPAT2 overexpression also promoted the expression of several key genes related to TAG assembly, sucrose metabolism, and glycolysis. These results demonstrate that the expression of AhLPAT2 plays an important role in glycerolipid production in peanuts.

Size-Dependent Affinity of Glycine and Its Short Oligomers to Pyrite Surface: A Model for Prebiotic Accumulation of Amino Acid Oligomers on a Mineral Surface

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Afrin, Rehana; Ganbaatar, Narangerel; Aono, Masashi; Cleaves, H. James; Yano, Taka-aki; Hara, Masahiko

2018-01-01

The interaction strength of progressively longer oligomers of glycine, (Gly), di-Gly, tri-Gly, and penta-Gly, with a natural pyrite surface was directly measured using the force mode of an atomic force microscope (AFM). In recent years, selective activation of abiotically formed amino acids on mineral surfaces, especially that of pyrite, has been proposed as an important step in many origins of life scenarios. To investigate such notions, we used AFM-based force measurements to probe possible non-covalent interactions between pyrite and amino acids, starting from the simplest amino acid, Gly. Although Gly itself interacted with the pyrite surface only weakly, progressively larger unbinding forces and binding frequencies were obtained using oligomers from di-Gly to penta-Gly. In addition to an expected increase of the configurational entropy and size-dependent van der Waals force, the increasing number of polar peptide bonds, among others, may be responsible for this observation. The effect of chain length was also investigated by performing similar experiments using l-lysine vs. poly-l-lysine (PLL), and l-glutamic acid vs. poly-l-glutamic acid. The results suggest that longer oligomers/polymers of amino acids can be preferentially adsorbed on pyrite surfaces. PMID:29370126

Major involvement of Na(+) -dependent multivitamin transporter (SLC5A6/SMVT) in uptake of biotin and pantothenic acid by human brain capillary endothelial cells.

Science.gov (United States)

Uchida, Yasuo; Ito, Katsuaki; Ohtsuki, Sumio; Kubo, Yoshiyuki; Suzuki, Takashi; Terasaki, Tetsuya

2015-07-01

The purpose of this study was to clarify the expression of Na(+) -dependent multivitamin transporter (SLC5A6/SMVT) and its contribution to the supply of biotin and pantothenic acid to the human brain via the blood-brain barrier. DNA microarray and immunohistochemical analyses confirmed that SLC5A6 is expressed in microvessels of human brain. The absolute expression levels of SLC5A6 protein in isolated human and monkey brain microvessels were 1.19 and 0.597 fmol/μg protein, respectively, as determined by a quantitative targeted absolute proteomics technique. Using an antibody-free method established by Kubo et al. (2015), we found that SLC5A6 was preferentially localized at the luminal membrane of brain capillary endothelium. Knock-down analysis using SLC5A6 siRNA showed that SLC5A6 accounts for 88.7% and 98.6% of total [(3) H]biotin and [(3) H]pantothenic acid uptakes, respectively, by human cerebral microvascular endothelial cell line hCMEC/D3. SLC5A6-mediated transport in hCMEC/D3 was markedly inhibited not only by biotin and pantothenic acid, but also by prostaglandin E2, lipoic acid, docosahexaenoic acid, indomethacin, ketoprofen, diclofenac, ibuprofen, phenylbutazone, and flurbiprofen. This study is the first to confirm expression of SLC5A6 in human brain microvessels and to provide evidence that SLC5A6 is a major contributor to luminal uptake of biotin and pantothenic acid at the human blood-brain barrier. In humans, it was unclear (not concluded) about what transport system at the blood-brain barrier (BBB) is responsible for the brain uptakes of two vitamins, biotin and pantothenic acid, which are necessary for brain proper function. This study clarified for the first time that the solute carrier 5A6/Na(+) -dependent multivitamin transporter SLC5A6/SMVT is responsible for the supplies of biotin and pantothenic acid into brain across the BBB in humans. DHA, docosahexaenoic acid; NSAID, non-steroidal anti-inflammatory drug; PGE2, prostaglandin E2. © 2015

Docosahexaenoic acid and other fatty acids induce a decrease in pHi in Jurkat T-cells

OpenAIRE

Aires, Virginie; Hichami, Aziz; Moutairou, Kabirou; Khan, Naim Akhtar

2003-01-01

Docosahexaenoic acid (DHA) induced rapid (t1/2=33 s) and dose-dependent decreases in pHi in BCECF-loaded human (Jurkat) T-cells. Addition of 5-(N,N-dimethyl)-amiloride, an inhibitor of Na+/H+ exchanger, prolonged DHA-induced acidification as a function of time, indicating that the exchanger is implicated in pHi recovery.Other fatty acids like oleic acid, arachidonic acid, eicosapentaenoic acid, but not palmitic acid, also induced a fall in pHi in these cells.To assess the role of calcium in t...

Dynamin-dependent amino acid endocytosis activates mechanistic target of rapamycin complex 1 (mTORC1).

Science.gov (United States)

Shibutani, Shusaku; Okazaki, Hana; Iwata, Hiroyuki

2017-11-03

The mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of protein synthesis and potential target for modifying cellular metabolism in various conditions, including cancer and aging. mTORC1 activity is tightly regulated by the availability of extracellular amino acids, and previous studies have revealed that amino acids in the extracellular fluid are transported to the lysosomal lumen. There, amino acids induce recruitment of cytoplasmic mTORC1 to the lysosome by the Rag GTPases, followed by mTORC1 activation by the small GTPase Ras homolog enriched in brain (Rheb). However, how the extracellular amino acids reach the lysosomal lumen and activate mTORC1 remains unclear. Here, we show that amino acid uptake by dynamin-dependent endocytosis plays a critical role in mTORC1 activation. We found that mTORC1 is inactivated when endocytosis is inhibited by overexpression of a dominant-negative form of dynamin 2 or by pharmacological inhibition of dynamin or clathrin. Consistently, the recruitment of mTORC1 to the lysosome was suppressed by the dynamin inhibition. The activity and lysosomal recruitment of mTORC1 were rescued by increasing intracellular amino acids via cycloheximide exposure or by Rag overexpression, indicating that amino acid deprivation is the main cause of mTORC1 inactivation via the dynamin inhibition. We further show that endocytosis inhibition does not induce autophagy even though mTORC1 inactivation is known to strongly induce autophagy. These findings open new perspectives for the use of endocytosis inhibitors as potential agents that can effectively inhibit nutrient utilization and shut down the upstream signals that activate mTORC1. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Transforming growth factor β-induced superficial zone protein accumulation in the surface zone of articular cartilage is dependent on the cytoskeleton.

Science.gov (United States)

McNary, Sean M; Athanasiou, Kyriacos A; Reddi, A Hari

2014-03-01

), and Y27632 (Rho effector Rho Kinase). On the other hand, lysophosphatidic acid, an upstream activator of Rho, increased SZP synthesis in response to TGFβ1. These results suggest that SZP production is dependent on the functional cytoskeleton, and Rho GTPases contribute to SZP accumulation by modulating the actions of TGFβ.

Antimicrobial medium- and long-chain free fatty acids prevent PrfA-dependent activation of virulence genes in Listeria monocytogenes.

Science.gov (United States)

Sternkopf Lillebæk, Eva Maria; Lambert Nielsen, Stine; Scheel Thomasen, Rikke; Færgeman, Nils J; Kallipolitis, Birgitte H

The foodborne pathogen Listeria monocytogenes is the causative agent of the invasive disease listeriosis. Infection by L. monocytogenes involves bacterial crossing of the intestinal barrier and intracellular replication in a variety of host cells. The PrfA protein is the master regulator of virulence factors required for bacterial entry, intracellular replication and cell-to-cell spread. PrfA-dependent activation of virulence genes occurs primarily in the blood and during intracellular infection. In contrast, PrfA does not play a significant role in regulation of virulence gene expression in the intestinal environment. In the gastrointestinal phase of infection, the bacterium encounters a variety of antimicrobial agents, including medium- and long-chain free fatty acids that are commonly found in our diet and as active components of bile. Here we show that subinhibitory concentrations of specific antimicrobial free fatty acids act to downregulate transcription of PrfA-activated virulence genes. Interestingly, the inhibitory effect is also evident in cells encoding a constitutively active variant of PrfA. Collectively, our data suggest that antimicrobial medium- and long-chain free fatty acids may act as signals to prevent PrfA-mediated activation of virulence genes in environments where PrfA activation is not required, such as in food and the gastrointestinal tract. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Regulation of phenylacetic acid uptake is sigma54 dependent in Pseudomonas putida CA-3.

LENUS (Irish Health Repository)

O' Leary, Niall D

2011-10-13

primer extension analysis. Comparative analyses of genomes encoding phenylacetyl CoA, (PACoA), catabolic operons identified a common association among styrene degradation linked PACoA catabolons in Pseudomonas species studied to date. Conclusions In summary, this is the first study to report RpoN dependent transcriptional activation of the PACoA catabolon paaL gene, encoding a transport protein essential for phenylacetic acid utilisation in P. putida CA-3. Bioinformatic analysis is provided to suggest this regulatory link may be common among styrene degrading Pseudomonads.

Hydrocortisone selectively inhibits IgE-dependent arachidonic acid release from rat peritoneal mast cells

International Nuclear Information System (INIS)

Heiman, A.S.; Crews, F.T.

1984-01-01

Purified rat mst cells were used to study the effects of antiinflammatory steroids on the release of [1-14C]-arachidonic acid ([1-14C]AA) and metabolites. Mast cell were incubated overnight with glucocorticoids, [1-14C]AA incorporated into cellular phospholipids and the release of [1-14C]AA, and metabolites determined using a variety of secretagogues. Release of [1-14C]AA and metabolites by concanavalin A, the antigen ovalbumin and anti-immunoglobulin E antibody was markedly reduced by glucocorticoid treatment. Neither the total incorporation of [1-14C]AA nor the distribution into phospholipids was altered by hydrocortisone pretreatment. Glucocorticoid pretreatment did not alter [1-14C]AA release stimulated by somatostatin, compound 48/80, or the calcium ionophore, A23187. These data indicate that antiinflammatory steroids selectively inhibit immunoglobulin dependent release of arachidonic acid from rat mast cells. These findings question the role of lipomodulin and macrocortin as general phospholipase inhibitors and suggest that they may be restricted to immunoglobulin stimuli

Dependence of the metabolic fecal amino acids on the amino acid content of the feed. 2

International Nuclear Information System (INIS)

Krawielitzki, K.; Schadereit, R.; Voelker, T.; Reichel, K.

1982-01-01

In an experiment with 20 15 N-labelled growing rats the excretion of amino acids as well as of metabolic fecal amino acids were investigated after feeding of soybean oil meal as sole protein source. A low, yet statistically significant increase of the excretion of amino acids and metabolic fecal amino acids was ascertained in accordance with a growing quota of soybean oil meal in the ration. The true digestibility of amino acids ascertained according to conventional methods is above 90% and, under consideration of the increase of metabolic fecal amino acids, on the average increases by 3.5 digestibility units (1.4 to 6.2). (author)

Hyaluronic acid-coated chitosan nanoparticles: molecular weight-dependent effects on morphology and hyaluronic acid presentation.

Science.gov (United States)

Almalik, Abdulaziz; Donno, Roberto; Cadman, Christopher J; Cellesi, Francesco; Day, Philip J; Tirelli, Nicola

2013-12-28

Chitosan nanoparticles are popular carriers for the delivery of macromolecular payloads, e.g. nucleic acids. In this study, nanoparticles were prepared via complexation with triphosphate (TPP) anions and were successively coated with hyaluronic acid (HA). Key variables of the preparative process (e.g. chitosan and HA molecular weight) were optimised in view of the maximisation of loading with DNA, of the Zeta potential and of the dimensional stability, and the resulting particles showed excellent storage stability. We have focused on the influence of chitosan molecular weight on nanoparticle properties. Larger molecular weight increased their porosity (=decreased cross-link density), and this caused also larger dimensional changes in response to variations in osmotic pressure or upon drying. The dependency of nanoparticle porosity on chitosan molecular weight had a profound effect on the adsorption of HA on the nanoparticles; HA was apparently able to penetrate deeply into the more porous high molecular weight (684 kDa) chitosan nanoparticles, while it formed a corona around those composed of more densely cross-linked low molecular weight (25 kDa) chitosan. Atomic Force Microscopy (AFM) allowed not only to highlight the presence of this corona, but also to estimate its apparent thickness to about 20-30 nm (in a dry state). The different morphology has a significant effect on the way HA is presented to biomolecules, and this has specific relevance in relation to interactions with HA receptors (e.g. CD44) that influence kinetics and mechanism of nanoparticle uptake. Finally, it is worth to mention that chitosan molecular weight did not appear to greatly affect the efficiency of nanoparticle loading with DNA, but significantly influenced its chitosanase-triggered release, with high molecular chitosan nanoparticles seemingly more prone to degradation by this enzyme. © 2013.

Gestational age dependent content, composition and intrauterine accretion rates of fatty acids in fetal white adipose tissue

NARCIS (Netherlands)

Kuipers, Remko S.; Luxwolda, Martine F.; Offringa, Pieter J.; Martini, Ingrid A.; Boersma, E. Rudy; Dijck-Brouwer, D. A. Janneke; Muskiet, Frits A. J.

2012-01-01

Background: Little is known about the gestational age (GA) dependent content, composition and intrauterine accretion rates of fatty acids (FA) in fetal white adipose tissue (WAT). Objective & design: To acquire this information, we collected abdominal subcutaneous WAT samples from 40 preterm and

Dose-dependent antiinflammatory effect of ursodeoxycholic acid in experimental colitis.

Science.gov (United States)

Martínez-Moya, Patricia; Romero-Calvo, Isabel; Requena, Pilar; Hernández-Chirlaque, Cristina; Aranda, Carlos J; González, Raquel; Zarzuelo, Antonio; Suárez, María Dolores; Martínez-Augustin, Olga; Marín, José Juan G; de Medina, Fermín Sánchez

2013-02-01

The denomination of inflammatory bowel disease comprises a group of chronic inflammatory diseases of the digestive tract, ulcerative colitis and Crohn's disease being the most important conditions. Bile acids may play a role both in etiology and pharmacology of this disease. Thus, although deoxycholic acid is regarded as a proinflammatory agent ursodeoxycholic acid, which is currently being used to treat certain types of cholestasis and primary biliary cirrhosis, because of their choleretic, cytoprotective and immunomodulatory effects, it has been reported to exert an anti-inflammatory activity. We aim to confirm and characterize the intestinal antiinflammatory activity of ursodeoxycholic acid. The experimental model trinitrobenzenesulfonic acid (TNBS)-induced colitis in rats has been used. Animal status was characterized by a number of macroscopic and biochemical parameters. Oral administration of ursodeoxycholic acid was able to ameliorate experimental colonic inflammation. This occurred only at a relatively high dose (50 mg/kg day), whereas ursodeoxycholic acid was without significant effect at doses of 10 and 25 mg/kg day. The therapeutic effect was evidenced, among others, by a higher body weight recovery, a diminished affected to total mucosal area and lower alkaline phosphatase activity in treated vs. control (TNBS treated) animals. These results indicate that, at the appropriate dose, ursodeoxycholic acid is a potentially useful drug to reduce intestinal inflammation and could be envisaged to be incorporated in the treatment of inflammatory bowel diseases. Copyright © 2012 Elsevier B.V. All rights reserved.

Equine arteritis virus is delivered to an acidic compartment of host cells via clathrin-dependent endocytosis

International Nuclear Information System (INIS)

Nitschke, Matthias; Korte, Thomas; Tielesch, Claudia; Ter-Avetisyan, Gohar; Tuennemann, Gisela; Cardoso, M. Cristina; Veit, Michael; Herrmann, Andreas

2008-01-01

Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae. Infection by EAV requires the release of the viral genome by fusion with the respective target membrane of the host cell. We have investigated the entry pathway of EAV into Baby Hamster Kindey cells (BHK). Infection of cells assessed by the plaque reduction assay was strongly inhibited by substances which interfere with clathrin-dependent endocytosis and by lysosomotropic compounds. Furthermore, infection of BHK cells was suppressed when clathrin-dependent endocytosis was inhibited by expression of antisense RNA of the clathrin-heavy chain before infection. These results strongly suggest that EAV is taken up via clathrin-dependent endocytosis and is delivered to acidic endosomal compartments

Intermittent apnea elicits inactivity-induced phrenic motor facilitation via a retinoic acid- and protein synthesis-dependent pathway.

Science.gov (United States)

Baertsch, Nathan A; Baker, Tracy L

2017-11-01

Respiratory motoneuron pools must provide rhythmic inspiratory drive that is robust and reliable, yet dynamic enough to respond to respiratory challenges. One form of plasticity that is hypothesized to contribute to motor output stability by sensing and responding to inadequate respiratory neural activity is inactivity-induced phrenic motor facilitation (iPMF), an increase in inspiratory output triggered by a reduction in phrenic synaptic inputs. Evidence suggests that mechanisms giving rise to iPMF differ depending on the pattern of reduced respiratory neural activity (i.e., neural apnea). A prolonged neural apnea elicits iPMF via a spinal TNF-α-induced increase in atypical PKC activity, but little is known regarding mechanisms that elicit iPMF following intermittent neural apnea. We tested the hypothesis that iPMF triggered by intermittent neural apnea requires retinoic acid and protein synthesis. Phrenic nerve activity was recorded in urethane-anesthetized and -ventilated rats treated intrathecally with an inhibitor of retinoic acid synthesis (4-diethlyaminobenzaldehyde, DEAB), a protein synthesis inhibitor (emetine), or vehicle (artificial cerebrospinal fluid) before intermittent (5 episodes, ~1.25 min each) or prolonged (30 min) neural apnea. Both DEAB and emetine abolished iPMF elicited by intermittent neural apnea but had no effect on iPMF elicited by a prolonged neural apnea. Thus different patterns of reduced respiratory neural activity elicit phenotypically similar iPMF via distinct spinal mechanisms. Understanding mechanisms that allow respiratory motoneurons to dynamically tune their output may have important implications in the context of respiratory control disorders that involve varied patterns of reduced respiratory neural activity, such as central sleep apnea and spinal cord injury. NEW & NOTEWORTHY We identify spinal retinoic acid and protein synthesis as critical components in the cellular cascade whereby repetitive reductions in respiratory

Coulometric bioelectrocatalytic reactions based on NAD-dependent dehydrogenases in tricarboxylic acid cycle

Energy Technology Data Exchange (ETDEWEB)

Fukuda, Jun [Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502 (Japan); Tsujimura, Seiya [Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502 (Japan)], E-mail: seiya@kais.kyoto-u.ac.jp; Kano, Kenji [Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502 (Japan)], E-mail: kkano@kais.kyoto-u.ac.jp

2008-12-30

This paper describes the characterization of mediated electro-enzymatic electrolysis systems based on NAD-dependent dehydrogenase reactions in the tricarboxylic acid (TCA) cycle. A micro-bulk electrolysis system with a carbon felt anode immersed in an electrolysis solution with a value of about 10 {mu}L was constructed for coulometric analysis of the substrate oxidation. Diaphorase (DI) was used to couple the NAD-dependent dehydrogenase reaction with the anode reaction of a suitable redox mediator. We focused on three types of NAD-dependant dehydrogenases reactions in this research: (1) isocitrate oxidation, in which the standard Gibbs energy change ({delta}G{sup o}') is negative; (2) {alpha}-ketoglutarate oxidation, which involves an electrochemically active coenzyme A (CoA); and (3) malate oxidation, which is thermodynamically unfavorable because of a large positive {delta}G{sup o}' value. The complete electrolysis of isocitrate was easily achieved, supporting the effective re-oxidation of NADH in the diaphorase-catalyzed electrochemical reaction. CoA was unfavorably oxidized at the electrodes in the presence of some mediators. The electrocatalytic oxidation of CoA was suppressed and the quantitative electrochemical oxidation of {alpha}-ketoglutarate was achieved by selecting a suitable mediator with negligibly slow electron transfer kinetics with CoA. The uphill malate oxidation was susceptible to product inhibition in the bioelectrochemical system, although NADH generated in the malate dehydrogenase reaction was immediately oxidized in the electrochemical system. The inhibition was successfully suppressed by linking citrate synthase to quench oxaloacetate and to make the total {delta}G{sup o}' value negative.

Coulometric bioelectrocatalytic reactions based on NAD-dependent dehydrogenases in tricarboxylic acid cycle

International Nuclear Information System (INIS)

Fukuda, Jun; Tsujimura, Seiya; Kano, Kenji

2008-01-01

This paper describes the characterization of mediated electro-enzymatic electrolysis systems based on NAD-dependent dehydrogenase reactions in the tricarboxylic acid (TCA) cycle. A micro-bulk electrolysis system with a carbon felt anode immersed in an electrolysis solution with a value of about 10 μL was constructed for coulometric analysis of the substrate oxidation. Diaphorase (DI) was used to couple the NAD-dependent dehydrogenase reaction with the anode reaction of a suitable redox mediator. We focused on three types of NAD-dependant dehydrogenases reactions in this research: (1) isocitrate oxidation, in which the standard Gibbs energy change (ΔG o ') is negative; (2) α-ketoglutarate oxidation, which involves an electrochemically active coenzyme A (CoA); and (3) malate oxidation, which is thermodynamically unfavorable because of a large positive ΔG o ' value. The complete electrolysis of isocitrate was easily achieved, supporting the effective re-oxidation of NADH in the diaphorase-catalyzed electrochemical reaction. CoA was unfavorably oxidized at the electrodes in the presence of some mediators. The electrocatalytic oxidation of CoA was suppressed and the quantitative electrochemical oxidation of α-ketoglutarate was achieved by selecting a suitable mediator with negligibly slow electron transfer kinetics with CoA. The uphill malate oxidation was susceptible to product inhibition in the bioelectrochemical system, although NADH generated in the malate dehydrogenase reaction was immediately oxidized in the electrochemical system. The inhibition was successfully suppressed by linking citrate synthase to quench oxaloacetate and to make the total ΔG o ' value negative

Site-specific antibodies distinguish single amino acid substitutions in position 57 in HLA-DQ beta-chain alleles associated with insulin-dependent diabetes

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Atar, D; Dyrberg, T; Michelsen, Birgitte

1989-01-01

The HLA-DQ beta-chain gene shows a close association with susceptibility or resistance to autoimmune insulin-dependent diabetes mellitus (IDDM) and it has been suggested that the amino acid in position 57 may be of pathogenetic importance. To study the expression of the IDDM associated HLA-DQ beta......-chain alleles, we immunized rabbits with 12 to 13 amino acid long peptides representing HLA-DQw7 and -DQw8 allelic sequences, differing only by one amino acid in position 57 being aspartic acid (Asp) and alanine (Ala), respectively. Immunoblot analysis of lymphoblastoid cells showed that several antisera...

A nucleic acid dependent chemical photocatalysis in live human cells

DEFF Research Database (Denmark)

Arian, Dumitru; Cló, Emiliano; Gothelf, Kurt V

2010-01-01

Only two nucleic acid directed chemical reactions that are compatible with live cells have been reported to date. Neither of these processes generate toxic species from nontoxic starting materials. Reactions of the latter type could be applied as gene-specific drugs, for example, in the treatment...

Effect of detergents, trypsin, and bivalent metal ions on interfacial activation and functioning of phospholipase D.

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Madyarov, Sh R

2014-07-01

The effects of detergents, trypsin, and bivalent metal ions on production of phosphatidic and lysophosphatidic acids by the action of phospholipase D (PLD) on lecithin and lysolecithin were studied. It was found that these reaction products and dodecyl sulfate ions activate PLD, whereas other anionic detergents are less effective. A protective effect of the functioning enzyme against its hydrolytic inactivation by trypsin was found. Bivalent metal ions can be arranged in the following sequence by their ability to activate PLD in the hydrolysis of lecithin and lysolecithin: Ca2+>Sr2+>Ba2+>Mg2+. These results are considered in relation to a proposed mechanism of activation and functioning of PLD with the participation of clusters of phosphatidates and lysophosphatidates. Such Me2+-induced formation of rafts or microdomains from the products of hydrolysis of phospholipids can rationalize not only PLD activation and self-regulation, but also the action of this mechanism on other components and properties of biomembranes. PLD and other lipolytic enzymes can be classified as lateral vector enzymes.

12(R)-hydroxyicosatetraenoic acid: a cytochrome P450-dependent arachidonate metabolite that inhibits Na/sup +/, K/sup +/-ATPase in the cornea

Energy Technology Data Exchange (ETDEWEB)

Schwartzman, M.L.; Balazy, M.; Masferrer, J.; Abraham, N.G.; McGiff, J.C.; Murphy, R.C.

1987-11-01

When corneal microsomes were incubated with arachidonic acid in the presence of an NADPH-generating system, four polar metabolites (compounds A-D) were formed. Synthesis of these metabolites could be inhibited by carbon monoxide, SKF 525A, and anti-cytochrome c reductase antibodies. One of the metabolites, compound C, was found to inhibit partially purified Na/sup +/, K/sup +/-ATPase from the corneal epithelium in a dose-dependent manner. After compound C was purified by TLC and HPLC, it was found to have a UV absorption spectrum with a maximum absorbance at 236 nm suggesting the presence of a conjugated diene. Mass spectrometric analysis using positive- and negative-ionization modes was carried out on derivatized compound C. Abundant fragment ions were consistent with compound C being a monooxygenated derivative of arachidonic acid with a hydroxyl substituent at carbon-12 of the icosanoid backbone; all deuterium atoms from (/sup 2/H/sub 8/)arachidonate were retained in the structure. Compound C was characterized as a 12-hydroxyicosatetraenoic acid. However, only 12(R) isomer was found to be an inhibitor of the Na/sup +/, K/sup +/-ATPase from the corneal epithelium, suggesting that the biologically active compound C was 12(R)-hydroxyy-5,8,10,14-icosatetraenoic acid. Such an inhibitor of Na/sup +/, K/sup +/-ATPase synthesized in the cornea may have an important role in regulating ocular transparency and aqueous human secretion.

Identification of a Transcription Factor Controlling pH-Dependent Organic Acid Response in Aspergillus niger

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Poulsen, Lars; Andersen, Mikael Rørdam; Lantz, Anna Eliasson

2012-01-01

exhibiting an oxalate overproducing phenotype were identified. The yield of oxalate was increased up to 158% compared to the wild type and the corresponding transcription factor was therefore entitled Oxalic Acid repression Factor, OafA. Detailed physiological characterization of one of the ΔoafA mutants......, compared to the wild type, showed that both strains produced substantial amounts of gluconic acid, but the mutant strain was more efficient in re-uptake of gluconic acid and converting it to oxalic acid, particularly at high pH (pH 5.0). Transcriptional profiles showed that 241 genes were differentially......Acid formation in Aspergillus niger is known to be subjected to tight regulation, and the acid production profiles are fine-tuned to respond to the ambient pH. Based on transcriptome data, putative trans-acting pH responding transcription factors were listed and through knock out studies, mutants...

Arginine-dependent acid-resistance pathway in Shigella boydii

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Ability to survive the low pH of the human stomach is considered be an important virulent determinant. Acid tolerance of Shigella boydii 18 CDPH, the strain implicated in an outbreak may have played an important role in surviving the acidic food (bean salad). The strain was capable of inducing arg...

Insulin-dependent signaling: regulation by amino acids and energy

NARCIS (Netherlands)

Meijer, A. J.

2004-01-01

Recent research has indicated that amino acids stimulate a signal-transduction pathway that is also used by insulin. Moreover, for insulin to exert its anabolic and anticatabolic effects on protein, there is an absolute requirement for amino acids. This signaling pathway becomes inhibited by

A review of acidity generation and consumption in acidic coal mine lakes and their watersheds.

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Blodau, Christian

2006-10-01

Lakes developing in former coal mine pits are often characterized by high concentrations of sulfate and iron and low pH. The review focuses on the causes for and fate of acidity in these lakes and their watersheds. Acidification is primarily caused by the generation of ferrous iron bearing and mineralized groundwater, transport through the groundwater-surface water interface, and subsequent iron oxidation and precipitation. Rates of acidity generation in mine tailings and dumps, and surface water are often similar (1 to >10 mol m(-2) yr(-1)). Weathering processes, however, often suffice to buffer groundwaters to only moderately acidic or neutral pH, depending on the suite of minerals present. In mine lakes, the acidity balance is further influenced by proton release from transformation of metastable iron hydroxysulfate minerals to goethite, and proton and ferrous iron sequestration by burial of iron sulfides and carbonates in sediments. These processes mostly cannot compensate acidity loading from the watershed, though. A master variable for almost all processes is the pH: rates of pyrite oxidation, ferrous iron oxidation, mineral dissolution, iron precipitation, iron hydroxide transformation, and iron and sulfate reduction are strongly pH dependent. While the principle mechanism of acidity generation and consumption and several controls are mostly understood, this cannot be said about the fate of acidity on larger spatial and temporal scales. Little is also known about critical loads and the internal regulation of biogeochemical iron, sulfur, and carbon cycling in acidic mine lakes.

A review of acidity generation and consumption in acidic coal mine lakes and their watersheds

International Nuclear Information System (INIS)

Blodau, Christian

2006-01-01

Lakes developing in former coal mine pits are often characterized by high concentrations of sulfate and iron and low pH. The review focuses on the causes for and fate of acidity in these lakes and their watersheds. Acidification is primarily caused by the generation of ferrous iron bearing and mineralized groundwater, transport through the groundwater-surface water interface, and subsequent iron oxidation and precipitation. Rates of acidity generation in mine tailings and dumps, and surface water are often similar (1 to >10 mol m -2 yr -1 ). Weathering processes, however, often suffice to buffer groundwaters to only moderately acidic or neutral pH, depending on the suite of minerals present. In mine lakes, the acidity balance is further influenced by proton release from transformation of metastable iron hydroxysulfate minerals to goethite, and proton and ferrous iron sequestration by burial of iron sulfides and carbonates in sediments. These processes mostly cannot compensate acidity loading from the watershed, though. A master variable for almost all processes is the pH: rates of pyrite oxidation, ferrous iron oxidation, mineral dissolution, iron precipitation, iron hydroxide transformation, and iron and sulfate reduction are strongly pH dependent. While the principle mechanism of acidity generation and consumption and several controls are mostly understood, this cannot be said about the fate of acidity on larger spatial and temporal scales. Little is also known about critical loads and the internal regulation of biogeochemical iron, sulfur, and carbon cycling in acidic mine lakes. (author)

Alterations to mitochondrial fatty-acid use in skeletal muscle after chronic exposure to hypoxia depend on metabolic phenotype.

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Malgoyre, Alexandra; Chabert, Clovis; Tonini, Julia; Koulmann, Nathalie; Bigard, Xavier; Sanchez, Hervé

2017-03-01

We investigated the effects of chronic hypoxia on the maximal use of and sensitivity of mitochondria to different substrates in rat slow-oxidative (soleus, SOL) and fast-glycolytic (extensor digitorum longus, EDL) muscles. We studied mitochondrial respiration in situ in permeabilized myofibers, using pyruvate, octanoate, palmitoyl-carnitine (PC), or palmitoyl-coenzyme A (PCoA). The hypophagia induced by hypoxia may also alter metabolism. Therefore, we used a group of pair-fed rats (reproducing the same caloric restriction, as observed in hypoxic animals), in addition to the normoxic control fed ad libitum. The resting respiratory exchange ratio decreased after 21 days of exposure to hypobaric hypoxia (simulated elevation of 5,500 m). The respiration supported by pyruvate and octanoate were unaffected. In contrast, the maximal oxidative respiratory rate for PCoA, the transport of which depends on carnitine palmitoyltransferase 1 (CPT-1), decreased in the rapid-glycolytic EDL and increased in the slow-oxidative SOL, although hypoxia improved affinity for this substrate in both muscle types. PC and PCoA were oxidized similarly in normoxic EDL, whereas chronic hypoxia limited transport at the CPT-1 step in this muscle. The effects of hypoxia were mediated by caloric restriction in the SOL and by hypoxia itself in the EDL. We conclude that improvements in mitochondrial affinity for PCoA, a physiological long-chain fatty acid, would facilitate fatty-acid use at rest after chronic hypoxia independently of quantitative alterations of mitochondria. Conversely, decreasing the maximal oxidation of PCoA in fast-glycolytic muscles would limit fatty-acid use during exercise. NEW & NOTEWORTHY Affinity for low concentrations of long-chain fatty acids (LCFA) in mitochondria skeletal muscles increases after chronic hypoxia. Combined with a lower respiratory exchange ratio, this suggests facility for fatty acid utilization at rest. This fuel preference is related to caloric

Stability of sonicated aqueous suspensions of phospholipids under air.

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Almog, R; Forward, R; Samsonoff, C

1991-12-01

The stability of phospholipids in liposomal aqueous suspension against oxidative degradation in air was investigated using spectrophotometric indices, glutathione peroxidase reactivity and thin layer chromatography. Zwitterionic phospholipid was found to be susceptible to degradation via oxidation of polyunsaturated hydrocarbon chains and ester hydrolysis, producing oxidized lysophosphatide and free fatty acid derivatives. These products were characterized as hydroperoxides based on their reactivity with the selenium-dependent glutathione peroxidase isolated from human erythrocytes. Lecithin in Tris buffer was more resistant to hydrolysis than in water. The sonication of 8.0 mM of soybean phosphatidylcholine (SB-PC) suspension in 0.1 M Tris (pH 7.5) in the presence of air produced relatively high concentration of conjugated diene hydroperoxide, but a small amount of hydrolyzed products. Anionic phospholipids, such as egg-phosphatidylglycerol (egg-PG), demonstrated higher resistance to air oxidation than the zwitterionic lecithin, but its oxidation was promoted by sonication.

Formulation and characterization of poly(propylacrylic acid)/poly(lactic-co-glycolic acid) blend microparticles for pH-dependent membrane disruption and cytosolic delivery.

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Fernando, Lawrence P; Lewis, Jamal S; Evans, Brian C; Duvall, Craig L; Keselowsky, Benjamin G

2018-04-01

Poly(lactic-co-glycolic acid) (PLGA) is widely used as a vehicle for delivery of pharmaceutically relevant payloads. PLGA is readily fabricated as a nano- or microparticle (MP) matrix to load both hydrophobic and hydrophilic small molecular drugs as well as biomacromolecules such as nucleic acids and proteins. However, targeting such payloads to the cell cytosol is often limited by MP entrapment and degradation within acidic endolysosomes. Poly(propylacrylic acid) (PPAA) is a polyelectrolyte polymer with the membrane disruptive capability triggered at low pH. PPAA has been previously formulated in various carrier configurations to enable cytosolic payload delivery, but requires sophisticated carrier design. Taking advantage of PPAA functionality, we have incorporated PPAA into PLGA MPs as a simple polymer mixture to enhance cytosolic delivery of PLGA-encapsulated payloads. Rhodamine loaded PLGA and PPAA/PLGA blend MPs were prepared by a modified nanoprecipitation method. Incorporation of PPAA into PLGA MPs had little to no effect on the size, shape, or loading efficiency, and evidenced no toxicity in Chinese hamster ovary epithelial cells. Notably, incorporation of PPAA into PLGA MPs enabled pH-dependent membrane disruption in a hemolysis assay, and a three-fold increased endosomal escape and cytosolic delivery in dendritic cells after 2 h of MP uptake. These results demonstrate that a simple PLGA/PPAA polymer blend is readily fabricated into composite MPs, enabling cytosolic delivery of an encapsulated payload. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1022-1033, 2018. © 2017 Wiley Periodicals, Inc.

RNASEK is required for internalization of diverse acid-dependent viruses.

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Hackett, Brent A; Yasunaga, Ari; Panda, Debasis; Tartell, Michael A; Hopkins, Kaycie C; Hensley, Scott E; Cherry, Sara

2015-06-23

Viruses must gain entry into cells to establish infection. In general, viruses enter either at the plasma membrane or from intracellular endosomal compartments. Viruses that use endosomal pathways are dependent on the cellular factors that control this process; however, these genes have proven to be essential for endogenous cargo uptake, and thus are of limited value for therapeutic intervention. The identification of genes that are selectively required for viral uptake would make appealing drug targets, as their inhibition would block an early step in the life cycle of diverse viruses. At this time, we lack pan-antiviral therapeutics, in part because of our lack of knowledge of such cellular factors. RNAi screening has begun to reveal previously unknown genes that play roles in viral infection. We identified dRNASEK in two genome-wide RNAi screens performed in Drosophila cells against West Nile and Rift Valley Fever viruses. Here we found that ribonuclease kappa (RNASEK) is essential for the infection of human cells by divergent and unrelated positive- and negative-strand-enveloped viruses from the Flaviviridae, Togaviridae, Bunyaviridae, and Orthomyxoviridae families that all enter cells from endosomal compartments. In contrast, RNASEK was dispensable for viruses, including parainfluenza virus 5 and Coxsackie B virus, that enter at the plasma membrane. RNASEK is dispensable for attachment but is required for uptake of these acid-dependent viruses. Furthermore, this requirement appears specific, as general endocytic uptake of transferrin is unaffected in RNASEK-depleted cells. Therefore, RNASEK is a potential host cell Achilles' heel for viral infection.

The General Amino Acid Permease FfGap1 of Fusarium fujikuroi Is Sorted to the Vacuole in a Nitrogen-Dependent, but Npr1 Kinase-Independent Manner.

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Andreas Pfannmüller

Full Text Available The rice pathogenic fungus Fusarium fujikuroi is well known for the production of a broad spectrum of secondary metabolites (SMs such as gibberellic acids (GAs, mycotoxins and pigments. The biosynthesis of most of these SMs strictly depends on nitrogen availability and of the activity of permeases of nitrogen sources, e.g. the ammonium and amino acid permeases. One of the three ammonium permeases, MepB, was recently shown to act not only as a transporter but also as a nitrogen sensor affecting the production of nitrogen-repressed SMs. Here we describe the identification of a general amino acid permease, FfGap1, among the 99 putative amino acid permeases (AAPs in the genome of F. fujikuroi. FfGap1 is able to fully restore growth of the yeast gap1∆ mutant on several amino acids including citrulline and tryptophane. In S. cerevisiae, Gap1 activity is regulated by shuttling between the plasma membrane (nitrogen limiting conditions and the vacuole (nitrogen sufficiency, which we also show for FfGap1. In yeast, the Npr1 serine/threonine kinase stabilizes the Gap1 position at the plasma membrane. Here, we identified and characterized three NPR1-homologous genes, encoding the putative protein kinases FfNpr1-1, FfNpr1-2 and FfNpr1-3 with significant similarity to yeast Npr1. Complementation of the yeast npr1Δ mutant with each of the three F. fujikuroi NPR1 homologues, resulted in partial restoration of ammonium, arginine and proline uptake by FfNPR1-1 while none of the three kinases affect growth on different nitrogen sources and nitrogen-dependent sorting of FfGap1 in F. fujikuroi. However, exchange of the putative ubiquitin-target lysine 9 (K9A and 15 (K15A residues of FfGap1 resulted in extended localization to the plasma membrane and increased protein stability independently of nitrogen availability. These data suggest a similar regulation of FfGap1 by nitrogen-dependent ubiquitination, but differences regarding the role of Fusarium Npr1

LPA is a chemorepellent for B16 melanoma cells: action through the cAMP-elevating LPA5 receptor.

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Maikel Jongsma

Full Text Available Lysophosphatidic acid (LPA, a lipid mediator enriched in serum, stimulates cell migration, proliferation and other functions in many cell types. LPA acts on six known G protein-coupled receptors, termed LPA(1-6, showing both overlapping and distinct signaling properties. Here we show that, unexpectedly, LPA and serum almost completely inhibit the transwell migration of B16 melanoma cells, with alkyl-LPA(18:1 being 10-fold more potent than acyl-LPA(18:1. The anti-migratory response to LPA is highly polarized and dependent on protein kinase A (PKA but not Rho kinase activity; it is associated with a rapid increase in intracellular cAMP levels and PIP3 depletion from the plasma membrane. B16 cells express LPA(2, LPA(5 and LPA(6 receptors. We show that LPA-induced chemorepulsion is mediated specifically by the alkyl-LPA-preferring LPA(5 receptor (GPR92, which raises intracellular cAMP via a noncanonical pathway. Our results define LPA(5 as an anti-migratory receptor and they implicate the cAMP-PKA pathway, along with reduced PIP3 signaling, as an effector of chemorepulsion in B16 melanoma cells.

Okadaic Acid, a Bioactive Fatty Acid from Halichondria okadai, Stimulates Lipolysis in Rat Adipocytes: The Pivotal Role of Perilipin Translocation

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Nen-Chung Chang

2013-01-01

Full Text Available Lipid metabolism in visceral fat cells is correlated with metabolic syndrome and cardiovascular diseases. Okadaic-acid, a 38-carbon fatty acid isolated from the black sponge Halichondria okadai, can stimulate lipolysis by promoting the phosphorylation of several proteins in adipocytes. However, the mechanism of okadaic acid-induced lipolysis and the effects of okadaic acid on lipid-droplet-associated proteins (perilipins and beta-actin remain unclear. We isolated adipocytes from rat epididymal fat pads and treated them with isoproterenol and/or okadaic acid to estimate lipolysis by measuring glycerol release. Incubating adipocytes with okadaic acid stimulated time-dependent lipolysis. Lipid-droplet-associated perilipins and beta-actin were analyzed by immunoblotting and immunofluorescence, and the association of perilipin A and B was found to be decreased in response to isoproterenol or okadaic acid treatment. Moreover, okadaic-acid treatment could enhance isoproterenol-mediated lipolysis, whereas treatment of several inhibitors such as KT-5720 (PKA inhibitor, calphostin C (PKC inhibitor, or KT-5823 (PKG inhibitor did not attenuate okadaic-acid-induced lipolysis. By contrast, vanadyl acetylacetonate (tyrosine phosphatase inhibitor blocked okadaic-acid-dependent lipolysis. These results suggest that okadaic acid induces the phosphorylation and detachment of lipid-droplet-associated perilipin A and B from the lipid droplet surface and thereby leads to accelerated lipolysis.

Characterization of Polyelectrolyte Complex Formation Between Anionic and Cationic Poly(amino acids) and Their Potential Applications in pH-Dependent Drug Delivery.

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Folchman-Wagner, Zoë; Zaro, Jennica; Shen, Wei-Chiang

2017-06-30

Polyelectrolyte complexes (PECs) are self-assembling nano-sized constructs that offer several advantages over traditional nanoparticle carriers including controllable size, biodegradability, biocompatibility, and lack of toxicity, making them particularly appealing as tools for drug delivery. Here, we discuss potential application of PECs for drug delivery to the slightly acidic tumor microenvironment, a pH in the range of 6.5-7.0. Poly(l-glutamic acid) (E n ), poly(l-lysine) (K n ), and a copolymer composed of histidine-glutamic acid repeats ((HE) n ) were studied for their ability to form PECs, which were analyzed for size, polydispersity, and pH sensitivity. PECs showed concentration dependent size variation at residue lengths of E 51 /K 55 and E 135 /K 127 , however, no complexes were observed when E 22 or K 21 were used, even in combination with the longer chains. (HE) 20 /K 55 PECs could encapsulate daunomycin, were stable from pH 7.4-6.5, and dissociated completely between pH 6.5-6.0. Conversely, the E 51-dauno /K 55 PEC dissociated between pH 4.0 and 3.0. These values for pH-dependent particle dissociation are consistent with the p K a 's of the ionizable groups in each formulation and indicate that the specific pH-sensitivity of (HE) 20-dauno /K 55 PECs is mediated by incorporation of histidine. This response within a pH range that is physiologically relevant to the acidic tumors suggests a potential application of these PECs in pH-dependent drug delivery.

The interrelationship between bile acid and vitamin A homeostasis

NARCIS (Netherlands)

Saeed, Ali; Hoekstra, Mark; Hoeke, Martijn Oscar; Heegsma, Janette; Faber, Klaas Nico

Vitamin A is a fat-soluble vitamin important for vision, reproduction, embryonic development, cell differentiation, epithelial barrier function and adequate immune responses. Efficient absorption of dietary vitamin A depends on the fat-solubilizing properties of bile acids. Bile acids are

Neighbor-dependent Ramachandran probability distributions of amino acids developed from a hierarchical Dirichlet process model.

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Daniel Ting

2010-04-01

Full Text Available Distributions of the backbone dihedral angles of proteins have been studied for over 40 years. While many statistical analyses have been presented, only a handful of probability densities are publicly available for use in structure validation and structure prediction methods. The available distributions differ in a number of important ways, which determine their usefulness for various purposes. These include: 1 input data size and criteria for structure inclusion (resolution, R-factor, etc.; 2 filtering of suspect conformations and outliers using B-factors or other features; 3 secondary structure of input data (e.g., whether helix and sheet are included; whether beta turns are included; 4 the method used for determining probability densities ranging from simple histograms to modern nonparametric density estimation; and 5 whether they include nearest neighbor effects on the distribution of conformations in different regions of the Ramachandran map. In this work, Ramachandran probability distributions are presented for residues in protein loops from a high-resolution data set with filtering based on calculated electron densities. Distributions for all 20 amino acids (with cis and trans proline treated separately have been determined, as well as 420 left-neighbor and 420 right-neighbor dependent distributions. The neighbor-independent and neighbor-dependent probability densities have been accurately estimated using Bayesian nonparametric statistical analysis based on the Dirichlet process. In particular, we used hierarchical Dirichlet process priors, which allow sharing of information between densities for a particular residue type and different neighbor residue types. The resulting distributions are tested in a loop modeling benchmark with the program Rosetta, and are shown to improve protein loop conformation prediction significantly. The distributions are available at http://dunbrack.fccc.edu/hdp.

A negative modulatory role for rho and rho-associated kinase signaling in delamination of neural crest cells

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Kalcheim Chaya

2008-10-01

Full Text Available Abstract Background Neural crest progenitors arise as epithelial cells and then undergo a process of epithelial to mesenchymal transition that precedes the generation of cellular motility and subsequent migration. We aim at understanding the underlying molecular network. Along this line, possible roles of Rho GTPases that act as molecular switches to control a variety of signal transduction pathways remain virtually unexplored, as are putative interactions between Rho proteins and additional known components of this cascade. Results We investigated the role of Rho/Rock signaling in neural crest delamination. Active RhoA and RhoB are expressed in the membrane of epithelial progenitors and are downregulated upon delamination. In vivo loss-of-function of RhoA or RhoB or of overall Rho signaling by C3 transferase enhanced and/or triggered premature crest delamination yet had no effect on cell specification. Consistently, treatment of explanted neural primordia with membrane-permeable C3 or with the Rock inhibitor Y27632 both accelerated and enhanced crest emigration without affecting cell proliferation. These treatments altered neural crest morphology by reducing stress fibers, focal adhesions and downregulating membrane-bound N-cadherin. Reciprocally, activation of endogenous Rho by lysophosphatidic acid inhibited emigration while enhancing the above. Since delamination is triggered by BMP and requires G1/S transition, we examined their relationship with Rho. Blocking Rho/Rock function rescued crest emigration upon treatment with noggin or with the G1/S inhibitor mimosine. In the latter condition, cells emigrated while arrested at G1. Conversely, BMP4 was unable to rescue cell emigration when endogenous Rho activity was enhanced by lysophosphatidic acid. Conclusion Rho-GTPases, through Rock, act downstream of BMP and of G1/S transition to negatively regulate crest delamination by modifying cytoskeleton assembly and intercellular adhesion.

Detection of Ca2+-dependent acid phosphatase activity identifies neuronal integrity in damaged rat central nervous system after application of bacterial melanin

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Tigran R Petrosyan

2016-01-01

Full Text Available The study aims to confirm the neuroregenerative effects of bacterial melanin (BM on central nervous system injury using a special staining method based on the detection of Ca2+-dependent acid phosphatase activity. Twenty-four rats were randomly assigned to undergo either unilateral destruction of sensorimotor cortex (group I; n = 12 or unilateral rubrospinal tract transection at the cervical level (C3–4 (group II; n = 12. In each group, six rats were randomly selected after surgery to undergo intramuscular injection of BM solution (BM subgroup and the remaining six rats were intramuscularly injected with saline (saline subgroup. Neurological testing confirmed that BM accelerated the recovery of motor function in rats from both BM and saline subgroups. Two months after surgery, Ca2+-dependent acid phosphatase activity detection in combination with Chilingarian's calcium adenoside triphosphate method revealed that BM stimulated the sprouting of fibers and dilated the capillaries in the brain and spinal cord. These results suggest that BM can promote the recovery of motor function of rats with central nervous system injury; and detection of Ca2+-dependent acid phosphatase activity is a fast and easy method used to study the regeneration-promoting effects of BM on the injured central nervous system.

Amino acids and carbohydrates absorption by Na+-dependent transporters in the pyloric ceca of Hoplias malabaricus (Erythrinidae

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Vieira Vania Lucia Pimentel

2001-01-01

Full Text Available Information about amino acids and carbohydrate absorption in fish is important to formulate an adequate diet to obtain optimal growth. Therefore, the objective of this study was to investigate if Na+-dependent transporters are involved on the absorption of glycine, L-glutamine, L-leucine, L-lysine, L-proline, L-alanine, and the carbohydrates fructose and glucose in the pyloric ceca of Hoplias malabaricus. The pyloric ceca were mounted in a system of continuous perfusion "in vitro". Amino acids and carbohydrates were placed on the mucosal side at concentrations of 10, 20, and 40mM. The serosal side of the pyloric ceca was positive in relation to the mucosal side. The addition of glycine, L-glutamine, L-leucine, L-lysine, L-proline (all tested concentrations, and glucose (at concentrations of 20 and 40mM increased the positivity of the serosal side, indicating the presence of Na+-dependent transporters in the absorption of these substances. L-alanine and fructose did not change the positivity of the serosal side. The pyloric ceca seem to be the main site of nutrient absorption in the digestive tract of H. malabaricus.

Akt-dependent NF-κB activation is required for bile acids to rescue colon cancer cells from stress-induced apoptosis

International Nuclear Information System (INIS)

Shant, Jasleen; Cheng, Kunrong; Marasa, Bernard S.; Wang Jianying; Raufman, Jean-Pierre

2009-01-01

Conjugated secondary bile acids promote human colon cancer cell proliferation by activating EGF receptors (EGFR). We hypothesized that bile acid-induced EGFR activation also mediates cell survival by downstream Akt-regulated activation of NF-κB. Deoxycholyltaurine (DCT) treatment attenuated TNF-α-induced colon cancer cell apoptosis, and stimulated rapid and sustained NF-κB nuclear translocation and transcriptional activity (detected by NF-κB binding to an oligonucleotide consensus sequence and by activation of luciferase reporter gene constructs). Both DCT-induced NF-κB nuclear translocation and attenuation of TNF-α-stimulated apoptosis were dependent on EGFR activation. Inhibitors of nuclear translocation, proteosome activity, and IκBα kinase attenuated NF-κB transcriptional activity. Cell transfection with adenoviral vectors encoding a non-degradable IκBα 'super-repressor' blocked the actions of DCT on both NF-κB activation and TNF-α-induced apoptosis. Likewise, transfection with mutant akt and treatment with a chemical inhibitor of Akt attenuated effects of DCT on NF-κB transcriptional activity and TNF-α-induced apoptosis. Chemical inhibitors of Akt and NF-κB activation also attenuated DCT-induced rescue of H508 cells from ultraviolet radiation-induced apoptosis. Collectively, these observations indicate that, downstream of EGFR, bile acid-induced colon cancer cell survival is mediated by Akt-dependent NF-κB activation. These findings provide a mechanism whereby bile acids increase resistance of colon cancer to chemotherapy and radiation

Biotin dependency due to a defect in biotin transport

OpenAIRE

Mardach, Rebecca; Zempleni, Janos; Wolf, Barry; Cannon, Martin J.; Jennings, Michael L.; Cress, Sally; Boylan, Jane; Roth, Susan; Cederbaum, Stephen; Mock, Donald M.

2002-01-01

We describe a 3-year-old boy with biotin dependency not caused by biotinidase, holocarboxylase synthetase, or nutritional biotin deficiency. We sought to define the mechanism of his biotin dependency. The child became acutely encephalopathic at age 18 months. Urinary organic acids indicated deficiency of several biotin-dependent carboxylases. Symptoms improved rapidly following biotin supplementation. Serum biotinidase activity and Biotinidase gene sequence were normal. Activities of biotin-d...

Identification of a transcription factor controlling pH-dependent organic acid response in Aspergillus niger.

Directory of Open Access Journals (Sweden)

Lars Poulsen

Full Text Available Acid formation in Aspergillus niger is known to be subjected to tight regulation, and the acid production profiles are fine-tuned to respond to the ambient pH. Based on transcriptome data, putative trans-acting pH responding transcription factors were listed and through knock out studies, mutants exhibiting an oxalate overproducing phenotype were identified. The yield of oxalate was increased up to 158% compared to the wild type and the corresponding transcription factor was therefore entitled Oxalic Acid repression Factor, OafA. Detailed physiological characterization of one of the ΔoafA mutants, compared to the wild type, showed that both strains produced substantial amounts of gluconic acid, but the mutant strain was more efficient in re-uptake of gluconic acid and converting it to oxalic acid, particularly at high pH (pH 5.0. Transcriptional profiles showed that 241 genes were differentially expressed due to the deletion of oafA and this supported the argument of OafA being a trans-acting transcription factor. Furthermore, expression of two phosphoketolases was down-regulated in the ΔoafA mutant, one of which has not previously been described in fungi. It was argued that the observed oxalate overproducing phenotype was a consequence of the efficient re-uptake of gluconic acid and thereby a higher flux through glycolysis. This results in a lower flux through the pentose phosphate pathway, demonstrated by the down-regulation of the phosphoketolases. Finally, the physiological data, in terms of the specific oxygen consumption, indicated a connection between the oxidative phosphorylation and oxalate production and this was further substantiated through transcription analysis.

Dependence of intestinal amino acid uptake on dietary protein or amino acid levels

International Nuclear Information System (INIS)

Karasov, W.H.; Solberg, D.H.; Diamond, J.M.

1987-01-01

To understand how intestinal amino acid (AA) transport is regulated by dietary substrate levels, the authors measured uptake of seven radioactively-labelled AAs and glucose across the jejunal brush-border membrane of mice kept on one of three isocaloric rations differing in nitrogen content. In the high-protein ration, uptake increased by 77-81% for the nonessential, less toxic AAs, proline, and aspartate but only by 32-61% for the more toxic essential AAs tested. In the nitrogen-deficient ration, uptake decreased for the nonessential aspartate and proline but stayed constant or increased for essential AAs and for the nonessential alanine. These patterns imply independent regulation of the intestine's various AA transporters. With decreasing dietary AA (or protein), the imino acid and acidic AA private transporters are repressed, while activities of the basic AA transporter and the neutral AA public transporter decrease to an asymptote or else go through a minimum. These regulatory patterns can be understood as a compromise among conflicting constraints imposed by protein's multiple roles as a source of calories, nitrogen, and essential AAs and by the toxicity of essential AAs at high concentrations

PPARalpha-dependent modulation of hepatic CYP1A by clofibric acid in rats.

Science.gov (United States)

Shaban, Zein; El-Shazly, Samir; Ishizuka, Mayumi; Kimura, Kazuhiro; Kazusaka, Akio; Fujita, Shoichi

2004-09-01

Fibrates, hypolipidemic drugs, have been reported to suppress the metabolic activities of cytochrome P450 1A1 and 1A2 in rats but the mechanism has not been elucidated. In the present study we tested the hypothesis that the inhibitory effect of fibrates on arylhydrocarbon receptor (AhR) function may be due to their stimulatory effects on PPARalpha. Sudan III (S.III) treatment induced CYP 1A1 and CYP 1A2 protein expression, mRNA and their metabolic activities, methoxyresorufin-O-demethylase (MROD) and ethoxyresorufin-O-deethylase (EROD), in Wistar rats higher than those in the control. Co-treatment of rats with S.III and clofibric acid (CA) caused a 40-50% decrease in the induced levels of CYP1A1 and CYP1A2 protein, mRNA expression and their metabolic activities and reduced AhR protein expression. When we treated HepG2 cells with S.III and/or CA, no suppressive effect on S.III-induced CYP1A1 protein expression due to CA was found. HepG2 cells were transiently transfected with increasing concentrations of PPARalpha mammalian expression vector and exposed to the same treatment. CA co-treatment with S.III decreased AhR protein and S.III-induced CYP1A1 protein expression with increasing dose of PPARalpha transfected into HepG2 cells. Our results demonstrate that the suppressive effect of fibrates on CYP1A is PPARalpha-dependent and suggest that PPARalpha has an inhibitory effect on AhR function.

Major Role of NAD-Dependent Lactate Dehydrogenases in the Production of l-Lactic Acid with High Optical Purity by the Thermophile Bacillus coagulans.

Science.gov (United States)

Wang, Limin; Cai, Yumeng; Zhu, Lingfeng; Guo, Honglian; Yu, Bo

2014-12-01

Bacillus coagulans 2-6 is an excellent producer of optically pure l-lactic acid. However, little is known about the mechanism of synthesis of the highly optically pure l-lactic acid produced by this strain. Three enzymes responsible for lactic acid production-NAD-dependent l-lactate dehydrogenase (l-nLDH; encoded by ldhL), NAD-dependent d-lactate dehydrogenase (d-nLDH; encoded by ldhD), and glycolate oxidase (GOX)-were systematically investigated in order to study the relationship between these enzymes and the optical purity of lactic acid. Lactobacillus delbrueckii subsp. bulgaricus DSM 20081 (a d-lactic acid producer) and Lactobacillus plantarum subsp. plantarum DSM 20174 (a dl-lactic acid producer) were also examined in this study as comparative strains, in addition to B. coagulans. The specific activities of key enzymes for lactic acid production in the three strains were characterized in vivo and in vitro, and the levels of transcription of the ldhL, ldhD, and GOX genes during fermentation were also analyzed. The catalytic activities of l-nLDH and d-nLDH were different in l-, d-, and dl-lactic acid producers. Only l-nLDH activity was detected in B. coagulans 2-6 under native conditions, and the level of transcription of ldhL in B. coagulans 2-6 was much higher than that of ldhD or the GOX gene at all growth phases. However, for the two Lactobacillus strains used in this study, ldhD transcription levels were higher than those of ldhL. The high catalytic efficiency of l-nLDH toward pyruvate and the high transcription ratios of ldhL to ldhD and ldhL to the GOX gene provide the key explanations for the high optical purity of l-lactic acid produced by B. coagulans 2-6. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Lewis acidity dependency of the electrochemical window of zinc chloride-1-ethyl-3-methylimidazolium chloride ionic liquids

International Nuclear Information System (INIS)

Hsiu, S-I; Huang, J.-F.; Sun, I-W.; Yuan, C.-H.; Shiea, Jantaie

2002-01-01

Negative ion fast atom bombardment mass spectra (FAB-MS) recorded for ZnCl 2 -1-ethyl-3-methylimidazolium chloride (ZnCl 2 -EMIC) ionic liquids with various compositions indicate that various Lewis acidic chlorozincate clusters (ZnCl 3 - , Zn 2 Cl 5 - and Zn 3 Cl 7 - ) are present in ZnCl 2 -EMIC ionic liquids depending on the percentage of ZnCl 2 used in preparing the ionic liquids; higher ZnCl 2 percentage favors the larger clusters. Cyclic voltammetry reveals that the potential limits for a basic 1:3 ZnCl 2 -EMIC melt correspond to the cathodic reduction of EMI + and anodic oxidation of Cl - , giving an electrochemical window of approximately 3.0 V which is the same as that observed for basic AlCl 3 -EMIC ionic liquids. For acidic ionic liquids that have a ZnCl 2 /EMIC molar ratio higher than 0.5:1, the negative potential limit is due to the deposition of metallic zinc, and the positive potential limit is due to the oxidation of the chlorozincate complexes. All the acidic ionic liquids exhibit an electrochemical window of approximately 2 V, although the potential limits shifted in the positive direction with increasing ZnCl 2 mole ratio. Underpotential deposition of zinc was observed on Pt and Ni electrodes in the acidic ionic liquids. At proper temperatures and potentials, crystalline zinc electrodeposits were obtained from the acidic ionic liquids

A single amino acid gates the KcsA channel

International Nuclear Information System (INIS)

Hirano, Minako; Okuno, Daichi; Onishi, Yukiko; Ide, Toru

2014-01-01

Highlights: • pH-dependent gating of the KcsA channel is regulated by the CPD. • E146 is the most essential amino acid for pH sensing by the KcsA. • The protonated-mimicking mutant, E146Q, is constitutively open independent of pH. • Minimal rearrangement of the CPD is sufficient for opening of the KcsA. - Abstract: The KcsA channel is a proton-activated potassium channel. We have previously shown that the cytoplasmic domain (CPD) acts as a pH-sensor, and the charged states of certain negatively charged amino acids in the CPD play an important role in regulating the pH-dependent gating. Here, we demonstrate the KcsA channel is constitutively open independent of pH upon mutating E146 to a neutrally charged amino acid. In addition, we found that rearrangement of the CPD following this mutation was not large. Our results indicate that minimal rearrangement of the CPD, particularly around E146, is sufficient for opening of the KcsA channel

A single amino acid gates the KcsA channel

Energy Technology Data Exchange (ETDEWEB)

Hirano, Minako, E-mail: hirano37@gpi.ac.jp [Bio Photonics Laboratory, The Graduate School for the Creation of New Photonics Industries, 1955-1 Kurematsu Nishi-ku Hamamatsu, Shizuoka 431-1202 (Japan); Laboratory for Cell Dynamics Observation, Quantitative Biology Center, RIKEN, 6-2-3 Furue-dai Suita, Osaka 565-0874 (Japan); Okuno, Daichi, E-mail: dokuno@riken.jp [Laboratory for Cell Dynamics Observation, Quantitative Biology Center, RIKEN, 6-2-3 Furue-dai Suita, Osaka 565-0874 (Japan); Onishi, Yukiko, E-mail: yonishi@riken.jp [Laboratory for Cell Dynamics Observation, Quantitative Biology Center, RIKEN, 6-2-3 Furue-dai Suita, Osaka 565-0874 (Japan); Ide, Toru, E-mail: ide@okayama-u.ac.jp [Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-naka Kita-ku Okayama-shi, Okayama 700-8530 (Japan)

2014-08-08

Highlights: • pH-dependent gating of the KcsA channel is regulated by the CPD. • E146 is the most essential amino acid for pH sensing by the KcsA. • The protonated-mimicking mutant, E146Q, is constitutively open independent of pH. • Minimal rearrangement of the CPD is sufficient for opening of the KcsA. - Abstract: The KcsA channel is a proton-activated potassium channel. We have previously shown that the cytoplasmic domain (CPD) acts as a pH-sensor, and the charged states of certain negatively charged amino acids in the CPD play an important role in regulating the pH-dependent gating. Here, we demonstrate the KcsA channel is constitutively open independent of pH upon mutating E146 to a neutrally charged amino acid. In addition, we found that rearrangement of the CPD following this mutation was not large. Our results indicate that minimal rearrangement of the CPD, particularly around E146, is sufficient for opening of the KcsA channel.

The Nitric Oxide Donor SNAP-Induced Amino Acid Neurotransmitter Release in Cortical Neurons. Effects of Blockers of Voltage-Dependent Sodium and Calcium Channels

Science.gov (United States)

Merino, José Joaquín; Arce, Carmen; Naddaf, Ahmad; Bellver-Landete, Victor; Oset-Gasque, Maria Jesús; González, María Pilar

2014-01-01

Background The discovery that nitric oxide (NO) functions as a signalling molecule in the nervous system has radically changed the concept of neuronal communication. NO induces the release of amino acid neurotransmitters but the underlying mechanisms remain to be elucidated. Findings The aim of this work was to study the effect of NO on amino acid neurotransmitter release (Asp, Glu, Gly and GABA) in cortical neurons as well as the mechanism underlying the release of these neurotransmitters. Cortical neurons were stimulated with SNAP, a NO donor, and the release of different amino acid neurotransmitters was measured by HPLC. The involvement of voltage dependent Na+ and Ca2+ channels as well as cGMP in its mechanism of action was evaluated. Conclusions Our results indicate that NO induces release of aspartate, glutamate, glycine and GABA in cortical neurons and that this release is inhibited by ODQ, an inhibitor of soluble guanylate cyclase. Thus, the NO effect on amino acid neurotransmission could be mediated by cGMP formation in cortical neurons. Our data also demonstrate that the Na+ and Ca2+ voltage- dependent calcium channels are involved in the NO effects on cortical neurons. PMID:24598811

The nitric oxide donor SNAP-induced amino acid neurotransmitter release in cortical neurons. Effects of blockers of voltage-dependent sodium and calcium channels.

Science.gov (United States)

Merino, José Joaquín; Arce, Carmen; Naddaf, Ahmad; Bellver-Landete, Victor; Oset-Gasque, Maria Jesús; González, María Pilar

2014-01-01

The discovery that nitric oxide (NO) functions as a signalling molecule in the nervous system has radically changed the concept of neuronal communication. NO induces the release of amino acid neurotransmitters but the underlying mechanisms remain to be elucidated. The aim of this work was to study the effect of NO on amino acid neurotransmitter release (Asp, Glu, Gly and GABA) in cortical neurons as well as the mechanism underlying the release of these neurotransmitters. Cortical neurons were stimulated with SNAP, a NO donor, and the release of different amino acid neurotransmitters was measured by HPLC. The involvement of voltage dependent Na+ and Ca2+ channels as well as cGMP in its mechanism of action was evaluated. Our results indicate that NO induces release of aspartate, glutamate, glycine and GABA in cortical neurons and that this release is inhibited by ODQ, an inhibitor of soluble guanylate cyclase. Thus, the NO effect on amino acid neurotransmission could be mediated by cGMP formation in cortical neurons. Our data also demonstrate that the Na+ and Ca2+ voltage- dependent calcium channels are involved in the NO effects on cortical neurons.

The nitric oxide donor SNAP-induced amino acid neurotransmitter release in cortical neurons. Effects of blockers of voltage-dependent sodium and calcium channels.

Directory of Open Access Journals (Sweden)

José Joaquín Merino

Full Text Available The discovery that nitric oxide (NO functions as a signalling molecule in the nervous system has radically changed the concept of neuronal communication. NO induces the release of amino acid neurotransmitters but the underlying mechanisms remain to be elucidated.The aim of this work was to study the effect of NO on amino acid neurotransmitter release (Asp, Glu, Gly and GABA in cortical neurons as well as the mechanism underlying the release of these neurotransmitters. Cortical neurons were stimulated with SNAP, a NO donor, and the release of different amino acid neurotransmitters was measured by HPLC. The involvement of voltage dependent Na+ and Ca2+ channels as well as cGMP in its mechanism of action was evaluated.Our results indicate that NO induces release of aspartate, glutamate, glycine and GABA in cortical neurons and that this release is inhibited by ODQ, an inhibitor of soluble guanylate cyclase. Thus, the NO effect on amino acid neurotransmission could be mediated by cGMP formation in cortical neurons. Our data also demonstrate that the Na+ and Ca2+ voltage- dependent calcium channels are involved in the NO effects on cortical neurons.

Purification, molecular cloning, and expression of 2-hydroxyphytanoyl- CoA lyase, a peroxisomal thiamine pyrophosphate-dependent enzyme that catalyzes the carbon-carbon bond cleavage during à-oxidation of 3- methyl-branched fatty acids

CERN Document Server

Foulon, V; Croes, K; Waelkens, E

1999-01-01

Purification, molecular cloning, and expression of 2-hydroxyphytanoyl- CoA lyase, a peroxisomal thiamine pyrophosphate-dependent enzyme that catalyzes the carbon-carbon bond cleavage during à-oxidation of 3- methyl-branched fatty acids

Salvianolic Acid-A Induces Apoptosis, Mitochondrial Membrane ...

African Journals Online (AJOL)

using Hoechst 33258 staining. The effect of the compound on mitochondrial membrane potential loss ... Fluorescence microscopy demonstrated that salvianolic acid-A induced dose- dependent ..... aggregation and anticancer properties. It has.

Effect of CLA and other C18 unsaturated fatty acids on DGAT in bovine milk fat biosynthetic systems.

Science.gov (United States)

Sørensen, Brent M; Chris Kazala, E; Murdoch, Gordon K; Keating, Aileen F; Cruz-Hernandez, Cristina; Wegner, Jochen; Kennelly, John J; Okine, Erasmus K; Weselake, Randall J

2008-10-01

Production of dairy products with increased amounts of nutraceutic FA such as conjugated linoleic acid (CLA) represents a recent approach for dairy producers and processors to increase the value of their products. The effect of CLA and other FA on the expression of diacylglycerol acyltransferase-1 (DGAT-1) and DGAT-2, and DGAT activity were investigated in bovine mammary gland epithelial (MAC-T) cells. DGAT gene expression analyses were also conducted using bovine mammary gland tissue from dairy cows. In the studies with MAC-T cells, there were no significant effects of CLA isomers or other FA on DGAT1 expression, whereas all FA tested showed enhanced DGAT2 expression (P < 0.05 to P < 0.001), with alpha-linolenic acid (alpha-18:3) having the greatest effect. Additionally, DGAT2 expression was co-ordinated with expression of lysophosphatidic acid acyltransferase (LPAAT), an observation that was also apparent in mammary gland from lactating dairy cows. In contrast, treatment of MAC-T cells with trans-10, cis-12 18:2 or alpha-18:3 resulted in a significant (P < 0.05) decrease in overall DGAT enzyme activity, although the mechanisms resulting in these effects are unclear. Competition assays using microsomes from bovine mammary gland tissue and 1-[(14)C]oleoyl-CoA suggested that DGAT activity was more selective for oleoyl (cis-9 18:1)-CoA than cis-9, trans-11 18:2-, trans-10, cis-12 18:2- or cis-9, cis-12 18:2-CoA. Collectively, the results suggest the relationship between trans-10, cis-12 18:2 and reduced TAG production in bovine milk is not linked to the production of DGAT1 or DGAT2 transcripts, but probably involves effects of this CLA isomer at events beyond transcription, such as post-translational and/or enzyme activity effects.

ATP-dependent paracrine communication between enteric neurons and glia in a primary cell culture derived from embryonic mice.

Science.gov (United States)

Gomes, P; Chevalier, J; Boesmans, W; Roosen, L; van den Abbeel, V; Neunlist, M; Tack, J; Vanden Berghe, P

2009-08-01

The importance of dynamic interactions between glia and neurons is increasingly recognized, both in the central and enteric nervous system. However, apart from their protective role, little is known about enteric neuro-glia interaction. The aim was to investigate neuro-glia intercellular communication in a mouse culture model using optical techniques. Complete embryonic (E13) guts were enzymatically dissociated, seeded on coverslips and studied with immunohistochemistry and Ca(2+)-imaging. Putative progenitor-like cells (expressing both PGP9.5 and S-100) differentiated over approximately 5 days into glia or neurons expressing typical cell-specific markers. The glia-neuron ratio could be manipulated by specific supplements (N2, G5). Neurons and glia were functionally identified both by their Ca(2+)-response to either depolarization (high K(+)) or lysophosphatidic acid and by the expression of typical markers. Neurons responded to ACh, DMPP, 5-HT, ATP and electrical stimulation, while glia responded to ATP and ADPbetas. Inhibition of glial responses by MRS2179 suggests involvement of P2Y1 receptors. Neuronal stimulation also caused delayed glial responses, which were reduced by suramin and by exogenous apyrases that catalyse nucleotide breakdown. Conversely, glial responses were enhanced by ARL-67156, an ecto-ATPase inhibitor. In this mouse enteric co-culture, functional glia and neurons can be easily monitored using optical techniques. Glial cells can be activated directly by ATP or ADPbetas. Activation of neuronal cells (DMPP, K(+)) causes secondary responses in glial cells, which can be modulated by tuning ATP and ADP breakdown. This strongly supports the involvement of paracrine purinergic communication between enteric neurons and glia.

Amino acids augment muscle protein synthesis in neonatal pigs during acute endotoxemia by stimulating mTOR-dependent translation initiation.

Science.gov (United States)

Orellana, Renán A; Jeyapalan, Asumthia; Escobar, Jeffery; Frank, Jason W; Nguyen, Hanh V; Suryawan, Agus; Davis, Teresa A

2007-11-01

In skeletal muscle of adults, sepsis reduces protein synthesis by depressing translation initiation and induces resistance to branched-chain amino acid stimulation. Normal neonates maintain a high basal muscle protein synthesis rate that is sensitive to amino acid stimulation. In the present study, we determined the effect of amino acids on protein synthesis in skeletal muscle and other tissues in septic neonates. Overnight-fasted neonatal pigs were infused with endotoxin (LPS, 0 and 10 microg.kg(-1).h(-1)), whereas glucose and insulin were maintained at fasting levels; amino acids were clamped at fasting or fed levels. In the presence of fasting insulin and amino acids, LPS reduced protein synthesis in longissimus dorsi (LD) and gastrocnemius muscles and increased protein synthesis in the diaphragm, but had no effect in masseter and heart muscles. Increasing amino acids to fed levels accelerated muscle protein synthesis in LD, gastrocnemius, masseter, and diaphragm. LPS stimulated protein synthesis in liver, lung, spleen, pancreas, and kidney in fasted animals. Raising amino acids to fed levels increased protein synthesis in liver of controls, but not LPS-treated animals. The increase in muscle protein synthesis in response to amino acids was associated with increased mTOR, 4E-BP1, and S6K1 phosphorylation and eIF4G-eIF4E association in control and LPS-infused animals. These findings suggest that amino acids stimulate skeletal muscle protein synthesis during acute endotoxemia via mTOR-dependent ribosomal assembly despite reduced basal protein synthesis rates in neonatal pigs. However, provision of amino acids does not further enhance the LPS-induced increase in liver protein synthesis.

Temperature dependent emission of formic and acetic acid from paper, and its consequences for the air quality in archives

DEFF Research Database (Denmark)

Hjerrild Smedemark, Signe; Ryhl-Svendsen, Morten; Vinther Hansen, Birgit

2014-01-01

Paper records affect the indoor environment in archives, because the paper may give off acid compounds to the air. These emission products origins from the decay of cellulose and lignin in the paper. As the off-gassing origins from chemical processes within the material (e.g., acid hydrolysis......) the reaction rate is therefore, among other things, depending on temperature. Some of the emission products from paper, e.g., acetic and formic acid, are problematic from a conservation point of view, because they will re-react with the paper and other archival materials and cause further deterioration....... The problem is amplified by an often very large loading (mass of material vs. volume) of paper in archival rooms. Furthermore, many archives have only little ventilation, so the exchange of polluted air with ambient air is low. The general trend; higher temperature accelerates material’s emission, and a high...

The DELLA Protein SLR1 Integrates and Amplifies Salicylic Acid- and Jasmonic Acid-Dependent Innate Immunity in Rice.

Science.gov (United States)

De Vleesschauwer, David; Seifi, Hamed Soren; Filipe, Osvaldo; Haeck, Ashley; Huu, Son Nguyen; Demeestere, Kristof; Höfte, Monica

2016-03-01

Gibberellins are a class of tetracyclic plant hormones that are well known to promote plant growth by inducing the degradation of a class of nuclear growth-repressing proteins, called DELLAs. In recent years, GA and DELLAs are also increasingly implicated in plant responses to pathogen attack, although our understanding of the underlying mechanisms is still limited, especially in monocotyledonous crop plants. Aiming to further decipher the molecular underpinnings of GA- and DELLA-modulated plant immunity, we studied the dynamics and impact of GA and DELLA during infection of the model crop rice (Oryza sativa) with four different pathogens exhibiting distinct lifestyles and infection strategies. Opposite to previous findings in Arabidopsis (Arabidopsis thaliana), our findings reveal a prominent role of the DELLA protein Slender Rice1 (SLR1) in the resistance toward (hemi)biotrophic but not necrotrophic rice pathogens. Moreover, contrary to the differential effect of DELLA on the archetypal defense hormones salicylic acid (SA) and jasmonic acid (JA) in Arabidopsis, we demonstrate that the resistance-promoting effect of SLR1 is due at least in part to its ability to boost both SA- and JA-mediated rice defenses. In a reciprocal manner, we found JA and SA treatment to interfere with GA metabolism and stabilize SLR1. Together, these findings favor a model whereby SLR1 acts as a positive regulator of hemibiotroph resistance in rice by integrating and amplifying SA- and JA-dependent defense signaling. Our results highlight the differences in hormone defense networking between rice and Arabidopsis and underscore the importance of GA and DELLA in molding disease outcomes. © 2016 American Society of Plant Biologists. All Rights Reserved.

Citric acid induces cell-cycle arrest and apoptosis of human immortalized keratinocyte cell line (HaCaT) via caspase- and mitochondrial-dependent signaling pathways.

Science.gov (United States)

Ying, Tsung-Ho; Chen, Chia-Wei; Hsiao, Yu-Ping; Hung, Sung-Jen; Chung, Jing-Gung; Yang, Jen-Hung

2013-10-01

Citric acid is an alpha-hydroxyacid (AHA) widely used in cosmetic dermatology and skincare products. However, there is concern regarding its safety for the skin. In this study, we investigated the cytotoxic effects of citric acid on the human keratinocyte cell line HaCaT. HaCaT cells were treated with citric acid at 2.5-12.5 mM for different time periods. Cell-cycle arrest and apoptosis were investigated by 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining, flow cytometry, western blot and confocal microscopy. Citric acid not only inhibited proliferation of HaCaT cells in a dose-dependent manner, but also induced apoptosis and cell cycle-arrest at the G2/M phase (before 24 h) and S phase (after 24 h). Citric acid increased the level of Bcl-2-associated X protein (BAX) and reduced the levels of B-cell lymphoma-2 (BCL-2), B-cell lymphoma-extra large (BCL-XL) and activated caspase-9 and caspase-3, which subsequently induced apoptosis via caspase-dependent and caspase-independent pathways. Citric acid also activated death receptors and increased the levels of caspase-8, activated BH3 interacting-domain death agonist (BID) protein, Apoptosis-inducing factor (AIF), and Endonuclease G (EndoG). Therefore, citric acid induces apoptosis through the mitochondrial pathway in the human keratinocyte cell line HaCaT. The study results suggest that citric acid is cytotoxic to HaCaT cells via induction of apoptosis and cell-cycle arrest in vitro.

Acetic acid treatment in S. cerevisiae creates significant energy deficiency and nutrient starvation that is dependent on the activity of the mitochondrial transcriptional complex Hap2-3-4-5

International Nuclear Information System (INIS)

Kitanovic, Ana; Bonowski, Felix; Heigwer, Florian; Ruoff, Peter; Kitanovic, Igor; Ungewiss, Christin; Wölfl, Stefan

2012-01-01

Metabolic pathways play an indispensable role in supplying cellular systems with energy and molecular building blocks for growth, maintenance and repair and are tightly linked with lifespan and systems stability of cells. For optimal growth and survival cells rapidly adopt to environmental changes. Accumulation of acetic acid in stationary phase budding yeast cultures is considered to be a primary mechanism of chronological aging and induction of apoptosis in yeast, which has prompted us to investigate the dependence of acetic acid toxicity on extracellular conditions in a systematic manner. Using an automated computer controlled assay system, we investigated and model the dynamic interconnection of biomass yield- and growth rate-dependence on extracellular glucose concentration, pH conditions and acetic acid concentration. Our results show that toxic concentrations of acetic acid inhibit glucose consumption and reduce ethanol production. In absence of carbohydrates uptake, cells initiate synthesis of storage carbohydrates, trehalose and glycogen, and upregulate gluconeogenesis. Accumulation of trehalose and glycogen, and induction of gluconeogenesis depends on mitochondrial activity, investigated by depletion of the Hap2-3-4-5 complex. Analyzing the activity of glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase (PYK), and glucose-6-phosphate dehydrogenase (G6PDH) we found that while high acetic acid concentration increased their activity, lower acetic acids concentrations significantly inhibited these enzymes. With this study we determined growth and functional adjustment of metabolism to acetic acid accumulation in a complex range of extracellular conditions. Our results show that substantial acidification of the intracellular environment, resulting from accumulation of dissociated acetic acid in the cytosol, is required for acetic acid toxicity, which creates a state of energy deficiency and nutrient starvation.

Acetic acid treatment in S. cerevisiae creates significant energy deficiency and nutrient starvation that is dependent on the activity of the mitochondrial transcriptional complex Hap2-3-4-5

Energy Technology Data Exchange (ETDEWEB)

Kitanovic, Ana; Bonowski, Felix; Heigwer, Florian [Institute for Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg (Germany); Ruoff, Peter [Faculty of Science and Technology, Centre for Organelle Research, University of Stavanger, Stavanger (Norway); Kitanovic, Igor; Ungewiss, Christin; Wölfl, Stefan, E-mail: wolfl@uni-hd.de [Institute for Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg (Germany)

2012-09-21

Metabolic pathways play an indispensable role in supplying cellular systems with energy and molecular building blocks for growth, maintenance and repair and are tightly linked with lifespan and systems stability of cells. For optimal growth and survival cells rapidly adopt to environmental changes. Accumulation of acetic acid in stationary phase budding yeast cultures is considered to be a primary mechanism of chronological aging and induction of apoptosis in yeast, which has prompted us to investigate the dependence of acetic acid toxicity on extracellular conditions in a systematic manner. Using an automated computer controlled assay system, we investigated and model the dynamic interconnection of biomass yield- and growth rate-dependence on extracellular glucose concentration, pH conditions and acetic acid concentration. Our results show that toxic concentrations of acetic acid inhibit glucose consumption and reduce ethanol production. In absence of carbohydrates uptake, cells initiate synthesis of storage carbohydrates, trehalose and glycogen, and upregulate gluconeogenesis. Accumulation of trehalose and glycogen, and induction of gluconeogenesis depends on mitochondrial activity, investigated by depletion of the Hap2-3-4-5 complex. Analyzing the activity of glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase (PYK), and glucose-6-phosphate dehydrogenase (G6PDH) we found that while high acetic acid concentration increased their activity, lower acetic acids concentrations significantly inhibited these enzymes. With this study we determined growth and functional adjustment of metabolism to acetic acid accumulation in a complex range of extracellular conditions. Our results show that substantial acidification of the intracellular environment, resulting from accumulation of dissociated acetic acid in the cytosol, is required for acetic acid toxicity, which creates a state of energy deficiency and nutrient starvation.

The DELLA Protein SLR1 Integrates and Amplifies Salicylic Acid- and Jasmonic Acid-Dependent Innate Immunity in Rice1

Science.gov (United States)

De Vleesschauwer, David; Seifi, Hamed Soren; Haeck, Ashley; Huu, Son Nguyen; Demeestere, Kristof

2016-01-01

Gibberellins are a class of tetracyclic plant hormones that are well known to promote plant growth by inducing the degradation of a class of nuclear growth-repressing proteins, called DELLAs. In recent years, GA and DELLAs are also increasingly implicated in plant responses to pathogen attack, although our understanding of the underlying mechanisms is still limited, especially in monocotyledonous crop plants. Aiming to further decipher the molecular underpinnings of GA- and DELLA-modulated plant immunity, we studied the dynamics and impact of GA and DELLA during infection of the model crop rice (Oryza sativa) with four different pathogens exhibiting distinct lifestyles and infection strategies. Opposite to previous findings in Arabidopsis (Arabidopsis thaliana), our findings reveal a prominent role of the DELLA protein Slender Rice1 (SLR1) in the resistance toward (hemi)biotrophic but not necrotrophic rice pathogens. Moreover, contrary to the differential effect of DELLA on the archetypal defense hormones salicylic acid (SA) and jasmonic acid (JA) in Arabidopsis, we demonstrate that the resistance-promoting effect of SLR1 is due at least in part to its ability to boost both SA- and JA-mediated rice defenses. In a reciprocal manner, we found JA and SA treatment to interfere with GA metabolism and stabilize SLR1. Together, these findings favor a model whereby SLR1 acts as a positive regulator of hemibiotroph resistance in rice by integrating and amplifying SA- and JA-dependent defense signaling. Our results highlight the differences in hormone defense networking between rice and Arabidopsis and underscore the importance of GA and DELLA in molding disease outcomes. PMID:26829979

A Molecular and Cellular Context-Dependent Role for Ir76b in Detection of Amino Acid Taste

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Anindya Ganguly

2017-01-01

Full Text Available Amino acid taste is expected to be a universal property among animals. Although sweet, bitter, salt, and water tastes have been well characterized in insects, the mechanisms underlying amino acid taste remain elusive. From a Drosophila RNAi screen, we identify an ionotropic receptor, Ir76b, as necessary for yeast preference. Using calcium imaging, we identify Ir76b+ amino acid taste neurons in legs, overlapping partially with sweet neurons but not those that sense other tastants. Ir76b mutants have reduced responses to amino acids, which are rescued by transgenic expression of Ir76b and a mosquito ortholog AgIr76b. Co-expression of Ir20a with Ir76b is sufficient for conferring amino acid responses in sweet-taste neurons. Notably, Ir20a also serves to block salt response of Ir76b. Our study establishes the role of a highly conserved receptor in amino acid taste and suggests a mechanism for mutually exclusive roles of Ir76b in salt- and amino-acid-sensing neurons.

Fibroblast Cluster Formation on 3D Collagen Matrices Requires Cell Contraction-Dependent Fibronectin Matrix Organization

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da Rocha-Azevedo, Bruno; Ho, Chin-Han; Grinnell, Frederick

2012-01-01

Fibroblasts incubated on 3D collagen matrices in serum or lysophosphatidic acid (LPA)-containing medium self-organize into clusters through a mechanism that requires cell contraction. However, in platelet-derived growth factor (PDGF)-containing medium, cells migrate as individuals and do not form clusters even though they constantly encounter each other. Here, we present evidence that a required function of cell contraction in clustering is formation of fibronectin fibrillar matrix. We found that in serum or LPA but not in PDGF or basal medium, cells organized FN (both serum and cellular) into a fibrillar, detergent-insoluble matrix. Cell clusters developed concomitant with FN matrix formation. FN fibrils accumulated beneath cells and along the borders of cell clusters in regions of cell-matrix tension. Blocking Rho kinase or myosin II activity prevented FN matrix assembly and cell clustering. Using siRNA silencing and function-blocking antibodies and peptides, we found that cell clustering and FN matrix assembly required α5β1 integrins and fibronectin. Cells were still able to exert contractile force and compact the collagen matrix under the latter conditions, which showed that contraction was not sufficient for cell clustering to occur. Our findings provide new insights into how procontractile (serum/LPA) and promigratory (PDGF) growth factor environments can differentially regulate FN matrix assembly by fibroblasts interacting with collagen matrices and thereby influence mesenchymal cell morphogenetic behavior under physiologic circumstances such as wound repair, morphogenesis and malignancy. PMID:23117111

Fibroblast cluster formation on 3D collagen matrices requires cell contraction dependent fibronectin matrix organization.

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da Rocha-Azevedo, Bruno; Ho, Chin-Han; Grinnell, Frederick

2013-02-15

Fibroblasts incubated on 3D collagen matrices in serum or lysophosphatidic acid (LPA)-containing medium self-organize into clusters through a mechanism that requires cell contraction. However, in platelet-derived growth factor (PDGF)-containing medium, cells migrate as individuals and do not form clusters even though they constantly encounter each other. Here, we present evidence that a required function of cell contraction in clustering is formation of fibronectin (FN) fibrillar matrix. We found that in serum or LPA but not in PDGF or basal medium, cells organized FN (both serum and cellular) into a fibrillar, detergent-insoluble matrix. Cell clusters developed concomitant with FN matrix formation. FN fibrils accumulated beneath cells and along the borders of cell clusters in regions of cell-matrix tension. Blocking Rho kinase or myosin II activity prevented FN matrix assembly and cell clustering. Using siRNA silencing and function-blocking antibodies and peptides, we found that cell clustering and FN matrix assembly required α5β1 integrins and fibronectin. Cells were still able to exert contractile force and compact the collagen matrix under the latter conditions, which showed that contraction was not sufficient for cell clustering to occur. Our findings provide new insights into how procontractile (serum/LPA) and promigratory (PDGF) growth factor environments can differentially regulate FN matrix assembly by fibroblasts interacting with collagen matrices and thereby influence mesenchymal cell morphogenetic behavior under physiologic circumstances such as wound repair, morphogenesis and malignancy. Copyright © 2012 Elsevier Inc. All rights reserved.

Jasmonic acid and salicylic acid activate a common defense system in rice.

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Tamaoki, Daisuke; Seo, Shigemi; Yamada, Shoko; Kano, Akihito; Miyamoto, Ayumi; Shishido, Hodaka; Miyoshi, Seika; Taniguchi, Shiduku; Akimitsu, Kazuya; Gomi, Kenji

2013-06-01

Jasmonic acid (JA) and salicylic acid (SA) play important roles in plant defense systems. JA and SA signaling pathways interact antagonistically in dicotyledonous plants, but, the status of crosstalk between JA and SA signaling is unknown in monocots. Our rice microarray analysis showed that more than half of the genes upregulated by the SA analog BTH are also upregulated by JA, suggesting that a major portion of the SA-upregulated genes are regulated by JA-dependent signaling in rice. A common defense system that is activated by both JA and SA is thus proposed which plays an important role in pathogen defense responses in rice.

A nuclear-receptor-dependent phosphatidylcholine pathway with antidiabetic effects.

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Lee, Jae Man; Lee, Yoon Kwang; Mamrosh, Jennifer L; Busby, Scott A; Griffin, Patrick R; Pathak, Manish C; Ortlund, Eric A; Moore, David D

2011-05-25

Nuclear hormone receptors regulate diverse metabolic pathways and the orphan nuclear receptor LRH-1 (also known as NR5A2) regulates bile acid biosynthesis. Structural studies have identified phospholipids as potential LRH-1 ligands, but their functional relevance is unclear. Here we show that an unusual phosphatidylcholine species with two saturated 12 carbon fatty acid acyl side chains (dilauroyl phosphatidylcholine (DLPC)) is an LRH-1 agonist ligand in vitro. DLPC treatment induces bile acid biosynthetic enzymes in mouse liver, increases bile acid levels, and lowers hepatic triglycerides and serum glucose. DLPC treatment also decreases hepatic steatosis and improves glucose homeostasis in two mouse models of insulin resistance. Both the antidiabetic and lipotropic effects are lost in liver-specific Lrh-1 knockouts. These findings identify an LRH-1 dependent phosphatidylcholine signalling pathway that regulates bile acid metabolism and glucose homeostasis.

Metal extraction by alkyl substituted diphosphonic acids. Part 1. P,P'-Di(2-ethylhexyl) methanediphosphonic acid

International Nuclear Information System (INIS)

Chiarizia, R.; Horwitz, E.P.; Rickert, P.G.; Herlinger, A.W.

1996-01-01

Two novel extractants, p,p'-di(2-ethylhexyl) methanediphosphonic acid (H 2 DEH[MDP]) and p,p'-dioctyl methanediphosphonic acid (H 2 DO[MDP]) have been synthesized at high purity and yield. H 2 DEH[MDP] was selected for metal extraction studies because of its better physical properties. An investigation of the extraction of alkaline earth cations, Fe(111) and representative tri-, tetra- and hexavalent actinide ions from nitric acid solutions into o-xylene solutions of H 2 DEH[MDP] at different concentrations was performed. With a few exceptions, the acid dependencies of the extraction of the above metal species strongly resembles those measured in the uptake of the same metals by the chelating ion exchange resin Diphonix R , which contains gem-diphosphonic acid groups chemically attached to a polymeric matrix. The almost lack of acid dependency observed with Fe(III) and tetra- and hexavalent actinides indicates that these ions are chelated by H 2 DEH[MDP] mostly through the P=O groups of the extractant. With Fe(111) and the actinides, variable slopes of the extractant dependencies were measured, their values being strongly dependent on the acidity of the aqueous phase. H 2 DEH[MDP] possesses an extraordinary affinity for actinides and Fe(111). 26 refs., 7 figs

On dependence of stability of lanthanum complexes with aminopolycarboxylic acids on the complex structure

International Nuclear Information System (INIS)

Poluehktov, N.S.; Meshkova, S.B.; Danilkovich, M.M.; Topilova, Z.M.

1985-01-01

Regularities in changes of stability constants of lanthanum complexes with aminopolycarboxylic acids (APA) versus their structure are studied, The stability of lathanum-APA complexes depends mainly on the number of carboxyl groups in a ligand molecule. At that, the highest stability constant is characteristic of a complex with a ligand, containing 3 nitrogen atoms and 5 carboxyl groups, in the presenoe of which the lanthanum ion coordination sphere gets satupated. The oxyethy group introduction into a ligand molecule also improves the lanthanum complex stability but to a lesser degree than during the introduction of a carboxyl group. The number of nitrogen atoms in a ligand polecule affects insignificantly the complex stability constant value, and the elongation of a chain of CH 2 groups, separating nitrogen atoms, reduces the constant to a -0.6 power

Activity-Dependent Bidirectional Regulation of GAD Expression in a Homeostatic Fashion Is Mediated by BDNF-Dependent and Independent Pathways

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Hanno-Iijima, Yoko; Tanaka, Masami; Iijima, Takatoshi

2015-01-01

Homeostatic synaptic plasticity, or synaptic scaling, is a mechanism that tunes neuronal transmission to compensate for prolonged, excessive changes in neuronal activity. Both excitatory and inhibitory neurons undergo homeostatic changes based on synaptic transmission strength, which could effectively contribute to a fine-tuning of circuit activity. However, gene regulation that underlies homeostatic synaptic plasticity in GABAergic (GABA, gamma aminobutyric) neurons is still poorly understood. The present study demonstrated activity-dependent dynamic scaling in which NMDA-R (N-methyl-D-aspartic acid receptor) activity regulated the expression of GABA synthetic enzymes: glutamic acid decarboxylase 65 and 67 (GAD65 and GAD67). Results revealed that activity-regulated BDNF (brain-derived neurotrophic factor) release is necessary, but not sufficient, for activity-dependent up-scaling of these GAD isoforms. Bidirectional forms of activity-dependent GAD expression require both BDNF-dependent and BDNF-independent pathways, both triggered by NMDA-R activity. Additional results indicated that these two GAD genes differ in their responsiveness to chronic changes in neuronal activity, which could be partially caused by differential dependence on BDNF. In parallel to activity-dependent bidirectional scaling in GAD expression, the present study further observed that a chronic change in neuronal activity leads to an alteration in neurotransmitter release from GABAergic neurons in a homeostatic, bidirectional fashion. Therefore, the differential expression of GAD65 and 67 during prolonged changes in neuronal activity may be implicated in some aspects of bidirectional homeostatic plasticity within mature GABAergic presynapses. PMID:26241953

Salicylic acid modulates levels of phosphoinositide dependent-phospholipase C substrates and products to remodel the Arabidopsis suspension cell transcriptome

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Eric eRuelland

2014-11-01

Full Text Available Basal phosphoinositide-dependent phospholipase C (PI-PLC activity controls gene expression in Arabidopsis suspension cells and seedlings. PI-PLC catalyzes the production of phosphorylated inositol and diacylglycerol (DAG from phosphoinositides. It is not known how PI-PLC regulates the transcriptome although the action of DAG-kinase (DGK on DAG immediately downstream from PI-PLC is responsible for some of the regulation. We previously established a list of genes whose expression is affected in the presence of PI-PLC inhibitors. Here this list of genes was used as a signature in similarity searches of curated plant hormone response transcriptome data. The strongest correlations obtained with the inhibited PI-PLC signature were with salicylic acid (SA treatments. We confirm here that in Arabidopsis suspension cells SA treatment leads to an increase in phosphoinositides, then demonstrate that SA leads to a significant 20% decrease in phosphatidic acid, indicative of a decrease in PI-PLC products. Previous sets of microarray data were re-assessed. The SA response of one set of genes was dependent on phosphoinositides. Alterations in the levels of a second set of genes, mostly SA-repressed genes, could be related to decreases in PI-PLC products that occur in response to SA action. Together, the two groups of genes comprise at least 40% of all SA-responsive genes. Overall these two groups of genes are distinct in the functional categories of the proteins they encode, their promoter cis-elements and their regulation by DGK or phospholipase D. SA-regulated genes dependent on phosphoinositides are typical SA response genes while those with an SA response that is possibly dependent on PI-PLC products are less SA-specific. We propose a model in which SA inhibits PI-PLC activity and alters levels of PI-PLC products and substrates, thereby regulating gene expression divergently.

Humidity-dependent compression-induced glass transition of the air-water interfacial Langmuir films of poly(D,L-lactic acid-ran-glycolic acid) (PLGA).

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Kim, Hyun Chang; Lee, Hoyoung; Jung, Hyunjung; Choi, Yun Hwa; Meron, Mati; Lin, Binhua; Bang, Joona; Won, You-Yeon

2015-07-28

Constant rate compression isotherms of the air-water interfacial Langmuir films of poly(D,L-lactic acid-ran-glycolic acid) (PLGA) show a distinct feature of an exponential increase in surface pressure in the high surface polymer concentration regime. We have previously demonstrated that this abrupt increase in surface pressure is linked to the glass transition of the polymer film, but the detailed mechanism of this process is not fully understood. In order to obtain a molecular-level understanding of this behavior, we performed extensive characterizations of the surface mechanical, structural and rheological properties of Langmuir PLGA films at the air-water interface, using combined experimental techniques including the Langmuir film balance, X-ray reflectivity and double-wall-ring interfacial rheometry methods. We observed that the mechanical and structural responses of the Langmuir PLGA films are significantly dependent on the rate of film compression; the glass transition was induced in the PLGA film only at fast compression rates. Surprisingly, we found that this deformation rate dependence is also dependent on the humidity of the environment. With water acting as a plasticizer for the PLGA material, the diffusion of water molecules through the PLGA film seems to be the key factor in the determination of the glass transformation properties and thus the mechanical response of the PLGA film against lateral compression. Based on our combined results, we hypothesize the following mechanism for the compression-induced glass transformation of the Langmuir PLGA film; (1) initially, a humidified/non-glassy PLGA film is formed in the full surface-coverage region (where the surface pressure shows a plateau) during compression; (2) further compression leads to the collapse of the PLGA chains and the formation of new surfaces on the air side of the film, and this newly formed top layer of the PLGA film is transiently glassy in character because the water evaporation rate

Molecular Characterization of Two Lysophospholipid:acyl-CoA Acyltransferases Belonging to the MBOAT Family in Nicotiana benthamiana.

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Donghui Zhang

Full Text Available In the remodeling pathway for the synthesis of phosphatidylcholine (PC, acyl-CoA-dependent lysophosphatidylcholine (lysoPC acyltransferase (LPCAT catalyzes the reacylation of lysoPC. A number of genes encoding LPCATs have been cloned and characterized from several plants in recent years. Using Arabidopsis and other plant LPCAT sequences to screen the genome database of Nicotiana benthamiana, we identified two cDNAs encoding the putative tobacco LPCATs (NbLPCAT1 and NbLPCAT2. Both of them were predicted to encode a protein of 463 amino acids with high similarity to LPCATs from other plants. Protein sequence features such as the presence of at least eight putative transmembrane regions, four highly conserved signature motifs and several invariant residues indicate that NbLPCATs belong to the membrane bound O-acyltransferase family. Lysophospholipid acyltransferase activity of NbLPCATs was confirmed by testing lyso-platelet-activating factor (lysoPAF sensitivity through heterologous expression of each full-length cDNA in a yeast mutant Y02431 (lca1△ disrupted in endogenous LPCAT enzyme activity. Analysis of fatty acid profiles of phospholipids from the NbLPCAT-expressing yeast mutant Y02431 cultures supplemented with polyunsaturated fatty acids suggested more incorporation of linoleic acid (18:2n6, LA and α-linolenic acid (18:3n3, ALA into PC compared to yeast mutant harbouring empty vector. In vitro enzymatic assay demonstrated that NbLPCAT1had high lysoPC acyltransferase activity with a clear preference for α-linolenoyl-CoA (18:3, while NbLPCAT2 showed a high lysophosphatidic acid (lysoPA acyltransferase activity towards α-linolenoyl-CoA and a weak lysoPC acyltransferase activity. Tissue-specific expression analysis showed a ubiquitous expression of NbLPCAT1 and NbLPCAT2 in roots, stems, leaves, flowers and seeds, and a strong expression in developing flowers. This is the first report on the cloning and characterization of lysophospholipid

A study of pH-dependent photodegradation of amiloride by a multivariate curve resolution approach to combined kinetic and acid-base titration UV data.

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De Luca, Michele; Ioele, Giuseppina; Mas, Sílvia; Tauler, Romà; Ragno, Gaetano

2012-11-21

Amiloride photostability at different pH values was studied in depth by applying Multivariate Curve Resolution Alternating Least Squares (MCR-ALS) to the UV spectrophotometric data from drug solutions exposed to stressing irradiation. Resolution of all degradation photoproducts was possible by simultaneous spectrophotometric analysis of kinetic photodegradation and acid-base titration experiments. Amiloride photodegradation showed to be strongly dependent on pH. Two hard modelling constraints were sequentially used in MCR-ALS for the unambiguous resolution of all the species involved in the photodegradation process. An amiloride acid-base system was defined by using the equilibrium constraint, and the photodegradation pathway was modelled taking into account the kinetic constraint. The simultaneous analysis of photodegradation and titration experiments revealed the presence of eight different species, which were differently distributed according to pH and time. Concentration profiles of all the species as well as their pure spectra were resolved and kinetic rate constants were estimated. The values of rate constants changed with pH and under alkaline conditions the degradation pathway and photoproducts also changed. These results were compared to those obtained by LC-MS analysis from drug photodegradation experiments. MS analysis allowed the identification of up to five species and showed the simultaneous presence of more than one acid-base equilibrium.

Bimodal voltage dependence of TRPA1: mutations of a key pore helix residue reveal strong intrinsic voltage-dependent inactivation.

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Wan, Xia; Lu, Yungang; Chen, Xueqin; Xiong, Jian; Zhou, Yuanda; Li, Ping; Xia, Bingqing; Li, Min; Zhu, Michael X; Gao, Zhaobing

2014-07-01

Transient receptor potential A1 (TRPA1) is implicated in somatosensory processing and pathological pain sensation. Although not strictly voltage-gated, ionic currents of TRPA1 typically rectify outwardly, indicating channel activation at depolarized membrane potentials. However, some reports also showed TRPA1 inactivation at high positive potentials, implicating voltage-dependent inactivation. Here we report a conserved leucine residue, L906, in the putative pore helix, which strongly impacts the voltage dependency of TRPA1. Mutation of the leucine to cysteine (L906C) converted the channel from outward to inward rectification independent of divalent cations and irrespective to stimulation by allyl isothiocyanate. The mutant, but not the wild-type channel, displayed exclusively voltage-dependent inactivation at positive potentials. The L906C mutation also exhibited reduced sensitivity to inhibition by TRPA1 blockers, HC030031 and ruthenium red. Further mutagenesis of the leucine to all natural amino acids individually revealed that most substitutions at L906 (15/19) resulted in inward rectification, with exceptions of three amino acids that dramatically reduced channel activity and one, methionine, which mimicked the wild-type channel. Our data are plausibly explained by a bimodal gating model involving both voltage-dependent activation and inactivation of TRPA1. We propose that the key pore helix residue, L906, plays an essential role in responding to the voltage-dependent gating.

Jaundice associated pruritis: a review of pathophysiology and treatment.

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Bassari, Ramez; Koea, Jonathan B

2015-02-07

To review the underlying pathophysiology and currently available treatments for pruritis associated with jaundice. English language literature was reviewed using MEDLINE, PubMed, EMBASE and clinicaltrials.gov for papers and trails addressing the pathophysiology and potential treatments for pruritis associated with jaundice. Recent advances in the understanding of the peripheral anatomy of itch transmission have defined a histamine stimulated pathway and a cowhage stimulated pathway with sensation conveyed centrally via the contralateral spinothalamic tract. Centrally, cowhage and histamine stimulated neurons terminate widely within the thalamus and sensorimotor cortex. The causative factors for itch in jaundice have not been clarified although endogenous opioids, serotonin, steroid and lysophosphatidic acid all play a role. Current guidelines for the treatment of itching in jaundice recommend initial management with biliary drainage where possible and medical management with ursodeoxycholic acid, followed by cholestyramine, rifampicin, naltrexone and sertraline. Other than biliary drainage no single treatment has proved universally effective. Pruritis associated with jaundice is a common but poorly understood condition for which biliary drainage is the most effective therapy. Pharmacological therapy has advanced but remains variably effective.

Response of tobacco to the Pseudomonas syringae pv. Tomato DC3000 is mainly dependent on salicylic acid signaling pathway.

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Liu, Yang; Wang, Li; Cai, Guohua; Jiang, Shanshan; Sun, Liping; Li, Dequan

2013-07-01

Pseudomonas syringae pv. Tomato DC3000 (Pst DC3000) was the first pathogen to be demonstrated to infect Arabidopsis and to cause disease symptoms in the laboratory setting. However, the defense response to Pst DC3000 was unclear in tobacco. In this report, the expression profiles of twelve defense response-related genes were analyzed after treatment with salicylic acid (SA), jasmonic acid (JA), and pathogen Pst DC3000 by qRT-PCR. According to our results, it could be presented that the genes primarily induced by SA were also induced to higher levels after Pst DC3000 infection. SA accumulation could be induced to a higher level than that of JA after Pst DC3000 infection. In addition, SA could result in hypersensitive response (HR), which did not completely depend on accumulation of reactive oxygen species. These results indicated that tobacco mainly depended on SA signaling pathway rather than on JA signaling pathway in response to Pst DC3000. Further study demonstrated that JA could significantly inhibit the accumulation of SA and the generation of the HR induced by Pst DC3000. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Effects of kainic acid lesions in lateral geniculate nucleus: activity dependence of retrograde axonal transport of fluorescent dyes.

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Woodward, W R; Coull, B M

1988-06-28

Kainic acid lesions in the dorsal lateral geniculate nucleus of rats block the retrograde axonal transport of fluorescent dyes in corticogeniculate neurons without affecting the retrograde transport of D-aspartate or the orthograde transport of radiolabelled proteins in these neurons. This blocking of dye transport does not appear to be a consequence of kainic acid-induced damage to axon terminals in the geniculate since retinal ganglion cells are still able to transport dyes retrograde. A more likely explanation for these results is that fluorescent dye transport requires electrical activity in neurons, and elimination of the geniculate afferents to visual cortex reduces impulse traffic in cortical output fibers to a level below that required to support detectable dye transport. This interpretation is supported by the observation that kainic acid lesions also reduce retrograde transport of dyes in cortical neurons which project to the superior colliculus. Electrical stimulation in the subcortical white matter restores the transport of dye compounds in corticogeniculate neurons: evidence consistent with an activity-dependent mechanism of retrograde transport for these substances. These results provide evidence that axon terminals of retinal ganglion cells and corticogeniculate neurons survive in kainate-lesioned geniculates and are capable of normal neuronal function.

Role of sialic acid in synaptosomal transport of amino acid transmitters

International Nuclear Information System (INIS)

Zaleska, M.M.; Erecinska, M.

1987-01-01

Active, high-affinity, sodium-dependent uptake of [ 14 C]-aminobutyric acid and of the acidic amino acid D-[ 3 H]-aspartate was inhibited by pretreatment of synaptosomes with neuraminidase from Vibrio cholerae. Inhibition was of a noncompetitive type and was related to the amount of sialic acid released. The maximum accumulation ratios of both amino acids (intracellular [amino acid]/extracellular [amino acid]) remained largely unaltered. Treatment with neuraminidase affected neither the synaptosomal energy levels nor the concentration of internal potassium. It is suggested that the γ-aminobutyric acid and acidic amino acid transporters are glycosylated and that sialic acid is involved in the operation of the carrier proteins directly and not through modification of driving forces responsible for amino acid uptake

Differences in chemical properties of humic acids depending on their origin

Energy Technology Data Exchange (ETDEWEB)

Kribek, B

1978-01-01

Humic acids of soils are characterized by high content of nitrogen and ash and low molecular mass. The molecular mass of humic acids of peat is high, while the nitrogen and ash residue in them are small. The humic acids of dirt occupy an intermediate position. For the caustobioliths, a high content of carbon, low nitrogen and ash residue and demethylation of the structure are characteristic. Differences in the chemical properties of humic acids are determined mainly by differences in the chemical composition of fractions with high molecular weight. The specific nature of chemical properties of the humic substances governed by the origin makes it possible to use them to solve questions of paleogeography and to investigate the degree of diagenesis of coals.

Sequential injection redox or acid-base titration for determination of ascorbic acid or acetic acid.

Science.gov (United States)

Lenghor, Narong; Jakmunee, Jaroon; Vilen, Michael; Sara, Rolf; Christian, Gary D; Grudpan, Kate

2002-12-06

Two sequential injection titration systems with spectrophotometric detection have been developed. The first system for determination of ascorbic acid was based on redox reaction between ascorbic acid and permanganate in an acidic medium and lead to a decrease in color intensity of permanganate, monitored at 525 nm. A linear dependence of peak area obtained with ascorbic acid concentration up to 1200 mg l(-1) was achieved. The relative standard deviation for 11 replicate determinations of 400 mg l(-1) ascorbic acid was 2.9%. The second system, for acetic acid determination, was based on acid-base titration of acetic acid with sodium hydroxide using phenolphthalein as an indicator. The decrease in color intensity of the indicator was proportional to the acid content. A linear calibration graph in the range of 2-8% w v(-1) of acetic acid with a relative standard deviation of 4.8% (5.0% w v(-1) acetic acid, n=11) was obtained. Sample throughputs of 60 h(-1) were achieved for both systems. The systems were successfully applied for the assays of ascorbic acid in vitamin C tablets and acetic acid content in vinegars, respectively.

Studies of the pH dependence of 13C shifts and carbon-carbon coupling constants of [U-13C]aspartic and -glutamic acids

International Nuclear Information System (INIS)

London, R.E.; Walker, T.E.; Kollman, V.H.; Matwiyoff, N.A.

1978-01-01

13 C NMR studies of the chemical shifts and carbon--carbon spin--spin coupling constants of 90% [U- 13 C]aspartic and -glutamic acids are reported. Effects of titration of the two carboxyl groups are separated computationally and the results compared with those for asparagine and glutamine, aspartate and glutamate containing peptides, and a series of amino-n-butyric acids. The results indicate that the carboxyl carbon shift resulting from titration of the carboxyl group is strongly dependent on its distance (number of bonds) from an amino group. Alternatively, remote methyl groups exhibit a much smaller titration induced shift than carboxyl groups in the corresponding position. Significant remote effects of pH titration on the one-bond carbon-carbon coupling are also observed, particularly for couplings involving the side-chain carboxyl carbons. These results are discussed in terms of polarization of the C--O bonds in response to titration of a remote carboxyl group. Values of 3 J/sub CC/ in asparate and glutamate indicate a strong conformational dependence. Rotamer populations predicted on the basis of the observed couplings and theoretical INDO calculations are in good agreement with values based on analysis of the 3 J/sub HH/ and 3 J/sub CH/ couplings. For a given conformation of glutamic acid, it is found that 3 J 14 is considerably smaller than 3 J 25 . This result is consistent with obsrvations on a number of other 13 C-labeled amino acids. 5 figures, 4 tables

Role of gallic and p-coumaric acids in the AHL-dependent expression of flgA gene and in the process of biofilm formation in food-associated Pseudomonas fluorescens KM120.

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Myszka, Kamila; Schmidt, Marcin T; Białas, Wojciech; Olkowicz, Mariola; Leja, Katarzyna; Czaczyk, Katarzyna

2016-09-01

In the process of Pseudomonas fluorescens biofilm formation, N-acyl-l-homoserine lactone (AHL)-mediated flagella synthesis plays a key role. Inhibition of AHL production may attenuate P. fluorescens biofilm on solid surfaces. This work validated the anti-biofilm properties of p-coumaric and gallic acids via the ability of phenolics to suppress AHL synthesis in P. fluorescens KM120. The dependence between synthesis of AHL molecules, expression of flagella gene (flgA) and the ability of biofilm formation by P. fluorescens KM120 on a stainless steel surface (type 304L) was also investigated. Research was carried out in a purpose-built flow cell device. Limitations on AHL synthesis in P. fluorescens KM120 were observed at concentrations of 120 and 240 µmol L(-1) of phenolic acids in medium. At such levels of gallic and p-coumaric acids the ability of P. fluorescens KM120 to synthesize 3-oxo-C6-homoserine lactone (HSL) was not observed. These concentrations caused decreased expression of flgA gene in P. fluorescens KM120. The changes in expression of AHL-dependent flgA gene significantly decreased the rate of microorganism colonization on the stainless steel surface. Phenolic acids are able to inhibit biofilm formation. The results obtained in the work may help to develop alternative techniques for anti-biofilm treatment in the food industry. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

HIC1 links retinoic acid signalling to group 3 innate lymphoid cell-dependent regulation of intestinal immunity and homeostasis

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Antignano, Frann; Korinek, Vladimir; Underhill, T. Michael

2018-01-01

The intestinal immune system must be able to respond to a wide variety of infectious organisms while maintaining tolerance to non-pathogenic microbes and food antigens. The Vitamin A metabolite all-trans-retinoic acid (atRA) has been implicated in the regulation of this balance, partially by regulating innate lymphoid cell (ILC) responses in the intestine. However, the molecular mechanisms of atRA-dependent intestinal immunity and homeostasis remain elusive. Here we define a role for the transcriptional repressor Hypermethylated in cancer 1 (HIC1, ZBTB29) in the regulation of ILC responses in the intestine. Intestinal ILCs express HIC1 in a vitamin A-dependent manner. In the absence of HIC1, group 3 ILCs (ILC3s) that produce IL-22 are lost, resulting in increased susceptibility to infection with the bacterial pathogen Citrobacter rodentium. Thus, atRA-dependent expression of HIC1 in ILC3s regulates intestinal homeostasis and protective immunity. PMID:29470558

Kinetic Studies on the Oxidation of Some para and meta-Substituted Cinnamic Acids by Pyridinium Bromochromate in the Presence of Oxalic Acid (A Co-oxidation Study

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G. Vanangamudi

2009-01-01

Full Text Available The kinetics of oxidation of cinnamic acids by pyridinium bromochromate (PBC in the presence of oxalic acid has been studied in acetic acid-water (60:40% medium. The reaction shows unit order dependence each with respect to oxidant as well as oxalic acid [OX], the order with respect to [H+] and [CA] are fractional. The reaction is acid catalyzed and a low dielectric constant favours the reaction. Increase the ionic strength has no effect on the reaction rate. In the case of substituted cinnamic acids the order with respect to substrate vary depending upon the nature of the substituent present in the ring. In general, the electron withdrawing substituents retard the rate while the electron releasing substituents enhance the rate of reaction. From the kinetic data obtained the activation parameters have been computed and a suitable mechanism has been proposed.

Inactivation of thiol-dependent enzymes by hypothiocyanous acid: role of sulfenyl thiocyanate and sulfenic acid intermediates

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Barrett, Tessa J.; Pattison, David I.; Leonard, Stephen E.; Carroll, Kate S.; Davies, Michael J.; Hawkins, Clare L.

2012-01-01

Myeloperoxidase (MPO) forms reactive oxidants including hypochlorous and hypothiocyanous acids (HOCl and HOSCN) under inflammatory conditions. HOCl causes extensive tissue damage and plays a role in the progression of many inflammatory-based diseases. Although HOSCN is a major MPO oxidant, particularly in smokers, who have elevated plasma thiocyanate, the role of this oxidant in disease is poorly characterized. HOSCN induces cellular damage by targeting thiols. However, the specific targets and mechanisms involved in this process are not well defined. We show that exposure of macrophages to HOSCN results in the inactivation of intracellular enzymes, including creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In each case, the active-site thiol residue is particularly sensitive to oxidation, with evidence for reversible inactivation and the formation of sulfenyl thiocyanate and sulfenic acid intermediates, on treatment with HOSCN (less than fivefold molar excess). Experiments with DAz-2, a cell-permeable chemical trap for sulfenic acids, demonstrate that these intermediates are formed on many cellular proteins, including GAPDH and CK, in macrophages exposed to HOSCN. This is the first direct evidence for the formation of protein sulfenic acids in HOSCN-treated cells and highlights the potential of this oxidant to perturb redox signaling processes. PMID:22248862

Perturbations of amino acid metabolism associated with glyphosate-dependent inhibition of shikimic acid metabolism affect cellular redox homeostasis and alter the abundance of proteins involved in photosynthesis and photorespiration.

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Vivancos, Pedro Diaz; Driscoll, Simon P; Bulman, Christopher A; Ying, Liu; Emami, Kaveh; Treumann, Achim; Mauve, Caroline; Noctor, Graham; Foyer, Christine H

2011-09-01

The herbicide glyphosate inhibits the shikimate pathway of the synthesis of amino acids such as phenylalanine, tyrosine, and tryptophan. However, much uncertainty remains concerning precisely how glyphosate kills plants or affects cellular redox homeostasis and related processes in glyphosate-sensitive and glyphosate-resistant crop plants. To address this issue, we performed an integrated study of photosynthesis, leaf proteomes, amino acid profiles, and redox profiles in the glyphosate-sensitive soybean (Glycine max) genotype PAN809 and glyphosate-resistant Roundup Ready Soybean (RRS). RRS leaves accumulated much more glyphosate than the sensitive line but showed relatively few changes in amino acid metabolism. Photosynthesis was unaffected by glyphosate in RRS leaves, but decreased abundance of photosynthesis/photorespiratory pathway proteins was observed together with oxidation of major redox pools. While treatment of a sensitive genotype with glyphosate rapidly inhibited photosynthesis and triggered the appearance of a nitrogen-rich amino acid profile, there was no evidence of oxidation of the redox pools. There was, however, an increase in starvation-associated and defense proteins. We conclude that glyphosate-dependent inhibition of soybean leaf metabolism leads to the induction of defense proteins without sustained oxidation. Conversely, the accumulation of high levels of glyphosate in RRS enhances cellular oxidation, possibly through mechanisms involving stimulation of the photorespiratory pathway.

Chemical mechanism of D-amino acid oxidase from Rhodotorula gracilis: pH dependence of kinetic parameters.

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Ramón, F; Castillón, M; De La Mata, I; Acebal, C

1998-01-01

The variation of kinetic parameters of d-amino acid oxidase from Rhodotorula gracilis with pH was used to gain information about the chemical mechanism of the oxidation of D-amino acids catalysed by this flavoenzyme. d-Alanine was the substrate used. The pH dependence of Vmax and Vmax/Km for alanine as substrate showed that a group with a pK value of 6.26-7.95 (pK1) must be unprotonated and a group with a pK of 10.8-9.90 (pK2) must be protonated for activity. The lower pK value corresponded to a group on the enzyme involved in catalysis and whose protonation state was not important for binding. The higher pK value was assumed to be the amino group of the substrate. Profiles of pKi for D-aspartate as competitive inhibitor showed that binding is prevented when a group on the enzyme with a pK value of 8.4 becomes unprotonated; this basic group was not detected in Vmax/Km profiles suggesting its involvement in binding of the beta-carboxylic group of the inhibitor. PMID:9461524

Dietary ascorbic acid and subsequent change in body weight and waist circumference: associations may depend on genetic predisposition to obesity - a prospective study of three independent cohorts

Science.gov (United States)

2014-01-01

Background Cross-sectional data suggests that a low level of plasma ascorbic acid positively associates with both Body Mass Index (BMI) and Waist Circumference (WC). This leads to questions about a possible relationship between dietary intake of ascorbic acid and subsequent changes in anthropometry, and whether such associations may depend on genetic predisposition to obesity. Hence, we examined whether dietary ascorbic acid, possibly in interaction with the genetic predisposition to a high BMI, WC or waist-hip ratio adjusted for BMI (WHR), associates with subsequent annual changes in weight (∆BW) and waist circumference (∆WC). Methods A total of 7,569 participants’ from MONICA, the Diet Cancer and Health study and the INTER99 study were included in the study. We combined 50 obesity associated single nucleotide polymorphisms (SNPs) in four genetic scores: a score of all SNPs and a score for each of the traits (BMI, WC and WHR) with which the SNPs associate. Linear regression was used to examine the association between ascorbic acid intake and ΔBW or ΔWC. SNP-score × ascorbic acid interactions were examined by adding product terms to the models. Results We found no significant associations between dietary ascorbic acid and ∆BW or ∆WC. Regarding SNP-score × ascorbic acid interactions, each additional risk allele of the 14 WHR associated SNPs associated with a ∆WC of 0.039 cm/year (P = 0.02, 95% CI: 0.005 to 0.073) per 100 mg/day higher ascorbic acid intake. However, the association to ∆WC only remained borderline significant after adjustment for ∆BW. Conclusion In general, our study does not support an association between dietary ascorbic acid and ∆BW or ∆WC, but a diet with a high content of ascorbic acid may be weakly associated to higher WC gain among people who are genetically predisposed to a high WHR. However, given the quite limited association any public health relevance is questionable. PMID:24886192

Effects of Fatty Acid Inclusion in a DMPC Bilayer Membrane

DEFF Research Database (Denmark)

Peters, Günther H.J.; Hansen, Flemming Yssing; Møller, Martin S.

2009-01-01

Free fatty acids in biomembranes have been proposed to be a central component in several cellular control and regulatory mechanisms. To elucidate some fundamental elements underlying this, we have applied molecular dynamics simulations and experimental density measurements to study the molecular...... packing and structure of oleic acid (HOA) and stearic acid (HSA) in fluid bilayers of dimyristoylphosphatidylcholine (DMPC). The experimental data show a small but consistent positive excess volume for fatty acid concentrations below 10 mol %. At higher concentrations the fatty acids mix ideally...... with fluid DMPC. The simulations, which were benchmarked against the densitometric data, revealed interesting differences in the structure and location of the fatty acids depending on their protonation status. Thus, the protonated (uncharged) acid is located rather deeply in the membrane with an average...

Amino Acid Availability Modulates Vacuolar H+-ATPase Assembly*

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Stransky, Laura A.; Forgac, Michael

2015-01-01

The vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump composed of a peripheral ATPase domain (V1) and a membrane-integral proton-translocating domain (V0) and is involved in many normal and disease processes. An important mechanism of regulating V-ATPase activity is reversible assembly of the V1 and V0 domains. Increased assembly in mammalian cells occurs under various conditions and has been shown to involve PI3K. The V-ATPase is necessary for amino acid-induced activation of mechanistic target of rapamycin complex 1 (mTORC1), which is important in controlling cell growth in response to nutrient availability and growth signals. The V-ATPase undergoes amino acid-dependent interactions with the Ragulator complex, which is involved in recruitment of mTORC1 to the lysosomal membrane during amino acid sensing. We hypothesized that changes in the V-ATPase/Ragulator interaction might involve amino acid-dependent changes in V-ATPase assembly. To test this, we measured V-ATPase assembly by cell fractionation in HEK293T cells treated with and without amino acids. V-ATPase assembly increases upon amino acid starvation, and this effect is reversed upon readdition of amino acids. Lysosomes from amino acid-starved cells possess greater V-ATPase-dependent proton transport, indicating that assembled pumps are catalytically active. Amino acid-dependent changes in both V-ATPase assembly and activity are independent of PI3K and mTORC1 activity, indicating the involvement of signaling pathways distinct from those implicated previously in controlling assembly. By contrast, lysosomal neutralization blocks the amino acid-dependent change in assembly and reactivation of mTORC1 after amino acid starvation. These results identify an important new stimulus for controlling V-ATPase assembly. PMID:26378229

Suppression of jasmonic acid-dependent defense in cotton plant by the mealybug Phenacoccus solenopsis.

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Pengjun Zhang

Full Text Available The solenopsis mealybug, Phenacoccus solenopsis, has been recently recognized as an aggressively invasive pest in China, and is now becoming a serious threat to the cotton industry in the country. Thus, it is necessary to investigate the molecular mechanisms employed by cotton for defending against P. solenopsis before the pest populations reach epidemic levels. Here, we examined the effects of exogenous jasmonic acid (JA, salicylic acid (SA, and herbivory treatments on feeding behavior and on development of female P. solenopsis. Further, we compared the volatile emissions of cotton plants upon JA, SA, and herbivory treatments, as well as the time-related changes in gossypol production and defense-related genes. Female adult P. solenopsis were repelled by leaves from JA-treated plant, but were not repelled by leaves from SA-treated plants. In contrast, females were attracted by leaves from plants pre-infested by P. solenopsis. The diverse feeding responses by P. solenopsis were due to the difference in volatile emission of plants from different treatments. Furthermore, we show that JA-treated plants slowed P. solenopsis development, but plants pre-infested by P. solenopsis accelerated its development. We also show that P. solenopsis feeding inhibited the JA-regulated gossypol production, and prevented the induction of JA-related genes. We conclude that P. solenopsis is able to prevent the activation of JA-dependent defenses associated with basal resistance to mealybugs.

Determination of the pK values of 5-aminosalicylic acid and N-acetylaminosalicylic acid and comparison of the pH dependent lipid-water partition coefficients of sulphasalazine and its metabolites.

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Allgayer, H; Sonnenbichler, J; Kruis, W; Paumgartner, G

1985-01-01

Sulphasalazine (SASP), used in the treatment of inflammatory bowel disease, is split into sulphapyridine (SP) and 5-aminosalicylic acid (5-ASA) in the colon. Lower plasma levels of SASP and 5-ASA as compared to those of SP may be due to different absorption rates from the colon because of different pK values and pH dependent lipid-water partition coefficients. In this study we determined the pK values of 5-ASA and its major metabolite, N-acetyl amino-salicylic acid (AcASA), by 13C-NMR spectroscopy and compared the pH dependent apparent benzene-water partition coefficients (Papp) of SASP, SP and 5-ASA with respect to their different plasma levels. The COOH group of 5-ASA had a pK value of 3.0, the -NH3+ group had 6.0, the -OH group 13.9; the -COOH group of AcASA had 2.7 and the -OH group 12.9; The Papp of SASP (0.042 +/- 0.004) and 5-ASA (0.059 +/- 0.01) were significantly lower than that of SP (0.092 +/- 0.03) (at pH 5.5).

Disinfection of wastewater with peracetic acid: a review.

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Kitis, Mehmet

2004-03-01

Peracetic acid is a strong disinfectant with a wide spectrum of antimicrobial activity. Due to its bactericidal, virucidal, fungicidal, and sporicidal effectiveness as demonstrated in various industries, the use of peracetic acid as a disinfectant for wastewater effluents has been drawing more attention in recent years. The desirable attributes of peracetic acid for wastewater disinfection are the ease of implementing treatment (without the need for expensive capital investment), broad spectrum of activity even in the presence of heterogeneous organic matter, absence of persistent toxic or mutagenic residuals or by-products, no quenching requirement (i.e., no dechlorination), small dependence on pH, short contact time, and effectiveness for primary and secondary effluents. Major disadvantages associated with peracetic acid disinfection are the increases of organic content in the effluent due to acetic acid (AA) and thus in the potential microbial regrowth (acetic acid is already present in the mixture and is also formed after peracetic acid decomposition). Another drawback to the use of peracetic acid is its high cost, which is partly due to limited production capacity worldwide. However, if the demand for peracetic acid increases, especially from the wastewater industry, the future mass production capacity might also be increased, thus lowering the cost. In such a case, in addition to having environmental advantages, peracetic acid may also become cost-competitive with chlorine.

Drosophila homologue of Diaphanous 1 (DIAPH1) controls the metastatic potential of colon cancer cells by regulating microtubule-dependent adhesion.

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Lin, Yuan-Na; Bhuwania, Ridhirama; Gromova, Kira; Failla, Antonio Virgilio; Lange, Tobias; Riecken, Kristoffer; Linder, Stefan; Kneussel, Matthias; Izbicki, Jakob R; Windhorst, Sabine

2015-07-30

Drosophila homologue of Diaphanous 1 (DIAPH1) regulates actin polymerization and microtubule (MT) stabilization upon stimulation with lysophosphatidic acid (LPA). Recently, we showed strongly reduced lung metastasis of DIAPH1-depleted colon cancer cells but we found accumulations of DIAPH1-depleted cells in bone marrow. Here, we analyzed possible organ- or tissue-specific metastasis of DIAPH1-depleted HCT-116 cells. Our data confirmed that depletion of DIAPH1 strongly inhibited lung metastasis and revealed that, in contrast to control cells, DIAPH1-depleted cells did not form metastases in further organs. Detailed mechanistic analysis on cells that were not stimulated with LPA to activate the cytoskeleton-modulating activity of DIAPH1, revealed that even under basal conditions DIAPH1 was essential for cellular adhesion to collagen. In non-stimulated cells DIAPH1 did not control actin dynamics but, interestingly, was essential for stabilization of microtubules (MTs). Additionally, DIAPH1 controlled directed vesicle trafficking and with this, local clustering of the adhesion protein integrin-β1 at the plasma membrane. Therefore, we conclude that under non-stimulating conditions DIAPH1 controls cellular adhesion by stabilizing MTs required for local clustering of integrin-β1 at the plasma membrane. Thus, blockade of DIAPH1-tubulin interaction may be a promising approach to inhibit one of the earliest steps in the metastatic cascade of colon cancer.

No Clinical or Biochemical Evidence for Essential Fatty Acid Deficiency in Home Patients Who Depend on Long-Term Mixed Olive Oil- and Soybean Oil-Based Parenteral Nutrition

NARCIS (Netherlands)

Olthof, E.D.; Roelofs, H.M.J.; Fisk, H.L.; Calder, P.C.; Wanten, G.J.A.

2016-01-01

BACKGROUND: Home parenteral nutrition (HPN) patients depend on lipid emulsions as part of their parenteral nutrition regimen to provide essential fatty acids (EFAs). Mixed-oil sources are used in modern lipid emulsions to decrease the amount of proinflammatory EFAs, mainly linoleic acid, which is

Stress Marker Signatures in Lesion Mimic Single and Double Mutants Identify a Crucial Leaf Age-Dependent Salicylic Acid Related Defense Signal.

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Kaurilind, Eve; Brosché, Mikael

2017-01-01

Plants are exposed to abiotic and biotic stress conditions throughout their lifespans that activates various defense programs. Programmed cell death (PCD) is an extreme defense strategy the plant uses to manage unfavorable environments as well as during developmentally induced senescence. Here we investigated the role of leaf age on the regulation of defense gene expression in Arabidopsis thaliana. Two lesion mimic mutants with misregulated cell death, catalase2 (cat2) and defense no death1 (dnd1) were used together with several double mutants to dissect signaling pathways regulating defense gene expression associated with cell death and leaf age. PCD marker genes showed leaf age dependent expression, with the highest expression in old leaves. The salicylic acid (SA) biosynthesis mutant salicylic acid induction deficient2 (sid2) had reduced expression of PCD marker genes in the cat2 sid2 double mutant demonstrating the importance of SA biosynthesis in regulation of defense gene expression. While the auxin- and jasmonic acid (JA)- insensitive auxin resistant1 (axr1) double mutant cat2 axr1 also led to decreased expression of PCD markers; the expression of several marker genes for SA signaling (ISOCHORISMATE SYNTHASE 1, PR1 and PR2) were additionally decreased in cat2 axr1 compared to cat2. The reduced expression of these SA markers genes in cat2 axr1 implicates AXR1 as a regulator of SA signaling in addition to its known role in auxin and JA signaling. Overall, the current study reinforces the important role of SA signaling in regulation of leaf age-related transcript signatures.

Single amino acids in the carboxyl terminal domain of aquaporin-1 contribute to cGMP-dependent ion channel activation

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Yool Andrea J

2003-10-01

Full Text Available Abstract Background Aquaporin-1 (AQP1 functions as an osmotic water channel and a gated cation channel. Activation of the AQP1 ion conductance by intracellular cGMP was hypothesized to involve the carboxyl (C- terminus, based on amino acid sequence alignments with cyclic-nucleotide-gated channels and cGMP-selective phosphodiesterases. Results Voltage clamp analyses of human AQP1 channels expressed in Xenopus oocytes demonstrated that the nitric oxide donor, sodium nitroprusside (SNP; 3–14 mM activated the ionic conductance response in a dose-dependent manner. Block of soluble guanylate cyclase prevented the response. Enzyme immunoassays confirmed a linear dose-dependent relationship between SNP and the resulting intracellular cGMP levels (up to 1700 fmol cGMP /oocyte at 14 mM SNP. Results here are the first to show that the efficacy of ion channel activation is decreased by mutations of AQP1 at conserved residues in the C-terminal domain (aspartate D237 and lysine K243. Conclusions These data support the idea that the limited amino acid sequence similarities found between three diverse classes of cGMP-binding proteins are significant to the function of AQP1 as a cGMP-gated ion channel, and provide direct evidence for the involvement of the AQP1 C-terminal domain in cGMP-mediated ion channel activation.

Fc-receptor-mediated phagocytosis is regulated by mechanical properties of the target

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Beningo, Karen A.; Wang, Yu-li

2002-01-01

Phagocytosis is an actin-based process used by macrophages to clear particles greater than 0.5 microm in diameter. In addition to its role in immunological responses, phagocytosis is also necessary for tissue remodeling and repair. To prevent catastrophic autoimmune reactions, phagocytosis must be tightly regulated. It is commonly assumed that the recognition/selection of phagocytic targets is based solely upon receptor-ligand binding. Here we report an important new criterion, that mechanical parameters of the target can dramatically affect the efficiency of phagocytosis. When presented with particles of identical chemical properties but different rigidity, macrophages showed a strong preference to engulf rigid objects. Furthermore, phagocytosis of soft particles can be stimulated with the microinjection of constitutively active Rac1 but not RhoA, and with lysophosphatidic acid, an agent known to activate the small GTP-binding proteins of the Rho family. These data suggest a Rac1-dependent mechanosensory mechanism for phagocytosis, which probably plays an important role in a number of physiological and pathological processes from embryonic development to autoimmune diseases.

Studies on the interference of hydrofluoric acid and phosphoric acid in the determination of uranium using Ti(III) reduction method-biamperometry end point

International Nuclear Information System (INIS)

Shiny, T.S.; Rajalakshmi, A.; Phal, D.G.; Charyulu, M.M.; Ramakumar, K.L.

2007-01-01

Accurate and precise determination of uranium in nuclear materials is necessary for chemical quality control as well as for nuclear material accounting purposes. Different types of uranium samples are received for the measurements. Depending upon the nature of the sample dissolution procedure is selected. Mixed oxide samples of uranium and plutonium, for example, are dissolved in nitric acid containing hydrofluoric acid under IR lamp. The fluoride ions are removed by repeated evaporation of the solution. However, some fluoride ions are left in the solutions depending on the conditions of evaporation. Uranium samples and alloy samples are dissolved in dilute hydrochloric acid. The rate of dissolution depends on concentration of acid. Sometimes a mixture of hydrochloric acid and hydrofluoric acid is used for the dissolution metal alloy samples, which may contain silica. Another method of dissolution of these samples is using a mixture of phosphoric acid and 1% hydrofluoric acid. It is necessary to study the interference of hydrofluoric acid and phosphoric acid on the determination of uranium

Amino acid analysis and cell cycle dependent phosphorylation of an H1-like, butyrate-enhanced protein (BEP; H10; IP25) from Chinese hamster cells

International Nuclear Information System (INIS)

D'Anna, J.A.; Gurley, L.R.; Becker, R.R.; Barham, S.S.; Tobey, R.A.; Walters, R.A.

1980-01-01

A fraction enriched in the butyrate-enhanced protein (BEP) has been isolated from Chinese hamster (line CHO) cells by perchloric acid extraction and Bio-Rex 70 chromatography. Amino acid analyses indicate that the composition of BEP resembles that of CHO H1; however, BEP contains 11% less alanine than H1, and, in contrast to H1, BEP contains methionine. Treatment of BEP with cyanogen bromide results in the cleavage of a small fragment of approx. 20 amino acids so that the large fragment seen in sodium dodecyl sulfate-acrylamide gels has a molecular weight of approx. 20,000. Radiolabeling and electrophoresis indicate that BEP is phosphorylated in a cell cycle dependent fashion. These data suggest that (1) BEP is a specialized histone of the H1 class and (2) BEP is the species equivalent of calf lung histone H1 0 , rat H1 0 , and IP 25 , a protein enhanced in differentiated Friend erythroleukemia cells. The data also indicate that putative HMG1 and HMG2 proteins do not undergo the extensive cell cycle dependent phosphorylations measured for histone H1 and BEP

A conserved aspartic acid is important for agonist (VUAA1 and odorant/tuning receptor-dependent activation of the insect odorant co-receptor (Orco.

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Brijesh N Kumar

Full Text Available Insect odorant receptors function as heteromeric odorant-gated cation channels comprising a conventional odorant-sensitive tuning receptor, and a conserved co-receptor (Orco. An Orco agonist, VUAA1, is able to activate both heteromeric and homomeric Orco-containing channels. Very little is known about specific residues in Orco that contribute to cation permeability and gating. We investigated the importance of two conserved Asp residues, one in each of transmembrane domains 5 and 7, for channel function by mutagenesis. Drosophila melanogaster Orco and its substitution mutants were expressed in HEK cells and VUAA1-stimulated channel activity was determined by Ca(2+ influx and whole-cell patch clamp electrophysiology. Substitution of D466 in transmembrane 7 with amino acids other than glutamic acid resulted in a substantial reduction in channel activity. The D466E Orco substitution mutant was ~2 times more sensitive to VUAA1. The permeability of the D466E Orco mutant to cations was unchanged relative to wild-type Orco. When D466E Orco is co-expressed with a conventional tuning odorant receptor, the heteromeric complex also shows increased sensitivity to an odorant. Thus, the effect of the D466E mutation is not specific to VUAA1 agonism or dependent on homomeric Orco assembly. We suggest the gain-of-activation characteristic of the D466E mutant identifies an amino acid that is likely to be important for activation of both heteromeric and homomeric insect odorant receptor channels.

Purification, properties, and N-terminal amino acid sequence of homogeneous Escherichia coli 2-amino-3-ketobutyrate CoA ligase, a pyridoxal phosphate-dependent enzyme.

Science.gov (United States)

Mukherjee, J J; Dekker, E E

1987-10-25

Starting with 100 g (wet weight) of a mutant of Escherichia coli K-12 forced to grow on L-threonine as sole carbon source, we developed a 6-step procedure that provides 30-40 mg of homogeneous 2-amino-3-ketobutyrate CoA ligase (also called aminoacetone synthetase or synthase). This ligase, which catalyzes the cleavage/condensation reaction between 2-amino-3-ketobutyrate (the presumed product of the L-threonine dehydrogenase-catalyzed reaction) and glycine + acetyl-CoA, has an apparent molecular weight approximately equal to 85,000 and consists of two identical (or nearly identical) subunits with Mr = 42,000. Computer analysis of amino acid composition data, which gives the best fit nearest integer ratio for each residue, indicates a total of 387 amino acids/subunit with a calculated Mr = 42,093. Stepwise Edman degradation provided the N-terminal sequence of the first 21 amino acids. It is a pyridoxal phosphate-dependent enzyme since (a) several carbonyl reagents caused greater than 90% loss of activity, (b) dialysis against buffer containing hydroxylamine resulted in 89% loss of activity coincident with an 86% decrease in absorptivity at 428 nm, (c) incubation of the apoenzyme with 20 microM pyridoxal phosphate showed a parallel recovery (greater than 90%) of activity and 428-nm absorptivity, and (d) reduction of the holoenzyme with NaBH4 resulted in complete inactivation, disappearance of a new absorption maximum at 333 nm. Strict specificity for glycine is shown but acetyl-CoA (100%), n-propionyl-CoA (127%), or n-butyryl-CoA (16%) is utilized in the condensation reaction. Apparent Km values for acetyl-CoA, n-propionyl-CoA, and glycine are 59 microM, 80 microM, and 12 mM, respectively; the pH optimum = 7.5. Added divalent metal ions or sulfhydryl compounds inhibited catalysis of the condensation reaction.

Ursolic acid mediates photosensitization by initiating mitochondrial-dependent apoptosis

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Lee, Yuan-Hao; Wang, Exing; Kumar, Neeru; Glickman, Randolph D.

2013-02-01

The signaling pathways PI3K/Akt and MAPK play key roles in transcription, translation and carcinogenesis, and may be activated by light exposure. These pathways may be modulated or inhibited by naturally-occurring compounds, such as the triterpenoid, ursolic acid (UA). Previously, the transcription factors p53 and NF-kB, which transactivate mitochondrial apoptosis-related genes, were shown to be differentially modulated by UA. Our current work indicates that UA causes these effects via the mTOR and insulin-mediated pathways. UA-modulated apoptosis, following exposure to UV radiation, is observed to correspond to differential levels of oxidative stress in retinal pigment epithelial (RPE) and skin melanoma (SM) cells. Flow cytometry analysis, DHE (dihydroethidium) staining and membrane permeability assay showed that UA pretreatment potentiated cell cycle arrest and radiation-induced apoptosis selectively on SM cells while DNA photo-oxidative damage (i.e. strand breakage) was reduced, presumably by some antioxidant activity of UA in RPE cells. The UA-mediated NF-κB activation in SM cells was reduced by rapamycin pretreatment, which indicates that these agents exert inter-antagonistic effects in the PI3K/Akt/mTOR pathway. In contrast, the antagonistic effect of UA on the PI3K/Akt pathway was reversed by insulin leading to greater NF-κB and p53 activation in RPE cells. MitoTracker, a mitochondrial functional assay, indicated that mitochondria in RPE cells experienced reduced oxidative stress while those in SM cells exhibited increased oxidative stress upon UA pretreatment. When rapamycin administration was followed by UA, mitochondrial oxidative stress was increased in RPE cells but decreased in SM cells. These results indicate that UA modulates p53 and NF-κB, initiating a mitogenic response to radiation that triggers mitochondria-dependent apoptosis.

Acyl-CoA-binding protein (ACBP) localizes to the endoplasmic reticulum and Golgi in a ligand-dependent manner in mammalian cells

DEFF Research Database (Denmark)

Hansen, Jesper S; Færgeman, Nils J; Kragelund, Birthe B

2008-01-01

showed that ACBP targeted to the ER (endoplasmic reticulum) and Golgi in a ligand-binding-dependent manner. A variant Y28F/K32A-FACI-50, which is unable to bind acyl-CoA, did no longer show association with the ER and became segregated from the Golgi, as analysed by intensity correlation calculations....... Depletion of fatty acids from cells by addition of FAFBSA (fatty-acid-free BSA) significantly decreased FACI-50 association with the Golgi, whereas fatty acid overloading increased Golgi association, strongly supporting that ACBP associates with the Golgi in a ligand-dependent manner. FRAP (fluorescence...... recovery after photobleaching) showed that the fatty-acid-induced targeting of FACI-50 to the Golgi resulted in a 5-fold reduction in FACI-50 mobility. We suggest that ACBP is targeted to the ER and Golgi in a ligand-binding-dependent manner in living cells and propose that ACBP may be involved...

Characterization of substrate preference for Slc1p and Cst26p in Saccharomyces cerevisiae using lipidomic approaches and an LPAAT activity assay.

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Guanghou Shui

Full Text Available BACKGROUND: Phosphatidic acid (PA is a key regulated intermediate and precursor for de novo biosynthesis of all glycerophospholipids. PA can be synthesized through the acylation of lysophosphatidic acid (LPA by 1-acyl-3-phosphate acyltransferase (also called lysophosphatidic acid acyltransferase, LPAAT. Recent findings have substantiated the essential roles of acyltransferases in various biological functions. METHODOLOGIES/PRINCIPAL FINDINGS: We used a flow-injection-based lipidomic approach with approximately 200 multiple reaction monitoring (MRM transitions to pre-screen fatty acyl composition of phospholipids in the yeast Saccharomyces cerevisiae mutants. Dramatic changes were observed in fatty acyl composition in some yeast mutants including Slc1p, a well-characterized LPAAT, and Cst26p, a recently characterized phosphatidylinositol stearoyl incorporating 1 protein and putative LPAAT in S. cerevisiae. A comprehensive high-performance liquid chromatography-based multi-stage MRM approach (more than 500 MRM transitions was developed and further applied to quantify individual phospholipids in both strains to confirm these changes. Our data suggest potential fatty acyl substrates as well as fatty acyls that compensate for defects in both Cst26p and Slc1p mutants. These results were consistent with those from a non-radioactive LPAAT enzymatic assay using C17-LPA and acyl-CoA donors as substrates. CONCLUSIONS: We found that Slc1p utilized fatty acid (FA 18:1 and FA 14:0 as substrates to synthesize corresponding PAs; moreover, it was probably the only acyltransferase responsible for acylation of saturated short-chain fatty acyls (12:0 and 10:0 in S. cerevisiae. We also identified FA 18:0, FA 16:0, FA 14:0 and exogenous FA 17:0 as preferred substrates for Cst26p because transformation with a GFP-tagged CST26 restored the phospholipid profile of a CST26 mutant. Our current findings expand the enzymes and existing scope of acyl-CoA donors for

Demonstration of disturbed free fatty acid metabolism of myocardium in patients with non-insulin-dependent diabetes mellitus as measured with iodine-123-heptadecanoic acid

International Nuclear Information System (INIS)

Kuikka, J.T.; Mustonen, J.N.; Uusitupa, M.I.J.; Rautio, P.; Vanninen, E.; Laakso, M.; Laensimies, E.; Kuopio Central Hospital

1991-01-01

Myocardial free fatty acid metabolism and left ventricular function were evaluated in 15 middle-aged patients with non-insulin dependent diabetes mellitus (NIDDM) and in 8 healthy control subjects. The study subjects had no evidence of coronary heart disease on the basis of clinical history, exercise ECG or myocardial perfusion scintigraphy. During peak exercise, iodine-123 hepatadecanoic acid (HDA) was intravenously injected. Myocardial activity distribution of 123 I-HDA was measured 10, 30, and 50 min after exercise using single-photon emission tomography (SPET); and then further corrected by free 123 I-iodine. Venous blood samples were drawn for detecting the plasma activity of 123 I. The net extraction of 123 I-HDA into the myocardium was obtained by dividing the corrected tissue 123 I concentration by the integral of the plasma time activity curve. The net extraction was 0.40±0.06 min -1 (mean±SD) patients with NIDDM and 0.38±0.006 min -1 in control subjects (P>0.1), respectively. The faster elimination rate of 123 I-HDA was found in patients with NIDDM (0.029±0.008 min -1 ) than in control subjects (0.022±0.004 min - 1); P 123 I-HDA and the change of LVEF, as well as with exercise load (r=0.68; P<0.01). In conclusion, evidence of an increased fatty acid utilization and triglyceride synthesis rate was observed in the diabetic myocardium. (orig.)

Angiopoietin-like 4 mediates PPAR delta effect on lipoprotein lipase-dependent fatty acid uptake but not on beta-oxidation in myotubes.

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Marius R Robciuc

Full Text Available Peroxisome proliferator-activated receptor (PPAR delta is an important regulator of fatty acid (FA metabolism. Angiopoietin-like 4 (Angptl4, a multifunctional protein, is one of the major targets of PPAR delta in skeletal muscle cells. Here we investigated the regulation of Angptl4 and its role in mediating PPAR delta functions using human, rat and mouse myotubes. Expression of Angptl4 was upregulated during myotubes differentiation and by oleic acid, insulin and PPAR delta agonist GW501516. Treatment with GW501516 or Angptl4 overexpression inhibited both lipoprotein lipase (LPL activity and LPL-dependent uptake of FAs whereas uptake of BSA-bound FAs was not affected by either treatment. Activation of retinoic X receptor (RXR, PPAR delta functional partner, using bexarotene upregulated Angptl4 expression and inhibited LPL activity in a PPAR delta dependent fashion. Silencing of Angptl4 blocked the effect of GW501516 and bexarotene on LPL activity. Treatment with GW501516 but not Angptl4 overexpression significantly increased palmitate oxidation. Furthermore, Angptl4 overexpression did not affect the capacity of GW501516 to increase palmitate oxidation. Basal and insulin stimulated glucose uptake, glycogen synthesis and glucose oxidation were not significantly modulated by Angptl4 overexpression. Our findings suggest that FAs-PPARdelta/RXR-Angptl4 axis controls the LPL-dependent uptake of FAs in myotubes, whereas the effect of PPAR delta activation on beta-oxidation is independent of Angptl4.

DIMETHYLARSINIC ACID ALTERS EXPRESSION OF OXIDATIVE STRESS AND DNA REPAIR GENES IN A DOSE DEPENDENT MANNER IN THE TRANSITIONAL EPITHELIUM OF THE URINARY BLADDER FROM FEMALE F344 RATS.

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Dose-dependent alteration of oxidative stress and DNA repair gene expression by Dimethylarsinic acid [DMA(V)] in transitional epithelium of urinary bladder from female F344 rats.Arsenic (As) is a major concern as millions of people are at risk from drinking arsenic contaminat...

A lower ratio of omega-6 to omega-3 fatty acids predicts better hippocampus-dependent spatial memory and cognitive status in older adults.

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Andruchow, Nadia D; Konishi, Kyoko; Shatenstein, Bryna; Bohbot, Véronique D

2017-10-01

Evidence from several cross-sectional studies indicates that an increase in omega-6 to omega-3 fatty acids (FAs) may negatively affect cognition in old age. The hippocampus is among the first neural structures affected by age and atrophy in this brain region is associated with cognitive decline. Therefore, we hypothesized that a lower omega-6:3 FA ratio would predict better hippocampus-dependent spatial memory, and a higher general cognitive status. Fifty-two healthy older adults completed a Food Frequency Questionnaire, the Montreal Cognitive Assessment test (MoCA; a test of global cognition) and virtual navigation tasks that assess navigational strategies and spatial memory. In this cross-sectional study, a lower ratio of omega-6 to omega-3 FA intake strongly predicted more accurate hippocampus-dependent spatial memory and faster learning on our virtual navigation tasks, as well as higher cognitive status overall. These results may help elucidate why certain dietary patterns with a lower omega-6:3 FA ratio, like the Mediterranean diet, are associated with reduced risk of cognitive decline. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

Identification of critical amino acids in the proximal C-terminal of TREK-2 K+ channel for activation by acidic pHi and ATP-dependent inhibition.

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Woo, Joohan; Jun, Young Keul; Zhang, Yin-Hua; Nam, Joo Hyun; Shin, Dong Hoon; Kim, Sung Joon

2018-02-01

TWIK-related two-pore domain K + channels (TREKs) are regulated by intracellular pH (pH i ) and Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ). Previously, Glu 306 in proximal C-terminal (pCt) of mouse TREK-1 was identified as the pH i -sensing residue. The direction of PI(4,5)P 2 sensitivity is controversial, and we have recently shown that TREKs are inhibited by intracellular ATP via endogenous PI(4,5)P 2 formation. Here we investigate the anionic and cationic residues of pCt for the pH i and ATP-sensitivity in human TREK-2 (hTREK-2). In inside-out patch clamp recordings (I TREK-2,i-o ), acidic pH i -induced activation was absent in E332A and was partly attenuated in E335A. Neutralization of cationic Lys (K330A) also eliminated the acidic pH i sensitivity of I TREK-2,i-o . Unlike the inhibition of wild-type (WT) I TREK-2,i-o by intracellular ATP, neither E332A nor K330A was sensitive to ATP. Nevertheless, exogenous PI(4,5)P 2 (10 μM) abolished I TREK-2 i-o in all the above mutants as well as in WT, indicating unspecific inhibition by exogenous PI(4,5)P 2 . In whole-cell recordings of TREK-2 (I TREK-2,w-c ), K330A and E332A showed higher or fully active basal activity, showing attenuated or insignificant activation by 2-APB, arachidonic acid, or acidic pH e 6.9. I TREK-1,w-c of WT is largely suppressed by pH e 6.9, and the inhibition is slightly attenuated in K312A and E315A. The results show concerted roles of the oppositely charged Lys and Glu in pCt for the ATP-dependent low basal activity and pH i sensitivity.

Cation-Size-Dependent Conformational Locking of Glutamic Acid by Alkali Ions: Infrared Photodissociation Spectroscopy of Cryogenic Ions.

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Klyne, Johanna; Bouchet, Aude; Ishiuchi, Shun-Ichi; Fujii, Masaaki; Dopfer, Otto

2018-03-01

Consolidated knowledge of conformation and stability of amino acids and their clusters is required to understand their biochemical recognition. Often, alkali ions interact with amino acids and proteins. Herein, infrared photodissociation (IRPD) spectra of cryogenic metalated glutamic acid ions (GluM + , M = Li-Cs) are systematically analyzed in the isomer-specific fingerprint and XH stretch ranges (1100-1900, 2600-3600 cm -1 ) to provide a direct measure for cation-size-dependent conformational locking. GluM + ions are generated by electrospray ionization and cooled down to 15 K in a cryogenic quadrupole ion trap. The assignment of the IRPD spectra is supported by density functional theory calculations at the dispersion-corrected B3LYP-D3/aug-cc-pVTZ level. In the global minimum of GluM + , the flexibility of Glu is strongly reduced by the formation of rigid ionic CO···M + ···OC metal bridges, corresponding to charge solvation. The M + binding energy decreases monotonically with increasing cation size from D 0 = 314 to 119 kJ/mol for Li-Cs. Whereas for Li and Na only the global minimum of GluM + is observed, for K-Cs at least three isomers exist at cryogenic temperature. The IRPD spectra of cold GluM + ions are compared to IR multiple-photon dissociation spectra measured at room temperature. Furthermore, we elucidate the differences of the impact of protonation and metalation on the structure and conformational locking of Glu.

Temperature-dependent dynamic control of the TCA cycle increases volumetric productivity of itaconic acid production by Escherichia coli.

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Harder, Björn-Johannes; Bettenbrock, Katja; Klamt, Steffen

2018-01-01

Based on the recently constructed Escherichia coli itaconic acid production strain ita23, we aimed to improve the productivity by applying a two-stage process strategy with decoupled production of biomass and itaconic acid. We constructed a strain ita32 (MG1655 ΔaceA Δpta ΔpykF ΔpykA pCadCs), which, in contrast to ita23, has an active tricarboxylic acid (TCA) cycle and a fast growth rate of 0.52 hr -1 at 37°C, thus representing an ideal phenotype for the first stage, the growth phase. Subsequently we implemented a synthetic genetic control allowing the downregulation of the TCA cycle and thus the switch from growth to itaconic acid production in the second stage. The promoter of the isocitrate dehydrogenase was replaced by the Lambda promoter (p R ) and its expression was controlled by the temperature-sensitive repressor CI857 which is active at lower temperatures (30°C). With glucose as substrate, the respective strain ita36A grew with a fast growth rate at 37°C and switched to production of itaconic acid at 28°C. To study the impact of the process strategy on productivity, we performed one-stage and two-stage bioreactor cultivations. The two-stage process enabled fast formation of biomass resulting in improved peak productivity of 0.86 g/L/hr (+48%) and volumetric productivity of 0.39 g/L/hr (+22%) in comparison to the one-stage process. With our dynamic production strain, we also resolved the glutamate auxotrophy of ita23 and increased the itaconic acid titer to 47 g/L. The temperature-dependent activation of gene expression by the Lambda promoters (p R /p L ) has been frequently used to improve protein or, in a few cases, metabolite production in two-stage processes. Here we demonstrate that the system can be as well used in the opposite direction to selectively knock-down an essential gene (icd) in E. coli to design a two-stage process for improved volumetric productivity. The control by temperature avoids expensive inducers and has the

Ursodeoxycholic and deoxycholic acids: A good and a bad bile acid for intestinal calcium absorption.

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Rodríguez, Valeria; Rivoira, María; Marchionatti, Ana; Pérez, Adriana; Tolosa de Talamoni, Nori

2013-12-01

The aim of this study was to investigate the effect of ursodeoxycholic acid (UDCA) on intestinal Ca(2+) absorption and to find out whether the inhibition of this process caused by NaDOC could be prevented by UDCA. Chicks were employed and divided into four groups: (a) controls, (b) treated with 10mM NaDOC, (c) treated with 60 μg UDCA/100g of b.w., and (d) treated with 10mM NaDOC and 60 μg UDCA/100g of b.w. UDCA enhanced intestinal Ca(2+) absorption, which was time and dose-dependent. UDCA avoided the inhibition of intestinal Ca(2+) absorption caused by NaDOC. Both bile acids altered protein and gene expression of molecules involved in the transcellular pathway of intestinal Ca(2+) absorption, but in the opposite way. UDCA aborted the oxidative stress produced by NaDOC in the intestine. UDCA and UDCA plus NaDOC increased vitamin D receptor protein expression. In conclusion, UDCA is a beneficial bile acid for intestinal Ca(2+) absorption. Contrarily, NaDOC inhibits the intestinal cation absorption through triggering oxidative stress. The use of UDCA in patients with cholestasis would be benefited because of the protective effect on the intestinal Ca(2+) absorption, avoiding the inhibition caused by hydrophobic bile acids and neutralizing the oxidative stress. Copyright © 2013 Elsevier Inc. All rights reserved.

The application of glutamic acid alpha-decarboxylase for the valorization of glutamic acid

NARCIS (Netherlands)

Lammens, T.M.; Biase, De Daniela; Franssen, M.C.R.; Scott, E.L.; Sanders, J.P.M.

2009-01-01

Glutamic acid is an important constituent of waste streams from biofuels production. It is an interesting starting material for the synthesis of nitrogen containing bulk chemicals, thereby decreasing the dependency on fossil fuels. On the pathway from glutamic acid to a range of molecules, the

Temperature dependences of saturated vapor pressure and the enthalpy of vaporization of n-pentyl esters of dicarboxylic acids

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Portnova, S. V.; Krasnykh, E. L.; Levanova, S. V.

2016-05-01

The saturated vapor pressures and enthalpies of vaporization of n-pentyl esters of linear C2-C6 dicarboxylic acids are determined by the transpiration method in the temperature range of 309.2-361.2 K. The dependences of enthalpies of vaporization on the number of carbon atoms in the molecule and on the retention indices have been determined. The predictive capabilities of the existing calculation schemes for estimation of enthalpy of vaporization of the studied compounds have been analyzed.

pH-, Lactic Acid-, and Non-Lactic Acid-Dependent Activities of Probiotic Lactobacilli against Salmonella enterica Serovar Typhimurium

OpenAIRE

Fayol-Messaoudi, Domitille; Berger, Cédric N.; Coconnier-Polter, Marie-Hélène; Liévin-Le Moal, Vanessa; Servin, Alain L.

2005-01-01

The mechanism(s) underlying the antibacterial activity of probiotic Lactobacillus strains appears to be multifactorial and includes lowering of the pH and the production of lactic acid and of antibacterial compounds, including bacteriocins and nonbacteriocin, non-lactic acid molecules. Addition of Dulbecco's modified Eagle's minimum essential medium to the incubating medium delays the killing activity of lactic acid. We found that the probiotic strains Lactobacillus johnsonii La1, Lactobacill...

Cell nanomechanics and focal adhesions are regulated by retinol and conjugated linoleic acid in a dose-dependent manner

International Nuclear Information System (INIS)

Silberberg, Yaron R; Horton, Michael A; Pelling, Andrew E; Yakubov, Gleb E

2009-01-01

Retinol and conjugated linoleic acid (CLA) have previously been shown to have an important role in gene expression and various cellular processes, including differentiation, proliferation and cell death. In this study we have investigated the effect of retinol and CLA, both individually and in combination, on the intracellular cytoskeleton, focal adhesions (FAs) and the nanomechanical properties of 3T3 fibroblasts. We observed a dose-dependent decrease in the formation of FAs following treatment with either compound, which was directly correlated to an increase in cell height (>30%) and a decrease in the measured Young's modulus (∼28%). Furthermore, treatments with both compounds demonstrated an increased effect and led to a reduction of>70% in the average number of FAs per cell and a decrease of >50% in average cell stiffness. These data reveal that retinol and CLA disrupt FA formation, leading to an increase in cell height and a significant decrease in stiffness. These results may broaden our understanding of the interplay between cell nanomechanics and cellular contact with the external microenvironment, and help to shed light on the important role of retinoids and CLA in health and disease.

A one-step colorimetric acid-base titration sensor using a complementary color changing coordination system.

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Cho, Hui Hun; Kim, Si Hyun; Heo, Jun Hyuk; Moon, Young Eel; Choi, Young Hun; Lim, Dong Cheol; Han, Kwon-Hoon; Lee, Jung Heon

2016-06-21

We report the development of a colorimetric sensor that allows for the quantitative measurement of the acid content via acid-base titration in a single-step. In order to create the sensor, we used a cobalt coordination system (Co-complex sensor) that changes from greenish blue colored Co(H2O)4(OH)2 to pink colored Co(H2O)6(2+) after neutralization. Greenish blue and pink are two complementary colors with a strong contrast. As a certain amount of acid is introduced to the Co-complex sensor, a portion of greenish blue colored Co(H2O)4(OH)2 changes to pink colored Co(H2O)6(2+), producing a different color. As the ratio of greenish blue and pink in the Co-complex sensor is determined by the amount of neutralization reaction occurring between Co(H2O)4(OH)2 and an acid, the sensor produced a spectrum of green, yellow green, brown, orange, and pink colors depending on the acid content. In contrast, the color change appeared only beyond the end point for normal acid-base titration. When we mixed this Co-complex sensor with different concentrations of citric acid, tartaric acid, and malic acid, three representative organic acids in fruits, we observed distinct color changes for each sample. This color change could also be observed in real fruit juice. When we treated the Co-complex sensor with real tangerine juice, it generated diverse colors depending on the concentration of citric acid in each sample. These results provide a new angle on simple but quantitative measurements of analytes for on-site usage in various applications, such as in food, farms, and the drug industry.

Determination and importance of temperature dependence of retention coefficient (RPHPLC) in QSAR model of nitrazepams' partition coefficient in bile acid micelles.

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Posa, Mihalj; Pilipović, Ana; Lalić, Mladena; Popović, Jovan

2011-02-15

Linear dependence between temperature (t) and retention coefficient (k, reversed phase HPLC) of bile acids is obtained. Parameters (a, intercept and b, slope) of the linear function k=f(t) highly correlate with bile acids' structures. Investigated bile acids form linear congeneric groups on a principal component (calculated from k=f(t)) score plot that are in accordance with conformations of the hydroxyl and oxo groups in a bile acid steroid skeleton. Partition coefficient (K(p)) of nitrazepam in bile acids' micelles is investigated. Nitrazepam molecules incorporated in micelles show modified bioavailability (depo effect, higher permeability, etc.). Using multiple linear regression method QSAR models of nitrazepams' partition coefficient, K(p) are derived on the temperatures of 25°C and 37°C. For deriving linear regression models on both temperatures experimentally obtained lipophilicity parameters are included (PC1 from data k=f(t)) and in silico descriptors of the shape of a molecule while on the higher temperature molecular polarisation is introduced. This indicates the fact that the incorporation mechanism of nitrazepam in BA micelles changes on the higher temperatures. QSAR models are derived using partial least squares method as well. Experimental parameters k=f(t) are shown to be significant predictive variables. Both QSAR models are validated using cross validation and internal validation method. PLS models have slightly higher predictive capability than MLR models. Copyright © 2010 Elsevier B.V. All rights reserved.

Anti-Diabetic Effects of Madecassic Acid and Rotundic Acid

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Yuan-Man Hsu

2015-12-01

Full Text Available Anti-diabetic effects of madecassic acid (MEA and rotundic acid (RA were examined. MEA or RA at 0.05% or 0.1% was supplied to diabetic mice for six weeks. The intake of MEA, not RA, dose-dependently lowered plasma glucose level and increased plasma insulin level. MEA, not RA, intake dose-dependently reduced plasminogen activator inhibitor-1 activity and fibrinogen level; as well as restored antithrombin-III and protein C activities in plasma of diabetic mice. MEA or RA intake decreased triglyceride and cholesterol levels in plasma and liver. Histological data agreed that MEA or RA intake lowered hepatic lipid droplets, determined by ORO stain. MEA intake dose-dependently declined reactive oxygen species (ROS and oxidized glutathione levels, increased glutathione content and maintained the activity of glutathione reductase and catalase in the heart and kidneys of diabetic mice. MEA intake dose-dependently reduced interleukin (IL-1β, IL-6, tumor necrosis factor-α and monocyte chemoattractant protein-1 levels in the heart and kidneys of diabetic mice. RA intake at 0.1% declined cardiac and renal levels of these inflammatory factors. These data indicated that MEA improved glycemic control and hemostatic imbalance, lowered lipid accumulation, and attenuated oxidative and inflammatory stress in diabetic mice. Thus, madecassic acid could be considered as an anti-diabetic agent.

Formaldehyde-induced mutations in Drosophila melanogaster in dependence of the presence of acids

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Stumm-Tegethoff, B F.A.

1969-01-01

The mutagenic activity of various combinations of formaldehyde, formic acid, acetic acid and hydrochloric acid was investigated by a sex-linked lethal test. All combinations were mutagenic and showed a mutation pattern from which it is concluded that in feeding experiments spermatocytes I are especially sensitive to the pairs of chemicals tested. In vapour experiments all germ cell stages were found to be susceptible. The presence of volatile acids was found to be necessary for the mutagenic activity of formaldehyde in the vapour state. Mutagenic effects were also observed in larval feeding experiments, in which only these acids were added to the medium. Experiments with stabilized pH at 7.5 did not show a significant mutagenic effect of formaldehyde. It is postulated that the tested agents are catalase inhibitors, which promote the formation of peroxides or free radicals which interfere with DNA replication, thus producing mutations.

Cluster shading modifies amino acids in grape (Vitis vinifera L.) berries in a genotype- and tissue-dependent manner.

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Guan, Le; Wu, Benhong; Hilbert, Ghislaine; Li, Shaohua; Gomès, Eric; Delrot, Serge; Dai, Zhanwu

2017-08-01

Amino acid composition of the grape berry at harvest is important for wine making. The present study investigates the complex interplay between tissue, cultivar and light conditions that determine berry amino acid content. Twenty amino acids were assessed in the berry skin and pulp of two grape cultivars (Gamay Noir and Gamay Fréaux), grown under either light exposure or cluster shading conditions. In all samples, cluster shading significantly reduced most amino acids, except gamma-aminobutyric acid (GABA) and phenylalanine. However, the magnitude of the decrease was stronger in the skin (67.0% decrease) than in the pulp (30.4%) and stronger in cv. Gamay Noir (69.7%) than in Gamay Fréaux (30.7%). Cluster shading also significantly modified amino acid composition by decreasing the proline content while increasing the GABA content. These results are of oenological interest for shaping the amino acid composition of the must and improving wine quality. Copyright © 2017 Elsevier Ltd. All rights reserved.

Conformational dynamics and role of the acidic pocket in ASIC pH-dependent gating.

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Vullo, Sabrina; Bonifacio, Gaetano; Roy, Sophie; Johner, Niklaus; Bernèche, Simon; Kellenberger, Stephan

2017-04-04

Acid-sensing ion channels (ASICs) are proton-activated Na + channels expressed in the nervous system, where they are involved in learning, fear behaviors, neurodegeneration, and pain sensation. In this work, we study the role in pH sensing of two regions of the ectodomain enriched in acidic residues: the acidic pocket, which faces the outside of the protein and is the binding site of several animal toxins, and the palm, a central channel domain. Using voltage clamp fluorometry, we find that the acidic pocket undergoes conformational changes during both activation and desensitization. Concurrently, we find that, although proton sensing in the acidic pocket is not required for channel function, it does contribute to both activation and desensitization. Furthermore, protonation-mimicking mutations of acidic residues in the palm induce a dramatic acceleration of desensitization followed by the appearance of a sustained current. In summary, this work describes the roles of potential pH sensors in two extracellular domains, and it proposes a model of acidification-induced conformational changes occurring in the acidic pocket of ASIC1a.

The old is new again: asparagine oxidation in calcium-dependent antibiotic biosynthesis.

Science.gov (United States)

Worthington, Andrew S; Burkart, Michael D

2007-03-20

Non-ribosomal peptides are built from both proteinogenic and non-proteinogenic amino acids. The latter resemble amino acids but contain modifications not found in proteins. The recent characterization of a non-heme Fe(2+) and alpha-ketoglutarate-dependent oxygenase that stereospecifically generates beta-hydroxyasparagine, an unnatural amino acid building block for the biosynthesis of calcium-dependent antibiotic, a lipopeptide antibiotic. This work improves our understanding of how these non-proteinogenic amino acids are synthesized.

Perturbations of Amino Acid Metabolism Associated with Glyphosate-Dependent Inhibition of Shikimic Acid Metabolism Affect Cellular Redox Homeostasis and Alter the Abundance of Proteins Involved in Photosynthesis and Photorespiration1[W][OA

Science.gov (United States)

Vivancos, Pedro Diaz; Driscoll, Simon P.; Bulman, Christopher A.; Ying, Liu; Emami, Kaveh; Treumann, Achim; Mauve, Caroline; Noctor, Graham; Foyer, Christine H.

2011-01-01

The herbicide glyphosate inhibits the shikimate pathway of the synthesis of amino acids such as phenylalanine, tyrosine, and tryptophan. However, much uncertainty remains concerning precisely how glyphosate kills plants or affects cellular redox homeostasis and related processes in glyphosate-sensitive and glyphosate-resistant crop plants. To address this issue, we performed an integrated study of photosynthesis, leaf proteomes, amino acid profiles, and redox profiles in the glyphosate-sensitive soybean (Glycine max) genotype PAN809 and glyphosate-resistant Roundup Ready Soybean (RRS). RRS leaves accumulated much more glyphosate than the sensitive line but showed relatively few changes in amino acid metabolism. Photosynthesis was unaffected by glyphosate in RRS leaves, but decreased abundance of photosynthesis/photorespiratory pathway proteins was observed together with oxidation of major redox pools. While treatment of a sensitive genotype with glyphosate rapidly inhibited photosynthesis and triggered the appearance of a nitrogen-rich amino acid profile, there was no evidence of oxidation of the redox pools. There was, however, an increase in starvation-associated and defense proteins. We conclude that glyphosate-dependent inhibition of soybean leaf metabolism leads to the induction of defense proteins without sustained oxidation. Conversely, the accumulation of high levels of glyphosate in RRS enhances cellular oxidation, possibly through mechanisms involving stimulation of the photorespiratory pathway. PMID:21757634

Effect of urine pH changed by dietary intervention on uric acid clearance mechanism of pH-dependent excretion of urinary uric acid

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Kanbara Aya

2012-06-01

Full Text Available Abstract Background The finding reported in a previous paper - alkalization of urine facilitates uric acid excretion - is contradictory to what one might expect to occur: because food materials for the alkalization of urine contain fewer purine bodies than those for acidification, less uric acid in alkaline urine should have been excreted than in acid urine. To make clear what component of uric acid excretion mechanisms is responsible for this unexpected finding, we simultaneously collected data for the concentration of both creatinine and uric acid in serum as well as in urine, in order to calculate both uric acid and creatinine clearances. Methods Within the framework of the Japanese government’s health promotion program, we made recipes which consisted of protein-rich and less vegetable-fruit food materials for H + -load (acidic diet and others composed of less protein and more vegetable-fruit rich food materials (alkaline diet. This is a crossover study within some limitations. Healthy female students, who had no medical problems at the regular physical examination provided by the university, were enrolled in this consecutive 5-day study for each test. From whole-day collected urine, total volume, pH, organic acid, creatinine, uric acid, titratable acid and all cations (Na+,K+,Ca2+,Mg2+,NH4+ and anions (Cl−,SO42−,PO4− necessary for the estimation of acid–base balance were measured. In the early morning before breakfast of the 1st, 3rd and 5th experimental day, we sampled 5 mL of blood to estimate the creatinine and uric acid concentration in serum. Results and discussion Urine pH reached a steady state 3 days after switching from ordinary daily diets to specified regimens. The amount of acid generated ([SO42−] + organic acid − gut alkaliwas linearly related with the excretion of acid (titratable acid + [NH4+] − [HCO3−], indicating that H + in urine is generated by the metabolic degradation of

Intravenous infusion of docosahexaenoic acid increases serum concentrations in a dose-dependent manner and increases seizure latency in the maximal PTZ model.

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Trépanier, Marc-Olivier; Kwong, Kei-Man; Domenichiello, Anthony F; Chen, Chuck T; Bazinet, Richard P; Burnham, W M

2015-09-01

Docosahexaenoic acid (DHA) is an omega-3 polyunsaturated fatty acid (n-3 PUFA) that has been shown to raise seizure thresholds in the maximal pentylenetetrazole model following acute subcutaneous (s.c.) administration in rats. Following s.c. administration, however, the dose-response relationship for DHA has shown an inverted U-pattern. The purposes of the present experiment were as follows: (1) to determine the pattern of serum unesterified concentrations resulting from the intravenous (i.v.) infusions of various doses of DHA, (2) to determine the time course of these concentrations following the discontinuation of the infusions, and (3) to determine whether seizure protection in the maximal PTZ model would correlate with serum unesterified DHA levels. Animals received 5-minute i.v. infusions of saline or 25, 50, 100, or 200mg/kg of DHA via a cannula inserted into one of the tail veins. Blood was collected during and after the infusions by means of a second cannula inserted into the other tail vein (Experiment 1). A separate group of animals received saline or 12.5-, 25-, 50-, 100-, or 200 mg/kg DHA i.v. via a cannula inserted into one of the tail veins and were then seizure-tested in the maximal PTZ model either during infusion or after the discontinuation of the infusions. Slow infusions of DHA increased serum unesterified DHA concentrations in a dose-dependent manner, with the 200-mg/kg dose increasing the concentration approximately 260-fold compared with saline-infused animals. Following discontinuation of the infusions, serum concentrations rapidly dropped toward baseline, with half-lives of approximately 40 and 11s for the 25-mg/kg dose and 100-mg/kg dose, respectively. In the seizure-tested animals, DHA significantly increased latency to seizure onset in a dose-dependent manner. Following the discontinuation of infusion, seizure latency rapidly decreased toward baseline. Overall, our study suggests that i.v. infusion of unesterified DHA results in

Unravelling the pH-dependence of a molecular photocatalytic system for hydrogen production.

Science.gov (United States)

Reynal, Anna; Pastor, Ernest; Gross, Manuela A; Selim, Shababa; Reisner, Erwin; Durrant, James R

2015-08-01

Photocatalytic systems for the reduction of aqueous protons are strongly pH-dependent, but the origin of this dependency is still not fully understood. We have studied the effect of different degrees of acidity on the electron transfer dynamics and catalysis taking place in a homogeneous photocatalytic system composed of a phosphonated ruthenium tris(bipyridine) dye ( RuP ) and a nickel bis(diphosphine) electrocatalyst ( NiP ) in an aqueous ascorbic acid solution. Our approach is based on transient absorption spectroscopy studies of the efficiency of photo-reduction of RuP and NiP correlated with pH-dependent photocatalytic H 2 production and the degree of catalyst protonation. The influence of these factors results in an observed optimum photoactivity at pH 4.5 for the RuP - NiP system. The electron transfer from photo-reduced RuP to NiP is efficient and independent of the pH value of the medium. At pH RuP photo-reduction by the sacrificial electron donor, ascorbic acid. At pH >4.5, the efficiency of the system is limited by the poor protonation of NiP , which inhibits its ability to reduce protons to hydrogen. We have therefore developed a rational strategy utilising transient absorption spectroscopy combined with bulk pH titration, electrocatalytic and photocatalytic experiments to disentangle the complex pH-dependent activity of the homogenous RuP - NiP photocatalytic system, which can be widely applied to other photocatalytic systems.

10-Oxo-trans-11-octadecenoic acid generated from linoleic acid by a gut lactic acid bacterium Lactobacillus plantarum is cytoprotective against oxidative stress.

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Furumoto, Hidehiro; Nanthirudjanar, Tharnath; Kume, Toshiaki; Izumi, Yasuhiko; Park, Si-Bum; Kitamura, Nahoko; Kishino, Shigenobu; Ogawa, Jun; Hirata, Takashi; Sugawara, Tatsuya

2016-04-01

Oxidative stress is a well-known cause of multiple diseases. The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway plays a central role in cellular antioxidative responses. In this study, we investigated the effects of novel fatty acid metabolite derivatives of linoleic acid generated by the gut lactic acid bacteria Lactobacillus plantarum on the Nrf2-ARE pathway. 10-Oxo-trans-11-octadecenoic acid (KetoC) protected HepG2 cells from cytotoxicity induced by hydrogen peroxide. KetoC also significantly increased cellular Nrf2 protein levels, ARE-dependent transcription, and the gene expression of antioxidative enzymes such as heme oxygenase-1 (HO-1), glutamate-cysteine ligase modifier subunit (GCLM), and quinone oxidoreductase 1 (NQO1) in HepG2 cells. Additionally, a single oral dose administration of KetoC also increased antioxidative gene expression and protein levels of Nrf2 and HO-1 in mouse organs. Since other fatty acid metabolites and linoleic acid did not affect cellular antioxidative responses, the cytoprotective effect of KetoC may be because of its α,β-unsaturated carbonyl moiety. Collectively, our data suggested that KetoC activated the Nrf2-ARE pathway to enhance cellular antioxidative responses in vitro and in vivo, which further suggests that KetoC may prevent multiple diseases induced by oxidative stress. Copyright © 2016. Published by Elsevier Inc.

Changes in population of S. enterica serotype Enteritidis in experimentally contaminated milk depending on temperature and acidity

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Rajić-Savić Nataša

2009-01-01

Full Text Available Salmonella bacteria are among the most frequent causes of food poisoning, and milk and milk products are food items through which these bacteria can be transmitted. More recent data indicate that the infective dose of Salmonella is very small - up to 101/g/ml food. The preservation of Salmonella in milk depends on the acidity, temperature and the duration of storage. The objective of the work was to examine the survival of Salmonella in milk depending on the changes of the total number of bacteria, pH and temperature. In the study, 0.1 ml of 24-hour bouillon culture of S. enteritidis was inoculated in raw collective milk (104 cfu/ml and in marketed pasteurized milk (102 cfu/ml each. The examined milk samples were maintained at room temperature and at a temperature of 7oC. At the beginning of the examinations, after 24h, 48h, 72h, after 7, 14 and 21 days, decimal solutions were made from the milk, and, following incubation, the number of S. enteritidis was determined (Rambach agar, the total number of bacteria (base for total number of bacteria by multiplying the number of grown colonies and the corresponding solution, and the milk pH was measured. There was a bigger reduction of S. enteritidis at room temperature because the pH value dropped more swiftly. The lowest pH at which S. enteritidis was isolated from experimentally contaminated milk was 3.88. The results of the examinations show that Salmonella can adapt to biochemical processes during milk acid fermentation of soft cheeses. No Salmonella bacteria were isolated from experimentally contaminated pasterurized milk at both maintenance temperatures after seven days of investigations.

Dependence of the expression of the radiation-induced gene conversion to arginine independence in diploid yeast on the amino acid concentration: effect on allelic mapping

International Nuclear Information System (INIS)

Murthy, M.S.S.; Rao, B.S.; Deorukhakar, V.V.

1976-01-01

The yield of radiation-induced gene conversion to arginine independence in diploid yeast depended on the concentration of the amino acid both in the plating medium and in the intracellular pool. By depletion of the level of arginine in the intracellular pool of amino acid or by provision of arginine at 0.4 mg/l of the plating medium, the yield was varied by a factor as high as 20. This may be important in studies of the genetic mapping of alleles based on the slope of conversion frequency versus dose line

Gallic Acid Induces a Reactive Oxygen Species-Provoked c-Jun NH2-Terminal Kinase-Dependent Apoptosis in Lung Fibroblasts

Science.gov (United States)

Chen, Chiu-Yuan; Chen, Kun-Chieh; Yang, Tsung-Ying; Liu, Hsiang-Chun; Hsu, Shih-Lan

2013-01-01

Idiopathic pulmonary fibrosis is a chronic lung disorder characterized by fibroblasts proliferation and extracellular matrix accumulation. Induction of fibroblast apoptosis therefore plays a crucial role in the resolution of this disease. Gallic acid (3,4,5-trihydroxybenzoic acid), a common botanic phenolic compound, has been reported to induce apoptosis in tumor cell lines and renal fibroblasts. The present study was undertaken to examine the role of mitogen-activated protein kinases (MAPKs) in lung fibroblasts apoptosis induced by gallic acid. We found that treatment with gallic acid resulted in activation of c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and protein kinase B (PKB, Akt), but not p38MAPK, in mouse lung fibroblasts. Inhibition of JNK using pharmacologic inhibitor (SP600125) and genetic knockdown (JNK specific siRNA) significantly inhibited p53 accumulation, reduced PUMA and Fas expression, and abolished apoptosis induced by gallic acid. Moreover, treatment with antioxidants (vitamin C, N-acetyl cysteine, and catalase) effectively diminished gallic acid-induced hydrogen peroxide production, JNK and p53 activation, and cell death. These observations imply that gallic acid-mediated hydrogen peroxide formation acts as an initiator of JNK signaling pathways, leading to p53 activation and apoptosis in mouse lung fibroblasts. PMID:23533505

Adipose tissue fatty acid patterns and changes in anthropometry: a cohort study.

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Christina Catherine Dahm

Full Text Available INTRODUCTION: Diets rich in n-3 long chain polyunsaturated fatty acids (LC-PUFA, but low in n-6 LC-PUFA and 18:1 trans-fatty acids (TFA, may lower the risk of overweight and obesity. These fatty acids have often been investigated individually. We explored associations between global patterns in adipose tissue fatty acids and changes in anthropometry. METHODS: 34 fatty acid species from adipose tissue biopsies were determined in a random sample of 1100 men and women from a Danish cohort study. We used sex-specific principal component analysis and multiple linear regression to investigate the associations of adipose tissue fatty acid patterns with changes in weight, waist circumference (WC, and WC controlled for changes in body mass index (WC(BMI, adjusting for confounders. RESULTS: 7 principal components were extracted for each sex, explaining 77.6% and 78.3% of fatty acid variation in men and women, respectively. Fatty acid patterns with high levels of TFA tended to be positively associated with changes in weight and WC for both sexes. Patterns with high levels of n-6 LC-PUFA tended to be negatively associated with changes in weight and WC in men, and positively associated in women. Associations with patterns with high levels of n-3 LC-PUFA were dependent on the context of the rest of the fatty acid pattern. CONCLUSIONS: Adipose tissue fatty acid patterns with high levels of TFA may be linked to weight gain, but patterns with high n-3 LC-PUFA did not appear to be linked to weight loss. Associations depended on characteristics of the rest of the pattern.

Obeticholic acid, a selective farnesoid X receptor agonist, regulates bile acid homeostasis in sandwich-cultured human hepatocytes.

Science.gov (United States)

Zhang, Yuanyuan; Jackson, Jonathan P; St Claire, Robert L; Freeman, Kimberly; Brouwer, Kenneth R; Edwards, Jeffrey E

2017-08-01

Farnesoid X receptor (FXR) is a master regulator of bile acid homeostasis through transcriptional regulation of genes involved in bile acid synthesis and cellular membrane transport. Impairment of bile acid efflux due to cholangiopathies results in chronic cholestasis leading to abnormal elevation of intrahepatic and systemic bile acid levels. Obeticholic acid (OCA) is a potent and selective FXR agonist that is 100-fold more potent than the endogenous ligand chenodeoxycholic acid (CDCA). The effects of OCA on genes involved in bile acid homeostasis were investigated using sandwich-cultured human hepatocytes. Gene expression was determined by measuring mRNA levels. OCA dose-dependently increased fibroblast growth factor-19 (FGF-19) and small heterodimer partner (SHP) which, in turn, suppress mRNA levels of cholesterol 7-alpha-hydroxylase (CYP7A1), the rate-limiting enzyme for de novo synthesis of bile acids. Consistent with CYP7A1 suppression, total bile acid content was decreased by OCA (1 μmol/L) to 42.7 ± 20.5% relative to control. In addition to suppressing de novo bile acids synthesis, OCA significantly increased the mRNA levels of transporters involved in bile acid homeostasis. The bile salt excretory pump (BSEP), a canalicular efflux transporter, increased by 6.4 ± 0.8-fold, and the basolateral efflux heterodimer transporters, organic solute transporter α (OST α ) and OST β increased by 6.4 ± 0.2-fold and 42.9 ± 7.9-fold, respectively. The upregulation of BSEP and OST α and OST β, by OCA reduced the intracellular concentrations of d 8 -TCA, a model bile acid, to 39.6 ± 8.9% relative to control. These data demonstrate that OCA does suppress bile acid synthesis and reduce hepatocellular bile acid levels, supporting the use of OCA to treat bile acid-induced toxicity observed in cholestatic diseases. © 2017 Intercept Pharmaceuticals. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and

Growth-Environment Dependent Modulation of Staphylococcus aureus Branched-Chain to Straight-Chain Fatty Acid Ratio and Incorporation of Unsaturated Fatty Acids.

Science.gov (United States)

Sen, Suranjana; Sirobhushanam, Sirisha; Johnson, Seth R; Song, Yang; Tefft, Ryan; Gatto, Craig; Wilkinson, Brian J

2016-01-01

The fatty acid composition of membrane glycerolipids is a major determinant of Staphylococcus aureus membrane biophysical properties that impacts key factors in cell physiology including susceptibility to membrane active antimicrobials, pathogenesis, and response to environmental stress. The fatty acids of S. aureus are considered to be a mixture of branched-chain fatty acids (BCFAs), which increase membrane fluidity, and straight-chain fatty acids (SCFAs) that decrease it. The balance of BCFAs and SCFAs in USA300 strain JE2 and strain SH1000 was affected considerably by differences in the conventional laboratory medium in which the strains were grown with media such as Mueller-Hinton broth and Luria broth resulting in high BCFAs and low SCFAs, whereas growth in Tryptic Soy Broth and Brain-Heart Infusion broth led to reduction in BCFAs and an increase in SCFAs. Straight-chain unsaturated fatty acids (SCUFAs) were not detected. However, when S. aureus was grown ex vivo in serum, the fatty acid composition was radically different with SCUFAs, which increase membrane fluidity, making up a substantial proportion of the total (37%) and BCFAs (>36%) making up the rest. Staphyloxanthin, an additional major membrane lipid component unique to S. aureus, tended to be greater in content in cells with high BCFAs or SCUFAs. Cells with high staphyloxanthin content had a lower membrane fluidity that was attributed to increased production of staphyloxanthin. S. aureus saves energy and carbon by utilizing host fatty acids for part of its total fatty acids when growing in serum, which may impact biophysical properties and pathogenesis given the role of SCUFAs in virulence. The nutritional environment in which S. aureus is grown in vitro or in vivo in an infection is likely to be a major determinant of membrane fatty acid composition.

Influence of fluorozirconic acid on sulfuric acid anodizing of aluminum

OpenAIRE

Elaish, R.; Curioni, M.; Gowers, K.; Kasuga, A.; Habazaki, H.; Hashimoto, T.; Skeldon, P.

2017-01-01

The effects of additions of fluorozirconic acid to sulfuric acid on the anodizing behavior of aluminum have been investigated under a constant voltage at temperatures of 0 and 20◦C. The fluoroacid increased the rate of film growth, with a dependence on the fluoroacid concentration, the electrolyte temperature and the anodizing time. Compositional analyses showed that fluorine species were present in the films. However, zirconium species were absent. The fluoroacid generally enhanced film diss...

Biosynthesis of triacylglycerols containing very long chain monounsaturated acyl moieties in developing seeds

International Nuclear Information System (INIS)

Fehling, E.; Murphy, D.J.; Mukherjee, K.D.

1990-01-01

Particulate (15,000g) fractions from developing seeds of honesty (Lunaria annua L.) and mustard (Sinapis alba L.) synthesize radioactive very long chain monounsaturated fatty acids (gadoleic, erucic, and nervonic) from [1- 14 C]oleoyl-CoA and malonyl-CoA or from oleoyl-CoA and [2- 14 C]malonyl-CoA. The very long chain monounsaturated fatty acids are rapidly channeled to triacylglycerols and other acyl lipids without intermediate accumulation of their CoA thioesters. When [1- 14 C]oleoyl-CoA is used as the radioactive substrate, phosphatidylcholines and other phospholipids are most extensively radiolabeled by oleoyl moieties rather than by very long chain monounsaturated acyl moieties. When [2- 14 C]malonyl-CoA is used as the radioactive substrate, no radioactive oleic acid is formed and the newly synthesized very long chain monounsaturated fatty acids are extensively incorporated into phosphatidylcholines and other phospholipids as well as triacylglycerols. The pattern of labeling of the key intermediates of the Kennedy pathway, e.g. lysophosphatidic acids, phosphatidic acids, and diacylglycerols by the newly synthesized very long chain monounsaturated fatty acids is consistent with the operation of this pathway in the biosynthesis of triacylglycerols

Temperature-dependent reactions of phthalic acid on Ag(100)

Czech Academy of Sciences Publication Activity Database

Franke, M.; Marchini, M.; Zhang, L.; Tariq, Q.; Tsud, N.; Vorokhta, M.; Vondráček, Martin; Prince, K.; Röckert, M.; Williams, F.J.; Steinrück, H.-P.; Lytken, O.

2015-01-01

Roč. 119, č. 41 (2015), 23580-23585 ISSN 1932-7447 Institutional support: RVO:68378271 Keywords : phthalic acid * NEXAFS * photoemission spectroscopy * temperature - programmed desoprtion Subject RIV: BM - Solid Matter Physics ; Magnetism Impact factor: 4.509, year: 2015

Microbiological study of lactic acid bacteria in kefir grains by culture-dependent and culture-independent methods.

Science.gov (United States)

Chen, Hsi-Chia; Wang, Sheng-Yao; Chen, Ming-Ju

2008-05-01

Lactic acid bacteria (LAB) in different original kefir grains were first assessed using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) by a culture-dependent way, and were further confirmed by DNA sequencing techniques. Results indicated that a combined method of cultivation with PCR-DGGE and subsequent DNA sequencing could successfully identify four LAB strains from three kefir grains from Taiwan (named Hsinchu, Mongolia and Ilan). Lactobacillus kefiri accounted, in the three kefir grains, for at least half of the isolated colonies while Lb. kefiranofaciens was the second most frequently isolated species. Leuconostoc mesenteroides was less frequently found but still in the three kefir grains conversely to Lactococcus lactis which based on culture-dependent isolation was only found in two of the kefir grains. It was interesting to find that all three kefir grains contain similar LAB species. Furthermore, the DGGE as a culture-independent method was also applied to detect the LAB strains. Results indicated that Lb. kefiranofaciens was found in all three kefir grains, whereas Lb. kefiri was only observed in Hsinchu kefir grain and Lc. lactis was found in both Mongolia and Ilan samples. Two additional strains, Pseudomonas spp. and E. coli, were also detected in kefir grains.

Voltage-Dependent Inhibition of Glycine Receptor Channels by Niflumic Acid

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Galyna Maleeva

2017-05-01

Full Text Available Niflumic acid (NFA is a member of the fenamate class of nonsteroidal anti-inflammatory drugs. This compound and its derivatives are used worldwide clinically for the relief of chronic and acute pain. NFA is also a commonly used blocker of voltage-gated chloride channels. Here we present evidence that NFA is an efficient blocker of chloride-permeable glycine receptors (GlyRs with subunit heterogeneity of action. Using the whole-cell configuration of patch-clamp recordings and molecular modeling, we analyzed the action of NFA on homomeric α1ΔIns, α2B, α3L, and heteromeric α1β and α2β GlyRs expressed in CHO cells. NFA inhibited glycine-induced currents in a voltage-dependent manner and its blocking potency in α2 and α3 GlyRs was higher than that in α1 GlyR. The Woodhull analysis suggests that NFA blocks α1 and α2 GlyRs at the fractional electrical distances of 0.16 and 0.65 from the external membrane surface, respectively. Thus, NFA binding site in α1 GlyR is closer to the external part of the membrane, while in α2 GlyR it is significantly deeper in the pore. Mutation G254A at the cytoplasmic part of the α1 GlyR pore-lining TM2 helix (level 2′ increased the NFA blocking potency, while incorporation of the β subunit did not have a significant effect. The Hill plot analysis suggests that α1 and α2 GlyRs are preferably blocked by two and one NFA molecules, respectively. Molecular modeling using Monte Carlo energy minimizations provides the structural rationale for the experimental data and proposes more than one interaction site along the pore where NFA can suppress the ion permeation.

Akt-dependent Activation of the Heart 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase (PFKFB2) Isoenzyme by Amino Acids*

Science.gov (United States)

Novellasdemunt, Laura; Tato, Irantzu; Navarro-Sabate, Aurea; Ruiz-Meana, Marisol; Méndez-Lucas, Andrés; Perales, Jose Carlos; Garcia-Dorado, David; Ventura, Francesc; Bartrons, Ramon; Rosa, Jose Luis

2013-01-01

Reciprocal regulation of metabolism and signaling allows cells to modulate their activity in accordance with their metabolic resources. Thus, amino acids could activate signal transduction pathways that control cell metabolism. To test this hypothesis, we analyzed the effect of amino acids on fructose-2,6-bisphosphate (Fru-2,6-P2) metabolism. We demonstrate that amino acids increase Fru-2,6-P2 concentration in HeLa and in MCF7 human cells. In conjunction with this, 6-phosphofructo-2-kinase activity, glucose uptake, and lactate concentration were increased. These data correlate with the specific phosphorylation of heart 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB2) isoenzyme at Ser-483. This activation was mediated by the PI3K and p38 signaling pathways. Furthermore, Akt inactivation blocked PFKFB2 phosphorylation and Fru-2,6-P2 production, thereby suggesting that the above signaling pathways converge at Akt kinase. In accordance with these results, kinase assays showed that amino acid-activated Akt phosphorylated PFKFB2 at Ser-483 and that knockdown experiments confirmed that the increase in Fru-2,6-P2 concentration induced by amino acids was due to PFKFB2. In addition, similar effects on Fru-2,6-P2 metabolism were observed in freshly isolated rat cardiomyocytes treated with amino acids, which indicates that these effects are not restricted to human cancer cells. In these cardiomyocytes, the glucose consumption and the production of lactate and ATP suggest an increase of glycolytic flux. Taken together, these results demonstrate that amino acids stimulate Fru-2,6-P2 synthesis by Akt-dependent PFKFB2 phosphorylation and activation and show how signaling and metabolism are inextricably linked. PMID:23457334

Salicylic acid-independent plant defence pathways

OpenAIRE

Pieterse, C.M.J.; Loon, L.C. van

1999-01-01

Salicylic acid is an important signalling molecule involved in both locally and systemically induced disease resistance responses. Recent advances in our understanding of plant defence signalling have revealed that plants employ a network of signal transduction pathways, some of which are independent of salicylic acid. Evidence is emerging that jasmonic acid and ethylene play key roles in these salicylic acid-independent pathways. Cross-talk between the salicylic acid-dependent and the salicy...

The random co-polymer glatiramer acetate rapidly kills primary human leukocytes through sialic-acid-dependent cell membrane damage

DEFF Research Database (Denmark)

Christiansen, Stig Hill; Zhang, Xianwei; Juul-Madsen, Kristian

2017-01-01

in innate immunity. It shares the positive charge and amphipathic character of GA, and, as shown here, also the ability to kill human leukocyte. The cytotoxicity of both compounds depends on sialic acid in the cell membrane. The killing was associated with the generation of CD45 + debris, derived from cell...... membrane deformation. Nanoparticle tracking analysis confirmed the formation of such debris, even at low GA concentrations. Electric cell-substrate impedance sensing measurements also recorded stable alterations in T lymphocytes following such treatment. LL-37 forms oligomers through weak hydrophobic...

Silymarin and caffeine combination ameliorates experimentally-induced hepatic fibrosis through down-regulation of LPAR1 expression.

Science.gov (United States)

Eraky, Salma M; El-Mesery, Mohamed; El-Karef, Amro; Eissa, Laila A; El-Gayar, Amal M

2018-05-01

Lysophosphatidic acid is a lipid mediator that is supposed to be implicated in hepatic fibrosis. Silymarin and caffeine are natural compounds known for their anti-inflammatory and antioxidant effects. Our study aimed to explore the effect of silymarin, caffeine, and their combination on lysophosphatidic acid receptor 1 (LPAR1) pathway in thioacetamide (TAA)-induced hepatic fibrosis. Hepatic fibrosis was induced in male Sprague-Dawley rats by intraperitoneal injection of 200 mg/kg of TAA twice a week for 8 weeks. Silymarin (50 mg/kg), caffeine (50 mg/kg), and their combination (50 mg/kg silymarin + 50 mg/kg caffeine) were orally given to rats every day for 8 weeks along with TAA injection. Liver functions were measured. Histopathological examination of liver tissues was performed using hematoxylin and eosin and Masson's trichrome staining. mRNA expressions of LPAR1, transforming growth factor beta 1 (TGF-β1), connective tissue growth factor (CTGF), and alpha smooth muscle actin (α-SMA) were measured using RT-PCR. LPAR1 tissue expression was scored using immunohistochemistry. Silymarin, caffeine, and their combination significantly improved liver function. They caused significant decrease in fibrosis and necro-inflammatory scores. Combination of silymain and caffeine caused a significant decrease in the necro-inflammatory score than the single treatment with silymarin or caffeine. In addition, silymarin, caffeine, and their combination significantly decreased hepatic LPAR1, TGF-β1, CTGF, and α-SMA gene expressions and LPAR1 tissue expression. Silymarin, caffeine, and their combination protect against liver fibrosis through down-regulation of LPAR1, TGF-β1, and CTGF. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

A charge-dependent mechanism is responsible for the dynamic accumulation of proteins inside nucleoli.

Science.gov (United States)

Musinova, Yana R; Kananykhina, Eugenia Y; Potashnikova, Daria M; Lisitsyna, Olga M; Sheval, Eugene V

2015-01-01

The majority of known nucleolar proteins are freely exchanged between the nucleolus and the surrounding nucleoplasm. One way proteins are retained in the nucleoli is by the presence of specific amino acid sequences, namely nucleolar localization signals (NoLSs). The mechanism by which NoLSs retain proteins inside the nucleoli is still unclear. Here, we present data showing that the charge-dependent (electrostatic) interactions of NoLSs with nucleolar components lead to nucleolar accumulation as follows: (i) known NoLSs are enriched in positively charged amino acids, but the NoLS structure is highly heterogeneous, and it is not possible to identify a consensus sequence for this type of signal; (ii) in two analyzed proteins (NF-κB-inducing kinase and HIV-1 Tat), the NoLS corresponds to a region that is enriched for positively charged amino acid residues; substituting charged amino acids with non-charged ones reduced the nucleolar accumulation in proportion to the charge reduction, and nucleolar accumulation efficiency was strongly correlated with the predicted charge of the tested sequences; and (iii) sequences containing only lysine or arginine residues (which were referred to as imitative NoLSs, or iNoLSs) are accumulated in the nucleoli in a charge-dependent manner. The results of experiments with iNoLSs suggested that charge-dependent accumulation inside the nucleoli was dependent on interactions with nucleolar RNAs. The results of this work are consistent with the hypothesis that nucleolar protein accumulation by NoLSs can be determined by the electrostatic interaction of positively charged regions with nucleolar RNAs rather than by any sequence-specific mechanism. Copyright © 2014 Elsevier B.V. All rights reserved.

Chemical transformations associated with neutronic irradiation of telluric acid; Transformations chimiques associees a l'irradiation neutronique de l'acide teliurique

Energy Technology Data Exchange (ETDEWEB)

Bertet, M [Commissariat a l' Energie Atomique, Saclay (France). Centre d' Etudes Nucleaires

1963-01-15

The chemical transformations which arise from irradiation of telluric acid with neutrons have been studied under several experimental conditions. The effects of the reaction (n, {gamma} ) on Te{sup VI} and of the isomeric transitions {sup 131m}Te{sup VI} {yields} {sup 131}Te and {sup 129m}Te{sup VI} {yields} {sup 129}Te, and the oxidation states of {sup 131}I formed by {beta} decay of {sup 131}Te have been investigated in detail. The Szilard-Chalmers effect has been put in evidence. Retention (R) depends on the isomeric state of Rd-Te and is higher for the metastable isotopes. R increases with the time of irradiation. R seems to be independent of the medium which is used for dissolving telluric acid irradiated in the solid state. Higher values of R are found if the acid is irradiated in neutral or alkaline solution; irradiation in acid solution leads to lower values for R. Retention for {sup 131}Te{sup VI} and {sup 129}Te{sup VI} formed by isomeric transition depends on the pH of the solution where this disintegration occurs. For instance, with {sup 129}Te, R is greater in 6 M NaOH (80 per cent) than in 3 M HCI (40 per cent). The relative amounts of the oxidation states of {sup 131}I (reduced fraction (I{sup -}, IO{sup -}, I{sub 2}), iodate and periodate) depend on the medium, both if the acid is irradiated in the solid state and it is irradiated in solution. In the first case, the reduced fraction increases from 12 to 89 per cent when the dissolving medium is changed from neutral to 0.8 M HNO{sub 3}. In the second case, the reduced fraction is 90 per cent in neutral or acid solution and 64 per cent in 0.5 M KOH. It has been shown, furthermore, that microamounts of Te{sup VI} are formed in certain cases. (author) [French] Les transformations chimiques accompagnant l'irradiation neutronique de l'acide tellurique ont ete etudiees dans differentes conditions experimentales. On a examine notamment les consequences de la reaction (n, {gamma}) sur Te{sup VI}, des

Cellulose with a High Fractal Dimension Is Easily Hydrolysable under Acid Catalysis

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Mariana Díaz

2017-05-01

Full Text Available The adsorption of three diverse amino acids couples onto the surface of microcrystalline cellulose was studied. Characterisation of modified celluloses included changes in the polarity and in roughness. The amino acids partially break down the hydrogen bonding network of the cellulose structure, leading to more reactive cellulose residues that were easily hydrolysed to glucose in the presence of hydrochloric acid or tungstophosphoric acid catalysts. The conversion of cellulose and selectivity for glucose was highly dependent on the self-assembled amino acids adsorbed onto the cellulose and the catalyst.

Fibrinolytic Activity and Dose-Dependent Effect of Incubating Human Blood Clots in Caffeic Acid Phenethyl Ester: In Vitro Assays

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Abuzar Elnager

2015-01-01

Full Text Available Background. Caffeic acid phenethyl ester (CAPE has been reported to possess time-dependent fibrinolytic activity by in vitro assay. This study is aimed at investigating fibrinolytic dose-dependent activity of CAPE using in vitro assays. Methods. Standardized human whole blood (WB clots were incubated in either blank controls or different concentrations of CAPE (3.75, 7.50, 15.00, 22.50, and 30.00 mM. After 3 hours, D-dimer (DD levels and WB clot weights were measured for each concentration. Thromboelastography (TEG parameters were recorded following CAPE incubation, and fibrin morphology was examined under a confocal microscope. Results. Overall, mean DD (μg/mL levels were significantly different across samples incubated with different CAPE concentrations, and the median pre- and postincubation WB clot weights (grams were significantly decreased for each CAPE concentration. Fibrin removal was observed microscopically and indicated dose-dependent effects. Based on the TEG test, the Ly30 fibrinolytic parameter was significantly different between samples incubated with two different CAPE concentrations (15.0 and 22.50 mM. The 50% effective dose (ED50 of CAPE (based on DD was 1.99 mg/mL. Conclusions. This study suggests that CAPE possesses fibrinolytic activity following in vitro incubation and that it has dose-dependent activities. Therefore, further investigation into CAPE as a potential alternative thrombolytic agent should be conducted.

Catalyzed oxidation reactions. IV. Picolinic acid catalysis of chromic acid oxidations

International Nuclear Information System (INIS)

Rocek, J.; Peng, T.Y.

1977-01-01

Picolinic acid and several closely related acids are effective catalysts in the chromic acid oxidation of primary and secondary alcohols; the oxidation of other substrates is accelerated only moderately. The reaction is first order in chromium-(VI), alcohol, and picolinic acid; it is second order in hydrogen ions at low acidity and approaches acidity independence at high perchloric acid concentrations. A primary deuterium kinetic isotope effect is observed at high but not at low acidities. At low acidity the reaction has a considerably lower activation energy and more negative activation entropy than at higher acidities. The reactive intermediate in the proposed mechanism is a negatively charged termolecular complex formed from chromic acid, picolinic acid, and alcohol. The rate-limiting step of the reaction changes with the acidity of the solution. At higher acidities the intermediate termolecular complex is formed reversibly and the overall reaction rate is determined by the rate of its decomposition into reaction products; at low acidities the formation of the complex is irreversible and hence rate limiting. Picolinic acids with a substituent in the 6 position show a greatly reduced catalytic activity. This observation is interpreted as suggesting a square pyramidal or octahedral structure for the reactive chromium (VI) intermediate. The temperature dependence of the deuterium isotope effect has been determined and the significance of the observed large values for E/sub a//sup D/ - E/sub a//sup H/ and A/sup D//A/sup H/ is discussed

Redox cycles of vitamin E: Hydrolysis and ascorbic acid dependent reduction of 8a-(alkyldioxy)tocopherones

International Nuclear Information System (INIS)

Liebler, D.C.; Kaysen, K.L.; Kennedy, T.A.

1989-01-01

Oxidation of the biological antioxidant α-tocopherol (vitamin E; TH) by peroxyl radicals yields 8a-(alkyldioxy)tocopherones, which either may hydrolyze to α-tocopheryl quinone (TQ) or may be reduced by ascorbic acid to regenerate TH. To define the chemistry of this putative two-electron TH redox cycle, we studied the hydrolysis and reduction of 8a-[(2,4-dimethyl-1-nitrilopent-2-yl)dioxyl]tocopherone (1) in acetonitrile/buffer mixtures and in phospholipid liposomes. TQ formation in acetonitrile/buffer mixtures, which was monitored spectrophotometrically, declined with increasing pH and could not be detected above pH 4. The rate of TQ formation from 1 first increased with time and then decreased in a first-order terminal phase. Rearrangement of 8a-hydroxy-α-tocopherone (2) to TQ displayed first-order kinetics identical with the terminal phase for TQ formation from 1. Both rate constants increased with decreasing pH. Hydrolysis of 1 in acetonitrile/H 2 18 O yielded [ 18 O]TQ. These observations suggest that 1 loses the 8a-(alkyldioxy) moiety to produce the tocopherone cation (T + ), which hydrolyzes to 2, the TQ-forming intermediate. Incubation of either 1 or 2 with ascorbic acid in acetonitrile/buffer yielded TH. Reduction of both 1 and 2 decreased with increasing pH. In phosphatidylcholine liposomes at pH 7, approximately 10% of the T + generated from 1 was reduced to TH by 5 mM ascorbic acid. The results collectively demonstrate that T + is the ascorbic acid reducible intermediate in a two-electron TH redox cycle, a process that probably would require biocatalysis to proceed in biological membranes

Dafachronic acid inhibits C. elegans germ cell proliferation in a DAF-12-dependent manner.

Science.gov (United States)

Mukherjee, Madhumati; Chaudhari, Snehal N; Balachandran, Riju S; Vagasi, Alexandra S; Kipreos, Edward T

2017-12-15

Dafachronic acid (DA) is a bile acid-like steroid hormone that regulates dauer formation, heterochrony, and lifespan in C. elegans. Here, we describe that DA is an inhibitor of C. elegans germ stem cell proliferation in adult hermaphrodites. Using a C. elegans germ cell primary culture system, we show that DA inhibits the proliferation of germ cells in vitro. Exogenous DA reduces the frequency of large tumors in adult tumorous germline mutants and decreases the proliferation of wild-type germ stem cells in adult hermaphrodites. In contrast, DA has no appreciable effect on the proliferation of larval-stage germ cells in wild type. The inhibition of adult germ cell proliferation by DA requires its canonical receptor DAF-12. Blocking DA production by inactivating the cytochrome P450 DAF-9 increases germ cell proliferation in wild-type adult hermaphrodites and the frequency of large tumors in germline tumorous mutants, suggesting that DA inhibits the rate of germ cell proliferation under normal growth conditions. Copyright © 2017 Elsevier Inc. All rights reserved.

Docosahexaenoic acid inhibits the growth of hormone-dependent prostate cancer cells by promoting the degradation of the androgen receptor.

Science.gov (United States)

Hu, Zhimei; Qi, Haixia; Zhang, Ruixue; Zhang, Kun; Shi, Zhemin; Chang, Yanan; Chen, Linfeng; Esmaeili, Mohsen; Baniahmad, Aria; Hong, Wei

2015-09-01

Epidemiological and preclinical data have demonstrated the preventative effects of ω-3 polyunsaturated fatty acids, including docosahexaenoic acid (DHA), on prostate cancer. However, there are inconsistencies in these previous studies and the underlying mechanisms remain to be elucidated. In the present study, the androgen receptor (AR), which is a transcription factor involved in cell proliferation and prostate carcinogenesis, was identified as a target of DHA. It was revealed that DHA inhibited hormone‑dependent growth of LNCaP prostate cancer cells. Reverse transcription-quantitative polymerase chain reaction analysis revealed that treatment with DHA caused no alteration in the transcribed mRNA expression levels of the AR gene. However, immunoblotting revealed that this treatment reduces the protein expression level of the AR. The androgen‑induced genes were subsequently repressed by treatment with DHA. It was demonstrated that DHA exhibits no effect on the translation process of the AR, however, it promotes the proteasome‑mediated degradation of the AR. Therefore, the present study provided a novel mechanism by which DHA exhibits an inhibitory effect on growth of prostate cancer cells.

Metabolic Effects of Cholecystectomy: Gallbladder Ablation Increases Basal Metabolic Rate through G-Protein Coupled Bile Acid Receptor Gpbar1-Dependent Mechanisms in Mice

Science.gov (United States)

Cortés, Víctor; Amigo, Ludwig; Zanlungo, Silvana; Galgani, José; Robledo, Fermín; Arrese, Marco; Bozinovic, Francisco; Nervi, Flavio

2015-01-01

Background & Aims Bile acids (BAs) regulate energy expenditure by activating G-protein Coupled Bile Acid Receptor Gpbar1/TGR5 by cAMP-dependent mechanisms. Cholecystectomy (XGB) increases BAs recirculation rates resulting in increased tissue exposure to BAs during the light phase of the diurnal cycle in mice. We aimed to determine: 1) the effects of XGB on basal metabolic rate (BMR) and 2) the roles of TGR5 on XGB-dependent changes in BMR. Methods BMR was determined by indirect calorimetry in wild type and Tgr5 deficient (Tgr5-/-) male mice. Bile flow and BAs secretion rates were measured by surgical diversion of biliary duct. Biliary BAs and cholesterol were quantified by enzymatic methods. BAs serum concentration and specific composition was determined by liquid chromatography/tandem mass spectrometry. Gene expression was determined by qPCR analysis. Results XGB increased biliary BAs and cholesterol secretion rates, and elevated serum BAs concentration in wild type and Tgr5-/- mice during the light phase of the diurnal cycle. BMR was ~25% higher in cholecystectomized wild type mice (p <0.02), whereas no changes were detected in cholecystectomized Tgr5-/- mice compared to wild-type animals. Conclusion XGB increases BMR by TGR5-dependent mechanisms in mice. PMID:25738495

Second site escape of a T20-dependent HIV-1 variant by a single amino acid change in the CD4 binding region of the envelope glycoprotein

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Berkhout Ben

2006-11-01

Full Text Available Abstract Background We previously described the selection of a T20-dependent human immunodeficiency virus type-1 (HIV-1 variant in a patient on T20 therapy. The fusion inhibitor T20 targets the viral envelope (Env protein by blocking a conformational switch that is critical for viral entry into the host cell. T20-dependent viral entry is the result of 2 mutations in Env (GIA-SKY, creating a protein that undergoes a premature conformational switch, and the presence of T20 prevents this premature switch and rescues viral entry. In the present study, we performed 6 independent evolution experiments with the T20-dependent HIV-1 variant in the absence of T20, with the aim to identify second site compensatory changes, which may provide new mechanistic insights into Env function and the T20-dependence mechanism. Results Escape variants with improved replication capacity appeared within 42 days in 5 evolution cultures. Strikingly, 3 cultures revealed the same single amino acid change in the CD4 binding region of Env (glycine at position 431 substituted for arginine: G431R. This mutation was sufficient to abolish the T20-dependence phenotype and restore viral replication in the absence of T20. The GIA-SKY-G431R escape variant produces an Env protein that exhibits reduced syncytia formation and reduced cell-cell fusion activity. The escape variant was more sensitive to an antibody acting on an early gp41 intermediate, suggesting that the G431R mutation helps preserve a pre-fusion Env conformation, similar to T20 action. The escape variant was also less sensitive to soluble CD4, suggesting a reduced CD4 receptor affinity. Conclusion The forced evolution experiments indicate that the premature conformational switch of the T20-dependent HIV-1 Env variant (GIA-SKY can be corrected by a second site mutation in Env (GIA-SKY-G431R that affects the interaction with the CD4 receptor.

Cloning and functional expression of a plant voltage-dependent chloride channel.

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Lurin, C; Geelen, D; Barbier-Brygoo, H; Guern, J; Maurel, C

1996-01-01

Plant cell membrane anion channels participate in basic physiological functions, such as cell volume regulation and signal transduction. However, nothing is known about their molecular structure. Using a polymerase chain reaction strategy, we have cloned a tobacco cDNA (CIC-Nt1) encoding a 780-amino acid protein with several putative transmembrane domains. CIC-Nt1 displays 24 to 32% amino acid identity with members of the animal voltage-dependent chloride channel (CIC) family, whose archetype is CIC-0 from the Torpedo marmorata electric organ. Injection of CIC-Nt1 complementary RNA into Xenopus oocytes elicited slowly activating inward currents upon membrane hyperpolarization more negative than -120 mV. These currents were carried mainly by anions, modulated by extracellular anions, and totally blocked by 10 mM extracellular calcium. The identification of CIC-Nt1 extends the CIC family to higher plants and provides a molecular probe for the study of voltage-dependent anion channels in plants. PMID:8624442

Regulation of autophagy by amino acids and MTOR-dependent signal transduction.

Science.gov (United States)

Meijer, Alfred J; Lorin, Séverine; Blommaart, Edward F; Codogno, Patrice

2015-10-01

Amino acids not only participate in intermediary metabolism but also stimulate insulin-mechanistic target of rapamycin (MTOR)-mediated signal transduction which controls the major metabolic pathways. Among these is the pathway of autophagy which takes care of the degradation of long-lived proteins and of the elimination of damaged or functionally redundant organelles. Proper functioning of this process is essential for cell survival. Dysregulation of autophagy has been implicated in the etiology of several pathologies. The history of the studies on the interrelationship between amino acids, MTOR signaling and autophagy is the subject of this review. The mechanisms responsible for the stimulation of MTOR-mediated signaling, and the inhibition of autophagy, by amino acids have been studied intensively in the past but are still not completely clarified. Recent developments in this field are discussed.

pH-Dependent Uptake of Fumaric Acid in Saccharomyces cerevisiae under Anaerobic Conditions

NARCIS (Netherlands)

Jamalzadeh, E.; Verheijen, P.J.; Heijnen, J.J.; Van Gulik, W.M.

2011-01-01

Microbial production of C4 dicarboxylic acids from renewable resources has gained renewed interest. The yeast Saccharomyces cerevisiae is known as a robust microorganism and is able to grow at low pH, which makes it a suitable candidate for biological production of organic acids. However, a

Rapid profiling of polymeric phenolic acids in Salvia miltiorrhiza by hybrid data-dependent/targeted multistage mass spectrometry acquisition based on expected compounds prediction and fragment ion searching.

Science.gov (United States)

Shen, Yao; Feng, Zijin; Yang, Min; Zhou, Zhe; Han, Sumei; Hou, Jinjun; Li, Zhenwei; Wu, Wanying; Guo, De-An

2018-04-01

Phenolic acids are the major water-soluble components in Salvia miltiorrhiza (>5%). According to previous studies, many of them contribute to the cardiovascular effects and antioxidant effects of S. miltiorrhiza. Polymeric phenolic acids can be considered as the tanshinol derived metabolites, e.g., dimmers, trimers, and tetramers. A strategy combined with tanshinol-based expected compounds prediction, total ion chromatogram filtering, fragment ion searching, and parent list-based multistage mass spectrometry acquisition by linear trap quadropole-orbitrap Velos mass spectrometry was proposed to rapid profile polymeric phenolic acids in S. miltiorrhiza. More than 480 potential polymeric phenolic acids could be screened out by this strategy. Based on the fragment information obtained by parent list-activated data dependent multistage mass spectrometry acquisition, 190 polymeric phenolic acids were characterized by comparing their mass information with literature data, and 18 of them were firstly detected from S. miltiorrhiza. Seven potential compounds were tentatively characterized as new polymeric phenolic acids from S. miltiorrhiza. This strategy facilitates identification of polymeric phenolic acids in complex matrix with both selectivity and sensitivity, which could be expanded for rapid discovery and identification of compounds from complex matrix. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

AtPP2CG1, a protein phosphatase 2C, positively regulates salt tolerance of Arabidopsis in abscisic acid-dependent manner

International Nuclear Information System (INIS)

Liu, Xin; Zhu, Yanming; Zhai, Hong; Cai, Hua; Ji, Wei; Luo, Xiao; Li, Jing; Bai, Xi

2012-01-01

Highlights: ► AtPP2CG1 positively regulates salt tolerance in ABA-dependent manner. ► AtPP2CG1 up-regulates the expression of marker genes in different pathways. ► AtPP2CG1 expresses in the vascular system and trichomes of Arabidopsis. -- Abstract: AtPP2CG1 (Arabidopsis thaliana protein phosphatase 2C G Group 1) was predicted as an abiotic stress candidate gene by bioinformatic analysis in our previous study. The gene encodes a putative protein phosphatase 2C that belongs to Group G of PP2C. There is no report of Group G genes involved in abiotic stress so far. Real-time RT-PCR analysis showed that AtPP2CG1 expression was induced by salt, drought, and abscisic acid (ABA) treatment. The expression levels of AtPP2CG1 in the ABA synthesis-deficient mutant abi2–3 were much lower than that in WT plants under salt stress suggesting that the expression of AtPP2CG1 acts in an ABA-dependent manner. Over-expression of AtPP2CG1 led to enhanced salt tolerance, whereas its loss of function caused decreased salt tolerance. These results indicate that AtPP2CG1 positively regulates salt stress in an ABA-dependent manner. Under salt treatment, AtPP2CG1 up-regulated the expression levels of stress-responsive genes, including RD29A, RD29B, DREB2A and KIN1. GUS activity was detected in roots, leaves, stems, flower, and trichomes of AtPP2CG1 promoter–GUS transgenic plants. AtPP2CG1 protein was localized in nucleus and cytoplasm via AtPP2CG1:eGFP and YFP:AtPP2CG1 fusion approaches.

AtPP2CG1, a protein phosphatase 2C, positively regulates salt tolerance of Arabidopsis in abscisic acid-dependent manner

Energy Technology Data Exchange (ETDEWEB)

Liu, Xin, E-mail: fangfei6073@126.com [Plant Bioengineering Laboratory, Northeast Agricultural University, Harbin 150030 (China); Zhu, Yanming, E-mail: ymzhu2001@neau.edu.cn [Plant Bioengineering Laboratory, Northeast Agricultural University, Harbin 150030 (China); Zhai, Hong, E-mail: Zhai.h@neigaehrb.ac.cn [Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Harbin 150040 (China); Cai, Hua, E-mail: small-big@sohu.com [Plant Bioengineering Laboratory, Northeast Agricultural University, Harbin 150030 (China); Ji, Wei, E-mail: iwei_j@hotmail.com [Plant Bioengineering Laboratory, Northeast Agricultural University, Harbin 150030 (China); Luo, Xiao, E-mail: luoxiao2010@yahoo.cn [Plant Bioengineering Laboratory, Northeast Agricultural University, Harbin 150030 (China); Li, Jing, E-mail: lijing@neau.edu.cn [Plant Secondary Metabolism Laboratory, Northeast Agricultural University, Harbin 150030 (China); Bai, Xi, E-mail: baixi@neau.edu.cn [Plant Bioengineering Laboratory, Northeast Agricultural University, Harbin 150030 (China)

2012-06-15

Highlights: Black-Right-Pointing-Pointer AtPP2CG1 positively regulates salt tolerance in ABA-dependent manner. Black-Right-Pointing-Pointer AtPP2CG1 up-regulates the expression of marker genes in different pathways. Black-Right-Pointing-Pointer AtPP2CG1 expresses in the vascular system and trichomes of Arabidopsis. -- Abstract: AtPP2CG1 (Arabidopsis thaliana protein phosphatase 2C G Group 1) was predicted as an abiotic stress candidate gene by bioinformatic analysis in our previous study. The gene encodes a putative protein phosphatase 2C that belongs to Group G of PP2C. There is no report of Group G genes involved in abiotic stress so far. Real-time RT-PCR analysis showed that AtPP2CG1 expression was induced by salt, drought, and abscisic acid (ABA) treatment. The expression levels of AtPP2CG1 in the ABA synthesis-deficient mutant abi2-3 were much lower than that in WT plants under salt stress suggesting that the expression of AtPP2CG1 acts in an ABA-dependent manner. Over-expression of AtPP2CG1 led to enhanced salt tolerance, whereas its loss of function caused decreased salt tolerance. These results indicate that AtPP2CG1 positively regulates salt stress in an ABA-dependent manner. Under salt treatment, AtPP2CG1 up-regulated the expression levels of stress-responsive genes, including RD29A, RD29B, DREB2A and KIN1. GUS activity was detected in roots, leaves, stems, flower, and trichomes of AtPP2CG1 promoter-GUS transgenic plants. AtPP2CG1 protein was localized in nucleus and cytoplasm via AtPP2CG1:eGFP and YFP:AtPP2CG1 fusion approaches.

Abscisic-acid-dependent basic leucine zipper (bZIP) transcription factors in plant abiotic stress.

Science.gov (United States)

Banerjee, Aditya; Roychoudhury, Aryadeep

2017-01-01

One of the major causes of significant crop loss throughout the world is the myriad of environmental stresses including drought, salinity, cold, heavy metal toxicity, and ultraviolet-B (UV-B) rays. Plants as sessile organisms have evolved various effective mechanism which enable them to withstand this plethora of stresses. Most of such regulatory mechanisms usually follow the abscisic-acid (ABA)-dependent pathway. In this review, we have primarily focussed on the basic leucine zipper (bZIP) transcription factors (TFs) activated by the ABA-mediated signalosome. Upon perception of ABA by specialized receptors, the signal is transduced via various groups of Ser/Thr kinases, which phosphorylate the bZIP TFs. Following such post-translational modification of TFs, they are activated so that they bind to specific cis-acting sequences called abscisic-acid-responsive elements (ABREs) or GC-rich coupling elements (CE), thereby influencing the expression of their target downstream genes. Several in silico techniques have been adopted so far to predict the structural features, recognize the regulatory modification sites, undergo phylogenetic analyses, and facilitate genome-wide survey of TF under multiple stresses. Current investigations on the epigenetic regulation that controls greater accessibility of the inducible regions of DNA of the target gene to the bZIP TFs exclusively under stress situations, along with the evolved stress memory responses via genomic imprinting mechanism, have been highlighted. The potentiality of overexpression of bZIP TFs, either in a homologous or in a heterologous background, in generating transgenic plants tolerant to various abiotic stressors have also been addressed by various groups. The present review will provide a coherent documentation on the functional characterization and regulation of bZIP TFs under multiple environmental stresses, with the major goal of generating multiple-stress-tolerant plant cultivars in near future.

Succinic acid production from Jerusalem artichoke

DEFF Research Database (Denmark)

Gunnarsson, Ingólfur Bragi; Karakashev, Dimitar Borisov; Angelidaki, Irini

In this work, A. succinogenes 130Z was used to produce succinic acid from Jerusalem artichoke tuber hydrolysate. Results showed that both fructose and glucose in the tuber hydrolysate were utilized for succinic acid production. The sugar utilization was found to be dependent on process control...... that Jerusalem artichoke tubers could be utilized for production of bio-succinic acid....

Oleanolic acid induces mitochondrial-dependent apoptosis and G0/G1 phase arrest in gallbladder cancer cells

Directory of Open Access Journals (Sweden)

Li HF

2015-06-01

Full Text Available Huai-Feng Li,1–3,* Xu-An Wang,1–3,* Shan-Shan Xiang,1–3,* Yun-Ping Hu,1–3 Lin Jiang,1–3 Yi-Jun Shu,1–3 Mao-Lan Li,1–3 Xiang-Song Wu,1–3 Fei Zhang,1–3 Yuan-Yuan Ye,1–3 Hao Weng,1–3 Run-Fa Bao,1–3 Yang Cao,1–3 Wei Lu,1–3 Qian Dong,1–3 Ying-Bin Liu1–3 1Department of General Surgery, 2Laboratory of General Surgery, 3Institute of Biliary Tract Disease, Xinhua Hospital, Affiliated to Shanghai Jiao Tong University, School of Medicine, Shanghai, People’s Republic of China *These authors contributed equally to this work Abstract: Oleanolic acid (OA, a naturally occurring triterpenoid, exhibits potential antitumor activity in many tumor cell lines. Gallbladder carcinoma is the most common malignancy of the biliary tract, and is a highly aggressive tumor with an extremely poor prognosis. Unfortunately, the effects of OA on gallbladder carcinoma are unknown. In this study, we investigated the effects of OA on gallbladder cancer cells and the underlying mechanism. The results showed that OA inhibits proliferation of gallbladder cancer cells in a dose-dependent and time-dependent manner on MTT and colony formation assay. A flow cytometry assay revealed apoptosis and G0/G1 phase arrest in GBC-SD and NOZ cells. Western blot analysis and a mitochondrial membrane potential assay demonstrated that OA functions through the mitochondrial apoptosis pathway. Moreover, this drug inhibited tumor growth in nude mice carrying subcutaneous NOZ tumor xenografts. These data suggest that OA inhibits proliferation of gallbladder cancer cells by regulating apoptosis and the cell cycle process. Thus, OA may be a promising drug for adjuvant chemotherapy in gallbladder carcinoma. Keywords: oleanolic acid, gallbladder carcinoma, apoptosis, cell cycle arrest, mitochondrial pathway

Dietary arachidonic acid in perinatal nutrition: a commentary.

Science.gov (United States)

Lauritzen, Lotte; Fewtrell, Mary; Agostoni, Carlo

2015-01-01

Arachidonic acid (AA) is supplied together with docosahexaenoic acid (DHA) in infant formulas, but we have limited knowledge about the effects of supplementation with either of these long-chain polyunsaturated fatty acids (LCPUFA) on growth and developmental outcomes. AA is present in similar levels in breast milk throughout the world, whereas the level of DHA is highly diet dependent. Autopsy studies show similar diet-dependent variation in brain DHA, whereas AA is little affected by intake. Early intake of DHA has been shown to affect visual development, but the effect of LCPUFA on neurodevelopment remains to be established. Few studies have found any functional difference between infants supplemented with DHA alone compared to DHA+AA, but some studies show neurodevelopmental advantages in breast-fed infants of mothers supplemented with n-3 LCPUFA alone. It also remains to be established whether the AA/DHA balance could affect allergic and inflammatory outcomes later in life. Disentangling effects of genetic variability and dietary intake on AA and DHA-status and on functional outcomes may be an important step in the process of determining whether AA-intake is of any physiological or clinical importance. However, based on the current evidence we hypothesize that dietary AA plays a minor role on growth and development relative to the impact of dietary DHA.

Inhibition studies of soybean (Glycine max) urease with heavy metals, sodium salts of mineral acids, boric acid, and boronic acids.

Science.gov (United States)

Kumar, Sandeep; Kayastha, Arvind M

2010-10-01

Various inhibitors were tested for their inhibitory effects on soybean urease. The K(i) values for boric acid, 4-bromophenylboronic acid, butylboronic acid, and phenylboronic acid were 0.20 +/- 0.05 mM, 0.22 +/- 0.04 mM, 1.50 +/- 0.10 mM, and 2.00 +/- 0.11 mM, respectively. The inhibition was competitive type with boric acid and boronic acids. Heavy metal ions including Ag(+), Hg(2+), and Cu(2+) showed strong inhibition on soybean urease, with the silver ion being a potent inhibitor (IC(50) = 2.3 x 10(-8) mM). Time-dependent inhibition studies exhibited biphasic kinetics with all heavy metal ions. Furthermore, inhibition studies with sodium salts of mineral acids (NaF, NaCl, NaNO(3), and Na(2)SO(4)) showed that only F(-) inhibited soybean urease significantly (IC(50) = 2.9 mM). Competitive type of inhibition was observed for this anion with a K(i) value of 1.30 mM.

Glycolic acid peel therapy – a current review

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Sharad J

2013-11-01

Full Text Available Jaishree Sharad Skinfiniti Aesthetic Skin and Laser Clinic, Mumbai, India Abstract: Chemical peels have been time-tested and are here to stay. Alpha-hydroxy peels are highly popular in the dermatologist's arsenal of procedures. Glycolic acid peel is the most common alpha-hydroxy acid peel, also known as fruit peel. It is simple, inexpensive, and has no downtime. This review talks about various studies of glycolic acid peels for various indications, such as acne, acne scars, melasma, postinflammatory hyperpigmentation, photoaging, and seborrhea. Combination therapies and treatment procedure are also discussed. Careful review of medical history, examination of the skin, and pre-peel priming of skin are important before every peel. Proper patient selection, peel timing, and neutralization on-time will ensure good results, with no side effects. Depth of the glycolic acid peel depends on the concentration of the acid used, the number of coats applied, and the time for which it is applied. Hence, it can be used as a very superficial peel, or even a medium depth peel. It has been found to be very safe with Fitzpatrick skin types I–IV. All in all, it is a peel that is here to stay. Keywords: acne scar, melasma, photoaging, chemical peel, alpha-hydroxy peel

pH dependence of the properties of waterborne pressure-sensitive adhesives containing acrylic acid.

Science.gov (United States)

Wang, Tao; Canetta, Elisabetta; Weerakkody, Tecla G; Keddie, Joseph L; Rivas, Urko

2009-03-01

Polymer colloids are often copolymerized with acrylic acid monomers in order to impart colloidal stability. Here, the effects of the pH on the nanoscale and macroscopic adhesive properties of waterborne poly(butyl acrylate-co-acrylic acid) films are reported. In films cast from acidic colloidal dispersions, hydrogen bonding between carboxylic acid groups dominates the particle-particle interactions, whereas ionic dipolar interactions are dominant in films cast from basic dispersions. Force spectroscopy using an atomic force microscope and macroscale mechanical measurements show that latex films with hydrogen-bonding interactions have lower elastic moduli and are more deformable. They yield higher adhesion energies. On the other hand, in basic latex, ionic dipolar interactions increase the moduli of the dried films. These materials are stiffer and less deformable and, consequently, exhibit lower adhesion energies. The rate of water loss from acidic latex is slower, perhaps because of hydrogen bonding with the water. Therefore, although acid latex offers greater adhesion, there is a limitation in the film formation.

Modifications of proteins by polyunsaturated fatty acid peroxidation products

DEFF Research Database (Denmark)

Refsgaard, Hanne; Tsai, Lin; Stadtman, Earl

2000-01-01

The ability of unsaturated fatty acid methyl esters to modify amino acid residues in bovine serum albumin (BSA), glutamine synthetase, and insulin in the presence of a metal-catalyzed oxidation system [ascorbate/Fe(lll)/O-2] depends on the degree of unsaturation of the fatty acid. The fatty acid......-dependent generation of carbonyl groups and loss of lysine residues increased in the order methyl linoleate fatty acids were oxidized in the presence...... in the formation of protein carbonyls, These results are consistent with the proposition that metal-catalyzed oxidation of polyunsaturated fatty acids can contribute to the generation of protein carbonyls by direct interaction of lipid oxidation products (alpha,beta-unsaturated aldehydes) with lysine residues...

Growth and instability of a phospholipid vesicle in a bath of fatty acids

Science.gov (United States)

Dervaux, J.; Noireaux, V.; Libchaber, A. J.

2017-06-01

Using a microfluidic trap, we study the behavior of individual phospholipid vesicles in contact with fatty acids. We show that spontaneous fatty acids insertion inside the bilayer is controlled by the vesicle size, osmotic pressure difference across the membrane and fatty acids concentration in the external bath. Depending on these parameters, vesicles can grow spherically or become unstable and fragment into several daughter vesicles. We establish the phase diagram for vesicle growth and we derive a simple thermodynamic model that reproduces the time evolution of the vesicle volume. Finally, we show that stable growth can be achieved on an artificial cell expressing a simple set of bacterial cytoskeletal proteins, paving the way toward artificial cell reproduction.

Activation of a peroxisomal Pichia pastoris d-amino acid oxidase, which uses d-alanine as a preferred substrate, depends on pyruvate carboxylase

NARCIS (Netherlands)

Klompmaker, Sandra H.; Kilic, Aysun; Baerends, Richard J.; Veenhuis, Marten; van der Klei, Ida J.; Goffeau, André

d-Amino acid oxidase (DAO) is an important flavo-enzyme that catalyzes the oxidative deamination of d-amino acids into the corresponding alpha-keto acid, ammonia and H(2)O(2). We identified two amino acid oxidases in the methylotrophic yeast Pichia pastoris: Dao1p, which preferentially uses

Enzymatic hydrolysis of 1-monoacyl-SN-glycerol-3-phosphoryl-choline (1-lysolecithin) by phospholipases from peanut seeds.

Science.gov (United States)

Strauss, H; Leibovitz-Ben Gershon, Z; Heller, M

1976-06-01

Hydrolysis of 1-lysolecithin (1-acyl glycerophosphorylcholine [1-acyl GPC]) by preparations of phospholipase D from peanut seeds was investigated. 1-Lysolecithin was hydrolyzed at a much slower rate than phosphatidylcholine (lecithin). Although Ca+2 ions are required for the cleavage of lecithin by the enzyme, their effect on the hydrolysis of lysolecithin depended upon the concentration of the substrate: at 0.2 mM 1-lysolecithin, Ca+2 ions increased the reaction rates, whereas at concentrations of the substrate lower than 0.1 mM, Ca+2 ions were inhibitory. A broad pH activity curve between 5 and 8 was obtained with higher rates in the alkaline range, both in the absence and presence of Ca+2 ions. The increased hydrolysis of lysolecithin due to Ca+2 was noticed over the entire pH range. Upon storage of the enzyme solutions at 4 C, decreased rates of hydrolysis of lecithin were observed, with t 1/2 values of ca. 50 and 100 days depending on the purity of the preparation. During the same period, no reduction occurred in the activity of these preparations on lysolecithin as substrate. The effects of Ca+2 ions and the analysis of the products of 1-acyl GPC cleavage by the enzyme preparations revealed the presence of more than one enzyme and the formation of the following compounds: lysophosphatidic acids (1 acyl glycerophosphoric acids), free fatty acids, glycerophosphorylcholine, and choline. The possible pathways leading to the degradation of lysolecithin and the formation of these products include reactions catalyzed by lysophospholipase A1 (lysophosphatidylcholine 1-acyl hydrolase, E.C. 3.1.1.5) and a phosphodiesterase (L-3-glycerylphosphorylcholine glycerophosphohydrolase, E.C.3.1.4.2), in addition to phospholipase D (phosphatidyl-choline phosphatidohydrolase, E.C. 3.1.4.4).

Microfiltration membranes prepared from polyethersulfone powder grafted with acrylic acid by simultaneous irradiation and their pH dependence

International Nuclear Information System (INIS)

Deng Bo; Li Jingye; Hou Zhengchi; Yao Side; Shi Liuqing; Liang Guoming; Sheng Kanglong

2008-01-01

Polyethersulfone (PES) powder was grafted with acrylic acid (AAc) by simultaneous γ-ray irradiation. The kinetics of the radiation induced graft polymerization was studied and the grafted PES powder was characterized. Then, microfiltration (MF) membranes were prepared from PES-g-PAAc powder with different degrees of grafting (DG) under phase inversion method. The swelling behavior and the mean pore size of MF membranes were measured, and the filtration property was tested. The results showed that the pore size and the flux of MF membranes increased with the increase in DG. And, MF membranes' properties were dependent on the pH value

Pb-induced responses in Zygophyllum fabago plants are organ-dependent and modulated by salicylic acid.

Science.gov (United States)

López-Orenes, Antonio; Martínez-Pérez, Ascensión; Calderón, Antonio A; Ferrer, María A

2014-11-01

Zygophyllum fabago is a promising species for restoring heavy metal (HM) polluted soils, although the mechanisms involved in HM tolerance in this non-model plant remain largely unknown. This paper analyses the extent to which redox-active compounds and enzymatic antioxidants in roots, stems and leaves are responsible for Pb tolerance in a metallicolous ecotype of Z. fabago and the possible influence of salicylic acid (SA) pretreatment (24 h, 0.5 mM SA) in the response to Pb stress. SA pretreatment reduced both the accumulation of Pb in roots and even more so the concentration of Pb in aerial parts of the plants, although a similar drop in the content of chlorophylls and in the maximum quantum yield of photosystem II was observed in both Pb- and SA-Pb-treated plants. Pb increased the endogenous free SA levels in all organs and this response was enhanced in root tissues upon SA pretreatment. Generally, Pb induced a reduction in catalase, ascorbate peroxidase and glutathione reductase specific activities, whereas dehydroascorbate reductase was increased in all organs of control plants. SA pretreatment enhanced the Pb-induced H2O2 accumulation in roots by up-regulating Fe-superoxide dismutase isoenzymes. Under Pb stress, the GSH redox ratio remained highly reduced in all organs while the ascorbic acid redox ratio dropped in leaf tissues where a rise in lipid peroxidation products and electrolyte leakage was observed. Finally, an organ-dependent accumulation of proline and β-carboline alkaloids was found, suggesting these nitrogen-redox-active compounds could play a role in the adaptation strategies of this species to Pb stress. Copyright © 2014. Published by Elsevier Masson SAS.

A review on methods of recovery of acid(s) from spent pickle liquor of steel industry.

Science.gov (United States)

Ghare, N Y; Wani, K S; Patil, V S

2013-04-01

Pickling is the process of removal of oxide layer and rust formed on metal surface. It also removes sand and corrosion products from the surface of metal. Acids such as sulfuric acid, hydrochloric acid are used for pickling. Hydrofluoric acid-Nitric acid mixture is used for stainless steel pickling. Pickling solutions are spent when acid concentration in pickling solutions decreases by 75-85%, which also has metal content up to 150-250 g/ dm3. Spent pickling liquor (SPL) should be dumped because the efficiency of pickling decreases with increasing content of dissolved metal in the bath. The SPL content depends on the plant of origin and the pickling method applied there. SPL from steel pickling in hot-dip galvanizing plants contains zinc(II), iron, traces of lead, chromium. and other heavy metals (max. 500 mg/dm3) and hydrochloric acid. Zinc(II) passes tothe spent solution after dissolution of this metal from zinc(II)-covered racks, chains and baskets used for transportation of galvanized elements. Unevenly covered zinc layers are usually removed in another pickling bath. Due to this, zinc(II) concentration increases even up to 110 g/dm3, while iron content may reach or exceed even 80 g/dm3 in the same solution. This review presents an overview on different aspects of generation and treatment of SPL with recourse to recovery of acid for recycling. Different processes are described in this review and higher weightage is given to membrane processes.

10-Oxo-trans-11-octadecenoic acid generated from linoleic acid by a gut lactic acid bacterium Lactobacillus plantarum is cytoprotective against oxidative stress

Energy Technology Data Exchange (ETDEWEB)

Furumoto, Hidehiro; Nanthirudjanar, Tharnath [Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502 (Japan); Kume, Toshiaki; Izumi, Yasuhiko [Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29, Simoadachi-cho, Sakyo-ku, Kyoto 606-8501 (Japan); Park, Si-Bum [Laboratory of Industrial Microbiology, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502 (Japan); Kitamura, Nahoko; Kishino, Shigenobu; Ogawa, Jun [Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502 (Japan); Hirata, Takashi [Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502 (Japan); Faculty of Rehabilitation, Shijonawategakuen University, 5-11-10, Hojo, Daitou-shi, Osaka 574-0011 (Japan); Sugawara, Tatsuya, E-mail: sugawara@kais.kyoto-u.ac.jp [Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502 (Japan)

2016-04-01

Oxidative stress is a well-known cause of multiple diseases. The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway plays a central role in cellular antioxidative responses. In this study, we investigated the effects of novel fatty acid metabolite derivatives of linoleic acid generated by the gut lactic acid bacteria Lactobacillus plantarum on the Nrf2-ARE pathway. 10-Oxo-trans-11-octadecenoic acid (KetoC) protected HepG2 cells from cytotoxicity induced by hydrogen peroxide. KetoC also significantly increased cellular Nrf2 protein levels, ARE-dependent transcription, and the gene expression of antioxidative enzymes such as heme oxygenase-1 (HO-1), glutamate-cysteine ligase modifier subunit (GCLM), and NAD(P)H:quinone oxidoreductase 1 (NQO1) in HepG2 cells. Additionally, a single oral dose administration of KetoC also increased antioxidative gene expression and protein levels of Nrf2 and HO-1 in mouse organs. Since other fatty acid metabolites and linoleic acid did not affect cellular antioxidative responses, the cytoprotective effect of KetoC may be because of its α,β-unsaturated carbonyl moiety. Collectively, our data suggested that KetoC activated the Nrf2-ARE pathway to enhance cellular antioxidative responses in vitro and in vivo, which further suggests that KetoC may prevent multiple diseases induced by oxidative stress. - Highlights: • We evaluated the effect of modified fatty acids generated by Lactobacillus plantarum. • 10-Oxo-trans-11-ocatadecenoic acid (KetoC) protected cells from oxidative stress. • KetoC activated the Nrf2-ARE pathway to promote antioxidative gene expression. • KetoC promoted the expression of antioxidative enzymes in mice organs. • The cytoprotective effect of KetoC was because of α,β-unsaturated carbonyl moiety.

10-Oxo-trans-11-octadecenoic acid generated from linoleic acid by a gut lactic acid bacterium Lactobacillus plantarum is cytoprotective against oxidative stress

International Nuclear Information System (INIS)

Furumoto, Hidehiro; Nanthirudjanar, Tharnath; Kume, Toshiaki; Izumi, Yasuhiko; Park, Si-Bum; Kitamura, Nahoko; Kishino, Shigenobu; Ogawa, Jun; Hirata, Takashi; Sugawara, Tatsuya

2016-01-01

Oxidative stress is a well-known cause of multiple diseases. The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway plays a central role in cellular antioxidative responses. In this study, we investigated the effects of novel fatty acid metabolite derivatives of linoleic acid generated by the gut lactic acid bacteria Lactobacillus plantarum on the Nrf2-ARE pathway. 10-Oxo-trans-11-octadecenoic acid (KetoC) protected HepG2 cells from cytotoxicity induced by hydrogen peroxide. KetoC also significantly increased cellular Nrf2 protein levels, ARE-dependent transcription, and the gene expression of antioxidative enzymes such as heme oxygenase-1 (HO-1), glutamate-cysteine ligase modifier subunit (GCLM), and NAD(P)H:quinone oxidoreductase 1 (NQO1) in HepG2 cells. Additionally, a single oral dose administration of KetoC also increased antioxidative gene expression and protein levels of Nrf2 and HO-1 in mouse organs. Since other fatty acid metabolites and linoleic acid did not affect cellular antioxidative responses, the cytoprotective effect of KetoC may be because of its α,β-unsaturated carbonyl moiety. Collectively, our data suggested that KetoC activated the Nrf2-ARE pathway to enhance cellular antioxidative responses in vitro and in vivo, which further suggests that KetoC may prevent multiple diseases induced by oxidative stress. - Highlights: • We evaluated the effect of modified fatty acids generated by Lactobacillus plantarum. • 10-Oxo-trans-11-ocatadecenoic acid (KetoC) protected cells from oxidative stress. • KetoC activated the Nrf2-ARE pathway to promote antioxidative gene expression. • KetoC promoted the expression of antioxidative enzymes in mice organs. • The cytoprotective effect of KetoC was because of α,β-unsaturated carbonyl moiety.

Substrate specificity and pH dependence of homogeneous wheat germ acid phosphatase.

Science.gov (United States)

Van Etten, R L; Waymack, P P

1991-08-01

The broad substrate specificity of a homogeneous isoenzyme of wheat germ acid phosphatase (WGAP) was extensively investigated by chromatographic, electrophoretic, NMR, and kinetic procedures. WGAP exhibited no divalent metal ion requirement and was unaffected upon incubation with EDTA or o-phenanthroline. A comparison of two catalytically homogeneous isoenzymes revealed little difference in substrate specificity. The specificity of WGAP was established by determining the Michaelis constants for a wide variety of substrates. p-Nitrophenyl phosphate, pyrophosphate, tripolyphosphate, and ATP were preferred substrates while lesser activities were seen toward sugar phosphates, trimetaphosphate, phosphoproteins, and (much less) phosphodiesters. An extensive table of Km and Vmax values is given. The pathway for the hydrolysis of trimetaphosphate was examined by colorimetric and 31P NMR methods and it was found that linear tripolyphosphate is not a free intermediate in the enzymatic reaction. In contrast to literature reports, homogeneous wheat germ acid phosphatase exhibits no measurable carboxylesterase activity, nor does it hydrolyze phenyl phosphonothioate esters or phytic acid at significant rates.

Amyloid fibril formation of peptides derived from the C-terminus of CETP modulated by lipids

Energy Technology Data Exchange (ETDEWEB)

García-González, Victor [Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, 04510 México, DF (Mexico); Mas-Oliva, Jaime, E-mail: jmas@ifc.unam.mx [Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, 04510 México, DF (Mexico); División de Investigación, Facultad de Medicina, Universidad Nacional Autónoma de México, 04510 México, DF (Mexico)

2013-04-26

Highlights: •The secondary structure of a C-terminal peptide derived from CETP was studied. •Lipids modulate secondary structure changes of a C-terminal peptide derived from CETP. •Lysophosphatidic acid maintains a functional α-helix and prevents fibril formation. •Transfer of lipids by CETP is related to the presence of an α-helix at its C-end. -- Abstract: Cholesteryl-ester transfer protein (CETP) is a plasmatic protein involved in neutral lipid transfer between lipoproteins. Focusing on the last 12 C-terminus residues we have previously shown that mutation D{sub 470}N promotes a conformational change towards a β-secondary structure. In turn, this modification leads to the formation of oligomers and fibrillar structures, which cause cytotoxic effects similar to the ones provoked by amyloid peptides. In this study, we evaluated the role of specific lipid arrangements on the structure of peptide helix-Z (D{sub 470}N) through the use of thioflavin T fluorescence, peptide bond absorbance, circular dichroism and electron microscopy. The results indicate that the use of micelles formed with lysophosphatidylcholine and lysophosphatidic acid (LPA) under neutral pH induce a conformational transition of peptide helix-Z containing a β-sheet conformation to a native α-helix structure, therefore avoiding the formation of amyloid fibrils. In contrast, incubation with phosphatidic acid does not change the profile for the β-sheet conformation. When the electrostatic charge at the surface of micelles or vesicles is regulated through the use of lipids such as phospholipid and LPA, minimal changes and the presence of β-structures were recorded. Mixtures with a positive net charge diminished the percentage of β-structure and the amount of amyloid fibrils. Our results suggest that the degree of solvation determined by the presence of a free hydroxyl group on lipids such as LPA is a key condition that can modulate the secondary structure and the consequent formation of

Amyloid fibril formation of peptides derived from the C-terminus of CETP modulated by lipids

International Nuclear Information System (INIS)

García-González, Victor; Mas-Oliva, Jaime

2013-01-01

Highlights: •The secondary structure of a C-terminal peptide derived from CETP was studied. •Lipids modulate secondary structure changes of a C-terminal peptide derived from CETP. •Lysophosphatidic acid maintains a functional α-helix and prevents fibril formation. •Transfer of lipids by CETP is related to the presence of an α-helix at its C-end. -- Abstract: Cholesteryl-ester transfer protein (CETP) is a plasmatic protein involved in neutral lipid transfer between lipoproteins. Focusing on the last 12 C-terminus residues we have previously shown that mutation D 470 N promotes a conformational change towards a β-secondary structure. In turn, this modification leads to the formation of oligomers and fibrillar structures, which cause cytotoxic effects similar to the ones provoked by amyloid peptides. In this study, we evaluated the role of specific lipid arrangements on the structure of peptide helix-Z (D 470 N) through the use of thioflavin T fluorescence, peptide bond absorbance, circular dichroism and electron microscopy. The results indicate that the use of micelles formed with lysophosphatidylcholine and lysophosphatidic acid (LPA) under neutral pH induce a conformational transition of peptide helix-Z containing a β-sheet conformation to a native α-helix structure, therefore avoiding the formation of amyloid fibrils. In contrast, incubation with phosphatidic acid does not change the profile for the β-sheet conformation. When the electrostatic charge at the surface of micelles or vesicles is regulated through the use of lipids such as phospholipid and LPA, minimal changes and the presence of β-structures were recorded. Mixtures with a positive net charge diminished the percentage of β-structure and the amount of amyloid fibrils. Our results suggest that the degree of solvation determined by the presence of a free hydroxyl group on lipids such as LPA is a key condition that can modulate the secondary structure and the consequent formation of amyloid

The Acid-Base Titration of a Very Weak Acid: Boric Acid