Leber’s congenital amaurosis and the role of gene therapy in congenital retinal disorders

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Walid Sharif

2017-03-01

Full Text Available Leber’s congenital amaurosis (LCA and recent gene therapy advancement for treating inherited retinopathies were extensive literature reviewed using MEDLINE, PubMed and EMBASE. Adeno-associated viral vectors were the most utilised vectors for ocular gene therapy. Cone photoreceptor cells might use an alternate pathway which was not reliant of the retinal pigment epithelium (RPE derived retinoid isomerohydrolase (RPE65 to access the 11-cis retinal dehydechromophore. Research efforts dedicated on the progression of a gene-based therapy for the treatment of LCA2. Such gene therapy approaches were extremely successful in canine, porcine and rodent LCA2 models. The recombinant AAV2.hRPE65v2 adeno-associated vector contained the RPE65 cDNA and was replication deficient. Its in vitro injection in target cells induced RPE65 protein production. The gene therapy trials that were so far conducted for inherited retinopathies have generated promising results. Phase I clinical trials to cure LCA and choroideremia demonstrated that adeno-associated viral vectors containing RPE genes and photoreceptors respectively, could be successfully administered to inherited retinopathy patients. A phase III trial is presently ongoing and if successful, it will lead the way to additional gene therapy attempts to cure monogenic, inherited retinopathies.

Leber’s congenital amaurosis and the role of gene therapy in congenital retinal disorders

Institute of Scientific and Technical Information of China (English)

Walid; Sharif; Zuhair; Sharif

2017-01-01

Leber’s congenital amaurosis(LCA)and recent gene therapy advancement for treating inherited retinopathies were extensive literature reviewed using MEDLINE,Pub Med and EMBASE. Adeno-associated viral vectors were the most utilised vectors for ocular gene therapy. Cone photoreceptor cells might use an alternate pathway which was not reliant of the retinal pigment epithelium(RPE)derived retinoid isomerohydrolase(RPE65)to access the 11-cis retinal dehydechromophore. Research efforts dedicated on the progression of a gene-based therapy for the treatment of LCA2. Such gene therapy approaches were extremely successful in canine,porcine and rodent LCA2 models. The recombinant AAV2.h RPE65v2 adenoassociated vector contained the RPE65 cDNA and was replication deficient. Its in vitro injection in target cells induced RPE65 protein production. The gene therapy trials that were so far conducted for inherited retinopathies have generated promising results. Phase I clinical trials to cure LCA and choroideremia demonstrated that adeno-associated viral vectors containing RPE genes and photoreceptors respectively,could be successfully administered to inherited retinopathy patients. A phase III trial is presently ongoing and if successful,it will lead the way to additional gene therapy attempts to cure monogenic,inherited retinopathies.

Uptake and processing of [3H]retinoids in rat liver studied by electron microscopic autoradiography

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Hendriks, H.F.; Elhanany, E.; Brouwer, A.; de Leeuw, A.M.; Knook, D.L.

1988-01-01

The role of rat liver cell organelles in retinoid uptake and processing was studied by electron microscopic autoradiography. [ 3 H]Retinoids were administered either orally, to make an inventory of the cell organelles involved, or intravenously as chylomicron remnant constituents to study retinoid processing by the liver with time. No qualitative differences were observed between the two routes of administration. Time-related changes in the distribution of grains were studied using chylomicron remnant [ 3 H]retinoids. The percentages of grains observed over cells and the space of Disse at 5 and 30 min after administration were, respectively: parenchymal cells, 72.6 and 70.4%; fat-storing cells, 5.0 and 18.1%, and the space of Disse, 14.4 and 8.9%. Low numbers of grains were observed over endothelial and Kupffer cells. The percentages of grains observed over parenchymal cell organelles were, respectively: sinusoidal area, 59.6 and 34.4%; smooth endoplasmic reticulum associated with glycogen, 13.8 and 13.4%; mitochondria, 5.4 and 13.6%; rough endoplasmic reticulum, 4.2 and 7.3%, and rough endoplasmic reticulum associated with mitochondria, 3.7 and 6.5%. It is concluded that chylomicron remnant [ 3 H]retinoids in combination with electron microscopic autoradiography provide a good system to study the liver processing of retinoids in vivo. These results, obtained in the intact liver under physiological conditions, further substantiate that retinoids are processed through parenchymal cells before storage occurs in fat-storing cell lipid droplets, that retinoid uptake is not mediated through lysosomes and that the endoplasmic reticulum is a major organelle in retinoid processing

Quantitation of biological retinoids by high-pressure liquid chromatography: primary internal standardization using tritiated retinoids

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Cullum, M.E.; Zile, M.H.

1986-01-01

A single method is described for quantitation of 14 retinoids found in biological material. The method consists of reversed-phase HPLC, internal standardization, and carrier extraction procedures with three synthetic retinoids. Primary standardization of HPLC uv detector is achieved using tritiated all-trans-retinoic acid, all-trans-retinol, all-trans-retinyl palmitate, and all-trans-retinyl acetate. Extraction methods are standardized by correlating the uv absorbance of retinoids at 340 nm with radioactivity of tritiated retinoids of known specific activity. Quantitation of 10 pg of tritiated or 5 ng of nonradioactive retinoid per 0.1 g sample in a polarity range from 4-oxo-retinoic acid to retinyl stearate can be achieved in a single, 50-min chromatographic run. A single HPLC pump, a C 18 reversed-phased analytical column, a multistep three-solvent gradient, and inexpensive solvents based on methanol, water, and chloroform comprise this cost-effective chromatographic system. Our primary standardization method allows investigators employing different procedures to compare results between laboratories by standardizing the HPLC uv detector with commercially available tritiated retinoids. With this method we were able to quantitate nanomolar amounts of endogenous retinoic acids and retinyl esters, that HPLC uv only conditions usually would not detect in the circulation and liver of rats under physiological conditions

Visual Cycle Modulation as an Approach toward Preservation of Retinal Integrity.

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Claes Bavik

Full Text Available Increased exposure to blue or visible light, fluctuations in oxygen tension, and the excessive accumulation of toxic retinoid byproducts places a tremendous amount of stress on the retina. Reduction of visual chromophore biosynthesis may be an effective method to reduce the impact of these stressors and preserve retinal integrity. A class of non-retinoid, small molecule compounds that target key proteins of the visual cycle have been developed. The first candidate in this class of compounds, referred to as visual cycle modulators, is emixustat hydrochloride (emixustat. Here, we describe the effects of emixustat, an inhibitor of the visual cycle isomerase (RPE65, on visual cycle function and preservation of retinal integrity in animal models. Emixustat potently inhibited isomerase activity in vitro (IC50 = 4.4 nM and was found to reduce the production of visual chromophore (11-cis retinal in wild-type mice following a single oral dose (ED50 = 0.18 mg/kg. Measure of drug effect on the retina by electroretinography revealed a dose-dependent slowing of rod photoreceptor recovery (ED50 = 0.21 mg/kg that was consistent with the pattern of visual chromophore reduction. In albino mice, emixustat was shown to be effective in preventing photoreceptor cell death caused by intense light exposure. Pre-treatment with a single dose of emixustat (0.3 mg/kg provided a ~50% protective effect against light-induced photoreceptor cell loss, while higher doses (1-3 mg/kg were nearly 100% effective. In Abca4-/- mice, an animal model of excessive lipofuscin and retinoid toxin (A2E accumulation, chronic (3 month emixustat treatment markedly reduced lipofuscin autofluorescence and reduced A2E levels by ~60% (ED50 = 0.47 mg/kg. Finally, in the retinopathy of prematurity rodent model, treatment with emixustat during the period of ischemia and reperfusion injury produced a ~30% reduction in retinal neovascularization (ED50 = 0.46mg/kg. These data demonstrate the ability of

Retinal Pigmented Epithelial Cells Obtained from Human Induced Pluripotent Stem Cells Possess Functional Visual Cycle Enzymes in Vitro and in Vivo*

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Maeda, Tadao; Lee, Mee Jee; Palczewska, Grazyna; Marsili, Stefania; Tesar, Paul J.; Palczewski, Krzysztof; Takahashi, Masayo; Maeda, Akiko

2013-01-01

Differentiated retinal pigmented epithelial (RPE) cells have been obtained from human induced pluripotent stem (hiPS) cells. However, the visual (retinoid) cycle in hiPS-RPE cells has not been adequately examined. Here we determined the expression of functional visual cycle enzymes in hiPS-RPE cells compared with that of isolated wild-type mouse primary RPE (mpRPE) cells in vitro and in vivo. hiPS-RPE cells appeared morphologically similar to mpRPE cells. Notably, expression of certain visual cycle proteins was maintained during cell culture of hiPS-RPE cells, whereas expression of these same molecules rapidly decreased in mpRPE cells. Production of the visual chromophore, 11-cis-retinal, and retinosome formation also were documented in hiPS-RPE cells in vitro. When mpRPE cells with luciferase activity were transplanted into the subretinal space of mice, bioluminance intensity was preserved for >3 months. Additionally, transplantation of mpRPE into blind Lrat−/− and Rpe65−/− mice resulted in the recovery of visual function, including increased electrographic signaling and endogenous 11-cis-retinal production. Finally, when hiPS-RPE cells were transplanted into the subretinal space of Lrat−/− and Rpe65−/− mice, their vision improved as well. Moreover, histological analyses of these eyes displayed replacement of dysfunctional RPE cells by hiPS-RPE cells. Together, our results show that hiPS-RPE cells can exhibit a functional visual cycle in vitro and in vivo. These cells could provide potential treatment options for certain blinding retinal degenerative diseases. PMID:24129572

TNF-α mediates choroidal neovascularization by upregulating VEGF expression in RPE through ROS-dependent β-catenin activation.

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Wang, Haibo; Han, Xiaokun; Wittchen, Erika S; Hartnett, M Elizabeth

2016-01-01

Inflammation, oxidative stress, and angiogenesis have been proposed to interact in age-related macular degeneration. It has been postulated that external stimuli that cause oxidative stress can increase production of vascular endothelial growth factor (VEGF) in retinal pigment epithelial (RPE) cells. In this study, we tested the hypothesis that the inflammatory cytokine, tumor necrosis factor alpha (TNF-α), contributed to choroidal neovascularization (CNV) by upregulating VEGF in RPE through intracellular reactive oxygen species (ROS)-dependent signaling and sought to understand the mechanisms involved. In a murine laser-induced CNV model, 7 days after laser treatment and intravitreal neutralizing mouse TNF-α antibody or isotype immunoglobulin G (IgG) control, the following measurements were made: 1) TNF-α protein and VEGF protein in RPE/choroids with western blot, 2) CNV volume in RPE/choroidal flatmounts, and 3) semiquantification of oxidized phospholipids stained with E06 antibody within CNV with immunohistochemistry (IHC). In cultured human RPE cells treated with TNF-α or PBS control, 1) ROS generation was measured using the 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence assay, and 2) NOX4 protein and VEGF protein or mRNA were measured with western blot or quantitative real-time PCR in cells pretreated with apocynin or nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) inhibitor, VAS 2870, or transfected with p22phox siRNA, and each was compared to its appropriate control. Western blots of phosphorylated p65 (p-p65), total p65 and β-actin, and quantitative real-time PCR of VEGF mRNA were measured in human RPE cells treated with TNF-α and pretreatment with the nuclear factor kappa B inhibitor, Bay 11-7082 or control. Western blots of β-catenin, VEGF, and p22phox and coimmunoprecipitation of β-catenin and T-cell transcriptional factor were performed in human RPE cells treated with TNF-α following pretreatment with �

Retinoid Signaling in Pancreatic Cancer, Injury and Regeneration

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Colvin, Emily K.; Susanto, Johana M.; Kench, James G.; Ong, Vivienna N.; Mawson, Amanda; Pinese, Mark; Chang, David K.; Rooman, Ilse; O'Toole, Sandra A.; Segara, Davendra; Musgrove, Elizabeth A.; Sutherland, Robert L.; Apte, Minoti V.; Scarlett, Christopher J.; Biankin, Andrew V.

2011-01-01

Background Activation of embryonic signaling pathways quiescent in the adult pancreas is a feature of pancreatic cancer (PC). These discoveries have led to the development of novel inhibitors of pathways such as Notch and Hedgehog signaling that are currently in early phase clinical trials in the treatment of several cancer types. Retinoid signaling is also essential for pancreatic development, and retinoid therapy is used successfully in other malignancies such as leukemia, but little is known concerning retinoid signaling in PC. Methodology/Principal Findings We investigated the role of retinoid signaling in vitro and in vivo in normal pancreas, pancreatic injury, regeneration and cancer. Retinoid signaling is active in occasional cells in the adult pancreas but is markedly augmented throughout the parenchyma during injury and regeneration. Both chemically induced and genetically engineered mouse models of PC exhibit a lack of retinoid signaling activity compared to normal pancreas. As a consequence, we investigated Cellular Retinoid Binding Protein 1 (CRBP1), a key regulator of retinoid signaling known to play a role in breast cancer development, as a potential therapeutic target. Loss, or significant downregulation of CRBP1 was present in 70% of human PC, and was evident in the very earliest precursor lesions (PanIN-1A). However, in vitro gain and loss of function studies and CRBP1 knockout mice suggested that loss of CRBP1 expression alone was not sufficient to induce carcinogenesis or to alter PC sensitivity to retinoid based therapies. Conclusions/Significance In conclusion, retinoid signalling appears to play a role in pancreatic regeneration and carcinogenesis, but unlike breast cancer, it is not mediated directly by CRBP1. PMID:22220202

Retinoid signaling in pancreatic cancer, injury and regeneration.

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Emily K Colvin

Full Text Available BACKGROUND: Activation of embryonic signaling pathways quiescent in the adult pancreas is a feature of pancreatic cancer (PC. These discoveries have led to the development of novel inhibitors of pathways such as Notch and Hedgehog signaling that are currently in early phase clinical trials in the treatment of several cancer types. Retinoid signaling is also essential for pancreatic development, and retinoid therapy is used successfully in other malignancies such as leukemia, but little is known concerning retinoid signaling in PC. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the role of retinoid signaling in vitro and in vivo in normal pancreas, pancreatic injury, regeneration and cancer. Retinoid signaling is active in occasional cells in the adult pancreas but is markedly augmented throughout the parenchyma during injury and regeneration. Both chemically induced and genetically engineered mouse models of PC exhibit a lack of retinoid signaling activity compared to normal pancreas. As a consequence, we investigated Cellular Retinoid Binding Protein 1 (CRBP1, a key regulator of retinoid signaling known to play a role in breast cancer development, as a potential therapeutic target. Loss, or significant downregulation of CRBP1 was present in 70% of human PC, and was evident in the very earliest precursor lesions (PanIN-1A. However, in vitro gain and loss of function studies and CRBP1 knockout mice suggested that loss of CRBP1 expression alone was not sufficient to induce carcinogenesis or to alter PC sensitivity to retinoid based therapies. CONCLUSIONS/SIGNIFICANCE: In conclusion, retinoid signalling appears to play a role in pancreatic regeneration and carcinogenesis, but unlike breast cancer, it is not mediated directly by CRBP1.

High-throughput screening (HTS) and modeling of the retinoid ...

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Presentation at the Retinoids Review 2nd workshop in Brussels, Belgium on the application of high throughput screening and model to the retinoid system Presentation at the Retinoids Review 2nd workshop in Brussels, Belgium on the application of high throughput screening and model to the retinoid system

Human RPE Stem Cells Grown into Polarized RPE Monolayers on a Polyester Matrix Are Maintained after Grafting into Rabbit Subretinal Space

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Boris V. Stanzel

2014-01-01

Full Text Available Transplantation of the retinal pigment epithelium (RPE is being developed as a cell-replacement therapy for age-related macular degeneration. Human embryonic stem cell (hESC and induced pluripotent stem cell (iPSC-derived RPE are currently translating toward clinic. We introduce the adult human RPE stem cell (hRPESC as an alternative RPE source. Polarized monolayers of adult hRPESC-derived RPE grown on polyester (PET membranes had near-native characteristics. Trephined pieces of RPE monolayers on PET were transplanted subretinally in the rabbit, a large-eyed animal model. After 4 days, retinal edema was observed above the implant, detected by spectral domain optical coherence tomography (SD-OCT and fundoscopy. At 1 week, retinal atrophy overlying the fetal or adult transplant was observed, remaining stable thereafter. Histology obtained 4 weeks after implantation confirmed a continuous polarized human RPE monolayer on PET. Taken together, the xeno-RPE survived with retained characteristics in the subretinal space. These experiments support that adult hRPESC-derived RPE are a potential source for transplantation therapies.

A method for the isolation and culture of adult rat retinal pigment epithelial (RPE cells to study retinal diseases

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Janosch Peter Heller

2015-11-01

Full Text Available Diseases such as age-related macular degeneration (AMD affect the retinal pigment epithelium (RPE and lead to the death of the epithelial cells and ultimately blindness. RPE transplantation is currently a major focus of eye research and clinical trials using human stem cell-derived RPE cells are ongoing. However, it remains to be established to which extent the source of RPE cells for transplantation affects their therapeutic efficacy and this needs to be explored in animal models. Autotransplantation of RPE cells has attractions as a therapy, but existing protocols to isolate adult RPE cells from rodents are technically difficult, time-consuming, have a low yield and are not optimized for long-term cell culturing. Here, we report a newly devised protocol which facilitates reliable and simple isolation and culture of RPE cells from adult rats. Incubation of a whole rat eyeball in 20 U/ml papain solution for 50 minutes yielded 4 x 104 viable RPE cells. These cells were hexagonal and pigmented upon culture. Using immunostaining, we demonstrated that the cells expressed RPE cell-specific marker proteins including cytokeratin 18 and RPE65, similar to RPE cells in vivo. Additionally, the cells were able to produce and secrete Bruch’s membrane matrix components similar to in vivo situation. Similarly, the cultured RPE cells adhered to isolated Bruch’s membrane as has previously been reported. Therefore, the protocol described in this article provides an efficient method for the rapid and easy isolation of high quantities of adult rat RPE cells. This provides a reliable platform for studying the therapeutic targets, testing the effects of drugs in a preclinical setup and to perform in vitro and in vivo transplantation experiments to study retinal diseases.

Validity and reliability of the session-RPE method for quantifying training load in karate athletes.

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Tabben, M; Tourny, C; Haddad, M; Chaabane, H; Chamari, K; Coquart, J B

2015-04-24

To test the construct validity and reliability of the session rating of perceived exertion (sRPE) method by examining the relationship between RPE and physiological parameters (heart rate: HR and blood lactate concentration: [La --] ) and the correlations between sRPE and two HR--based methods for quantifying internal training load (Banister's method and Edwards's method) during karate training camp. Eighteen elite karate athletes: ten men (age: 24.2 ± 2.3 y, body mass: 71.2 ± 9.0 kg, body fat: 8.2 ± 1.3% and height: 178 ± 7 cm) and eight women (age: 22.6 ± 1.2 y, body mass: 59.8 ± 8.4 kg, body fat: 20.2 ± 4.4%, height: 169 ± 4 cm) were included in the study. During training camp, subjects participated in eight karate--training sessions including three training modes (4 tactical--technical, 2 technical--development, and 2 randori training), during which RPE, HR, and [La -- ] were recorded. Significant correlations were found between RPE and physiological parameters (percentage of maximal HR: r = 0.75, 95% CI = 0.64--0.86; [La --] : r = 0.62, 95% CI = 0.49--0.75; P training load ( r = 0.65--0.95; P reliability of the same intensity across training sessions (Cronbach's α = 0.81, 95% CI = 0.61--0.92). This study demonstrates that the sRPE method is valid for quantifying internal training load and intensity in karate.

Age-related changes in the retinal pigment epithelium (RPE.

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Xiaorong Gu

Full Text Available Age-related changes in the retina are often accompanied by visual impairment but their mechanistic details remain poorly understood.Proteomic studies were pursued toward a better molecular understanding of retinal pigment epithelium (RPE aging mechanisms. RPE cells were isolated from young adults (3-4 month-old and old (24-25 month-old F344BN rats, and separated into subcellular fractions containing apical microvilli (MV and RPE cell bodies (CB lacking their apical microvilli. Proteins were extracted in detergent, separated by SDS-PAGE, digested in situ with trypsin and analyzed by LC MS/MS. Select proteins detected in young and old rat RPE were further studied using immunofluorescence and Western blot analysis.A total of 356 proteins were identified in RPE MV from young and 378 in RPE MV from old rats, 48% of which were common to each age group. A total of 897 proteins were identified in RPE CB from young rats and 675 in old CB, 56% of which were common to each age group. Several of the identified proteins, including proteins involved in response to oxidative stress, displayed both quantitative and qualitative changes in overall abundance during RPE aging. Numerous proteins were identified for the first time in the RPE. One such protein, collectrin, was localized to the apical membrane of apical brush border of proximal tubules where it likely regulates several amino acid transporters. Elsewhere, collectrin is involved in pancreatic β cell proliferation and insulin secretion. In the RPE, collectrin expression was significantly modulated during RPE aging. Another age-regulated, newly described protein was DJ-1, a protein extensively studied in brain where oxidative stress-related functions have been described.The data presented here reveals specific changes in the RPE during aging, providing the first protein database of RPE aging, which will facilitate future studies of age-related retinal diseases.

Signalling with retinoids in the human lung: validation of new tools for the expression study of retinoid receptors

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Poulain, Stéphane; Lacomme, Stéphanie; Battaglia-Hsu, Shyue-Fang; Manoir, Stanislas du; Brochin, Lydia; Vignaud, Jean-Michel; Martinet, Nadine

2009-01-01

Retinoid Receptors are involved in development and cell homeostasis. Alterations of their expressions have been observed in lung cancer. However, retinoid chemoprevention trials in populations at risk to develop such tumors have failed. Therefore, the pertinence of new clinical trials using second generation retinoid requires prior better understanding of retinoid signalling. This is our aim when validating extensively research tools, focused on Retinoic Acid Receptor beta, whose major role in lung cancer is documented. Biocomputing was used to assess the genomic organization of RAR beta. Its putative RAR-beta1' promoter features were investigated experimentally. Specific measures realized, with qRT-PCR Syber Green assays and a triplex of Taqman probes, were extensively validated to establish Retinoid Receptors mRNAs reference values for in vivo normal human bronchial cells, lung tumors and cell lines. Finally, a pan-RAR-beta antibody was generated and extensively validated by western-blot and immunoprecipitation. No promoter-like activity was found for RAR-beta1'. RAR-beta2 mRNAs increase signs the normal differentiation of the human bronchial epithelium while a decrease is observed in most lung cancer cell lines. Accordingly, it is also, along with RXR beta, down-regulated in lung tumors. When using nuclear extracts of BEAS-2B and normal lung cells, only the RAR-beta2 long protein isoform was recognized by our antibody. Rigorous samples processing and extensive biocomputing, were the key factors for this study. mRNA reference values and validated tools can now be used to advance researches on retinoid signalling in the lung

Molecular pharmacodynamics of emixustat in protection against retinal degeneration.

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Zhang, Jianye; Kiser, Philip D; Badiee, Mohsen; Palczewska, Grazyna; Dong, Zhiqian; Golczak, Marcin; Tochtrop, Gregory P; Palczewski, Krzysztof

2015-07-01

Emixustat is a visual cycle modulator that has entered clinical trials as a treatment for age-related macular degeneration (AMD). This molecule has been proposed to inhibit the visual cycle isomerase RPE65, thereby slowing regeneration of 11-cis-retinal and reducing production of retinaldehyde condensation byproducts that may be involved in AMD pathology. Previously, we reported that all-trans-retinal (atRAL) is directly cytotoxic and that certain primary amine compounds that transiently sequester atRAL via Schiff base formation ameliorate retinal degeneration. Here, we have shown that emixustat stereoselectively inhibits RPE65 by direct active site binding. However, we detected the presence of emixustat-atRAL Schiff base conjugates, indicating that emixustat also acts as a retinal scavenger, which may contribute to its therapeutic effects. Using agents that lack either RPE65 inhibitory activity or the capacity to sequester atRAL, we assessed the relative importance of these 2 modes of action in protection against retinal phototoxicity in mice. The atRAL sequestrant QEA-B-001-NH2 conferred protection against phototoxicity without inhibiting RPE65, whereas an emixustat derivative incapable of atRAL sequestration was minimally protective, despite direct inhibition of RPE65. These data indicate that atRAL sequestration is an essential mechanism underlying the protective effects of emixustat and related compounds against retinal phototoxicity. Moreover, atRAL sequestration should be considered in the design of next-generation visual cycle modulators.

That Escalated Quickly—Planning to Ignore RPE Can Backfire

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Maik Bieleke

2017-09-01

Full Text Available Ratings of perceived exertion (RPE are routinely assessed in exercise science and RPE is substantially associated with physiological criterion measures. According to the psychobiological model of endurance, RPE is a central limiting factor in performance. While RPE is known to be affected by psychological manipulations, it remains to be examined whether RPE can be self-regulated during static muscular endurance exercises to enhance performance. In this experiment, we investigate the effectiveness of the widely used and recommended self-regulation strategy of if-then planning (i.e., implementation intentions in down-regulating RPE and improving performance in a static muscular endurance task. 62 female students (age: M = 23.7 years, SD = 4.0 were randomly assigned to an implementation intention or a control condition and performed a static muscular endurance task. They held two intertwined rings as long as possible while avoiding contacts between the rings. In the implementation intention condition, participants had an if-then plan: “If the task becomes too strenuous for me, then I ignore the strain and tell myself: Keep going!” Every 25 ± 10 s participants reported their RPE along with their perceived pain. Endurance performance was measured as time to failure, along with contact errors as a measure of performance quality. No differences emerged between implementation intention and control participants regarding time to failure and performance quality. However, mixed-effects model analyses revealed a significant Time-to-Failure × Condition interaction for RPE. Compared to the control condition, participants in the implementation intention condition reported substantially greater increases in RPE during the second half of the task and reached higher total values of RPE before task termination. A similar but weaker pattern evinced for perceived pain. Our results demonstrate that RPE during an endurance task can be self-regulated with if

Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase that increases the production rate of D-tagatose.

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Kim, H-J; Kim, J-H; Oh, H-J; Oh, D-K

2006-07-01

Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase used to increase the production rate of D-tagatose. A mutated gene was obtained by an error-prone polymerase chain reaction using L-arabinose isomerase gene from G. stearothermophilus as a template and the gene was expressed in Escherichia coli. The expressed mutated L-arabinose isomerase exhibited the change of three amino acids (Met322-->Val, Ser393-->Thr, and Val408-->Ala), compared with the wild-type enzyme and was then purified to homogeneity. The mutated enzyme had a maximum galactose isomerization activity at pH 8.0, 65 degrees C, and 1.0 mM Co2+, while the wild-type enzyme had a maximum activity at pH 8.0, 60 degrees C, and 1.0-mM Mn2+. The mutated L-arabinose isomerase exhibited increases in D-galactose isomerization activity, optimum temperature, catalytic efficiency (kcat/Km) for D-galactose, and the production rate of D-tagatose from D-galactose. The mutated L-arabinose isomerase from G. stearothermophilus is valuable for the commercial production of D-tagatose. This work contributes knowledge on the characterization of a mutated L-arabinose isomerase, and allows an increased production rate for D-tagatose from D-galactose using the mutated enzyme.

Effect of pH on simultaneous saccharification and isomerization by glucoamylase and glucose isomerase.

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Mishra, Abha; Debnath Das, Meera

2002-01-01

pH and temperature play critical roles in multistep enzymatic conversions. In such conversions, the optimal pH for individual steps differs greatly. In this article, we describe the production of glucoamylase (from Aspergillus oryzae MTCC152 in solid-state fermentation) and glucose isomerase (from Streptomyces griseus NCIM2020 in submerged fermentation), used in industries for producing high-fructose syrup. Optimum pH for glucoamylase was found to be 5.0. For glucose isomerase, the optimum pH ranged between 7.0 and 8.5, depending on the type of buffer used. Optimum temperature for glucoamylase and glucose isomerase was 50 and 60 degrees C, respectively. When both the enzymatic conversions were performed simultaneously at a compromised pH of 6.5, both the enzymes showed lowered activity. We also studied the kinetics at different pHs, which allows the two-step reaction to take place simultaneously. This was done by separating two steps by a thin layer of urease. Ammonia generated by the hydrolysis of urea consumed the hydrogen ions, thereby allowing optimal activity of glucose isomerase at an acidic pH of 5.0.

Isolation of key retinoid signalling and metabolic modules in invertebrates

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Ana André

2014-05-01

Full Text Available Retinoids are a class of molecules related to vitamin A (Retinol that are required for regulation of critical chordate ndocrine-mediated process, such as embryonic development, reproduction, and vision. To maintain such physiological process, chordates have a complex mechanism to regulate the spatial and temporal distribution of retinoids that includes metabolic and signalling modules. Initially, retinoid modules were seen as a chordate novelty. However, emerging biochemical and genomic evidences have challenged this view, clearly pointing to a more basal ancestry than previously thought. However, for the majority of non-chordate invertebrate lineages a clearly characterization of the main enzymatic/molecular players is still missing. Despite limited, the available evidence supports the presence of biologically active retinoid pathways in invertebrates. In order to enhance our insights on retinoid biology, evolution, and its putative disruption by environmental chemicals, the isolation and functional characterization of key retinoid metabolic players in marine invertebrates has been carried out.

Retinoid quantification by HPLC/MS(n)

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McCaffery, Peter; Evans, James; Koul, Omanand; Volpert, Amy; Reid, Kevin; Ullman, M. David

2002-01-01

Retinoic acid (RA) mediates most of the biological effects of vitamin A that are essential for vertebrate survival. It acts through binding to receptors that belong to the nuclear receptor transcription factor superfamily (Mangelsdorf et al. 1994). It is also a highly potent vertebrate teratogen. To determine the function and effects of endogenous and exogenous RA, it is important to have a highly specific, sensitive, accurate, and precise analytical procedure. Current analyses of RA and other retinoids are labor intensive, of poor sensitivity, have limited specificity, or require compatibility with RA reporter cell lines (Chen et al. 1995. BIOCHEM: Pharmacol. 50: 1257-1264; Creech Kraft et al. 1994. BIOCHEM: J. 301: 111-119; Lanvers et al. 1996. J. Chromatogr. B Biomed. Appl. 685: 233-240; Maden et al. 1998. DEVELOPMENT: 125: 4133-4144; Wagner et al. 1992. DEVELOPMENT: 116: 55-66). This paper describes an HPLC/mass spectrometry/mass spectrometry product ion scan (HPLC/MS(n)) procedure for the analysis of retinoids that employs atmospheric pressure chemical ionization MS. The retinoids are separated by normal-phase column chromatography with a linear hexane-isopropanol-dioxane gradient. Each retinoid is detected by a unique series of MS(n) functions set at optimal collision-induced dissociation energy (30% to 32%) for all MS(n) steps. The scan events are divided into three segments, based on HPLC elution order, to maximize the mass spectrometer duty cycle. The all-trans, 9-cis, and 13-cis RA isomers are separated, if desired, by an isocratic hexane-dioxane-isopropanol mobile phase. This paper describes an HPLC/MS(n) procedure possessing high sensitivity and specificity for retinoids.

TNF-α decreases VEGF secretion in highly polarized RPE cells but increases it in non-polarized RPE cells related to crosstalk between JNK and NF-κB pathways.

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Hiroto Terasaki

Full Text Available Asymmetrical secretion of vascular endothelial growth factor (VEGF by retinal pigment epithelial (RPE cells in situ is critical for maintaining the homeostasis of the retina and choroid. VEGF is also involved in the development and progression of age-related macular degeneration (AMD. We studied the effect of tumor necrosis factor-α (TNF-α on the secretion of VEGF in polarized and non-polarized RPE cells (P-RPE cells and N-RPE cells, respectively in culture and in situ in rats. A subretinal injection of TNF-α caused a decrease in VEGF expression and choroidal atrophy. Porcine RPE cells were seeded on Transwell™ filters, and their maturation and polarization were confirmed by the asymmetrical VEGF secretion and trans electrical resistance. Exposure to TNF-α decreased the VEGF secretion in P-RPE cells but increased it in N-RPE cells in culture. TNF-α inactivated JNK in P-RPE cells but activated it in N-RPE cells, and TNF-α activated NF-κB in P-RPE cells but not in N-RPE cells. Inhibition of NF-κB activated JNK in both types of RPE cells indicating crosstalk between JNK and NF-κB. TNF-α induced the inhibitory effects of NF-κB on JNK in P-RPE cells because NF-κB is continuously inactivated. In N-RPE cells, however, it was not evident because NF-κB was already activated. The basic activation pattern of JNK and NF-κB and their crosstalk led to opposing responses of RPE cells to TNF-α. These results suggest that VEGF secretion under inflammatory conditions depends on cellular polarization, and the TNF-α-induced VEGF down-regulation may result in choroidal atrophy in polarized physiological RPE cells. TNF-α-induced VEGF up-regulation may cause neovascularization by non-polarized or non-physiological RPE cells.

Cadherins in the retinal pigment epithelium (RPE revisited: P-cadherin is the highly dominant cadherin expressed in human and mouse RPE in vivo.

Directory of Open Access Journals (Sweden)

Xue Yang

Full Text Available The retinal pigment epithelium (RPE supports the health and function of retinal photoreceptors and is essential for normal vision. RPE cells are post-mitotic, terminally differentiated, and polarized epithelial cells. In pathological conditions, however, they lose their epithelial integrity, become dysfunctional, even dedifferentiate, and ultimately die. The integrity of epithelial cells is maintained, in part, by adherens junctions, which are composed of cadherin homodimers and p120-, β-, and α-catenins linking to actin filaments. While E-cadherin is the major cadherin for forming the epithelial phenotype in most epithelial cell types, it has been reported that cadherin expression in RPE cells is different from other epithelial cells based on results with cultured RPE cells. In this study, we revisited the expression of cadherins in the RPE to clarify their relative contribution by measuring the absolute quantity of cDNAs produced from mRNAs of three classical cadherins (E-, N-, and P-cadherins in the RPE in vivo. We found that P-cadherin (CDH3 is highly dominant in both mouse and human RPE in situ. The degree of dominance of P-cadherin is surprisingly large, with mouse Cdh3 and human CDH3 accounting for 82-85% and 92-93% of the total of the three cadherin mRNAs, respectively. We confirmed the expression of P-cadherin protein at the cell-cell border of mouse RPE in situ by immunofluorescence. Furthermore, we found that oxidative stress induces dissociation of P-cadherin and β-catenin from the cell membrane and subsequent translocation of β-catenin into the nucleus, resulting in activation of the canonical Wnt/β-catenin pathway. This is the first report of absolute comparison of the expression of three cadherins in the RPE, and the results suggest that the physiological role of P-cadherin in the RPE needs to be reevaluated.

Mefloquine use, psychosis, and violence: a retinoid toxicity hypothesis.

Science.gov (United States)

Mawson, Anthony

2013-07-15

Mefloquine use has been linked to severe gastrointestinal and neuropsychiatric adverse effects, including cognitive disturbances, anxiety, depression, psychosis, and violence. The adverse effects of the drug are thought to result from the secondary consequences of hepatocellular injury; in fact, mefloquine is known to cause a transient, anicteric chemical hepatitis. However, the mechanism of mefloquine-associated liver damage and the associated neuropsychiatric and behavioral effects of the drug are not well understood. Mefloquine and other 8-amino-quinolines are the only antimalarial drugs that target the liver-stage malaria parasites, which selectively absorb vitamin A from the host. Vitamin A is also stored mainly in the liver, in potentially poisonous concentrations. These observations suggest that both the therapeutic effectiveness of mefloquine and its adverse effects are related to the ability of the 8-aminoquinolines to alter the metabolism of retinoids (vitamin A and its congeners). Several lines of evidence support the hypothesis that mefloquine neurotoxicity and other adverse effects reflect an endogenous form of hypervitaminosis A due to a process involving: mefloquine-induced dehydrogenase inhibition; the accumulation of retinoids in the liver; retinoid-induced hepatocellular damage; the spillage of stored retinoids into the circulation; and the transport of these compounds to the gut and brain in toxic concentrations. The retinoid hypothesis could be tested clinically by comparing cases of mefloquine toxicity and untreated controls in terms of retinoid profiles (retinol, retinyl esters, percent retinyl esters, and retinoic acid). Subject to such tests, retinoid profiling could provide an indicator for assessing mefloquine-associated adverse effects.

Rational design of Bacillus stearothermophilus US100 L-arabinose isomerase: potential applications for D-tagatose production.

Science.gov (United States)

Rhimi, Moez; Aghajari, Nushin; Juy, Michel; Chouayekh, Hichem; Maguin, Emmanuelle; Haser, Richard; Bejar, Samir

2009-05-01

L-arabinose isomerases catalyze the bioconversion of D-galactose into D-tagatose. With the aim of producing an enzyme optimized for D-tagatose production, three Bacillus stearothermophilus US100 L-arabinose isomerase mutants were constructed, purified and characterized. Our results indicate that mutant Q268K was significantly more acidotolerant and more stable at acidic pH than the wild-type enzyme. The N175H mutant has a broad optimal temperature range from 50 to 65 degrees C. With the aim of constructing an acidotolerant mutant working at relatively low temperatures we generated the Q268K/N175H construct. This double mutant displays an optimal pH in the range 6.0-7.0 and an optimal activity around 50-65 degrees C, temperatures at which the enzyme was stable without addition of metal ions.

Does the Timing of Measurement Alter Session-RPE in Boxers?

Science.gov (United States)

Uchida, Marco C; Teixeira, Luis F M; Godoi, Vladmir J; Marchetti, Paulo H; Conte, Marcelo; Coutts, Aaron J; Bacurau, Reury F P

2014-01-01

The purpose of this study was to compare the influence of measuring the overall session rating of perceived exertion (session-RPE) at 10 vs. 30 minutes following exercise. Eight boxers completed three different standardized training sessions of different intensities (easy, moderate and hard) in a matchedpairs, randomized research design. Exercise intensity was assessed during each bout by measuring heart rate, blood lactate concentration and session-RPE. To assess the effect of measurement timing on session-RPE, RPE data were collected either 10 or 30 minutes post-exercise. There was no significant effect of measurement time on session-RPE values following easy (10 minutes: session-RPE = 1.3 ± 1.0 Arbitrary Unit (AU), %Heart Rate Reserve (HRR) = 49.5 ± 11.1, and ∆Blood lactate = -2.3 ± 16.3%; 30 minutes: session-RPE = 1.7 ± 1.0 AU, %HRR = 51.3 ± 10.8, and ∆Blood lactate = 0.7 ± 25.2%), moderate (10 minutes: session-RPE = 2.7 ± 1.6 AU, %HRR = 67.2 ± 10.8, and ∆Blood lactate = 2.2 ± 19%; 30 minutes: session-RPE = 2.5 ± 0.9 AU, %HRR = 67.2 ± 5.9, and ∆Blood lactate = 24.5 ± 17.1%) and hard (10 minutes: session-RPE = 5.7 ± 1.0 AU, %HRR = 88.1 ± 6.3, and ∆Blood lactate = 146.3 ± 87.9%; 30 minutes: session-RPE = 5.8 ± 1.9 AU, %HRR> = 83.3 ± 8.0, and ∆Blood lactate = 91.6 ± 39%) sessions. In conclusion, our findings suggest that session-RPE can be used in boxing training routines across a range of intensities and accurate measurements can be determined as early as 10 minutes after exercise. Key PointsIt is difficult to quantify and monitoring the external training load in martial arts (e.g. Aikido, Kung Fu, Judo) and physical combat sports (e.g. Boxing, Muay Thai), session RPE method appears to be a reliable method to quantifying training load in those sports.For many athletes it is impractical to wait 30 minutes after training session to provide a session-RPE. The present findings show that collecting ses-sion-RPE measures at 10 min

Breastfeeding, retinoids, and postpartum depression: a new theory.

Science.gov (United States)

Mawson, Anthony R; Xueyuan, Wang

2013-09-25

Postpartum depression (PPD) is an international public health problem affecting at least 1 in 8 mothers. Known risk factors include: giving birth to a preterm or low birth weight infant, babies with greater symptoms of illness at age 4-6 weeks, formula feeding, younger maternal age, smoking, and fatigue. Prolonged breastfeeding is associated with a reduced risk of PPD but the mechanisms are not well understood. Interventions for PPD focusing on psychosocial risk factors have been largely unsuccessful, suggesting that the condition has a mainly biological basis. The hypothesis proposed for consideration is that breastfeeding protects against PPD by maintaining endogenous retinoids (vitamin A-related compounds) below a threshold concentration. In fact, breast milk is rich in retinoids; pregnant women accumulate retinoids in liver and breast in preparation for lactation; there is increasing evidence that retinoids in higher concentration are associated with cognitive disturbances and mood disorders, including depression and suicide; and prolonged lactation reduces maternal stores of retinoids. Consistent with this hypothesis, it is estimated that an amount of vitamin A is transferred from mother to infant during the first six months of exclusive breastfeeding equivalent to 76% of a dose known to cause acute vitamin A poisoning in an adult. Breastfeeding may thus have evolutionary-adaptive functions for both mother and infant, transferring vital nutrients to an infant unable to feed itself, yet at the same time providing a natural means of reducing potentially toxic concentrations of retinoids in the mother. © 2013 Elsevier B.V. All rights reserved.

Retinoid receptor signaling and autophagy in acute promyelocytic leukemia.

LENUS (Irish Health Repository)

Orfali, Nina

2014-05-15

Retinoids are a family of signaling molecules derived from vitamin A with well established roles in cellular differentiation. Physiologically active retinoids mediate transcriptional effects on cells through interactions with retinoic acid (RARs) and retinoid-X (RXR) receptors. Chromosomal translocations involving the RARα gene, which lead to impaired retinoid signaling, are implicated in acute promyelocytic leukemia (APL). All-trans-retinoic acid (ATRA), alone and in combination with arsenic trioxide (ATO), restores differentiation in APL cells and promotes degradation of the abnormal oncogenic fusion protein through several proteolytic mechanisms. RARα fusion-protein elimination is emerging as critical to obtaining sustained remission and long-term cure in APL. Autophagy is a degradative cellular pathway involved in protein turnover. Both ATRA and ATO also induce autophagy in APL cells. Enhancing autophagy may therefore be of therapeutic benefit in resistant APL and could broaden the application of differentiation therapy to other cancers. Here we discuss retinoid signaling in hematopoiesis, leukemogenesis, and APL treatment. We highlight autophagy as a potential important regulator in anti-leukemic strategies.

Off-label uses of retinoids in dermatology

Directory of Open Access Journals (Sweden)

Wei Li

2012-09-01

Full Text Available Retinoids has been used widely in the topical and systemic treatments of various dermatoses: psoriasis, disorders of keratinization (DOK, keratotic genodermatosis, and severe acne. Moreover, it is also used in the treatment and/or chemoprevention of skin cancer and other neoplasms. Retinoids display key regulatory functions and most dermatologists are familiar with the FDA-approved indication of this medication. Retinoic acid is a potent signaling molecule that is essential for many biological processes, and its levels are tightly regulated by mechanisms that are only partially understood. This article will review these recent findings and attempt to synthesize their meaning to provide a view into the off-label uses of retinoids in dermatology with an emphasis on oral isotretinoin and acitrein.

Alcohol and retinoids

DEFF Research Database (Denmark)

Crabb, D.W.; Pinairs, J.; Hasanadka, R.

2001-01-01

, M. Fang, and David W. Crabb; (2) Alcohol, vitamin A, and beta-carotene: Adverse interactions, by M. A. Leo and Charles S. Lieber; (3) Retinoic acid, hepatic stellate cells, and Kupffer cells, by Hidekazu Tsukamoto, K. Motomura, T. Miyahara, and M. Ohata; (4) Retinoid storage and metabolism in liver...

Retinoid receptor signaling and autophagy in acute promyelocytic leukemia

International Nuclear Information System (INIS)

Orfali, Nina; McKenna, Sharon L.; Cahill, Mary R.; Gudas, Lorraine J.; Mongan, Nigel P.

2014-01-01

Retinoids are a family of signaling molecules derived from vitamin A with well established roles in cellular differentiation. Physiologically active retinoids mediate transcriptional effects on cells through interactions with retinoic acid (RARs) and retinoid-X (RXR) receptors. Chromosomal translocations involving the RARα gene, which lead to impaired retinoid signaling, are implicated in acute promyelocytic leukemia (APL). All-trans-retinoic acid (ATRA), alone and in combination with arsenic trioxide (ATO), restores differentiation in APL cells and promotes degradation of the abnormal oncogenic fusion protein through several proteolytic mechanisms. RARα fusion-protein elimination is emerging as critical to obtaining sustained remission and long-term cure in APL. Autophagy is a degradative cellular pathway involved in protein turnover. Both ATRA and ATO also induce autophagy in APL cells. Enhancing autophagy may therefore be of therapeutic benefit in resistant APL and could broaden the application of differentiation therapy to other cancers. Here we discuss retinoid signaling in hematopoiesis, leukemogenesis, and APL treatment. We highlight autophagy as a potential important regulator in anti-leukemic strategies. - Highlights: • Normal and aberrant retinoid signaling in hematopoiesis and leukemia is reviewed. • We suggest a novel role for RARα in the development of X-RARα gene fusions in APL. • ATRA therapy in APL activates transcription and promotes onco-protein degradation. • Autophagy may be involved in both onco-protein degradation and differentiation. • Pharmacologic autophagy induction may potentiate ATRA's therapeutic effects

Retinoid receptor signaling and autophagy in acute promyelocytic leukemia

Energy Technology Data Exchange (ETDEWEB)

Orfali, Nina [Cork Cancer Research Center, University College Cork, Cork (Ireland); Department of Pharmacology, Weill Cornell Medical College, New York, NY 10065, USA. (United States); McKenna, Sharon L. [Cork Cancer Research Center, University College Cork, Cork (Ireland); Cahill, Mary R. [Department of Hematology, Cork University Hospital, Cork (Ireland); Gudas, Lorraine J., E-mail: ljgudas@med.cornell.edu [Department of Pharmacology, Weill Cornell Medical College, New York, NY 10065, USA. (United States); Mongan, Nigel P., E-mail: nigel.mongan@nottingham.ac.uk [Faculty of Medicine and Health Science, School of Veterinary Medicine and Science, University of Nottingham, LE12 5RD (United Kingdom); Department of Pharmacology, Weill Cornell Medical College, New York, NY 10065, USA. (United States)

2014-05-15

Retinoids are a family of signaling molecules derived from vitamin A with well established roles in cellular differentiation. Physiologically active retinoids mediate transcriptional effects on cells through interactions with retinoic acid (RARs) and retinoid-X (RXR) receptors. Chromosomal translocations involving the RARα gene, which lead to impaired retinoid signaling, are implicated in acute promyelocytic leukemia (APL). All-trans-retinoic acid (ATRA), alone and in combination with arsenic trioxide (ATO), restores differentiation in APL cells and promotes degradation of the abnormal oncogenic fusion protein through several proteolytic mechanisms. RARα fusion-protein elimination is emerging as critical to obtaining sustained remission and long-term cure in APL. Autophagy is a degradative cellular pathway involved in protein turnover. Both ATRA and ATO also induce autophagy in APL cells. Enhancing autophagy may therefore be of therapeutic benefit in resistant APL and could broaden the application of differentiation therapy to other cancers. Here we discuss retinoid signaling in hematopoiesis, leukemogenesis, and APL treatment. We highlight autophagy as a potential important regulator in anti-leukemic strategies. - Highlights: • Normal and aberrant retinoid signaling in hematopoiesis and leukemia is reviewed. • We suggest a novel role for RARα in the development of X-RARα gene fusions in APL. • ATRA therapy in APL activates transcription and promotes onco-protein degradation. • Autophagy may be involved in both onco-protein degradation and differentiation. • Pharmacologic autophagy induction may potentiate ATRA's therapeutic effects.

USING SESSION RPE TO MONITOR DIFFERENT METHODS OF RESISTANCE EXERCISE

Directory of Open Access Journals (Sweden)

Alison D. Egan

2006-06-01

Full Text Available The purpose of this study was to compare session rating of perceived exertion for different resistance training techniques in the squat exercise. These techniques included traditional resistance training, super slow, and maximal power training. Fourteen college-age women (Mean ± SD; age = 22 ± 3 years; height = 1.68 ± 0. 07 m completed three experimental trials in a randomized crossover design. The traditional resistance training protocol consisted of 6 sets of 6 repetitions of squats using 80% of 1-RM. The super slow protocol consisted of 6 sets of 6 repetitions using 55% of 1-RM. The maximal power protocol consisted of 6 sets of 6 repetitions using 30% of 1-RM. Rating of perceived exertion (RPE measures were obtained following each set using Borg's CR-10 scale. In addition, a session RPE value was obtained 30 minutes following each exercise session. When comparing average RPE and session RPE, no significant difference was found. However, power training had significantly lower (p < 0.05 average and session RPE (4.50 ± 1.9 and 4.5 ± 2.1 compared to both super slow training (7.81 ± 1.75 and 7.43 ± 1.73 and traditional training (7.33 ± 1.52 and 7.13 ± 1.73. The results indicate that session RPE values are not significantly different from the more traditional methods of measuring RPE during exercise bouts. It does appear that the resistance training mode that is used results in differences in perceived exertion that does not relate directly to the loading that is used. Using session RPE provides practitioners with the same information about perceived exertion as the traditional RPE measures. Taking a single measure following a training session would appear to be much easier than using multiple measures of RPE throughout a resistance training workout. However, practitioners should also be aware that the RPE does not directly relate to the relative intensity used and appears to be dependent on the mode of resistance exercise that is used

Chromatographic analysis of endogenous retinoids in tissues and serum

NARCIS (Netherlands)

Schmidt, C.K.; Brouwer, A.; Nau, H.

2003-01-01

We present a reliable, highly sensitive, and versatile method for the simultaneous determination of endogenous polar (acidic) and apolar (retinol, retinal, and retinyl esters) retinoids in various biological matrices. Following a single liquid extraction of retinoids from tissues or plasma with

Retinoid-binding proteins: similar protein architectures bind similar ligands via completely different ways.

Directory of Open Access Journals (Sweden)

Yu-Ru Zhang

Full Text Available BACKGROUND: Retinoids are a class of compounds that are chemically related to vitamin A, which is an essential nutrient that plays a key role in vision, cell growth and differentiation. In vivo, retinoids must bind with specific proteins to perform their necessary functions. Plasma retinol-binding protein (RBP and epididymal retinoic acid binding protein (ERABP carry retinoids in bodily fluids, while cellular retinol-binding proteins (CRBPs and cellular retinoic acid-binding proteins (CRABPs carry retinoids within cells. Interestingly, although all of these transport proteins possess similar structures, the modes of binding for the different retinoid ligands with their carrier proteins are different. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we analyzed the various retinoid transport mechanisms using structure and sequence comparisons, binding site analyses and molecular dynamics simulations. Our results show that in the same family of proteins and subcellular location, the orientation of a retinoid molecule within a binding protein is same, whereas when different families of proteins are considered, the orientation of the bound retinoid is completely different. In addition, none of the amino acid residues involved in ligand binding is conserved between the transport proteins. However, for each specific binding protein, the amino acids involved in the ligand binding are conserved. The results of this study allow us to propose a possible transport model for retinoids. CONCLUSIONS/SIGNIFICANCE: Our results reveal the differences in the binding modes between the different retinoid-binding proteins.

Potential role of retinoids in ovarian physiology and pathogenesis of polycystic ovary syndrome.

Science.gov (United States)

Jiang, Yanwen; Li, Chunjin; Chen, Lu; Wang, Fengge; Zhou, Xu

2017-06-01

Retinoids (retinol and its derivatives) are required for maintaining vision, immunity, barrier function, reproduction, embryogenesis, cell proliferation and differentiation. Furthermore, retinoid signaling plays a key role in initiating meiosis of germ cells of the mammalian fetal ovary. Recently, studies indicated that precise retinoid level regulation in the ovary provides a molecular control of ovarian development, steroidogenesis and oocyte maturation. Besides, abnormal retinoid signaling may be involved in the pathogenesis of polycystic ovary syndrome (PCOS), one of the most common ovarian endocrinopathies in reproductive-aged women worldwide. This review primarily summarizes recent advancements made in investigating the action of retinoid signaling in ovarian physiology as well as the abnormal retinoid signaling in PCOS. Copyright © 2017. Published by Elsevier B.V.

l-Arabinose Isomerase and d-Xylose Isomerase from Lactobacillus reuteri: Characterization, Coexpression in the Food Grade Host Lactobacillus plantarum, and Application in the Conversion of d-Galactose and d-Glucose

Science.gov (United States)

2014-01-01

The l-arabinose isomerase (l-AI) and the d-xylose isomerase (d-XI) encoding genes from Lactobacillus reuteri (DSMZ 17509) were cloned and overexpressed in Escherichia coli BL21 (DE3). The proteins were purified to homogeneity by one-step affinity chromatography and characterized biochemically. l-AI displayed maximum activity at 65 °C and pH 6.0, whereas d-XI showed maximum activity at 65 °C and pH 5.0. Both enzymes require divalent metal ions. The genes were also ligated into the inducible lactobacillal expression vectors pSIP409 and pSIP609, the latter containing a food grade auxotrophy marker instead of an antibiotic resistance marker, and the l-AI- and d-XI-encoding sequences/genes were coexpressed in the food grade host Lactobacillus plantarum. The recombinant enzymes were tested for applications in carbohydrate conversion reactions of industrial relevance. The purified l-AI converted d-galactose to d-tagatose with a maximum conversion rate of 35%, and the d-XI isomerized d-glucose to d-fructose with a maximum conversion rate of 48% at 60 °C. PMID:24443973

Glucose (xylose) isomerase production from thermotolerant and ...

African Journals Online (AJOL)

Owner

2012-11-13

Nov 13, 2012 ... in the production of the high fructose corn syrup (HFCS) from corn starch. ... Key words: Glucose isomerase, xylose isomerase, enzyme activity, Klebsiella, ... Soil, water, and manure (five samples each) were collected from.

Acyclic retinoid in chemoprevention of hepatocellular carcinoma: Targeting phosphorylated retinoid X receptor-α for prevention of liver carcinogenesis

Directory of Open Access Journals (Sweden)

Masahito Shimizu

2012-01-01

Full Text Available One of the key features of hepatocellular carcinoma (HCC is the high rate of intrahepatic recurrence that correlates with poor prognosis. Therefore, in order to improve the clinical outcome for patients with HCC, development of a chemopreventive agent that can decrease or delay the incidence of recurrence is a critical issue for urgent investigation. Acyclic retinoid (ACR, a synthetic retinoid, successfully improves HCC patient survival by preventing recurrence and the formation of secondary tumors. A malfunction of the retinoid X receptor-α (RXRα due to phosphorylation by the Ras-MAPK signaling pathway plays a critical role in liver carcinogenesis, and ACR exerts chemopreventive effects on HCC development by inhibiting RXRα phosphorylation. Here, we review the relationship between retinoid signaling abnormalities and liver disease, the mechanisms of how RXRα phosphorylation contributes to liver carcinogenesis, and the detailed effects of ACR on preventing HCC development, especially based on the results of our basic and clinical research. We also outline the concept of "clonal deletion and inhibition" therapy, which is defined as the removal and inhibition of latent malignant clones from the liver before they expand into clinically detectable HCC, because ACR prevents the development of HCC by implementing this concept. Looking toward the future, we discuss "combination chemoprevention" using ACR as a key drug since it can generate a synergistic effect, and may thus be an effective new strategy for the prevention of HCC.

Effects of retinoids on ultraviolet-induced carcinogenesis

International Nuclear Information System (INIS)

Epstein, J.H.

1981-01-01

The evidence for effects of the retinoids on UV-induced carcinogenesis is sparse. Clinical observations indicate that topical RA can cause significant regression of premalignant actinic keratoses. Also there is some evidence that this agent can cause dissolution of some basal cell epitheliomas. However this latter effect does not appear to be of therapeutic value. Systemic retinoids are of little value in the treatment of premalignant and malignant cutaneous lesions though 13-cis-retinoic acid might be of use in the basal cell nevus syndrome. Examination of the influence of the retinoids on photocarcinogenesis essentially has been confined to RA and animal experimentation. RA in nontoxic concentrations can both stimulate and inhibit photocarcinogenesis depending upon the circumstances of the study. The mechanisms of these responses are not clear. Influences on DNA synthesis directly and/or indirectly or on immune responses may be involved in both effects. Preliminary studies with oral 13-cis-retinoic acid have not demonstrated any effects to date on UV-induced skin cancer formation

Enhancing Understanding of the Visual Cycle by Applying CRISPR/Cas9 Gene Editing in Zebrafish

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Rebecca Ward

2018-04-01

Full Text Available During the vertebrate visual cycle, all-trans-retinal is exported from photoreceptors to the adjacent RPE or Müller glia wherein 11-cis-retinal is regenerated. The 11-cis chromophore is returned to photoreceptors, forming light-sensitive visual pigments with opsin GPCRs. Dysfunction of this process perturbs phototransduction because functional visual pigment cannot be generated. Mutations in visual cycle genes can result in monogenic inherited forms of blindness. Though key enzymatic processes are well characterized, questions remain as to the physiological role of visual cycle proteins in different retinal cell types, functional domains of these proteins in retinoid biochemistry and in vivo pathogenesis of disease mutations. Significant progress is needed to develop effective and accessible treatments for inherited blindness arising from mutations in visual cycle genes. Here, we review opportunities to apply gene editing technology to two crucial visual cycle components, RPE65 and CRALBP. Expressed exclusively in the human RPE, RPE65 enzymatically converts retinyl esters into 11-cis retinal. CRALBP is an 11-cis-retinal binding protein expressed in human RPE and Muller glia. Loss-of-function mutations in either protein results in autosomal recessive forms of blindness. Modeling these human conditions using RPE65 or CRALBP murine knockout models have enhanced our understanding of their biochemical function, associated disease pathogenesis and development of therapeutics. However, rod-dominated murine retinae provide a challenge to assess cone function. The cone-rich zebrafish model is amenable to cost-effective maintenance of a variety of strains. Interestingly, gene duplication in zebrafish resulted in three Rpe65 and two Cralbp isoforms with differential temporal and spatial expression patterns. Functional investigations of zebrafish Rpe65 and Cralbp were restricted to gene knockdown with morpholino oligonucleotides. However, transient

Side effects of retinoid therapy on the quality of vision

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Bergler-Czop Beata

2016-12-01

Full Text Available Retinoids are compounds chemically related to vitamin A, which are frequently used in dermatological practice (1. They are characterized by numerous mechanisms of action leading to normalization of keratinocyte proliferation and maturation. They have anti-seborrhoeic, immunomodulatory and anti-inflammatory effects (1, 2. A number of side effects to retinoid treatment have been recorded; one group of such side effects relates to eyes and vision. Dry eye syndrome and blepharoconjunctivitis are the most common side effects, appearing in 20-50 % of patients treated with retinoids. They often contribute to the occurrence of other side-effects such as eye discomfort and contact lens intolerance. Due to the widespread use in clinical practice, the adverse effects, including ocular side effects, should be studied. To confirm the variety of adverse effects of retinoids, several case reports of rare side-effects are presented.

Mouse Retinal Pigmented Epithelial Cell Lines retain their phenotypic characteristics after transfection with Human Papilloma Virus: A new tool to further the study of RPE biology

Science.gov (United States)

Catanuto, Paola; Espinosa-Heidmann, Diego; Pereira-Simon, Simone; Sanchez, Patricia; Salas, Pedro; Hernandez, Eleut; Cousins, Scott W.; Elliot, Sharon J.

2009-01-01

Development of immortalized mouse retinal pigmented epithelial cell (RPE) lines that retain many of their in vivo phenotypic characteristics, would aid in studies of ocular diseases including age related macular degeneration (AMD). RPE cells were isolated from 16 month old (estrogen receptor knockout) ERKOα and ERKOβ mice and their C57Bl/6 wild type littermates. RPE65 and cellular retinaldehyde binding protein (CRALBP) expression, in vivo markers of RPE cells, were detected by real-time RT-PCR and western analysis. We confirmed the presence of epithelial cell markers, ZO1, cytokeratin 8 and 18 by immunofluorescence staining. In addition, we confirmed the distribution of actin filaments and the expression of ezrin. To develop cell lines, RPE cells were isolated, propagated and immortalized using human papilloma virus (HPV) 16 (E6/E7). RPE-specific markers and morphology were assessed before and after immortalization. In wildtype littermate controls, there was no evidence of any alterations in the parameters that we examined including MMP-2, TIMP-2, collagen type IV, and estrogen receptor (ER) α and ERβ protein expression and ER copy number ratio. Therefore, immortalized mouse RPE cell lines that retain their in vivo phenotype can be isolated from either pharmacologically or genetically manipulated mice, and may be used to study RPE cell biology. PMID:19013153

MTH1745, a protein disulfide isomerase-like protein from thermophilic archaea, Methanothermobacter thermoautotrophicum involving in stress response.

Science.gov (United States)

Ding, Xia; Lv, Zhen-Mei; Zhao, Yang; Min, Hang; Yang, Wei-Jun

2008-01-01

MTH1745 is a putative protein disulfide isomerase characterized with 151 amino acid residues and a CPAC active-site from the anaerobic archaea Methanothermobacter thermoautotrophicum. The potential functions of MTH1745 are not clear. In the present study, we show a crucial role of MTH1745 in protecting cells against stress which may be related to its functions as a disulfide isomerase and its chaperone properties. Using real-time polymerase chain reaction analyses, the level of MTH1745 messenger RNA (mRNA) in the thermophilic archaea M. thermoautotrophicum was found to be stress-induced in that it was significantly higher under low (50 degrees C) and high (70 degrees C) growth temperatures than under the optimal growth temperature for the organism (65 degrees C). Additionally, the expression of MTH1745 mRNA was up-regulated by cold shock (4 degrees C). Furthermore, the survival of MTH1745 expressing Escherichia coli cells was markedly higher than that of control cells in response to heat shock (51.0 degrees C). These results indicated that MTH1745 plays an important role in the resistance of stress. By assay of enzyme activities in vitro, MTH1745 also exhibited a chaperone function by promoting the functional folding of citrate synthase after thermodenaturation. On the other hand, MTH1745 was also shown to function as a disulfide isomerase on the refolding of denatured and reduced ribonuclease A. On the basis of its single thioredoxin domain, function as a disulfide isomerase, and its chaperone activity, we suggest that MTH1745 may be an ancient protein disulfide isomerase. These studies may provide clues to the understanding of the function of protein disulfide isomerase in archaea.

Methodologies for analysis of patterning in the mouse RPE sheet

Science.gov (United States)

Boatright, Jeffrey H.; Dalal, Nupur; Chrenek, Micah A.; Gardner, Christopher; Ziesel, Alison; Jiang, Yi; Grossniklaus, Hans E.

2015-01-01

Purpose Our goal was to optimize procedures for assessing shapes, sizes, and other quantitative metrics of retinal pigment epithelium (RPE) cells and contact- and noncontact-mediated cell-to-cell interactions across a large series of flatmount RPE images. Methods The two principal methodological advances of this study were optimization of a mouse RPE flatmount preparation and refinement of open-access software to rapidly analyze large numbers of flatmount images. Mouse eyes were harvested, and extra-orbital fat and muscles were removed. Eyes were fixed for 10 min, and dissected by puncturing the cornea with a sharp needle or a stab knife. Four radial cuts were made with iridectomy scissors from the puncture to near the optic nerve head. The lens, iris, and the neural retina were removed, leaving the RPE sheet exposed. The dissection and outcomes were monitored and evaluated by video recording. The RPE sheet was imaged under fluorescence confocal microscopy after staining for ZO-1 to identify RPE cell boundaries. Photoshop, Java, Perl, and Matlab scripts, as well as CellProfiler, were used to quantify selected parameters. Data were exported into Excel spreadsheets for further analysis. Results A simplified dissection procedure afforded a consistent source of images that could be processed by computer. The dissection and flatmounting techniques were illustrated in a video recording. Almost all of the sheet could be routinely imaged, and substantial fractions of the RPE sheet (usually 20–50% of the sheet) could be analyzed. Several common technical problems were noted and workarounds developed. The software-based analysis merged 25 to 36 images into one and adjusted settings to record an image suitable for large-scale identification of cell-to-cell boundaries, and then obtained quantitative descriptors of the shape of each cell, its neighbors, and interactions beyond direct cell–cell contact in the sheet. To validate the software, human- and computer

Retinoid production using metabolically engineered Escherichia coli with a two-phase culture system.

Science.gov (United States)

Jang, Hui-Jeong; Yoon, Sang-Hwal; Ryu, Hee-Kyung; Kim, Jung-Hun; Wang, Chong-Long; Kim, Jae-Yean; Oh, Deok-Kun; Kim, Seon-Won

2011-07-29

Retinoids are lipophilic isoprenoids composed of a cyclic group and a linear chain with a hydrophilic end group. These compounds include retinol, retinal, retinoic acid, retinyl esters, and various derivatives of these structures. Retinoids are used as cosmetic agents and effective pharmaceuticals for skin diseases. Retinal, an immediate precursor of retinoids, is derived by β-carotene 15,15'-mono(di)oxygenase (BCM(D)O) from β-carotene, which is synthesized from the isoprenoid building blocks isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Retinoids are chemically unstable and biologically degraded via retinoic acid. Although extensive studies have been performed on the microbial production of carotenoids, retinoid production using microbial metabolic engineering has not been reported. Here, we report retinoid production using engineered Escherichia coli that express exogenous BCM(D)O and the mevalonate (MVA) pathway for the building blocks synthesis in combination with a two-phase culture system using a dodecane overlay. Among the BCM(D)O tested in E. coli, the synthetic retinoid synthesis protein (SR), based on bacteriorhodopsin-related protein-like homolog (Blh) of the uncultured marine bacteria 66A03, showed the highest β-carotene cleavage activity with no residual intracellular β-carotene. By introducing the exogenous MVA pathway, 8.7 mg/L of retinal was produced, which is 4-fold higher production than that of augmenting the MEP pathway (dxs overexpression). There was a large gap between retinal production and β-carotene consumption using the exogenous MVA pathway; therefore, the retinal derivatives were analyzed. The derivatives, except for retinoic acid, that formed were identified, and the levels of retinal, retinol, and retinyl acetate were measured. Amounts as high as 95 mg/L retinoids were obtained from engineered E. coli DH5α harboring the synthetic SR gene and the exogenous MVA pathway in addition to dxs overexpression, which

Analysis of the RPE sheet in the rd10 retinal degeneration model

Energy Technology Data Exchange (ETDEWEB)

Jiang, Yi [Los Alamos National Laboratory

2011-01-04

The normal RPE sheet in the C57Bl/6J mouse is subclassified into two major tiling patterns: A regular generally hexagonal array covering most of the surface and a 'soft network' near the ciliary body made of irregularly shaped cells. Physics models predict these two patterns based on contractility and elasticity of the RPE cell, and strength of cellular adhesion between cells. We hypothesized and identified major changes in RPE regular hexagonal tiling pattern in rdl0 compared to C57BL/6J mice. RPE sheet damage was extensive but occurred in rd10 later than expected, after most retinal degeneration. RPE sheet changes occur in zones with a bullseye pattern. In the posterior zone around the optic nerve RPE cells take on larger irregular and varied shapes to form an intact monolayer. In mid periphery, there is a higher than normal density of cells that progress into involuted layers of RPE under the retina. The periphery remains mostly normal until late stages of degeneration. The number of neighboring cells varies widely depending on zone and progression. RPE morphology continues to deteriorate long after the photoreceptors have degenerated. The RPE cells are bystanders to the rd10 degeneration within photo receptors, and the collateral damage to the RPE sheet resembles stimulation of migration or chemotaxis. Quantitative measures of the tiling patterns and histopathology detected here, scripted in a pipeline written in Perl and Cell Profiler (an open source Matlab plugin), are directly applicable to RPE sheet images from noninvasive fundus autofluorescence (FAF), adaptive optics confocal scanning laser ophthalmoscope (AO-cSLO), and spectral domain optical coherence tomography (SD-OCT) of patients with early stage AMD or RP.

Organochlorine contaminant and retinoid levels in blubber of common dolphins (Delphinus delphis) off northwestern Spain

International Nuclear Information System (INIS)

Tornero, Victoria; Borrell, Assumpcio; Aguilar, Alex; Forcada, Jaume; Lockyer, Christina

2006-01-01

The effect of age, sex, nutritive condition and organochlorine concentration on blubber retinoid concentrations was examined in 74 common dolphins incidentally caught off northwestern Spain. Age and blubber lipid content were strong determinants of the retinoid concentrations in males, while these variables did not account for the variation found in females. Retinoids were positively correlated with organochlorines in males and negatively in females. However, pollution levels were moderate and likely to be below threshold levels above that a toxicological response is to be expected. Thus, a cause-effect relationship between organochlorine and retinoid concentrations could not be properly established, and the observed correlation may be the result of an independent association of the two variables with age. Further research on the influence of the best predictor variables on retinoid dynamics is required to implement the use of retinoids as biomarkers of pollutant exposure in cetaceans. - Organochlorine contaminants and retinoids in common dolphins

Retinoids, race and the pathogenesis of dengue hemorrhagic fever.

Science.gov (United States)

Mawson, Anthony R

2013-12-01

Dengue hemorrhagic fever (DHF) is the most significant mosquito-borne viral disease worldwide in terms of illness, mortality and economic cost, but the pathogenesis of DHF is not well understood and there is no specific treatment or vaccine. Based on evidence of liver involvement, it is proposed that dengue virus and retinoids interact to cause cholestatic liver damage, resulting in the spillage of stored retinoids into the circulation and in an endogenous form of hypervitaminosisis A manifested by the signs and symptoms of the disease, including: fever, severe joint and bone pain, capillary leakage, thrombocytopenia, headache, and gastrointestinal symptoms. While retinoids in low concentration are essential for numerous biological functions, they are prooxidant, cytotoxic, mutagenic and teratogenic in higher concentration, especially when unbound to protein, and an endogenous form of vitamin A intoxication is recognized in cholestasis. The model tentatively explains the observations that 1) repeat infections are more severe than initial dengue virus infections; 2) the incidence of denue has increased dramatically worldwide in recent decades; 3) DHF is less prevalent in people of African ancestry than those of other racial backgrounds; and 4) infants are protected from dengue. The retinoid toxicity hypothesis of DHF predicts the co-existence of low serum concentrations of retinol coupled with high concentrations of retinoic acid and an increased percentage of retinyl esters to total vitamin A. Subject to such tests, it may be possible to treat DHF effectively using drugs that target the metabolism and expression of retinoids. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

Physiological and Pathogenic Roles of Prolyl Isomerase Pin1 in Metabolic Regulations via Multiple Signal Transduction Pathway Modulations

Directory of Open Access Journals (Sweden)

Yusuke Nakatsu

2016-09-01

Full Text Available Prolyl isomerases are divided into three groups, the FKBP family, Cyclophilin and the Parvulin family (Pin1 and Par14. Among these isomerases, Pin1 is a unique prolyl isomerase binding to the motif including pSer/pThr-Pro that is phosphorylated by kinases. Once bound, Pin1 modulates the enzymatic activity, protein stability or subcellular localization of target proteins by changing the cis- and trans-formations of proline. Several studies have examined the roles of Pin1 in the pathogenesis of cancers and Alzheimer’s disease. On the other hand, recent studies have newly demonstrated Pin1 to be involved in regulating glucose and lipid metabolism. Interestingly, while Pin1 expression is markedly increased by high-fat diet feeding, Pin1 KO mice are resistant to diet-induced obesity, non-alcoholic steatohepatitis and diabetic vascular dysfunction. These phenomena result from the binding of Pin1 to several key factors regulating metabolic functions, which include insulin receptor substrate-1, AMPK, Crtc2 and NF-κB p65. In this review, we focus on recent advances in elucidating the physiological roles of Pin1 as well as the pathogenesis of disorders involving this isomerase, from the viewpoint of the relationships between signal transductions and metabolic functions.

Calcium-independent phospholipase A₂, group VIA, is critical for RPE cell survival

DEFF Research Database (Denmark)

Kolko, Miriam; Vohra, Rupali; Westlund, Barbro S.

2014-01-01

PURPOSE: To investigate the significance of calcium-independent phospholipase A₂, group VIA (iPLA2-VIA), in RPE cell survival following responses to sodium iodate (SI) in cell cultures. METHODS: The human retinal pigment epithelium (RPE) cell line (ARPE-19) cells and primary mouse-RPE cultures were...

Purification and characterization of the d-xylose isomerase gene from Escherichia coli

Energy Technology Data Exchange (ETDEWEB)

Ho, N W.Y.; Rosenfeld, S; Stevis, P; Tsao, G T

1983-11-01

A DNA fragment containing both the Escherichia coli D-xylose isomerase (D-xylose ketol-isomerase, EC 5.3.1.5) gene and the D-xylulokinase (ATP: D-xylulose 5-phosphotransferase, EC 2.7.1.17) gene has been cloned on an E. coli plasmid. The D-xylose isomerase gene was separated from the D-xylulokinase gene by the construction of a new deletion plasmid, pLX7. The D-xylose isomerase gene cloned on pLX7 was found still to be an intact gene. The precise location of the D-xylose isomerase gene on the plasmid pLX7 was further determined by the construction of two more plasmids, pLX8 and pLX9. This is believed to be the first D-xylose isomerase gene that has been isolated and extensively purified from any organism. D-Xylose isomerase, the enzyme product of the D-xylose isomerase gene, is responsible for the conversion of D-xylose to D-xylulose, as well as D-glucose to D-fructose. It is widely believed that yeast cannot ferment D-xylose to ethanol primarily because of the lack of D-xylose isomerase in yeast. D-Xylose isomerase (also known as D-glucose isomerase) is also used for the commercial production of high-fructose syrups. The purification of the D-xylose isomerase gene may lead to the following industrial applications: (1) cloning and expression of the gene in yeast to make the latter organism capable of directly fermenting D-xylose to ethanol, and (2) cloning of the gene on a high-copy-number plasmid in a proper host to overproduce the enzyme, which should have a profound impact on the high-fructose syrup technology. 14 references.

Effects of aflibercept on primary RPE cells: toxicity, wound healing, uptake and phagocytosis.

Science.gov (United States)

Klettner, Alexa; Tahmaz, Nihat; Dithmer, Michaela; Richert, Elisabeth; Roider, Johann

2014-10-01

Anti-VEGF treatment is the therapy of choice in age-related macular degeneration, and is also applied in diabetic macular oedema or retinal vein occlusion. Recently, the fusion protein, aflibercept, has been approved for therapeutic use. In this study, we investigate the effects of aflibercept on primary RPE cells. Primary RPE cells were prepared from freshly slaughtered pigs' eyes. The impact of aflibercept on cell viability was investigated with MTT and trypan blue exclusion assay. The influence of aflibercept on wound healing was assessed with a scratch assay. Intracellular uptake of aflibercept was investigated in immunohistochemistry and its influence on phagocytosis with a phagocytosis assay using opsonised latex beads. Aflibercept displays no cytotoxicity on RPE cells but impairs its wound healing ability. It is taken up into RPE cells and can be intracellularly detected for at least 7 days. Intracellular aflibercept impairs the phagocytic capacity of RPE cells. Aflibercept interferes with the physiology of RPE cells, as it is taken up into RPE cells, which is accompanied by a reduction of the phagocytic ability. Additionally, it impairs the wound healing capacity of RPE cells. These effects on the physiology of RPE cells may indicate possible side effects. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Differentiation of RPE cells from integration-free iPS cells and their cell biological characterization.

Science.gov (United States)

Hazim, Roni A; Karumbayaram, Saravanan; Jiang, Mei; Dimashkie, Anupama; Lopes, Vanda S; Li, Douran; Burgess, Barry L; Vijayaraj, Preethi; Alva-Ornelas, Jackelyn A; Zack, Jerome A; Kohn, Donald B; Gomperts, Brigitte N; Pyle, April D; Lowry, William E; Williams, David S

2017-10-02

Dysfunction of the retinal pigment epithelium (RPE) is implicated in numerous forms of retinal degeneration. The readily accessible environment of the eye makes it particularly suitable for the transplantation of RPE cells, which can now be derived from autologous induced pluripotent stem cells (iPSCs), to treat retinal degeneration. For RPE transplantation to become feasible in the clinic, patient-specific somatic cells should be reprogrammed to iPSCs without the introduction of reprogramming genes into the genome of the host cell, and then subsequently differentiated into RPE cells that are well characterized for safety and functionality prior to transplantation. We have reprogrammed human dermal fibroblasts to iPSCs using nonintegrating RNA, and differentiated the iPSCs toward an RPE fate (iPSC-RPE), under Good Manufacturing Practice (GMP)-compatible conditions. Using highly sensitive assays for cell polarity, structure, organelle trafficking, and function, we found that iPSC-RPE cells in culture exhibited key characteristics of native RPE. Importantly, we demonstrate for the first time with any stem cell-derived RPE cell that live cells are able to support dynamic organelle transport. This highly sensitive test is critical for RPE cells intended for transplantation, since defects in intracellular motility have been shown to promote RPE pathogenesis akin to that found in macular degeneration. To test their capabilities for in-vivo transplantation, we injected the iPSC-RPE cells into the subretinal space of a mouse model of retinal degeneration, and demonstrated that the transplanted cells are capable of rescuing lost RPE function. This report documents the successful generation, under GMP-compatible conditions, of human iPSC-RPE cells that possess specific characteristics of healthy RPE. The report adds to a growing literature on the utility of human iPSC-RPE cells for cell culture investigations on pathogenicity and for therapeutic transplantation, by

A possible contribution of retinoids to regulation of fetal B lymphopoiesis.

Science.gov (United States)

Chen, Xinrong; Welner, Robert S; Kincade, Paul W

2009-09-01

We recently found that all trans retinoic acid (ATRA) accelerated B lymphocyte formation. In the current study, we address the question whether retinoids account for the rapid lymphopoiesis that is characteristic of fetal progenitors. Surprisingly, addition of ATRA to fetal liver cultures actually reduced B lymphopoiesis. A pan-retinoid receptor antagonist selectively suppressed lymphocyte formation from fetal and adult progenitors, suggesting some normal contribution of retinoids to this process. Consistent with this role, B lymphopoiesis was compromised in the marrow of mice with prolonged vitamin A deficiency. Recently identified B1 progenitors from adult marrow were similar to adult B2 progenitors in that their differentiation was stimulated by ATRA. The inhibitory response observed with fetal cells was seen when adult progenitors were exposed to high doses in culture or when adult mice were treated with ATRA for 2 wk. In addition to explosive lymphocyte generation, fetal progenitors tend to be less IL-7 dependent than their adult counterparts, but ATRA did not make fetal progenitors IL-7 independent. We conclude that all known categories of B lineage progenitors are responsive to retinoids and probably regulated by these compounds under physiological conditions. Retinoids may account in part for rapid differentiation in fetal life, but not all unique features of fetal progenitors.

Changes in the skeleton after long-term treatment with retinoids

Energy Technology Data Exchange (ETDEWEB)

Montag, M.; Reiser, M.; Hamm, H.; Traupe, H.; Vogt, H.J.

1988-07-01

The synthetic retinoids, the vitamin-A-derivatives ethretinate and isotretinoin, have substantially enlarged the therapeutic arsenal in dermatology. They are primarily used in severe cases of acne and cornification disorders. In the majority of cases, long-term treatment is necessary. Certain side effects in the skeletal system can occur, e.g., osteoporosis, premature epiphyseal closure, and changes similar to DISH (diffuse idiopathic skeletal hyperostosis). We discuss the reports in the literature and our own observations in 31 patients treated at the Westphalian Wilhelms University in Muenster, as well as at the Technical University in Munich. In 3 out of 31 patients treated by retinoids on a long-term basis, skeletal changes were found radiologically as a result of the retinoid medication.

Retinoids in the treatment of glioma: a new perspective

Directory of Open Access Journals (Sweden)

Mawson AR

2012-08-01

Full Text Available Anthony R MawsonDepartment of Health Policy and Management, School of Health Sciences, College of Public Service, Jackson State University, Jackson, MS, USAAbstract: Primary brain tumors are among the top ten causes of cancer-related deaths in the US. Malignant gliomas account for approximately 70% of the 22,500 new cases of malignant primary brain tumors diagnosed in adults each year and are associated with high morbidity and mortality. Despite optimal treatment, the prognosis for patients with gliomas remains poor. The use of retinoids (vitamin A and its congeners in the treatment of certain tumors was originally based on the assumption that these conditions were associated with an underlying deficiency of vitamin A and that supplementation with pharmacological doses would correct the deficiency. Yet the results of retinoid treatment have been only modestly beneficial and usually short-lived. Studies also indicate that vitamin A excess and supplementation have pro-oxidant effects and are associated with increased risks of mortality from cancer and other diseases. The therapeutic role of vitamin A in cancer thus remains uncertain and a new perspective on the facts is needed. The modest and temporary benefits of retinoid treatment could result from a process of feedback inhibition, whereby exogenous retinoid temporarily inhibits the endogenous synthesis of these compounds. In fact, repeated and/or excessive exposure of the tissues to endogenous retinoic acid may contribute to carcinogenesis. Gliomas, in particular, may result from an imbalance in retinoid receptor expression initiated by environmental factors that increase the endogenous production of retinoic acid in glia. At the receptor level, it is proposed that this imbalance is characterized by excessive expression of retinoic acid receptor-α(RARα and reduced expression of retinoic acid receptor-β (RARβ. This suggests a potential new treatment strategy for gliomas, possibly even at a

Evolution of Retinoid and Steroid Signaling: Vertebrate Diversification from an Amphioxus Perspective

Science.gov (United States)

Albalat, Ricard; Brunet, Frédéric; Laudet, Vincent; Schubert, Michael

2011-01-01

Although the physiological relevance of retinoids and steroids in vertebrates is very well established, the origin and evolution of the genetic machineries implicated in their metabolic pathways is still very poorly understood. We investigated the evolution of these genetic networks by conducting an exhaustive survey of components of the retinoid and steroid pathways in the genome of the invertebrate chordate amphioxus (Branchiostoma floridae). Due to its phylogenetic position at the base of chordates, amphioxus is a very useful model to identify and study chordate versus vertebrate innovations, both on a morphological and a genomic level. We have characterized more than 220 amphioxus genes evolutionarily related to vertebrate components of the retinoid and steroid pathways and found that, globally, amphioxus has orthologs of most of the vertebrate components of these two pathways, with some very important exceptions. For example, we failed to identify a vertebrate-like machinery for retinoid storage, transport, and delivery in amphioxus and were also unable to characterize components of the adrenal steroid pathway in this invertebrate chordate. The absence of these genes from the amphioxus genome suggests that both an elaboration and a refinement of the retinoid and steroid pathways took place at the base of the vertebrate lineage. In stark contrast, we also identified massive amplifications in some amphioxus gene families, most extensively in the short-chain dehydrogenase/reductase superfamily, which, based on phylogenetic and genomic linkage analyses, were likely the result of duplications specific to the amphioxus lineage. In sum, this detailed characterization of genes implicated in retinoid and steroid signaling in amphioxus allows us not only to reconstruct an outline of these pathways in the ancestral chordate but also to discuss functional innovations in retinoid homeostasis and steroid-dependent regulation in both cephalochordate and vertebrate evolution

Session-RPE Method for Training Load Monitoring: Validity, Ecological Usefulness, and Influencing Factors

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Monoem Haddad

2017-11-01

Full Text Available Purpose: The aim of this review is to (1 retrieve all data validating the Session-rating of perceived exertion (RPE-method using various criteria, (2 highlight the rationale of this method and its ecological usefulness, and (3 describe factors that can alter RPE and users of this method should take into consideration.Method: Search engines such as SPORTDiscus, PubMed, and Google Scholar databases in the English language between 2001 and 2016 were consulted for the validity and usefulness of the session-RPE method. Studies were considered for further analysis when they used the session-RPE method proposed by Foster et al. in 2001. Participants were athletes of any gender, age, or level of competition. Studies using languages other than English were excluded in the analysis of the validity and reliability of the session-RPE method. Other studies were examined to explain the rationale of the session-RPE method and the origin of RPE.Results: A total of 950 studies cited the Foster et al. study that proposed the session RPE-method. 36 studies have examined the validity and reliability of this proposed method using the modified CR-10.Conclusion: These studies confirmed the validity and good reliability and internal consistency of session-RPE method in several sports and physical activities with men and women of different age categories (children, adolescents, and adults among various expertise levels. This method could be used as “standing alone” method for training load (TL monitoring purposes though some recommend to combine it with other physiological parameters as heart rate.

Therapy of psoriasis with retinoid plus PUVA

International Nuclear Information System (INIS)

Heidbreder, G.; Christophers, E.

1979-01-01

In a group of 40 patients suffering from wide-spread psoriasis oral administration of a retinoid (Ro 10-9359) was followed by PUVA therapy. The clearance rate was increased by 30% as compared to PUVA alone. Except for cheilitis no side effects were seen. Histological analysis in 20 patients before, during and after therapy revealed an intensification of psoriatic tissue changes after retinoid treatment. Loss of corneal layers, massive exoserosis, and neutrophil migration were prominent features. Mitotic counts were not increased by the pretreatment. The increased susceptibility of diseased skin to PUVA as produced by this drug appears to be based on several factors related to the tissue changes revealed by histology. (orig.) [de

Liposomal Formulation of Retinoids Designed for Enzyme Triggered Release

DEFF Research Database (Denmark)

Pedersen, Palle Jacob; Adolph, Sidsel Kramshøj; Subramanian, Arun Kumar

2010-01-01

The design of retinoid phospholipid prodrugs is described based on molecular dynamics simulations and cytotoxicity studies of synthetic retinoid esters. The prodrugs are degradable by secretory phospholipase A(2) IIA and have potential in liposomal drug delivery targeting tumors. We have synthesi...... displayed IC50 values in the range of 3-19 mu M toward HT-29 and Colo205 colon cancer cells in the presence of phospholipase A(2), while no significant cell death was observed in the absence of the enzyme....

Synthesis of retinoid vitamin A-vitamin B6 conjugate analogues for antiviral chemotherapy

International Nuclear Information System (INIS)

Kesel, Andreas J.

2003-01-01

The synthesis of retinoid vitamin A-vitamin B 6 conjugate analogues from a vitamin B 6 coenzyme analogue and putative HIV-1 trans-activating transcriptional regulatory protein Tat antagonist (Z)-5 ' -O-phosphono-pyridoxylidenerhodanine (B6PR) monosodium salt hemiheptadecahydrate [(Z)-B6PRNa8.5H 2 O] is discussed here. All-trans-retinoic acid (ATRA) is coupled to B6PR by a modified Stork enamine acylation. It results in a product library of more than eight compounds, each with at least one intact all-trans or 13-cis vitamin A double bond system. This yellow oily concentrate mixture was subjected to matrix-assisted laser desorption/ionization-time-of-flight (MALDI-ToF) mass spectrometry (MS), UV/VIS-spectrophotometry, and proton nuclear magnetic resonance spectroscopy ( 1 H-NMR). The chemical structures of six components of the concentrate mixture could be established by combination of these analytical methods. The two main components are 65% 2 ' C,3O-(all-trans-retinylidyne)B6PT (B6RA) and 25% 2 ' C-(all-trans-retinoyl)B6PT, chemically derived from (5RS)-5-(5 ' -O-phosphono-pyridoxyl)-2,4-thiazolidinedione (B6PT). This new retinoid selection could be of further interest in antiviral applications, especially treating conditions caused by RNA viruses like HIV

RPE cell surface proteins in normal and dystrophic rats

International Nuclear Information System (INIS)

Clark, V.M.; Hall, M.O.

1986-01-01

Membrane-bound proteins in plasma membrane enriched fractions from cultured rat RPE were analyzed by two-dimensional gel electrophoresis. Membrane proteins were characterized on three increasingly specific levels. Total protein was visualized by silver staining. A maximum of 102 separate proteins were counted in silver-stained gels. Glycoproteins were labeled with 3H-glucosamine or 3H-fucose and detected by autoradiography. Thirty-eight fucose-labeled and 61-71 glucosamine-labeled proteins were identified. All of the fucose-labeled proteins were labeled with glucosamine-derived radioactivity. Proteins exposed at the cell surface were labeled by lactoperoxidase-catalyzed radioiodination prior to preparation of membranes for two-dimensional analysis. Forty separate 125I-labeled surface proteins were resolved by two-dimensional electrophoresis/autoradiography. Comparison with the glycoprotein map showed that a number of these surface labeled proteins were glycoproteins. Two-dimensional maps of total protein, fucose-labeled, and glucosamine-labeled glycoproteins, and 125I-labeled surface proteins of membranes from dystrophic (RCS rdy-p+) and normal (Long Evans or RCS rdy+p+) RPE were compared. No differences in the total protein or surface-labeled proteins were observed. However, the results suggest that a 183K glycoprotein is more heavily glycosylated with glucosamine and fucose in normal RPE membranes as compared to membranes from dystrophic RPE

Enzymatic conversion of D-galactose to D-tagatose: heterologous expression and characterisation of a thermostable L-arabinose isomerase from Thermoanaerobacter mathranii.

Science.gov (United States)

Jørgensen, F; Hansen, O C; Stougaard, P

2004-06-01

The ability to convert D-galactose into D-tagatose was compared among a number of bacterial L-arabinose isomerases ( araA). One of the most efficient enzymes, from the anaerobic thermophilic bacterium Thermoanaerobacter mathranii, was produced heterologously in Escherichia coli and characterised. Amino acid sequence comparisons indicated that this enzyme is only distantly related to the group of previously known araA sequences in which the sequence similarity is evident. The substrate specificity and the Michaelis-Menten constants of the enzyme determined with L-arabinose, D-galactose and D-fucose also indicated that this enzyme is an unusual, versatile L-arabinose isomerase which is able to isomerise structurally related sugars. The enzyme was immobilised and used for production of D-tagatose at 65 degrees C. Starting from a 30% solution of D-galactose, the yield of D-tagatose was 42% and no sugars other than D-tagatose and D-galactose were detected. Direct conversion of lactose to D-tagatose in a single reactor was demonstrated using a thermostable beta-galactosidase together with the thermostable L-arabinose isomerase. The two enzymes were also successfully combined with a commercially available glucose isomerase for conversion of lactose into a sweetening mixture comprising lactose, glucose, galactose, fructose and tagatose.

Cumulative irritation potential of topical retinoid formulations.

Science.gov (United States)

Leyden, James J; Grossman, Rachel; Nighland, Marge

2008-08-01

Localized irritation can limit treatment success with topical retinoids such as tretinoin and adapalene. The factors that influence irritant reactions have been shown to include individual skin sensitivity, the particular retinoid and concentration used, and the vehicle formulation. To compare the cutaneous tolerability of tretinoin 0.04% microsphere gel (TMG) with that of adapalene 0.3% gel and a standard tretinoin 0.025% cream. The results of 2 randomized, investigator-blinded studies of 2 to 3 weeks' duration, which utilized a split-face method to compare cumulative irritation scores induced by topical retinoids in subjects with healthy skin, were combined. Study 1 compared TMG 0.04% with adapalene 0.3% gel over 2 weeks, while study 2 compared TMG 0.04% with tretinoin 0.025% cream over 3 weeks. In study 1, TMG 0.04% was associated with significantly lower cumulative scores for erythema, dryness, and burning/stinging than adapalene 0.3% gel. However, in study 2, there were no significant differences in cumulative irritation scores between TMG 0.04% and tretinoin 0.025% cream. Measurements of erythema by a chromameter showed no significant differences between the test formulations in either study. Cutaneous tolerance of TMG 0.04% on the face was superior to that of adapalene 0.3% gel and similar to that of a standard tretinoin cream containing a lower concentration of the drug (0.025%).

Retinoids in the treatment of glioma: a new perspective.

Science.gov (United States)

Mawson, Anthony R

2012-01-01

Primary brain tumors are among the top ten causes of cancer-related deaths in the US. Malignant gliomas account for approximately 70% of the 22,500 new cases of malignant primary brain tumors diagnosed in adults each year and are associated with high morbidity and mortality. Despite optimal treatment, the prognosis for patients with gliomas remains poor. The use of retinoids (vitamin A and its congeners) in the treatment of certain tumors was originally based on the assumption that these conditions were associated with an underlying deficiency of vitamin A and that supplementation with pharmacological doses would correct the deficiency. Yet the results of retinoid treatment have been only modestly beneficial and usually short-lived. Studies also indicate that vitamin A excess and supplementation have pro-oxidant effects and are associated with increased risks of mortality from cancer and other diseases. The therapeutic role of vitamin A in cancer thus remains uncertain and a new perspective on the facts is needed. The modest and temporary benefits of retinoid treatment could result from a process of feedback inhibition, whereby exogenous retinoid temporarily inhibits the endogenous synthesis of these compounds. In fact, repeated and/or excessive exposure of the tissues to endogenous retinoic acid may contribute to carcinogenesis. Gliomas, in particular, may result from an imbalance in retinoid receptor expression initiated by environmental factors that increase the endogenous production of retinoic acid in glia. At the receptor level, it is proposed that this imbalance is characterized by excessive expression of retinoic acid receptor-α (RARα) and reduced expression of retinoic acid receptor-β (RARβ). This suggests a potential new treatment strategy for gliomas, possibly even at a late stage of the disease, ie, to combine the use of a RARα antagonist and a RARβ agonist. According to this hypothesis, the RARα antagonist would be expected to inhibit RAR�

Retinoids in the treatment of glioma: a new perspective

International Nuclear Information System (INIS)

Mawson, Anthony R

2012-01-01

Primary brain tumors are among the top ten causes of cancer-related deaths in the US. Malignant gliomas account for approximately 70% of the 22,500 new cases of malignant primary brain tumors diagnosed in adults each year and are associated with high morbidity and mortality. Despite optimal treatment, the prognosis for patients with gliomas remains poor. The use of retinoids (vitamin A and its congeners) in the treatment of certain tumors was originally based on the assumption that these conditions were associated with an underlying deficiency of vitamin A and that supplementation with pharmacological doses would correct the deficiency. Yet the results of retinoid treatment have been only modestly beneficial and usually short-lived. Studies also indicate that vitamin A excess and supplementation have pro-oxidant effects and are associated with increased risks of mortality from cancer and other diseases. The therapeutic role of vitamin A in cancer thus remains uncertain and a new perspective on the facts is needed. The modest and temporary benefits of retinoid treatment could result from a process of feedback inhibition, whereby exogenous retinoid temporarily inhibits the endogenous synthesis of these compounds. In fact, repeated and/or excessive exposure of the tissues to endogenous retinoic acid may contribute to carcinogenesis. Gliomas, in particular, may result from an imbalance in retinoid receptor expression initiated by environmental factors that increase the endogenous production of retinoic acid in glia. At the receptor level, it is proposed that this imbalance is characterized by excessive expression of retinoic acid receptor-α (RARα) and reduced expression of retinoic acid receptor-β (RARβ). This suggests a potential new treatment strategy for gliomas, possibly even at a late stage of the disease, ie, to combine the use of a RARα antagonist and a RARβ agonist. According to this hypothesis, the RARα antagonist would be expected to inhibit RAR�

The anterior lens capsule used as support material in RPE cell-transplantation

DEFF Research Database (Denmark)

Nicolini, J; Kiilgaard, Jens Folke; Wiencke, A K

2000-01-01

To investigate the use of an ocular basement membrane as support material for transplanted porcine RPE cells.......To investigate the use of an ocular basement membrane as support material for transplanted porcine RPE cells....

Abnormal retinoid and TrkB signaling in the prefrontal cortex in mood disorders

NARCIS (Netherlands)

Qi, Xin-Rui; Zhao, Juan; Liu, Ji; Fang, Hui; Swaab, Dick F; Zhou, Jiang-Ning

The prefrontal cortex shows structural and functional alterations in mood disorders. Retinoid signaling, brain-derived neurotrophic factor (BDNF), and its receptor TrkB are reported to be involved in depression. Here, we found that mRNA levels of key elements of retinoid signaling were significantly

[Molecular genetics of pigmentary retinopathies: identification of mutations in CHM, RDS, RHO, RPE65, USH2A and XLRS1 genes].

Science.gov (United States)

Hamel, C P; Griffoin, J M; Bazalgette, C; Lasquellec, L; Duval, P A; Bareil, C; Beaufrère, L; Bonnet, S; Eliaou, C; Marlhens, F; Schmitt-Bernard, C F; Tuffery, S; Claustres, M; Arnaud, B

2000-12-01

To evaluate the occurrence and inheritance of various types of pigmentary retinopathy in patients followed at the outpatient clinic in the university hospital, Montpellier, France. To characterize genes and mutations causing these conditions. Ophthalmic examination and various visual tests were performed. Mutations were sought from genomic DNA by PCR amplification of exons associated with single-strand conformation analysis and/or direct sequencing. Among 315 patients over an 8-year period, cases of retinitis pigmentosa (63.2%), Usher's syndrome (10.2%), Stargardt's disease (5.4%), choroideremia (3.2%), Leber's congenital amaurosis (3.2%), congenital stationary night blindness (2.9%), cone dystrophy (2.5%), dominant optic atrophy (1.9%), X-linked juvenile retinoschisis (1.6%), Best's disease (1.6%), and others (4.3%) were diagnosed. In retinitis pigmentosa, inheritance could be determined in 54.2% of the cases including dominant autosomic (26.6%), recessive autosomic (22.6%), and X-linked cases (5%) while it could not be confirmed in 45.7% of the cases (simplex cases in the majority). For the 6 examined genes, mutations were found in 22 out of 182 propositus (12.1%). Analysis of phenotype-genotype correlations indicates that in retinitis pigmentosa, RDS is more frequently associated with macular involvement and retinal flecks, RHO with regional disease, and RPE65 with the great severity of the disease with some cases of Leber's congenital amaurosis. Identification of genes may help in diagnosis and in genetic counseling, especially in simplex cases with retinitis pigmentosa. In this latter condition, molecular diagnosis will be necessary to rationalize future treatments.

A single and two step isomerization process for d-tagatose and l-ribose bioproduction using l-arabinose isomerase and d-lyxose isomerase.

Science.gov (United States)

Patel, Manisha J; Akhani, Rekha C; Patel, Arti T; Dedania, Samir R; Patel, Darshan H

2017-02-01

l-ribose and d-tagatose are biochemically synthesized using sugar isomerases. The l-arabinose isomerase gene from Shigella flexneri (Sf-AI) was cloned and expressed in Escherichia coli BL-21. Sf-AI was applied for the bioproduction of d-tagatose from d-galactose. l-ribose synthesis was performed by two step isomerization using Sf-AI and d-lyxose/ribose isomerase from Cohnella laevoribosii. The overall 22.3% and 25% conversion rate were observed for d-tagatose and l-ribose production from d-galactose and l-arabinose respectively. In the present manuscript, synthesis of rare sugars from naturally available sugars is discussed along with the biochemical characterization of Sf-AI and its efficiency. Copyright © 2016 Elsevier Inc. All rights reserved.

RPE and RPO in the revised Swiss radiation protection legislation; RPE und RPO in der revidierten schweizerischen Strahlenschutzgesetzgebung

Energy Technology Data Exchange (ETDEWEB)

Jahn, S.G. [Eidgenoessisches Nuklearsicherheitsinspektorat (ENSI), Brugg (Switzerland); Stritt, N.; Bogni, S. [Bundesamt fuer Gesundheit (BAG), Bern (Switzerland); Hammans, M. [Schweizerische Unfallversicherungsanstalt (SUVA), Luzern (Switzerland); Duerst, B. [Departement fuer Verteidigung, Bevoelkerungsschutz und Sport (VBS), Bern (Switzerland); Kuehne, G. [Paul-Scherrer-Institut (PSI), Wuerenlingen (Switzerland); Besancon, A. [Institut de Radiophysique (IRA), Lausanne (Switzerland)

2013-07-01

Currently the Swiss 'Ordinance on Radiation Protection' and the 'Ordinance about Education and Training in RP' are undergoing a revision process. Concerning responsibilities and obligations as well as the capabilities and skills in RP the new ordinances are supposed to take account on the new EU Basic Safety Standard on the one hand and on the other hand to resume the approved approaches on RP in Switzerland. An overview of the planned content of the revised ordinances will be given in this report. In the Swiss system the education, training and retraining of RPO has a higher priority. Particularly it will be described which groups of persons assume the roles of RPE and therefore the definition of RPE in the new Swiss legislation is needless. (orig.)

Retinoid inhibition of in vitro invasion of human amnion basement membrane by human tumor cells

International Nuclear Information System (INIS)

Fazely, F.; Ledinko, N.; Smith, D.J.

1986-01-01

The biological activity of retinoids was assayed in an in vitro quantitative assay of human tumor cell invasion using human amnion basement membrane (BM). The effects measured were the inhibition of tumor cell migration through the BM and tumor cell degradative enzyme activity on 14 C-proline labeled collagenous and noncollagenous components of the BM. The human lung carcinoma A549 or the human Ewing's sarcoma TC-106 cell lines treated with retinoids for two days were incubated on the BM in the absence of retinoids. A dose-dependent inhibition of cell invasion was produced by retinoids. Among the retinoids tested, the most powerful was retinol acetate which inhibited invasion by 50% of A549 cells at a concentration of 0.009 μg/mL, and of TC-106 cells at 0.07 μg/mL. Retinol acetate inhibited A549 and TC-106 cell growth by approximately 50% at levels over 100-fold higher than those needed for antiinvasive activity. Retinol acetate was about 20 times more potent than retinoic acid and 30 times more potent than retinol palmitate. The model system will be useful for investigating antiinvasive activity of other retinoids as well as other compounds

Biochemistry and pharmacology of rhodopsin regeneration in the vertebrate eye

International Nuclear Information System (INIS)

Bernstein, P.S.

1988-01-01

In this thesis, the missing reaction of the vertebrate visual cycle, the energy-dependent isomerization of all-trans-retinoids to 11-cis-retinoids, is investigated through biochemical and pharmacological means. The biochemical processes of vision are first probed through the use of 1,5-di-(p-aminophenoxy) pentane (DAPP), the most powerful and selective pharmacological inhibitor of dark adaptation known. Next, the biochemical pathway of isomerization of retinoids in living animals is examined through double-label radioisotope studies in normal animals and in animals treated with DAPP or other inhibitors of dark adaptation. Finally, a novel retinoid isomerase activity was discovered in homogenates of frog retina/pigment epithelium that catalyzes the endergonic isomerization of all-trans-retinoids to 11-cis-retinoids in darkness. In partially purified preparations, added [11,12- 3 H]-all-trans-retinol is converted to 11-cis-retinol and other 11-cis-retinoids, while added labeled all-trans-retinal and all-trans-retinyl palmitate are not isomerized to a significant extent

Identification of Novel Retinoid Targets in Prostate Cancer

National Research Council Canada - National Science Library

Piedrafita, F. J

2006-01-01

.... However except for the efficient treatment of acute promyelocytic leukemia and certain skin disorders most natural and synthetic retinoids have failed in clinical trials because of toxicity and limited activity...

highroad Is a Carboxypetidase Induced by Retinoids to Clear Mutant Rhodopsin-1 in Drosophila Retinitis Pigmentosa Models

Directory of Open Access Journals (Sweden)

Huai-Wei Huang

2018-02-01

Full Text Available Rhodopsins require retinoid chromophores for their function. In vertebrates, retinoids also serve as signaling molecules, but whether these molecules similarly regulate gene expression in Drosophila remains unclear. Here, we report the identification of a retinoid-inducible gene in Drosophila, highroad, which is required for photoreceptors to clear folding-defective mutant Rhodopsin-1 proteins. Specifically, knockdown or genetic deletion of highroad blocks the degradation of folding-defective Rhodopsin-1 mutant, ninaEG69D. Moreover, loss of highroad accelerates the age-related retinal degeneration phenotype of ninaEG69D mutants. Elevated highroad transcript levels are detected in ninaEG69D flies, and interestingly, deprivation of retinoids in the fly diet blocks this effect. Consistently, mutations in the retinoid transporter, santa maria, impairs the induction of highroad in ninaEG69D flies. In cultured S2 cells, highroad expression is induced by retinoic acid treatment. These results indicate that cellular quality-control mechanisms against misfolded Rhodopsin-1 involve regulation of gene expression by retinoids.

Repressed SIRT1/PGC-1α pathway and mitochondrial disintegration in iPSC-derived RPE disease model of age-related macular degeneration.

Science.gov (United States)

Golestaneh, Nady; Chu, Yi; Cheng, Shuk Kei; Cao, Hong; Poliakov, Eugenia; Berinstein, Daniel M

2016-12-20

Study of age related macular degeneration (AMD) has been hampered by lack of human models that represent the complexity of the disease. Here we have developed a human in vitro disease model of AMD to investigate the underlying AMD disease mechanisms. Generation of iPSCs from retinal pigment epithelium (RPE) of AMD donors, age-matched normal donors, skin fibroblasts of a dry AMD patient, and differentiation of iPSCs into RPE (AMD RPE-iPSC-RPE, normal RPE-iPSC-RPE and AMD Skin-iPSC-RPE, respectively). Immunostaining, cell viability assay and reactive oxygen species (ROS) production under oxidative stress conditions, electron microscopy (EM) imaging, ATP production and glycogen concentration assays, quantitative real time PCR, western blot, karyotyping. The AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE present functional impairment and exhibit distinct disease phenotypes compared to RPE-iPSC-RPE generated from normal donors (Normal RPE-iPSC-RPE). The AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE show increased susceptibility to oxidative stress and produced higher levels of reactive oxygen species (ROS) under stress in accordance with recent reports. The susceptibility to oxidative stress-induced cell death in AMD RPE-iPSC-RPE and Skin-iPSC-RPE was consistent with inability of the AMD RPE-iPSC-RPE and Skin-iPSC-RPE to increase SOD2 expression under oxidative stress. Phenotypic analysis revealed disintegrated mitochondria, accumulation of autophagosomes and lipid droplets in AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE. Mitochondrial activity was significantly lower in AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE compared to normal cells and glycogen concentration was significantly increased in the diseased cells. Furthermore, Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a regulator of mitochondrial biogenesis and function was repressed, and lower expression levels of NAD-dependent deacetylase sirtuin1 (SIRT1) were found in AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE

Crystal Structure and Substrate Specificity of D-Galactose-6-Phosphate Isomerase Complexed with Substrates

Science.gov (United States)

Lee, Jung-Kul; Pan, Cheol-Ho

2013-01-01

D-Galactose-6-phosphate isomerase from Lactobacillus rhamnosus (LacAB; EC 5.3.1.26), which is encoded by the tagatose-6-phosphate pathway gene cluster (lacABCD), catalyzes the isomerization of D-galactose-6-phosphate to D-tagatose-6-phosphate during lactose catabolism and is used to produce rare sugars as low-calorie natural sweeteners. The crystal structures of LacAB and its complex with D-tagatose-6-phosphate revealed that LacAB is a homotetramer of LacA and LacB subunits, with a structure similar to that of ribose-5-phosphate isomerase (Rpi). Structurally, LacAB belongs to the RpiB/LacAB superfamily, having a Rossmann-like αβα sandwich fold as has been identified in pentose phosphate isomerase and hexose phosphate isomerase. In contrast to other family members, the LacB subunit also has a unique α7 helix in its C-terminus. One active site is distinctly located at the interface between LacA and LacB, whereas two active sites are present in RpiB. In the structure of the product complex, the phosphate group of D-tagatose-6-phosphate is bound to three arginine residues, including Arg-39, producing a different substrate orientation than that in RpiB, where the substrate binds at Asp-43. Due to the proximity of the Arg-134 residue and backbone Cα of the α6 helix in LacA to the last Asp-172 residue of LacB with a hydrogen bond, a six-carbon sugar-phosphate can bind in the larger pocket of LacAB, compared with RpiB. His-96 in the active site is important for ring opening and substrate orientation, and Cys-65 is essential for the isomerization activity of the enzyme. Two rare sugar substrates, D-psicose and D-ribulose, show optimal binding in the LacAB-substrate complex. These findings were supported by the results of LacA activity assays. PMID:24015281

Crystal structure and substrate specificity of D-galactose-6-phosphate isomerase complexed with substrates.

Directory of Open Access Journals (Sweden)

Woo-Suk Jung

Full Text Available D-Galactose-6-phosphate isomerase from Lactobacillus rhamnosus (LacAB; EC 5.3.1.26, which is encoded by the tagatose-6-phosphate pathway gene cluster (lacABCD, catalyzes the isomerization of D-galactose-6-phosphate to D-tagatose-6-phosphate during lactose catabolism and is used to produce rare sugars as low-calorie natural sweeteners. The crystal structures of LacAB and its complex with D-tagatose-6-phosphate revealed that LacAB is a homotetramer of LacA and LacB subunits, with a structure similar to that of ribose-5-phosphate isomerase (Rpi. Structurally, LacAB belongs to the RpiB/LacAB superfamily, having a Rossmann-like αβα sandwich fold as has been identified in pentose phosphate isomerase and hexose phosphate isomerase. In contrast to other family members, the LacB subunit also has a unique α7 helix in its C-terminus. One active site is distinctly located at the interface between LacA and LacB, whereas two active sites are present in RpiB. In the structure of the product complex, the phosphate group of D-tagatose-6-phosphate is bound to three arginine residues, including Arg-39, producing a different substrate orientation than that in RpiB, where the substrate binds at Asp-43. Due to the proximity of the Arg-134 residue and backbone Cα of the α6 helix in LacA to the last Asp-172 residue of LacB with a hydrogen bond, a six-carbon sugar-phosphate can bind in the larger pocket of LacAB, compared with RpiB. His-96 in the active site is important for ring opening and substrate orientation, and Cys-65 is essential for the isomerization activity of the enzyme. Two rare sugar substrates, D-psicose and D-ribulose, show optimal binding in the LacAB-substrate complex. These findings were supported by the results of LacA activity assays.

Bovine cumulus-granulosa cells contain biologically active retinoid receptors that can respond to retinoic acid

Directory of Open Access Journals (Sweden)

Malayer Jerry

2003-11-01

Full Text Available Abstract Retinoids, a class of compounds that include retinol and its metabolite, retinoic acid, are absolutely essential for ovarian steroid production, oocyte maturation, and early embryogenesis. Previous studies have detected high concentrations of retinol in bovine large follicles. Further, administration of retinol in vivo and supplementation of retinoic acid during in vitro maturation results in enhanced embryonic development. In the present study, we hypothesized that retinoids administered either in vivo previously or in vitro can exert receptor-mediated effects in cumulus-granulosa cells. Total RNA extracted from in vitro cultured cumulus-granulosa cells was subjected to reverse transcription polymerase chain reaction (RT-PCR and mRNA expression for retinol binding protein (RBP, retinoic acid receptor alpha (RARalpha, retinoic acid receptor beta (RARbeta, retinoic acid receptor gamma (RARgamma, retinoid X receptor alpha (RXRalpha, retinoid X receptor beta (RXRbeta, retinaldehyde dehydrogenase-2 (RALDH-2, and peroxisome proliferator activated receptor gamma (PPARgamma. Transcripts were detected for RBP, RARalpha, RARgamma, RXRalpha, RXRbeta, RALDH-2, and PPARgamma. Expression of RARbeta was not detected in cumulus-granulosa cells. Using western blotting, immunoreactive RARalpha, and RXRbeta protein was also detected in bovine cumulus-granulosa cells. The biological activity of these endogenous retinoid receptors was tested using a transient reporter assay using the pAAV-MCS-betaRARE-Luc vector. Addition of 0.5 and 1 micro molar all-trans retinoic acid significantly (P trans retinol stimulated a mild increase in reporter activity, however, the increase was not statistically significant. Based on these results we conclude that cumulus cells contain endogenously active retinoid receptors and may also be competent to synthesize retinoic acid using the precursor, retinol. These results also indirectly provide evidence that retinoids

Keratoacanthoma centrifugum marginatum: unresponsive to oral retinoid and successfully treated with wide local excision.

Directory of Open Access Journals (Sweden)

Kapildev Das

2010-01-01

Full Text Available We describe a case of a 65-year-old male presenting with a large plaque with a rolled-out interrupted margin, atrophic center, and island of normal skin over the left arm. It grew peripherally with central healing, and there was a history of recurrence after inadequate excision. Investigations ruled out other clin­ical mimickers; namely, squamous cell carcinoma, lupus vulgaris, botryomycosis, and blastomycosis-like pyoderma. Histopathological sections showed irregularly shaped craters filled with keratin and epithelial pearl but no evidence of granuloma or cellular atypia. Clinico­pathological correlation proved the lesion to be keratoacanthoma centrifugum marginatum (KCM, a rare variant of keratoacanthoma, which spreads centrifugally, attains a huge size, and never involutes spontaneously. Treatment of KCM has been a problem always and, in our case, systemic retinoid (acitretin for three months proved ineffective. The patient also had a history of recurrence following surgical intervention previously, necessitating wide excision to achieve complete clearance of tumor cells. Hence, after failure of retinoid therapy, the decision of excision with a 1-centimeter margin was taken and the large defect was closed by a split thickness skin graft. The graft uptake was satisfactory, and the patient is being followed-up presently and shows no signs of recurrence after six months, highlighting wide local excision as a useful treatment option.

Retinoids induce integrin-independent lymphocyte adhesion through RAR-α nuclear receptor activity

International Nuclear Information System (INIS)

Whelan, Jarrett T.; Wang, Lei; Chen, Jianming; Metts, Meagan E.; Nasser, Taj A.; McGoldrick, Liam J.; Bridges, Lance C.

2014-01-01

Highlights: • Transcription and translation are required for retinoid-induced lymphocyte adhesion. • RAR activation is sufficient to induced lymphocyte cell adhesion. • Vitamin D derivatives inhibit RAR-prompted lymphocyte adhesion. • Adhesion occurs through a novel binding site within ADAM disintegrin domains. • RARα is a key nuclear receptor for retinoid-dependent lymphocyte cell adhesion. - Abstract: Oxidative metabolites of vitamin A, in particular all-trans-retinoic acid (atRA), have emerged as key factors in immunity by specifying the localization of immune cells to the gut. Although it is appreciated that isomers of retinoic acid activate the retinoic acid receptor (RAR) and retinoid X receptor (RXR) family of nuclear receptors to elicit cellular changes, the molecular details of retinoic acid action remain poorly defined in immune processes. Here we employ a battery of agonists and antagonists to delineate the specific nuclear receptors utilized by retinoids to evoke lymphocyte cell adhesion to ADAM (adisintegrin and metalloprotease) protein family members. We report that RAR agonism is sufficient to promote immune cell adhesion in both immortal and primary immune cells. Interestingly, adhesion occurs independent of integrin function, and mutant studies demonstrate that atRA-induced adhesion to ADAM members required a distinct binding interface(s) as compared to integrin recognition. Anti-inflammatory corticosteroids as well as 1,25-(OH) 2 D 3 , a vitamin D metabolite that prompts immune cell trafficking to the skin, potently inhibited the observed adhesion. Finally, our data establish that induced adhesion was specifically attributable to the RAR-α receptor isotype. The current study provides novel molecular resolution as to which nuclear receptors transduce retinoid exposure into immune cell adhesion

Retinoids induce integrin-independent lymphocyte adhesion through RAR-α nuclear receptor activity

Energy Technology Data Exchange (ETDEWEB)

Whelan, Jarrett T.; Wang, Lei; Chen, Jianming; Metts, Meagan E.; Nasser, Taj A.; McGoldrick, Liam J. [Department of Biochemistry and Molecular Biology, The Brody School of Medicine at East Carolina University, Greenville, NC 27834 (United States); Bridges, Lance C., E-mail: bridgesl@ecu.edu [Department of Biochemistry and Molecular Biology, The Brody School of Medicine at East Carolina University, Greenville, NC 27834 (United States); East Carolina Diabetes and Obesity Institute, The Brody School of Medicine at East Carolina University, Greenville, NC 27834 (United States)

2014-11-28

Highlights: • Transcription and translation are required for retinoid-induced lymphocyte adhesion. • RAR activation is sufficient to induced lymphocyte cell adhesion. • Vitamin D derivatives inhibit RAR-prompted lymphocyte adhesion. • Adhesion occurs through a novel binding site within ADAM disintegrin domains. • RARα is a key nuclear receptor for retinoid-dependent lymphocyte cell adhesion. - Abstract: Oxidative metabolites of vitamin A, in particular all-trans-retinoic acid (atRA), have emerged as key factors in immunity by specifying the localization of immune cells to the gut. Although it is appreciated that isomers of retinoic acid activate the retinoic acid receptor (RAR) and retinoid X receptor (RXR) family of nuclear receptors to elicit cellular changes, the molecular details of retinoic acid action remain poorly defined in immune processes. Here we employ a battery of agonists and antagonists to delineate the specific nuclear receptors utilized by retinoids to evoke lymphocyte cell adhesion to ADAM (adisintegrin and metalloprotease) protein family members. We report that RAR agonism is sufficient to promote immune cell adhesion in both immortal and primary immune cells. Interestingly, adhesion occurs independent of integrin function, and mutant studies demonstrate that atRA-induced adhesion to ADAM members required a distinct binding interface(s) as compared to integrin recognition. Anti-inflammatory corticosteroids as well as 1,25-(OH){sub 2}D{sub 3}, a vitamin D metabolite that prompts immune cell trafficking to the skin, potently inhibited the observed adhesion. Finally, our data establish that induced adhesion was specifically attributable to the RAR-α receptor isotype. The current study provides novel molecular resolution as to which nuclear receptors transduce retinoid exposure into immune cell adhesion.

Session-RPE for quantifying load of different youth taekwondo training sessions.

Science.gov (United States)

Lupo, Corrado; Capranica, Laura; Cortis, Cristina; Guidotti, Flavia; Bianco, Antonino; Tessitore, Antonio

2017-03-01

The session rating of perceived exertion (session-RPE) proved to be a valuable method to quantify the internal training load (ITL) in taekwondo. However, no study validated this method in youth taekwondo athletes performing different training sessions. Thus this study aimed at evaluating the reliability of the session-RPE to monitor the ITL of prepubescent taekwondo athletes during pre-competitive (PC) and competitive (C) training sessions. Five female (age: 12.0±0.7 y; height: 1.54±0.08 m; body mass: 48.8±7.3 kg) and four male (age: 12.0±0.8 yrs; height: 1.55±0.07 m; body mass: 47.3±5.3 kg) taekwondo athletes were monitored during 100 individual sessions (PC: N.=33; C: N.=67). The Edwards' HR method was used as reference measure of ITL; the CR-10 RPE scale was administered at 1- and 30-minutes from the end of each session. No difference for gender emerged. The ITLs of C (Edwards: 228±40 arbitrary units, AU) resulted higher than that of PC (192±26 AU; P=0.04). Although all training typologies and data collections achieved significant correlations between Edwards' and session-RPE methods, a large relationship (r =0.71, Psessions evaluated at 30 minutes of the recovery phases. Findings support coaches of prepubescent taekwondo athletes to successfully use session-RPE to monitor the ITL of different training typologies. However, PC training evaluated at 30 minutes of the recovery phase represents the best condition for a highly reliable ITL perception.

Triosephosphate isomerase is a common crystallization contaminant of soluble His-tagged proteins produced in Escherichia coli

International Nuclear Information System (INIS)

Kozlov, Guennadi; Vinaik, Roohi; Gehring, Kalle

2013-01-01

Crystals of E. coli triosephosphate isomerase were obtained as a contaminant and its structure was determined to 1.85 Å resolution. Attempts to crystallize several mammalian proteins overexpressed in Escherichia coli revealed a common contaminant, triosephosphate isomerase, a protein involved in glucose metabolism. Even with triosephosphate isomerase present in very small amounts, similarly shaped crystals appeared in the crystallization drops in a number of polyethylene glycol-containing conditions. All of the target proteins were His-tagged and their purification involved immobilized metal-affinity chromatography (IMAC), a step that was likely to lead to triosephosphate isomerase contamination. Analysis of the triosephosphate isomerase crystals led to the structure of E. coli triosephosphate isomerase at 1.85 Å resolution, which is a significant improvement over the previous structure

Immobilization of Recombinant Glucose Isomerase for Efficient Production of High Fructose Corn Syrup.

Science.gov (United States)

Jin, Li-Qun; Xu, Qi; Liu, Zhi-Qiang; Jia, Dong-Xu; Liao, Cheng-Jun; Chen, De-Shui; Zheng, Yu-Guo

2017-09-01

Glucose isomerase is the important enzyme for the production of high fructose corn syrup (HFCS). One-step production of HFCS containing more than 55% fructose (HFCS-55) is receiving much attention for its industrial applications. In this work, the Escherichia coli harboring glucose isomerase mutant TEGI-W139F/V186T was immobilized for efficient production of HFCS-55. The immobilization conditions were optimized, and the maximum enzyme activity recovery of 92% was obtained. The immobilized glucose isomerase showed higher pH, temperature, and operational stabilities with a K m value of 272 mM and maximum reaction rate of 23.8 mM min -1 . The fructose concentration still retained above 55% after the immobilized glucose isomerase was reused for 10 cycles, and more than 85% of its initial activity was reserved even after 15 recycles of usage at temperature of 90 °C. The results highlighted the immobilized glucose isomerase as a potential biocatalyst for HFCS-55 production.

Session-RPE for quantifying the load of different youth basketball training sessions.

Science.gov (United States)

Lupo, C; Tessitore, A; Gasperi, L; Gomez, Mar

2017-03-01

The aim of the study was to evaluate youth basketball training, verifying the reliability of the session-RPE method in relation to session duration (session) categories. Six male youth basketball players (age, 16.5±0.5 years; height, 195.5±6.75 cm; body mass, 93.9±10.9 kg; and body mass index, 23.6±2.8 kg.m -2 ) were monitored (HR, type and duration of workouts) during 15 (66 individual) training sessions (80±26 minutes). Edwards' HR method was used as a reference measure of internal training load (ITL); the CR-10 RPE scale was administered 30 minutes after the end of each session. The results obtained showed that all comparisons between different session durations and workout portions revealed effects in term of Edwards' ITLs except for warm-up portions. Moderate to strong relationships between Edwards' and session- RPE methods emerged for all sessions (r = .85, P sessions (r range = .79 - .95, P session durations (session-RPE to monitor the ITL, regardless of session durations and workout portions.

Exploring (novel) gene expression during retinoid-induced maturation and cell death of acute promyelocytic leukemia.

Science.gov (United States)

Benoit, G R; Tong, J H; Balajthy, Z; Lanotte, M

2001-01-01

During recent years, reports have shown that biological responses of acute promyelocytic leukemia (APL) cells to retinoids are more complex than initially envisioned. PML-RARalpha chimeric protein disturbs various biological processes such as cell proliferation, differentiation, and apoptosis. The distinct biological programs that regulate these processes stem from specific transcriptional activation of distinct (but overlapping) sets of genes. These programs are sometimes mutually exclusive and depend on whether the signals are delivered by RAR or RXR agonists. Furthermore, evidence that retinoid nuclear signaling by retinoid, on its own, is not enough to trigger these cellular responses is rapidly accumulating. Indeed, work with NB4 cells show that the fate of APL cells treated by retinoid depends on complex signaling cross-talk. Elucidation of the sequence of events and cascades of transcriptional regulation necessary for APL cell maturation will be an additional tool with which to further improve therapy by retinoids. In this task, the classical techniques used to analyze gene expression have proved time consuming, and their yield has been limited. Global analyses of the APL cell transcriptome are needed. We review the technical approaches currently available (differential display, complementary DNA microarrays), to identify novel genes involved in the determination of cell fate.

Performance measurement of a new concept reciprocating piston expander (RPE using a newly developed small-scale dynamometer unit

Directory of Open Access Journals (Sweden)

Mohamad MNA

2017-01-01

Full Text Available This paper presents the progress of a small-scale dynamometer prototype development for performance measurement of a reciprocating piston expander (RPE. Since the available dynamometer systems in the market are limited to specific applications that require for the customization, their price normally very expensive. Since the current study on the RPE required a dynamometer unit, therefore, a new and cheaper dynamometer prototype that was suitable for RPE application has been developed. Using air as RPE working fluid, a case study has been carried out to measure its performance at different inlet fluid conditions, i.e., within 20°C–140°C and 3–5 bars. The results observed that the performance of RPE was proportionally increased to the increased of inlet fluid pressure and temperature. The maximum brake power produced was 27 Watt when the RPE operated at 140°C, 5 bars and the speed of 820 rpm. It also revealed that the changes in the pressure of inlet fluid can give significant change on the performance of the RPE due to its direct relation to the RPE actual rotating force. Although the RPE and dynamometer seem good being adapted to each other, both of them require some improvements to ensure both systems well operated and reliable.

Neurological findings in triosephosphate isomerase deficiency

NARCIS (Netherlands)

Poll-The, B. T.; Aicardi, J.; Girot, R.; Rosa, R.

1985-01-01

Two siblings with hemolytic anemia caused by triosephosphate isomerase deficiency developed a progressive neurological syndrome featuring dystonic movements, tremor, pyramidal tract signs, and evidence of spinal motor neuron involvement. Intelligence was unaffected. The findings in these patients

Action spectrum for photochemical retinal pigment epithelium (RPE) disruption in an in vivo monkey model

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Zhang, Jie; Sabarinathan, Ranjani; Bubel, Tracy; Williams, David R.; Hunter, Jennifer J.

2016-03-01

Observations of RPE disruption and autofluorescence (AF) photobleaching at light levels below the ANSI photochemical maximum permissible exposure (MPE) (Morgan et al., 2008) indicates a demand to modify future light safety standards to protect the retina from harm. To establish safe light exposures, we measured the visible light action spectrum for RPE disruption in an in vivo monkey model with fluorescence adaptive optics retinal imaging. Using this high resolution imaging modality can provide insight into the consequences of light on a cellular level and allow for longitudinal monitoring of retinal changes. The threshold retinal radiant exposures (RRE) for RPE disruption were determined for 4 wavelengths (460, 488, 544, and 594 nm). The anaesthetized macaque retina was exposed to a uniform 0.5° × 0.5° field of view (FOV). Imaging within a 2° × 2° FOV was performed before, immediately after and at 2 week intervals for 10 weeks. At each wavelength, multiple RREs were tested with 4 repetitions each to determine the threshold for RPE disruption. For qualitative analysis, RPE disruption is defined as any detectable change from the pre exposure condition in the cell mosaic in the exposed region relative to the corresponding mosaic in the immediately surrounding area. We have tested several metrics to evaluate the RPE images obtained before and after exposure. The measured action spectrum for photochemical RPE disruption has a shallower slope than the current ANSI photochemical MPE for the same conditions and suggests that longer wavelength light is more hazardous than other measurements would suggest.

Increase in D-tagatose production rate by site-directed mutagenesis of L-arabinose isomerase from Geobacillus thermodenitrificans.

Science.gov (United States)

Oh, Hyo-Jung; Kim, Hye-Jung; Oh, Deok-Kun

2006-02-01

Among single-site mutations of L-arabinose isomerase derived from Geobacillus thermodenitrificans, two mutants were produced having the lowest and highest activities of D-tagatose production. Site-directed mutagenesis at these sites showed that the aromatic ring at amino acid 164 and the size of amino acid 475 were important for D-tagatose production. Among double-site mutations, one mutant converted D-galactose into D-tagatose with a yield of 58% whereas the wild type gave 46% D-tagatose conversion after 300 min at 65 degrees C.

Overexpression, purification, crystallization and preliminary X-ray crystal analysis of Bacillus pallidusd-arabinose isomerase

International Nuclear Information System (INIS)

Takeda, Kosei; Yoshida, Hiromi; Takada, Goro; Izumori, Ken; Kamitori, Shigehiro

2008-01-01

Recombinant B. pallidusd-arabinose isomerase was crystallized and diffraction data were collected to 2.3 Å resolution. d-Arabinose isomerase catalyzes the isomerization of d-arabinose to d-ribulose. Bacillus pallidusd-arabinose isomerase has broad substrate specificity and can catalyze the isomerization of d-arabinose, l-fucose, l-xylose, l-galactose and d-altrose. Recombinant B. pallidusd-arabinose isomerase was overexpressed, purified and crystallized. A crystal of the enzyme was obtained by the sitting-drop method at room temperature and belonged to the orthorhombic space group P2 1 2 1 2, with unit-cell parameters a = 144.9, b = 127.9, c = 109.5 Å. Diffraction data were collected to 2.3 Å resolution

Odorant Receptor 51E2 Agonist β-ionone Regulates RPE Cell Migration and Proliferation

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Nikolina Jovancevic

2017-11-01

Full Text Available The odorant receptor 51E2 (OR51E2, which is well-characterized in prostate cancer cells and epidermal pigment cells, was identified for the first time as the most highly expressed OR in human fetal and adult retinal pigment epithelial (RPE cells. Immunofluorescence staining and Western blot analysis revealed OR51E2 localization throughout the cytosol and in the plasma membrane. Additionally, immunohistochemical staining of diverse layers of the eye showed that the expression of OR51E2 is restricted to the pigment cells of the RPE and choroid. The results of Ca2+-imaging experiments demonstrate that activation of OR51E2 triggers a Ca2+ dependent signal pathway in RPE cells. Downstream signaling of OR51E2 involves the activation of adenylyl cyclase, ERK1/2 and AKT. The activity of these protein kinases likely accounts for the demonstrated increase in the migration and proliferation of RPE cells upon stimulation with the OR51E2 ligand β-ionone. These findings suggest that OR51E2 is involved in the regulation of RPE cell growth. Thus, OR51E2 represents a potential target for the treatment of proliferative disorders.

Screening and selection of wild strains for L-arabinose isomerase production

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R. M. Manzo

2013-12-01

Full Text Available The majority of L-arabinose isomerases have been isolated by recombinant techniques, but this methodology implies a reduced technological application. For this reason, 29 bacterial strains, some of them previously characterized as L-arabinose isomerase producers, were assayed as L-arabinose fermenting strains by employing conveniently designed culture media with 0.5% (w/v L-arabinose as main carbon source. From all evaluated bacterial strains, Enterococcus faecium DBFIQ ID: E36, Enterococcus faecium DBFIQ ID: ETW4 and Pediococcus acidilactici ATCC ID: 8042 were, in this order, the best L-arabinose fermenting strains. Afterwards, to assay L-arabinose metabolization and L-arabinose isomerase activity, cell-free extract and saline precipitated cell-free extract of the three bacterial cultures were obtained and the production of ketoses was determined by the cysteine carbazole sulfuric acid method. Results showed that the greater the L-arabinose metabolization ability, the higher the enzymatic activity achieved, so Enterococcus faecium DBFIQ ID: E36 was selected to continue with production, purification and characterization studies. This work thus describes a simple microbiological method for the selection of L-arabinose fermenting bacteria for the potential production of the enzyme L-arabinose isomerase.

Retinoids: literature review and suggested algorithm for use prior to facial resurfacing procedures

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Patrick J Buchanan

2016-01-01

Full Text Available Vitamin A-containing products have been used topically since the early 1940s to treat various skin conditions. To date, there are four generations of retinoids, a family of Vitamin A-containing compounds. Tretinoin, all-trans-retinoic acid, is a first-generation, naturally occurring, retinoid. It is available, commercially, as a gel or cream. The authors conducted a complete review of all studies, clinical- and basic science-based studies, within the literature involving tretinoin treatment recommendations for impending facial procedures. The literature currently lacks definitive recommendations for the use of tretinoin-containing products prior to undergoing facial procedures. Tretinoin pretreatment regimens vary greatly in terms of the strength of retinoid used, the length of the pre-procedure treatment, and the ideal time to stop treatment before the procedure. Based on the current literature and personal experience, the authors set forth a set of guidelines for the use of tretinoin prior to various facial procedures.

Coexpression of β-D-galactosidase and L-arabinose isomerase in the production of D-tagatose: a functional sweetener.

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Zhan, Yijing; Xu, Zheng; Li, Sha; Liu, Xiaoliu; Xu, Lu; Feng, Xiaohai; Xu, Hong

2014-03-19

The functional sweetener, d-tagatose, is commonly transformed from galactose by l-arabinose isomerase. To make use of a much cheaper starting material, lactose, hydrolization, and isomerization are required to take place collaboratively. Therefore, a single-step method involving β-d-galactosidase was explored for d-tagatose production. The two vital genes, β-d-galactosidase gene (lacZ) and l-arabinose isomerase mutant gene (araA') were extracted separately from Escherichia coli strains and incorporated into E. coli simultaneously. This gave us E. coli-ZY, a recombinant producing strain capable of coexpressing the two key enzymes. The resulted cells exhibited maximum d-tagatose producing activity at 34 °C and pH 6.5 and in the presence of borate, 10 mM Fe(2+), and 1 mM Mn(2+). Further monitoring showed that the recombinant cells could hydrolyze more than 95% lactose and convert 43% d-galactose into d-tagatose. This research has verified the feasibility of single-step d-tagatose fermentation, thereby laying down the foundation for industrial usage of lactose.

Effect of SOCS1 overexpression on RPE cell activation by proinflammatory cytokines.

Science.gov (United States)

Bazewicz, Magdalena; Draganova, Dafina; Makhoul, Maya; Chtarto, Abdel; Elmaleh, Valerie; Tenenbaum, Liliane; Caspers, Laure; Bruyns, Catherine; Willermain, François

2016-09-06

The purpose of this study was to investigate the in vitro effect of Suppressor Of Cytokine Signaling 1 (SOCS1) overexpression in retinal pigment epithelium (RPE) cells on their activation by pro-inflammatory cytokines IFNγ, TNFα and IL-17. Retinal pigment epithelium cells (ARPE-19) were stably transfected with the control plasmid pIRES2-AcGFP1 or the plasmid pSOCS1-IRES2-AcGFP1. They were stimulated by IFNγ (150ng/ml), TNFα (30ng/ml) or IL-17 (100ng/ml). The levels of SOCS1 mRNA were measured by real-time PCR. Signal Transducer and Activator of Transcription 1 (STAT1) phosphorylation and IκBα expression were analysed by western Blot (WB). IL-8 secretion was analysed by ELISA and expression of MHCII molecules and ICAM-1/CD54 by flow cytometry. Our data show that SOCS1 mRNA overexpression in RPE cells prevents IFNγ-induced SOCS1 mRNA increase and IFNγ-mediated STAT1 phosphorylation. Moreover, SOCS1 overexpression in RPE cells inhibits IFNγ-induced decrease of IL-8 secretion and prevents IFNγ-induced MHC II and ICAM1/CD54 upregulation. However, SOCS1 overexpression does not affect TNFα-induced IκBα degradation nor block TNFα-induced or IL-17-induced IL-8 secretion. On the contrary, IL-17-induced secretion is increased by SOCS1 overexpression. In conclusion, SOCS1 overexpression in RPE cells inhibits some IFNγ-mediated responses that lead to uveitis development. This notion raises the possibility that SOCS1 overexpression could be a novel target for treating non-infectious uveitis. However, some proinflammatory effects of TNFα and IL-17 stimulation on RPE are not blocked by SOCS1 overexpression. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Shape Analysis of the Peripapillary RPE Layer in Papilledema and Ischemic Optic Neuropathy

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Kupersmith, Mark J.; Rohlf, F. James

2011-01-01

Purpose. Geometric morphometrics (GM) was used to analyze the shape of the peripapillary retinal pigment epithelium–Bruch's membrane (RPE/BM) layer imaged on the SD-OCT 5-line raster in normal subjects and in patients with papilledema and ischemic optic neuropathy. Methods. Three groups of subjects were compared: 30 normals, 20 with anterior ischemic optic neuropathy (AION), and 25 with papilledema and intracranial hypertension. Twenty equidistant semilandmarks were digitized on OCT images of the RPE/BM layer spanning 2500 μm on each side of the neural canal opening (NCO). The data were analyzed using standard GM techniques, including a generalized least-squares Procrustes superimposition, principal component analysis, thin-plate spline (to visualize deformations), and permutation statistical analysis to evaluate differences in shape variables. Results. The RPE/BM layer in normals and AION have a characteristic V shape pointing away from the vitreous; the RPE/BM layer in papilledema has an inverted U shape, skewed nasally inward toward the vitreous. The differences were statistically significant. There was no significant difference in shapes between normals and AION. Pre- and posttreatment OCTs, in select cases of papilledema, showed that the inverted U-shaped RPE/BM moved posteriorly into a normal V shape as the papilledema resolved with weight loss or shunting. Conclusions. The shape difference in papilledema, absent in AION, cannot be explained by disc edema alone. The difference is a consequence of both the translaminar pressure gradient and the material properties of the peripapillary sclera. GM offers a novel way of statistically assessing shape differences of the peripapillary optic nerve head. PMID:21896851

Xylose isomerase improves growth and ethanol production rates from biomass sugars for both Saccharomyces pastorianus and Saccharomyces cerevisiae.

Science.gov (United States)

Miller, Kristen P; Gowtham, Yogender Kumar; Henson, J Michael; Harcum, Sarah W

2012-01-01

The demand for biofuel ethanol made from clean, renewable nonfood sources is growing. Cellulosic biomass, such as switch grass (Panicum virgatum L.), is an alternative feedstock for ethanol production; however, cellulosic feedstock hydrolysates contain high levels of xylose, which needs to be converted to ethanol to meet economic feasibility. In this study, the effects of xylose isomerase on cell growth and ethanol production from biomass sugars representative of switch grass were investigated using low cell density cultures. The lager yeast species Saccharomyces pastorianus was grown with immobilized xylose isomerase in the fermentation step to determine the impact of the glucose and xylose concentrations on the ethanol production rates. Ethanol production rates were improved due to xylose isomerase; however, the positive effect was not due solely to the conversion of xylose to xylulose. Xylose isomerase also has glucose isomerase activity, so to better understand the impact of the xylose isomerase on S. pastorianus, growth and ethanol production were examined in cultures provided fructose as the sole carbon. It was observed that growth and ethanol production rates were higher for the fructose cultures with xylose isomerase even in the absence of xylose. To determine whether the positive effects of xylose isomerase extended to other yeast species, a side-by-side comparison of S. pastorianus and Saccharomyces cerevisiae was conducted. These comparisons demonstrated that the xylose isomerase increased ethanol productivity for both the yeast species by increasing the glucose consumption rate. These results suggest that xylose isomerase can contribute to improved ethanol productivity, even without significant xylose conversion. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

DJ-1-dependent regulation of oxidative stress in the retinal pigment epithelium (RPE.

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Karen G Shadrach

Full Text Available DJ-1 is found in many tissues, including the brain, where it has been extensively studied due to its association with Parkinson's disease. DJ-1 functions as a redox-sensitive molecular chaperone and transcription regulator that robustly protects cells from oxidative stress.Retinal pigment epithelial (RPE cultures were treated with H2O2 for various times followed by biochemical and immunohistological analysis. Cells were transfected with adenoviruses carrying the full-length human DJ-1 cDNA and a mutant construct, which has the cysteine residues at amino acid 46, 53 and 106 mutated to serine (C to S prior to stress experiments. DJ-1 localization, levels of expression and reactive oxygen species (ROS generation were also analyzed in cells expressing exogenous DJ-1 under baseline and oxidative stress conditions. The presence of DJ-1 and oxidized DJ-1 was evaluated in human RPE total lysates. The distribution of DJ-1 was assessed in AMD and non-AMD cryosectionss and in isolated human Bruch's membrane (BM/choroid from AMD eyes.DJ-1 in RPE cells under baseline conditions, displays a diffuse cytoplasmic and nuclear staining. After oxidative challenge, more DJ-1 was associated with mitochondria. Increasing concentrations of H2O2 resulted in a dose-dependent increase in DJ-1. Overexpression of DJ-1 but not the C to S mutant prior to exposure to oxidative stress led to significant decrease in the generation of ROS. DJ-1 and oxDJ-1 intensity of immunoreactivity was significantly higher in the RPE lysates from AMD eyes. More DJ-1 was localized to RPE cells from AMD donors with geographic atrophy and DJ-1 was also present in isolated human BM/choroid from AMD eyes.DJ-1 regulates RPE responses to oxidative stress. Most importantly, increased DJ-1 expression prior to oxidative stress leads to decreased generation of ROS, which will be relevant for future studies of AMD since oxidative stress is a known factor affecting this disease.

21 CFR 862.1570 - Phosphohexose isomerase test system.

Science.gov (United States)

2010-04-01

.... Measurements of phosphohexose isomerase are used in the diagnosis and treatment of muscle diseases such as muscular dystrophy, liver diseases such as hepatitis or cirrhosis, and metastatic carcinoma. (b...

A new class of synthetic retinoid antibiotics effective against bacterial persisters.

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Kim, Wooseong; Zhu, Wenpeng; Hendricks, Gabriel Lambert; Van Tyne, Daria; Steele, Andrew D; Keohane, Colleen E; Fricke, Nico; Conery, Annie L; Shen, Steven; Pan, Wen; Lee, Kiho; Rajamuthiah, Rajmohan; Fuchs, Beth Burgwyn; Vlahovska, Petia M; Wuest, William M; Gilmore, Michael S; Gao, Huajian; Ausubel, Frederick M; Mylonakis, Eleftherios

2018-04-05

A challenge in the treatment of Staphylococcus aureus infections is the high prevalence of methicillin-resistant S. aureus (MRSA) strains and the formation of non-growing, dormant 'persister' subpopulations that exhibit high levels of tolerance to antibiotics and have a role in chronic or recurrent infections. As conventional antibiotics are not effective in the treatment of infections caused by such bacteria, novel antibacterial therapeutics are urgently required. Here we used a Caenorhabditis elegans-MRSA infection screen to identify two synthetic retinoids, CD437 and CD1530, which kill both growing and persister MRSA cells by disrupting lipid bilayers. CD437 and CD1530 exhibit high killing rates, synergism with gentamicin, and a low probability of resistance selection. All-atom molecular dynamics simulations demonstrated that the ability of retinoids to penetrate and embed in lipid bilayers correlates with their bactericidal ability. An analogue of CD437 was found to retain anti-persister activity and show an improved cytotoxicity profile. Both CD437 and this analogue, alone or in combination with gentamicin, exhibit considerable efficacy in a mouse model of chronic MRSA infection. With further development and optimization, synthetic retinoids have the potential to become a new class of antimicrobials for the treatment of Gram-positive bacterial infections that are currently difficult to cure.

A new class of synthetic retinoid antibiotics effective against bacterial persisters

Science.gov (United States)

Kim, Wooseong; Zhu, Wenpeng; Hendricks, Gabriel Lambert; van Tyne, Daria; Steele, Andrew D.; Keohane, Colleen E.; Fricke, Nico; Conery, Annie L.; Shen, Steven; Pan, Wen; Lee, Kiho; Rajamuthiah, Rajmohan; Fuchs, Beth Burgwyn; Vlahovska, Petia M.; Wuest, William M.; Gilmore, Michael S.; Gao, Huajian; Ausubel, Frederick M.; Mylonakis, Eleftherios

2018-04-01

A challenge in the treatment of Staphylococcus aureus infections is the high prevalence of methicillin-resistant S. aureus (MRSA) strains and the formation of non-growing, dormant ‘persister’ subpopulations that exhibit high levels of tolerance to antibiotics and have a role in chronic or recurrent infections. As conventional antibiotics are not effective in the treatment of infections caused by such bacteria, novel antibacterial therapeutics are urgently required. Here we used a Caenorhabditis elegans–MRSA infection screen to identify two synthetic retinoids, CD437 and CD1530, which kill both growing and persister MRSA cells by disrupting lipid bilayers. CD437 and CD1530 exhibit high killing rates, synergism with gentamicin, and a low probability of resistance selection. All-atom molecular dynamics simulations demonstrated that the ability of retinoids to penetrate and embed in lipid bilayers correlates with their bactericidal ability. An analogue of CD437 was found to retain anti-persister activity and show an improved cytotoxicity profile. Both CD437 and this analogue, alone or in combination with gentamicin, exhibit considerable efficacy in a mouse model of chronic MRSA infection. With further development and optimization, synthetic retinoids have the potential to become a new class of antimicrobials for the treatment of Gram-positive bacterial infections that are currently difficult to cure.

Continuous D-tagatose production by immobilized thermostable L-arabinose isomerase in a packed-bed bioreactor.

Science.gov (United States)

Ryu, Se-Ah; Kim, Chang Sup; Kim, Hye-Jung; Baek, Dae Heoun; Oh, Deok-Kun

2003-01-01

D-Tagatose was continuously produced using thermostable L-arabinose isomerase immobilized in alginate with D-galactose solution in a packed-bed bioreactor. Bead size, L/D (length/diameter) of reactor, dilution rate, total loaded enzyme amount, and substrate concentration were found to be optimal at 0.8 mm, 520/7 mm, 0.375 h(-1), 5.65 units, and 300 g/L, respectively. Under these conditions, the bioreactor produced about 145 g/L tagatose with an average productivity of 54 g tagatose/L x h and an average conversion yield of 48% (w/w). Operational stability of the immobilized enzyme was demonstrated, with a tagatose production half-life of 24 days.

The secreted l-arabinose isomerase displays anti-hyperglycemic effects in mice

OpenAIRE

Rhimi, Moez; Bermudez-Humaran, Luis G.; Huang, Yuan; Boudebbouze, Samira; Gaci, Nadia; Garnier, Alexandrine; Gratadoux, Jean-Jacques; Mkaouar, H?la; Langella, Philippe; Maguin, Emmanuelle

2015-01-01

Background The l-arabinose isomerase is an intracellular enzyme which converts l-arabinose into l-ribulose in living systems and d-galactose into d-tagatose in industrial processes and at industrial scales. d-tagatose is a natural ketohexose with potential uses in pharmaceutical and food industries. The d-galactose isomerization reaction is thermodynamically equilibrated, and leads to secondary subproducts at high pH. Therefore, an attractive l-arabinose isomerase should be thermoactive and a...

Memory, reconsolidation and extinction in Lymnaea require the soma of RPeD1.

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Sangha, Susan; Varshney, Nishi; Fras, Mary; Smyth, Kim; Rosenegger, David; Parvez, Kashif; Sadamoto, Hisayo; Lukowiak, Ken

2004-01-01

The central pattern generator (CPG) that drives aerial respiratory behaviour in Lymnaea consists of 3 neurons. One of these, RPeD1--the cell that initiates activity in the circuit, plays an absolutely necessary role as a site for memory formation, memory reconsolidation, and extinction. Using an operant conditioning training procedure that results in a long-term non-declarative memory (LTM), we decrease the occurrence of aerial respiratory behaviour. Since snails can still breathe cutaneously learning this procedure is not harmful. Concomitant with behavioural memory are changes in the spiking activity of RPeD1. Going beyond neural correlates of memory we directly show that RPeD1 is a necessary site for LTM formation. Expanding on this finding we show that this neuron is also a necessary site for memory reconsolidation and 'Pavlovian' extinction. As far as we can determine, this is the first time a single neuron has been shown to be a necessary site for these different aspects memory. RPeD1 is thus a key neuron mediating different hierarchical aspects of memory. We are now in a position to determine the necessary neuronal, molecular and proteomic events in this neuron that are causal to memory formation, reconsolidation and extinction.

Subretinal posterior pole injury induces selective proliferation of RPE cells in the periphery in in vivo studies in pigs

DEFF Research Database (Denmark)

Kiilgaard, Jens Folke; Prause, Jan U; Prause, Michala

2007-01-01

PURPOSE: To study topographical differences in porcine retinal pigment epithelial (RPE) cell proliferation (1) in vivo, after experimental central surgical subretinal injury, and (2) in vitro. METHODS: Domestic pigs underwent either experimental RPE debridement (n = 5), subretinal amniotic membrane...... cells. This observation was true of both types of experimental surgery performed. In vitro, RPE isolates from the pre-equatorial region consistently yielded higher cell densities than did RPE cell isolates from more central parts of the epithelium. This was apparent through the three first passages...

The influence of rAAV2-mediated SOX2 delivery into neonatal and adult human RPE cells; a comparative study.

Science.gov (United States)

Ezati, Razie; Etemadzadeh, Azadeh; Soheili, Zahra-Soheila; Samiei, Shahram; Ranaei Pirmardan, Ehsan; Davari, Malihe; Najafabadi, Hoda Shams

2018-02-01

Cell replacement is a promising therapy for degenerative diseases like age-related macular degeneration (AMD). Since the human retina lacks regeneration capacity, much attention has been directed toward persuading for cells that can differentiate into retinal neurons. In this report, we have investigated reprogramming of the human RPE cells and concerned the effect of donor age on the cellular fate as a critical determinant in reprogramming competence. We evaluated the effect of SOX2 over-expression in human neonatal and adult RPE cells in cultures. The coding region of human SOX2 gene was cloned into adeno-associated virus (AAV2) and primary culture of human neonatal/adult RPE cells were infected by recombinant virus. De-differentiation of RPE to neural/retinal progenitor cells was investigated by quantitative real-time PCR and ICC for neural/retinal progenitor cells' markers. Gene expression analysis showed 80-fold and 12-fold over-expression for SOX2 gene in infected neonatal and adult hRPE cells, respectively. The fold of increase for Nestin in neonatal and adult hRPE cells was 3.8-fold and 2.5-fold, respectively. PAX6 expression was increased threefold and 2.5-fold in neonatal/adult treated cultures. Howbeit, we could not detect rhodopsin, and CHX10 expression in neonatal hRPE cultures and expression of rhodopsin in adult hRPE cells. Results showed SOX2 induced human neonatal/adult RPE cells to de-differentiate toward retinal progenitor cells. However, the increased number of PAX6, CHX10, Thy1, and rhodopsin positive cells in adult hRPE treated cultures clearly indicated the considerable generation of neuro-retinal terminally differentiated cells. © 2017 Wiley Periodicals, Inc.

AAV delivery of GRP78/BiP promotes adaptation of human RPE cell to ER stress.

Science.gov (United States)

Ghaderi, Shima; Ahmadian, Shahin; Soheili, Zahra-Soheila; Ahmadieh, Hamid; Samiei, Shahram; Kheitan, Samira; Pirmardan, Ehsan R

2018-02-01

Adeno associated virus (AAV)-mediated gene delivery of GRP78 (78 kDa glucose-regulated protein) attenuates the condition of endoplasmic reticulum (ER) stress and prevents apoptotic loss of photoreceptors in Retinitis pigmentosa (RP) rats. In the current study we overexpressed Grp78 with the help of AAV-2 in primary human retinal pigmented epithelium (hRPE) cell cultures and examined its effect on cell response to ER stress. The purpose of this work was studying potential stimulating effect of GRP78 on adaptation/pro-survival of hRPE cells under ER stress, as an in vitro model for RPE degeneration. To investigate the effect of Grp78 overexpression on unfolded protein response (UPR) markers under ER stress, hRPE primary cultures were transduced by recombinant virus rAAV/Grp78, and treated with ER stressor drug, tunicamycin. Expression changes of four UPR markers including GRP78, PERK, ATF6α, and GADD153/CHOP, were assessed by real-time PCR and western blotting. We found that GRP78 has a great contribution in modulation of UPR markers to favor adaptive response in ER-stressed hRPE cells. In fact, GRP78 overexpression affected adaptation and apoptotic phases of early UPR, through enhancement of two master regulators/ER stress sensors (PERK and ATF6α) and down-regulation of a key pro-apoptotic cascade activator (GADD153/CHOP). Together these findings demonstrate the promoting effect of GRP78 on adaptation/pro-survival of hRPE cells under ER stress. This protein with anti-apoptotic actions in the early UPR and important role in cell fate regulation, can be recruited as a useful candidate for future investigations of RPE degenerative diseases. © 2017 Wiley Periodicals, Inc.

Retinoid-induced expression and activity of an immediate early tumor suppressor gene in vascular smooth muscle cells.

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Jeffrey W Streb

2011-04-01

Full Text Available Retinoids are used clinically to treat a number of hyper-proliferative disorders and have been shown in experimental animals to attenuate vascular occlusive diseases, presumably through nuclear receptors bound to retinoic acid response elements (RARE located in target genes. Here, we show that natural or synthetic retinoids rapidly induce mRNA and protein expression of a specific isoform of A-Kinase Anchoring Protein 12 (AKAP12β in cultured smooth muscle cells (SMC as well as the intact vessel wall. Expression kinetics and actinomycin D studies indicate Akap12β is a retinoid-induced, immediate-early gene. Akap12β promoter analyses reveal a conserved RARE mildly induced with atRA in a region that exhibits hyper-acetylation. Immunofluorescence microscopy and protein kinase A (PKA regulatory subunit overlay assays in SMC suggest a physical association between AKAP12β and PKA following retinoid treatment. Consistent with its designation as a tumor suppressor, inducible expression of AKAP12β attenuates SMC growth in vitro. Further, immunohistochemistry studies establish marked decreases in AKAP12 expression in experimentally-injured vessels of mice as well as atheromatous lesions in humans. Collectively, these results demonstrate a novel role for retinoids in the induction of an AKAP tumor suppressor that blocks vascular SMC growth thus providing new molecular insight into how retiniods may exert their anti-proliferative effects in the injured vessel wall.

Lutein Inhibits the Migration of Retinal Pigment Epithelial Cells via Cytosolic and Mitochondrial Akt Pathways (Lutein Inhibits RPE Cells Migration

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Ching-Chieh Su

2014-08-01

Full Text Available During the course of proliferative vitreoretinopathy (PVR, the retinal pigment epithelium (RPE cells will de-differentiate, proliferate, and migrate onto the surfaces of the sensory retina. Several studies have shown that platelet-derived growth factor (PDGF can induce migration of RPE cells via an Akt-related pathway. In this study, the effect of lutein on PDGF-BB-induced RPE cells migration was examined using transwell migration assays and Western blot analyses. We found that both phosphorylation of Akt and mitochondrial translocation of Akt in RPE cells induced by PDGF-BB stimulation were suppressed by lutein. Furthermore, the increased migration observed in RPE cells with overexpressed mitochondrial Akt could also be suppressed by lutein. Our results demonstrate that lutein can inhibit PDGF-BB induced RPE cells migration through the inhibition of both cytoplasmic and mitochondrial Akt activation.

Retinoid inhibition of in vitro invasion of human amnion basement membrane by human tumor cells

International Nuclear Information System (INIS)

Fazely, F.

1988-01-01

The effects measured were the inhibition of tumor cell migration through the basement membrane (BM) and tumor cell degradative enzyme activity on 3 H-proline labeled collagenous and non collagenous components of the BM. The human lung carcinoma A549 or the human Ewing's sarcoma TC-106 cell lines treated with retinoids for two days were incubated on the BM in the absence of retinoids. A dose-dependent inhibition of cell invasion was produced by retinoids. Among the retinoids tested the most powerful was retinol acetate which inhibited invasion by 50% of A549 cells at a concentration of 0.09 μg/ml, and TC-106 cells at 0.08 μg/ml. Retinol acetate inhibited A549 and TC-106 cell growth by approximately 50% at levels almost 100-fold higher than those needed for antiinvasive activity. Retinol acetate was about 20 times more potent than retinoic acid and 30 times more than retinol palmitate. Furthermore, A549 cells treated with retinol acetate, under conditions whereby an anti-invasive state was induced,showed an increase in the number of cellular retinoic acid binding proteins (CRABP), a decrease in the activity of type IV collagenase and ectosialyltransferase, and no change in the activity of transglutaminase

Session-RPE for quantifying the load of different youth basketball training sessions

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C Lupo

2016-12-01

Full Text Available The aim of the study was to evaluate youth basketball training, verifying the reliability of the session-RPE method in relation to session duration (< and ≥ 80 minutes and workout typology (reduced and high warm-up, conditioning, technical, tactical, game portions within a single session categories. Six male youth basketball players (age, 16.5±0.5 years; height, 195.5±6.75 cm; body mass, 93.9±10.9 kg; and body mass index, 23.6±2.8 kg.m-2 were monitored (HR, type and duration of workouts during 15 (66 individual training sessions (80±26 minutes. Edwards’ HR method was used as a reference measure of internal training load (ITL; the CR-10 RPE scale was administered 30 minutes after the end of each session. The results obtained showed that all comparisons between different session durations and workout portions revealed effects in term of Edwards’ ITLs except for warm-up portions. Moderate to strong relationships between Edwards’ and session- RPE methods emerged for all sessions (r = .85, P < .001, player’s sessions (r range = .79 - .95, P < .001, session durations (< 80 minutes: r = .67, P < .001; ≥ 80 minutes: r = .75, P < .001, and workout portions (r range = .78 - .89, P range = .002 - < .001. The findings indicated that coaches of youth basketball players can successfully use session-RPE to monitor the ITL, regardless of session durations and workout portions.

Purification, crystallization and preliminary crytallographic analysis of phosphoglucose isomerase from the hyperthermophilic archaeon Pyrococcus furiosus

NARCIS (Netherlands)

Akerboom, A.P.; Turnbull, A.P.; Hargreaves, D.; Fischer, M.; Geus, de D.; Sedelnikova, S.E.; Berrisford, J.M.; Baker, P.J.; Verhees, C.H.; Oost, van der J.; Rice, D.W.

2003-01-01

The glycolytic enzyme phosphoglucose isomerase catalyses the reversible isomerization of glucose 6-phosphate to fructose 6-phosphate. The phosphoglucose isomerase from the hyperthermophilic archaeon Pyrococcus furiosus, which shows no sequence similarity to any known bacterial or eukaryotic

Structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC118

International Nuclear Information System (INIS)

Lobley, Carina M. C.; Aller, Pierre; Douangamath, Alice; Reddivari, Yamini; Bumann, Mario; Bird, Louise E.; Nettleship, Joanne E.; Brandao-Neto, Jose; Owens, Raymond J.; O’Toole, Paul W.; Walsh, Martin A.

2012-01-01

The crystal structure of ribose 5-phosphate isomerase has been determined to 1.72 Å resolution and is presented with a brief comparison to other known ribose 5-phosphate isomerase A structures. The structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC188 has been determined at 1.72 Å resolution. The structure was solved by molecular replacement, which identified the functional homodimer in the asymmetric unit. Despite only showing 57% sequence identity to its closest homologue, the structure adopted the typical α and β d-ribose 5-phosphate isomerase fold. Comparison to other related structures revealed high homology in the active site, allowing a model of the substrate-bound protein to be proposed. The determination of the structure was expedited by the use of in situ crystallization-plate screening on beamline I04-1 at Diamond Light Source to identify well diffracting protein crystals prior to routine cryocrystallography

Acute and session RPE responses during resistance training: Bouts ...

African Journals Online (AJOL)

Journal Home > Vol 21, No 1 (2009) > ... On separate days in a counterbalanced order, subjects performed 3 sets of each exercise to volitional failure at a ... total work and acute RPE were significantly greater (p=0.01) for LI for all exercises.

Preparation of fluorescence quenched libraries containing interchain disulphide bonds for studies of protein disulphide isomerases

DEFF Research Database (Denmark)

Spetzler, J C; Westphal, V; Winther, Jakob R.

1998-01-01

Protein disulphide isomerase is an enzyme that catalyses disulphide redox reactions in proteins. In this paper, fluorogenic and interchain disulphide bond containing peptide libraries and suitable substrates, useful in the study of protein disulphide isomerase, are described. In order to establish...... the quenching chromophore (Tyr(NO2)) and Cys(pNpys) activated for reaction with a second thiol. The formation and cleavage of the interchain disulphide bonds in the library were monitored under a fluorescence microscope. Substrates to investigate the properties of protein disulphide isomerase in solution were...

X-ray microprobe analysis of the retina and RPE in sheep with ovine ceroid-lipofuscinosis

International Nuclear Information System (INIS)

Samuelson, D.A.; Armstrong, D.; Jolly, R.

1990-01-01

Ovine ceroid-lipofuscinosis (OCL) is one animal model for the human condition, and because autofluorescent lipopigments are prominent in the brain and eye, it may also prove useful as a model for aging. For example, a progressive decline in electrical recording from brain and retina are observed in both aging and OCL. Samples of retinal and retinal pigment epithelial (RPE) tissues were obtained from a young control. 2 animals with OCL and a normal aged sheep. Specimens were cryo-fractured and examined by scanning electron microscopy/x-ray microanalysis. Measurements made of 6 individual cells in the ganglion layer of OCL specimens, the remainder of the retina, and RPE showed age-related changes in zinc, iron, and copper which were associated with lipopigment accumulation in the RPE. There was marked decrease in phosphate, sulfur, and manganese levels, as photoreceptor cells and their outer segments are lost in the disease process. This is the first report of metal analysis in the retina and RPE in a disease entity, and as a function of normal aging

Study of Short-Pulsed Laser Retinal Injury Mechanisms By Time-Resolved Imaging of Photomechanical Transients in RPE

National Research Council Canada - National Science Library

Lin, Charles

2000-01-01

We studied RPE cell damage mechanism for laser duration from 100 femtosec to 5 microsec, and we have investigated the dependence of threshold fluence for cell damage on the laser spot size on the RPE...

Functional differences in yeast protein disulfide isomerases

DEFF Research Database (Denmark)

Nørgaard, P; Westphal, V; Tachibana, C

2001-01-01

PDI1 is the essential gene encoding protein disulfide isomerase in yeast. The Saccharomyces cerevisiae genome, however, contains four other nonessential genes with homology to PDI1: MPD1, MPD2, EUG1, and EPS1. We have investigated the effects of simultaneous deletions of these genes. In several...

CASTING A BROAD NETWORK: FISHING FOR MECHANISMS OF RETINOID TERATOGENICITY

Science.gov (United States)

This is a short essay that serves to introduce a featured paper for an issue of Toxicological Sciences. The paper being introduced describes a study of mechanisms of retinoid induced abnormal limb development in mice. The paper was notable because the authors used gene expressi...

Comparing the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting Saccharomyces cerevisiae strains

Directory of Open Access Journals (Sweden)

Hahn-Hägerdal Bärbel

2008-10-01

Full Text Available Abstract Background Ethanolic fermentation of lignocellulosic biomass is a sustainable option for the production of bioethanol. This process would greatly benefit from recombinant Saccharomyces cerevisiae strains also able to ferment, besides the hexose sugar fraction, the pentose sugars, arabinose and xylose. Different pathways can be introduced in S. cerevisiae to provide arabinose and xylose utilisation. In this study, the bacterial arabinose isomerase pathway was combined with two different xylose utilisation pathways: the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways, respectively, in genetically identical strains. The strains were compared with respect to aerobic growth in arabinose and xylose batch culture and in anaerobic batch fermentation of a mixture of glucose, arabinose and xylose. Results The specific aerobic arabinose growth rate was identical, 0.03 h-1, for the xylose reductase/xylitol dehydrogenase and xylose isomerase strain. The xylose reductase/xylitol dehydrogenase strain displayed higher aerobic growth rate on xylose, 0.14 h-1, and higher specific xylose consumption rate in anaerobic batch fermentation, 0.09 g (g cells-1 h-1 than the xylose isomerase strain, which only reached 0.03 h-1 and 0.02 g (g cells-1h-1, respectively. Whereas the xylose reductase/xylitol dehydrogenase strain produced higher ethanol yield on total sugars, 0.23 g g-1 compared with 0.18 g g-1 for the xylose isomerase strain, the xylose isomerase strain achieved higher ethanol yield on consumed sugars, 0.41 g g-1 compared with 0.32 g g-1 for the xylose reductase/xylitol dehydrogenase strain. Anaerobic fermentation of a mixture of glucose, arabinose and xylose resulted in higher final ethanol concentration, 14.7 g l-1 for the xylose reductase/xylitol dehydrogenase strain compared with 11.8 g l-1 for the xylose isomerase strain, and in higher specific ethanol productivity, 0.024 g (g cells-1 h-1 compared with 0.01 g (g cells-1 h-1

The neurobehavioral teratology of retinoids: a 50-year history.

Science.gov (United States)

Adams, Jane

2010-10-01

This review of the central nervous system (CNS) and behavioral teratology of the retinoids over the last 50 years is a commemorative retrospective organized by decade to show the prominent research focus within each period and the most salient findings. In the 1960s, research focused on the gross CNS malformations associated with exposure and the delineation of dose-response and stage-specific responses in rodent models. Relevant scientific events before and during the 1960s are also discussed to provide the zeitgeist in which the field of neurobehavioral teratology emerged in the 1970s. During this period, studies demonstrated that adverse effects on postnatal behavior could be produced in animals exposed to doses of vitamin A lower than those that were teratogenic or impacted growth. Work during the 1980s showed an overrepresentation of behavioral studies focused on the reliability of screening methods, while the marked effects of human exposure were illustrated in children born to women treated with isotretinoin during pregnancy. The human catastrophe invigorated research during the 1990s, a period when technological advances allowed more elegant examinations of the developing CNS, of biochemical, cellular, and molecular developmental events and regulatory actions, and of the effects of direct genetic manipulations. Likewise, research in the 1990s reflected a reinvigoration of research in neurobehavioral teratology evinced in studies that used animal models to try to better understand human vulnerability. These foci continued in the 2000-2010 period while examinations of the role of retinoids in brain development and lifelong functioning became increasingly sophisticated and broader in scope. This review of the work on retinoids also provides a lens on the more general ontogeny of the field of neurobehavioral teratology. Birth Defects Research (Part A), 2010. © 2010 Wiley-Liss, Inc.

rpe v5: an emulator for reduced floating-point precision in large numerical simulations

Science.gov (United States)

Dawson, Andrew; Düben, Peter D.

2017-06-01

This paper describes the rpe (reduced-precision emulator) library which has the capability to emulate the use of arbitrary reduced floating-point precision within large numerical models written in Fortran. The rpe software allows model developers to test how reduced floating-point precision affects the result of their simulations without having to make extensive code changes or port the model onto specialized hardware. The software can be used to identify parts of a program that are problematic for numerical precision and to guide changes to the program to allow a stronger reduction in precision.The development of rpe was motivated by the strong demand for more computing power. If numerical precision can be reduced for an application under consideration while still achieving results of acceptable quality, computational cost can be reduced, since a reduction in numerical precision may allow an increase in performance or a reduction in power consumption. For simulations with weather and climate models, savings due to a reduction in precision could be reinvested to allow model simulations at higher spatial resolution or complexity, or to increase the number of ensemble members to improve predictions. rpe was developed with a particular focus on the community of weather and climate modelling, but the software could be used with numerical simulations from other domains.

Retinal pigment epithelial cells secrete neurotrophic factors and synthesize dopamine: possible contribution to therapeutic effects of RPE cell transplantation in Parkinson's disease

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Gu Qing

2009-06-01

Full Text Available Abstract Background New strategies for the treatment of Parkinson's disease (PD are shifted from dopamine (DA replacement to regeneration or restoration of the nigro-striatal system. A cell therapy using human retinal pigment epithelial (RPE cells as substitution for degenerated dopaminergic (DAergic neurons has been developed and showed promising prospect in clinical treatment of PD, but the exact mechanism underlying this therapy is not fully elucidated. In the present study, we investigated whether the beneficial effects of this therapy are related to the trophic properties of RPE cells and their ability to synthesize DA. Methods We evaluated the protective effects of conditioned medium (CM from cultured RPE cells on the DAergic cells against 6-hydroxydopamine (6-OHDA- and rotenone-induced neurotoxicity and determined the levels of glial cell derived neurotrophic factor (GDNF and brain derived neurotrophic factor (BDNF released by RPE cells. We also measured the DA synthesis and release. Finally we transplanted microcarriers-RPE cells into 6-OHDA lesioned rats and observed the improvement in apomorphine-induced rotations (AIR. Results We report here: (1 CM from RPE cells can secret trophic factors GDNF and BDNF, and protect DAergic neurons against the 6-OHDA- and rotenone-induced cell injury; (2 cultured RPE cells express L-dopa decarboxylase (DDC and synthesize DA; (3 RPE cells attached to microcarriers can survive in the host striatum and improve the AIR in 6-OHDA-lesioned animal model of PD; (4 GDNF and BDNF levels are found significantly higher in the RPE cell-grafted tissues. Conclusion These findings indicate the RPE cells have the ability to secret GDNF and BDNF, and synthesize DA, which probably contribute to the therapeutic effects of RPE cell transplantation in PD.

Chromosomal locations and modes of action of genes of the retinoid (vitamin A) system support their involvement in the etiology of schizophrenia

Energy Technology Data Exchange (ETDEWEB)

Goodman, A.B. [Columbia Univ. School of Public Health, New York, NY (United States)

1995-08-14

Vitamin A (retinoid), an essential nutrient for fetal and subsequent mammalian development, is involved in gene expression, cell differentiation, proliferation, migration, and death. Retinoic acid (RA) the morphogenic derivative of vitamin A is highly teratogenic. In humans retinoid excess or deficit can result in brain anomalies and psychosis. This review discusses chromosomal loci of genes that control the retinoid cascade in relation to some candidate genes in schizophrenia. The paper relates the knowledge about the transport, delivery, and action of retinoids to what is presently known about the pathology of schizophrenia, with particular reference to the dopamine hypothesis, neurotransmitters, the glutamate hypothesis, neurotransmitters, the glutamate hypothesis, retinitis pigmentosa, dermatologic disorders, and craniofacial anomalies. 201 refs., 1 tab.

acute and session rPe responses during resistance training: Bouts ...

African Journals Online (AJOL)

tive measure of aerobic exercise intensity. While correlating well ... ing intensity in aerobic-type exercise. 7 ... While original work dealt with RPE during an exercise bout, ..... Borg G. Perceived exertion as an indicator of somatic stress. Scan J.

The Use of Session RPE to Monitor the Intensity of Weight Training in Older Women: Acute Responses to Eccentric, Concentric, and Dynamic Exercises

Directory of Open Access Journals (Sweden)

Sandro S. Ferreira

2014-01-01

Full Text Available The rating of perceived exertion (RPE is ability to detect and interpret organic sensations while performing exercises. This method has been used to measure the level of effort that is felt during weight-training at a given intensity. The purpose of this investigation was to compare session RPE values with those of traditional RPE measurements for different weight-training muscle actions, performed together or separately. Fourteen women with no former weight-training experience were recruited for the investigation. All participants completed five sessions of exercise: familiarization, maximum force, concentric-only (CONC-only, eccentric-only (ECC-only, and dynamic (DYN = CONC + ECC. The traditional RPE method was measured after each series of exercises, and the session RPE was measured 30 min after the end of the training session. The statistical analyses used were the paired t-test, one-way analysis of variance, and repeated measures analysis of variance. Significant differences between traditional RPE and session RPE for DYN, CONC, and ECC exercises were not found. This investigation demonstrated that session RPE is similar to traditional RPE in terms of weight-training involving concentric, eccentric, or dynamic muscle exercises, and that it can be used to prescribe and monitor weight-training sessions in older subjects.

Pharmacotherapy of retinal disease with visual cycle modulators.

Science.gov (United States)

Hussain, Rehan M; Gregori, Ninel Z; Ciulla, Thomas A; Lam, Byron L

2018-04-01

Pharmacotherapy with visual cycle modulators (VCMs) is under investigation for retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), Stargardt macular dystrophy (SMD) and nonexudative age-related macular degeneration (AMD), all blinding diseases that lack effective treatment options. Areas covered: The authors review investigational VCMs, including oral retinoids, 9-cis-retinyl-acetate (zuretinol) and 9-cis-β-carotene, which restore 11-cis-retinal levels in RP and LCA caused by LRAT and RPE65 gene mutations, and may improve visual acuity and visual fields. Therapies for SMD aiming to decrease accumulation of toxic Vitamin A dimers and lipofuscin in the retina and retinal pigment epithelium (RPE) include C20-D3-vitamin A (ALK-001), isotretinoin, VM200, emixustat, and A1120. Mouse models of SMD show promising data for these treatments, though proof of efficacy in humans is currently lacking. Fenretinide and emixustat are investigational VCMs for dry AMD, though neither has been shown to reduce geographic atrophy or improve vision in human trials. A1120 prevents retinol transport into the RPE and may spare the side effects typically seen in VCMs (nyctalopia and chromatopsia) per mouse studies. Expert opinion: Oral VCMs may be feasible treatment options for degenerative retinal diseases based on pre-clinical and some early clinical studies. Further trials are warranted to assess their efficacy and safety in humans.

Identifying the receptor subtype selectivity of retinoid X and retinoic acid receptors via quantum mechanics.

Science.gov (United States)

Tsuji, Motonori; Shudo, Koichi; Kagechika, Hiroyuki

2017-03-01

Understanding and identifying the receptor subtype selectivity of a ligand is an important issue in the field of drug discovery. Using a combination of classical molecular mechanics and quantum mechanical calculations, this report assesses the receptor subtype selectivity for the human retinoid X receptor (hRXR) and retinoic acid receptor (hRAR) ligand-binding domains (LBDs) complexed with retinoid ligands. The calculated energies show good correlation with the experimentally reported binding affinities. The technique proposed here is a promising method as it reveals the origin of the receptor subtype selectivity of selective ligands.

High production of D-tagatose, a potential sugar substitute, using immobilized L-arabinose isomerase.

Science.gov (United States)

Kim, P; Yoon, S H; Roh, H J; Choi, J H

2001-01-01

An L-arabinose isomerase of Escherichia coli was immobilized using covalent binding to agarose to produce D-tagatose, a bulking sweetener that can be economically used as a sugar substitute. The immobilized L-arabinose isomerase stably produced an average of 7.5 g-tagatose/L.day for 7 days with a productivity exceeding that of the free enzyme (0.47 vs 0.30 mg/U.day). Using a scaled-up immobilized enzyme system, 99.9 g-tagatose/L was produced from galactose with 20% equilibrium in 48 h. The process was repeated two more times with production of 104.1 and 103.5 g-tagatose/L. D-Tagatose production using an immobilized L-arabinose isomerase has a high potential for commercial application.

Ultraviolet (UV and Hydrogen Peroxide Activate Ceramide-ER Stress-AMPK Signaling Axis to Promote Retinal Pigment Epithelium (RPE Cell Apoptosis

Directory of Open Access Journals (Sweden)

Jin Yao

2013-05-01

Full Text Available Ultraviolet (UV radiation and reactive oxygen species (ROS impair the physiological functions of retinal pigment epithelium (RPE cells by inducing cell apoptosis, which is the main cause of age-related macular degeneration (AMD. The mechanism by which UV/ROS induces RPE cell death is not fully addressed. Here, we observed the activation of a ceramide-endoplasmic reticulum (ER stress-AMP activated protein kinase (AMPK signaling axis in UV and hydrogen peroxide (H2O2-treated RPE cells. UV and H2O2 induced an early ceramide production, profound ER stress and AMPK activation. Pharmacological inhibitors against ER stress (salubrinal, ceramide production (fumonisin B1 and AMPK activation (compound C suppressed UV- and H2O2-induced RPE cell apoptosis. Conversely, cell permeable short-chain C6 ceramide and AMPK activator AICAR (5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide mimicked UV and H2O2’s effects and promoted RPE cell apoptosis. Together, these results suggest that UV/H2O2 activates the ceramide-ER stress-AMPK signaling axis to promote RPE cell apoptosis.

Bioproduction of D-Tagatose from D-Galactose Using Phosphoglucose Isomerase from Pseudomonas aeruginosa PAO1.

Science.gov (United States)

Patel, Manisha J; Patel, Arti T; Akhani, Rekha; Dedania, Samir; Patel, Darshan H

2016-07-01

Pseudomonas aeruginosa PAO1 phosphoglucose isomerase was purified as an active soluble form by a single-step purification using Ni-NTA chromatography that showed homogeneity on SDS-PAGE with molecular mass ∼62 kDa. The optimum temperature and pH for the maximum isomerization activity with D-galactose were 60 °C and 7.0, respectively. Generally, sugar phosphate isomerases show metal-independent activity but PA-PGI exhibited metal-dependent isomerization activity with aldosugars and optimally catalyzed the D-galactose isomerization in the presence of 1.0 mM MnCl2. The apparent Km and Vmax for D-galactose under standardized conditions were calculated to be 1029 mM (±31.30 with S.E.) and 5.95 U/mg (±0.9 with S.E.), respectively. Equilibrium reached after 180 min with production of 567.51 μM D-tagatose from 1000 mM of D-galactose. Though, the bioconversion ratio is low but it can be increased by immobilization and enzyme engineering. Although various L-arabinose isomerases have been characterized for bioproduction of D-tagatose, P. aeruginosa glucose phosphate isomerase is distinguished from the other L-arabinose isomerases by its optimal temperature (60 °C) for D-tagatose production being mesophilic bacteria, making it an alternate choice for bulk production.

Bacterial L-arabinose isomerases: industrial application for D-tagatose production.

Science.gov (United States)

Boudebbouze, Samira; Maguin, Emmanuelle; Rhimi, Moez

2011-12-01

D-tagatose is a natural monosaccharide with a low caloric value and has an anti-hyperglycemiant effect. This hexose has potential applications both in pharmaceutical and agro-food industries. However, the use of D-tagatose remains limited by its production cost. Many production procedures including chemical and biological processes were developed and patented. The most profitable production way is based on the use of L-arabinose isomerase which allows the manufacture of D-tagatose with an attractive rate. Future developments are focused on the generation of L-arabinose isomerases having biochemical properties satisfying the industrial applications. This report provides a brief review of the most recent patents that have been published relating to this area.

Galectin-3 induces clustering of CD147 and integrin-β1 transmembrane glycoprotein receptors on the RPE cell surface.

Directory of Open Access Journals (Sweden)

Claudia S Priglinger

Full Text Available Proliferative vitreoretinopathy (PVR is a blinding disease frequently occurring after retinal detachment surgery. Adhesion, migration and matrix remodeling of dedifferentiated retinal pigment epithelial (RPE cells characterize the onset of the disease. Treatment options are still restrained and identification of factors responsible for the abnormal behavior of the RPE cells will facilitate the development of novel therapeutics. Galectin-3, a carbohydrate-binding protein, was previously found to inhibit attachment and spreading of retinal pigment epithelial cells, and thus bares the potential to counteract PVR-associated cellular events. However, the identities of the corresponding cell surface glycoprotein receptor proteins on RPE cells are not known. Here we characterize RPE-specific Gal-3 containing glycoprotein complexes using a proteomic approach. Integrin-β1, integrin-α3 and CD147/EMMPRIN, a transmembrane glycoprotein implicated in regulating matrix metalloproteinase induction, were identified as potential Gal-3 interactors on RPE cell surfaces. In reciprocal immunoprecipitation experiments we confirmed that Gal-3 associated with CD147 and integrin-β1, but not with integrin-α3. Additionally, association of Gal-3 with CD147 and integrin-β1 was observed in co-localization analyses, while integrin-α3 only partially co-localized with Gal-3. Blocking of CD147 and integrin-β1 on RPE cell surfaces inhibited binding of Gal-3, whereas blocking of integrin-α3 failed to do so, suggesting that integrin-α3 is rather an indirect interactor. Importantly, Gal-3 binding promoted pronounced clustering and co-localization of CD147 and integrin-β1, with only partial association of integrin-α3. Finally, we show that RPE derived CD147 and integrin-β1, but not integrin-α3, carry predominantly β-1,6-N-actyl-D-glucosamine-branched glycans, which are high-affinity ligands for Gal-3. We conclude from these data that extracellular Gal-3 triggers

Galectin-3 Induces Clustering of CD147 and Integrin-β1 Transmembrane Glycoprotein Receptors on the RPE Cell Surface

Science.gov (United States)

Priglinger, Claudia S.; Szober, Christoph M.; Priglinger, Siegfried G.; Merl, Juliane; Euler, Kerstin N.; Kernt, Marcus; Gondi, Gabor; Behler, Jennifer; Geerlof, Arie; Kampik, Anselm; Ueffing, Marius; Hauck, Stefanie M.

2013-01-01

Proliferative vitreoretinopathy (PVR) is a blinding disease frequently occurring after retinal detachment surgery. Adhesion, migration and matrix remodeling of dedifferentiated retinal pigment epithelial (RPE) cells characterize the onset of the disease. Treatment options are still restrained and identification of factors responsible for the abnormal behavior of the RPE cells will facilitate the development of novel therapeutics. Galectin-3, a carbohydrate-binding protein, was previously found to inhibit attachment and spreading of retinal pigment epithelial cells, and thus bares the potential to counteract PVR-associated cellular events. However, the identities of the corresponding cell surface glycoprotein receptor proteins on RPE cells are not known. Here we characterize RPE-specific Gal-3 containing glycoprotein complexes using a proteomic approach. Integrin-β1, integrin-α3 and CD147/EMMPRIN, a transmembrane glycoprotein implicated in regulating matrix metalloproteinase induction, were identified as potential Gal-3 interactors on RPE cell surfaces. In reciprocal immunoprecipitation experiments we confirmed that Gal-3 associated with CD147 and integrin-β1, but not with integrin-α3. Additionally, association of Gal-3 with CD147 and integrin-β1 was observed in co-localization analyses, while integrin-α3 only partially co-localized with Gal-3. Blocking of CD147 and integrin-β1 on RPE cell surfaces inhibited binding of Gal-3, whereas blocking of integrin-α3 failed to do so, suggesting that integrin-α3 is rather an indirect interactor. Importantly, Gal-3 binding promoted pronounced clustering and co-localization of CD147 and integrin-β1, with only partial association of integrin-α3. Finally, we show that RPE derived CD147 and integrin-β1, but not integrin-α3, carry predominantly β-1,6-N-actyl-D-glucosamine-branched glycans, which are high-affinity ligands for Gal-3. We conclude from these data that extracellular Gal-3 triggers clustering of CD147 and

Directed evolution of xylose isomerase for improved xylose catabolism and fermentation in the yeast Saccharomyces cerevisiae.

Science.gov (United States)

Lee, Sun-Mi; Jellison, Taylor; Alper, Hal S

2012-08-01

The heterologous expression of a highly functional xylose isomerase pathway in Saccharomyces cerevisiae would have significant advantages for ethanol yield, since the pathway bypasses cofactor requirements found in the traditionally used oxidoreductase pathways. However, nearly all reported xylose isomerase-based pathways in S. cerevisiae suffer from poor ethanol productivity, low xylose consumption rates, and poor cell growth compared with an oxidoreductase pathway and, additionally, often require adaptive strain evolution. Here, we report on the directed evolution of the Piromyces sp. xylose isomerase (encoded by xylA) for use in yeast. After three rounds of mutagenesis and growth-based screening, we isolated a variant containing six mutations (E15D, E114G, E129D, T142S, A177T, and V433I) that exhibited a 77% increase in enzymatic activity. When expressed in a minimally engineered yeast host containing a gre3 knockout and tal1 and XKS1 overexpression, the strain expressing this mutant enzyme improved its aerobic growth rate by 61-fold and both ethanol production and xylose consumption rates by nearly 8-fold. Moreover, the mutant enzyme enabled ethanol production by these yeasts under oxygen-limited fermentation conditions, unlike the wild-type enzyme. Under microaerobic conditions, the ethanol production rates of the strain expressing the mutant xylose isomerase were considerably higher than previously reported values for yeast harboring a xylose isomerase pathway and were also comparable to those of the strains harboring an oxidoreductase pathway. Consequently, this study shows the potential to evolve a xylose isomerase pathway for more efficient xylose utilization.

Methods of measuring Protein Disulfide Isomerase activity: a critical overview

Science.gov (United States)

Watanabe, Monica; Laurindo, Francisco; Fernandes, Denise

2014-09-01

Protein disulfide isomerase is an essential redox chaperone from the endoplasmic reticulum (ER) and is responsible for correct disulfide bond formation in nascent proteins. PDI is also found in other cellular locations in the cell, particularly the cell surface. Overall, PDI contributes to ER and global cell redox homeostasis and signaling. The knowledge about PDI structure and function progressed substantially based on in vitro studies using recombinant PDI and chimeric proteins. In these experimental scenarios, PDI reductase and chaperone activities are readily approachable. In contrast, assays to measure PDI isomerase activity, the hallmark of PDI family, are more complex. Assessment of PDI roles in cells and tissues mainly relies on gain- or loss-of-function studies. However, there is limited information regarding correlation of experimental readouts with the distinct types of PDI activities. In this mini-review, we evaluate the main methods described for measuring the different kinds of PDI activity: thiol reductase, thiol oxidase, thiol isomerase and chaperone. We emphasize the need to use appropriate controls and the role of critical interferents (e.g., detergent, presence of reducing agents). We also discuss the translation of results from in vitro studies with purified recombinant PDI to cellular and tissue samples, with critical comments on the interpretation of results.

Topical retinoids in the management of photodamaged skin: from theory to evidence-based practical approach.

Science.gov (United States)

Darlenski, R; Surber, C; Fluhr, J W

2010-12-01

Skin, being exposed directly to the environment, represents a unique model for demonstrating the synergistic effects of intrinsic and extrinsic factors on the ageing process. Ultraviolet radiation (UVR) is the major factor among exogenous stressors responsible for premature skin ageing. The problem of skin ageing has captured public attention and has an important social impact. Different therapeutic approaches have been developed to treat cutaneous ageing and to diminish or prevent the negative effects of UVR. Topical retinoids represent an important and powerful class of molecules in the dermatologist's hands for the treatment of photodamaged skin. Since their introduction more than 20 years ago, topical retinoids have shown beneficial efficacy and good safety profiles in the management of photodamaged skin, and as therapeutic anti-ageing agents. This review provides a brief retrospective of the development of topical retinoids in the treatment of photodamaged skin, elucidates their mechanism of action, delineates their use and addresses clinical, pharmaceutical and regulatory issues in connection with their intended use. © 2010 The Authors. BJD © 2010 British Association of Dermatologists.

The secreted L-arabinose isomerase displays anti-hyperglycemic effects in mice.

Science.gov (United States)

Rhimi, Moez; Bermudez-Humaran, Luis G; Huang, Yuan; Boudebbouze, Samira; Gaci, Nadia; Garnier, Alexandrine; Gratadoux, Jean-Jacques; Mkaouar, Héla; Langella, Philippe; Maguin, Emmanuelle

2015-12-21

The L-arabinose isomerase is an intracellular enzyme which converts L-arabinose into L-ribulose in living systems and D-galactose into D-tagatose in industrial processes and at industrial scales. D-tagatose is a natural ketohexose with potential uses in pharmaceutical and food industries. The D-galactose isomerization reaction is thermodynamically equilibrated, and leads to secondary subproducts at high pH. Therefore, an attractive L-arabinose isomerase should be thermoactive and acidotolerant with high catalytic efficiency. While many reports focused on the set out of a low cost process for the industrial production of D-tagatose, these procedures remain costly. When compared to intracellular enzymes, the production of extracellular ones constitutes an interesting strategy to increase the suitability of the biocatalysts. The L-arabinose isomerase (L-AI) from Lactobacillus sakei was expressed in Lactococcus lactis in fusion with the signal peptide of usp45 (SP(Usp45)). The L-AI protein and activity were detected only in the supernatant of the induced cultures of the recombinant L. lactis demonstrating the secretion in the medium of the intracellular L. sakei L-AI in an active form. Moreover, we showed an improvement in the enzyme secretion using either (1) L. lactis strains deficient for their two major proteases, ClpP and HtrA, or (2) an enhancer of protein secretion in L. lactis fused to the recombinant L-AI with the SP(Usp45). Th L-AI enzyme secreted by the recombinant L. lactis strains or produced intracellularly in E. coli, showed the same functional properties than the native enzyme. Furthermore, when mice are fed with the L. lactis strain secreting the L-AI and galactose, tagatose was produced in vivo and reduced the glycemia index. We report for the first time the secretion of the intracellular L-arabinose isomerase in the supernatant of food grade L. lactis cultures with hardly display other secreted proteins. The secreted L-AI originated from the food

Structural and Biochemical Characterization of the Human Cyclophilin Family of Peptidyl-Prolyl Isomerases

Energy Technology Data Exchange (ETDEWEB)

Davis, Tara L.; Walker, John R.; Campagna-Slater, Valérie; Finerty, Jr., Patrick J.; Paramanathan, Ragika; Bernstein, Galina; MacKenzie, Farrell; Tempel, Wolfram; Ouyang, Hui; Lee, Wen Hwa; Eisenmesser, Elan Z.; Dhe-Paganon, Sirano (Toronto); (Colorado)

2011-12-14

Peptidyl-prolyl isomerases catalyze the conversion between cis and trans isomers of proline. The cyclophilin family of peptidyl-prolyl isomerases is well known for being the target of the immunosuppressive drug cyclosporin, used to combat organ transplant rejection. There is great interest in both the substrate specificity of these enzymes and the design of isoform-selective ligands for them. However, the dearth of available data for individual family members inhibits attempts to design drug specificity; additionally, in order to define physiological functions for the cyclophilins, definitive isoform characterization is required. In the current study, enzymatic activity was assayed for 15 of the 17 human cyclophilin isomerase domains, and binding to the cyclosporin scaffold was tested. In order to rationalize the observed isoform diversity, the high-resolution crystallographic structures of seven cyclophilin domains were determined. These models, combined with seven previously solved cyclophilin isoforms, provide the basis for a family-wide structure:function analysis. Detailed structural analysis of the human cyclophilin isomerase explains why cyclophilin activity against short peptides is correlated with an ability to ligate cyclosporin and why certain isoforms are not competent for either activity. In addition, we find that regions of the isomerase domain outside the proline-binding surface impart isoform specificity for both in vivo substrates and drug design. We hypothesize that there is a well-defined molecular surface corresponding to the substrate-binding S2 position that is a site of diversity in the cyclophilin family. Computational simulations of substrate binding in this region support our observations. Our data indicate that unique isoform determinants exist that may be exploited for development of selective ligands and suggest that the currently available small-molecule and peptide-based ligands for this class of enzyme are insufficient for isoform

Characteristics of chalcone isomerase promoter in crabapple leaves ...

African Journals Online (AJOL)

Anthocyanins are secondary metabolites found in higher plants that contribute to the colors of plants and chalcone isomerase (CHI) is one of the key enzymes in anthocyanin biosynthetic pathway. What characteristic is CHI promoter known as the regulation sequence of CHI gene, has been rarely investigated. We isolated A ...

Simultaneous application of bevacizumab and anti-CTGF antibody effectively suppresses proangiogenic and profibrotic factors in human RPE cells.

Science.gov (United States)

Bagheri, Abouzar; Soheili, Zahra-Soheila; Ahmadieh, Hamid; Samiei, Shahram; Sheibani, Nader; Astaneh, Shamila Darvishalipour; Kanavi, Mozhgan Rezaei; Mohammadian, Azam

2015-01-01

Retinal pigment epithelial (RPE) cells play key roles in the development of choroidal neovascularization and subsequent fibrosis. We investigated the impact of bevacizumab, antihuman vascular endothelial growth factor (VEGF) antibody, and anticonnective tissue growth factor (anti-CTGF) neutralizing antibody, individually or in combination, on proangiogenic and profibrotic properties of RPE cells. Primary cultures of human RPE cells were incubated with different concentrations of bevacizumab (0.25, 0.5, and 0.8 mg/ml) and/or anti-CTGF (10 μg/ml), and cell proliferation and apoptosis were determined. Expression and activity of proangiogenic and profibrotic genes including matrix metalloproteinases (MMP)-2 and 9, VEGFA, CTGF, vascular endothelial growth factor receptor-1 (VEGFR-1), cathepsin D, tissue inhibitor of metalloproteinases (TIMP) -1 and -2, and alpha smooth muscle actin (α-SMA) were assessed with slot blot, real-time RT-PCR, and zymography. Bevacizumab alone inhibited proliferation of RPE cells while anti-CTGF or bevacizumab and anti-CTGF combined had no inhibitory effect in this regard. Bevacizumab increased MMP-2, MMP-9, and cathepsin D but decreased VEGFA and VEGFR-1 expression. The CTGF level was increased by using 0.25 mg/ml bevacizumab but decreased at the 0.8 mg/ml concentration of bevacizumab. Treatment with anti-CTGF antibody decreased MMP-2 expression whereas combined treatment with bevacizumab and anti-CTGF resulted in decreased expression of MMP-2, TIMP-1, cathepsin D, VEGFA, CTGF, and α-SMA in the treated cultures. Treatment of RPE cells with the combination of bevacizumab and anti-CTGF could effectively suppress the proangiogenic and profibrotic activity of RPE cells.

Consequences of the natural retinoid/retinoid X receptor ligands action in human breast cancer MDA-MB-231 cell line: Focus on functional proteomics

Czech Academy of Sciences Publication Activity Database

Flodrová, Dana; Toporová, L.; Laštovičková, Markéta; Macejová, D.; Hunaková, L.; Brtko, J.; Bobálová, Janette

2017-01-01

Roč. 281, NOV (2017), s. 26-34 ISSN 0378-4274 R&D Projects: GA ČR(CZ) GA15-15479S Grant - others:AV ČR(CZ) SAV-15-01 Program:Bilaterální spolupráce Institutional support: RVO:68081715 Keywords : breast cancer * MDA-MB-231 * biomarker * retinoids Subject RIV: CB - Analytical Chemistry, Separation OBOR OECD: Analytical chemistry Impact factor: 3.858, year: 2016

Singlet Oxygen and Free Radical Reactions of Retinoids and Carotenoids—A Review

Science.gov (United States)

Truscott, T. George

2018-01-01

We report on studies of reactions of singlet oxygen with carotenoids and retinoids and a range of free radical studies on carotenoids and retinoids with emphasis on recent work, dietary carotenoids and the role of oxygen in biological processes. Many previous reviews are cited and updated together with new data not previously reviewed. The review does not deal with computational studies but the emphasis is on laboratory-based results. We contrast the ease of study of both singlet oxygen and polyene radical cations compared to neutral radicals. Of particular interest is the switch from anti- to pro-oxidant behavior of a carotenoid with change of oxygen concentration: results for lycopene in a cellular model system show total protection of the human cells studied at zero oxygen concentration, but zero protection at 100% oxygen concentration. PMID:29301252

Fundus autofluorescence and the bisretinoids of retina.

Science.gov (United States)

Sparrow, Janet R; Wu, Yalin; Nagasaki, Takayuki; Yoon, Kee Dong; Yamamoto, Kazunori; Zhou, Jilin

2010-11-01

Imaging of the human fundus of the eye with excitation wavelengths in the visible spectrum reveals a natural autofluorescence, that in a healthy retina originates primarily from the bisretinoids that constitute the lipofuscin of retinal pigment epithelial (RPE) cells. Since the intensity and distribution of fundus autofluorescence is altered in the presence of retinal disease, we have examined the fluorescence properties of the retinal bisretinoids with a view to aiding clinical interpretations. As is also observed for fundus autofluorescence, fluorescence emission from RPE lipofuscin was generated with a wide range of exciting wavelengths; with increasing excitation wavelength, the emission maximum shifted towards longer wavelengths and spectral width was decreased. These features are consistent with fluorescence generation from a mixture of compounds. While the bisretinoids that constitute RPE lipofuscin all fluoresced with maxima that were centered around 600 nm, fluorescence intensities varied when excited at 488 nm, the excitation wavelength utilized for fundus autofuorescence imaging. For instance the fluorescence efficiency of the bisretinoid A2-dihydropyridine-phosphatidylethanolamine (A2-DHP-PE) was greater than A2E and relative to both of the latter, all-trans-retinal dimer-phosphatidylethanolamine was weakly fluorescent. On the other hand, certain photooxidized forms of the bisretinoids present in both RPE and photoreceptor cells were more strongly fluorescent than the parent compound. We also sought to evaluate whether diffuse puncta of autofluorescence observed in some retinal disorders of monogenic origin are attributable to retinoid accumulation. However, two retinoids of the visual cycle, all-trans-retinyl ester and all-trans-retinal, did not exhibit fluorescence at 488 nm excitation.

RPE and Velocity Relationships for the Back Squat, Bench Press, and Deadlift in Powerlifters.

Science.gov (United States)

Helms, Eric R; Storey, Adam; Cross, Matt R; Brown, Scott R; Lenetsky, Seth; Ramsay, Hamish; Dillen, Carolina; Zourdos, Michael C

2017-02-01

Helms, ER, Storey, A, Cross, MR, Browm, SR, Lenetsky, S, Ramsay, H, Dillen, C, and Zourdos, MC. RPE and velocity relationships for the back squat, bench press, and deadlift in powerlifters. J Strength Cond Res 31(2): 292-297, 2017-The purpose of this study was to compare average concentric velocity (ACV) and rating of perceived exertion (RPE) based on repetitions in reserve on the squat, bench press, and deadlift. Fifteen powerlifters (3 women and 12 men, mean age 28.4 ± 8.5 years) worked up to a one repetition maximum (1RM) on each lift. Rating of perceived exertion was recorded on all sets, and the ACV was recorded for all sets performed at 80% of estimated 1RM and higher, up to 1RM. Rating of perceived exertion at 1RM on squat, bench press, and deadlift was 9.6 ± 0.5, 9.7 ± 0.4, and 9.6 ± 0.5, respectively and was not significantly different (p > 0.05). The ACV at 1RM on squat, bench press and deadlift was 0.23 ± 0.05, 0.10 ± 0.04, and 0.14 ± 0.05 m·second, respectively. Squat was faster than both bench press and deadlift (p > 0.001), and deadlift was faster than bench press (p = 0.05). Very strong relationships (r = 0.88-0.91) between percentage 1RM and RPE were observed on each lift. The ACV showed strong (r = -0.79 to -0.87) and very strong (r = -0.90 to 92) inverse relationships with RPE and percentage 1RM on each lift, respectively. We conclude that RPE may be a useful tool for prescribing intensity for squat, bench press, and deadlift in powerlifters, in addition to traditional methods such as percentage of 1RM. Despite high correlations between percentage 1RM and ACV, a "velocity load profile" should be developed to prescribe intensity on an individual basis with appropriate accuracy.

Bcl-2, Bax, and c-Fos expression correlates to RPE cell apoptosis induced by UV-light and daunorubicin

DEFF Research Database (Denmark)

Liang, Y G; Jorgensen, A G; Kaestel, C G

2000-01-01

PURPOSE. The aim of this study was to determine the role of Bcl-2, Bcl-X L, Bax, and c-Fos in regulation of apoptosis, induced by ultraviolet-light A (UV-A) and daunorubicin (DNR), in retinal pigment epithelium (RPE) cells grown on bovine extracellular matrix (ECM)-coated or uncoated plastic dishes....... METHODS. Apoptosis in confluent RPE cells cultured on ECM-coated or uncoated dishes was induced by UV-A or DNR. Apoptosis was detected by 7-amino-actinomycin D labeling followed by flow cytometry and by terminal deoxy-transferase mediated X-dUTP nick end labeling (TUNEL). Cellular expression of Bcl-2, Bcl......-X L, Bax, and c-Fos was determined by the use of antibodies and flow cytometry, Western blot analysis, and immunocytochemical staining. RESULTS. Both UV-A and DNR induce apoptosis in human RPE cells in vitro. Human fetal RPE cells grown on ECM-coated dishes were significantly more resistant to UV...

Voltage-dependent ion channels in the mouse RPE: comparison with Norrie disease mice.

Science.gov (United States)

Wollmann, Guido; Lenzner, Steffen; Berger, Wolfgang; Rosenthal, Rita; Karl, Mike O; Strauss, Olaf

2006-03-01

We studied electrophysiological properties of cultured retinal pigment epithelial (RPE) cells from mouse and a mouse model for Norrie disease. Wild-type RPE cells revealed the expression of ion channels known from other species: delayed-rectifier K(+) channels composed of Kv1.3 subunits, inward rectifier K(+) channels, Ca(V)1.3 L-type Ca(2+) channels and outwardly rectifying Cl(-) channels. Expression pattern and the ion channel characteristics current density, blocker sensitivity, kinetics and voltage-dependence were compared in cells from wild-type and Norrie mice. Although no significant differences were observed, our study provides a base for future studies on ion channel function and dysfunction in transgenic mouse models.

Overexpression of miR-183/-96/-182 triggers neuronal cell fate in Human Retinal Pigment Epithelial (hRPE) cells in culture.

Science.gov (United States)

Davari, Maliheh; Soheili, Zahra-Soheila; Samiei, Shahram; Sharifi, Zohreh; Pirmardan, Ehsan Ranaei

2017-01-29

miR-183 cluster, composed of miR-183/-96/-182 genes, is highly expressed in the adult retina, particularly in photoreceptors. It involves in development, maturation and normal function of neuroretina. Ectopic overexpression of miR-183/-96/-182 genes was performed to assess reprogramming of hRPE cells. They were amplified from genomic DNA and cloned independently or in tandem configuration into pAAV.MCS vector. hRPE cells were then transfected with the recombinant constructs. Real-Time PCR was performed to measure the expression levels of miR-183/-96/-182 and that of several retina-specific neuronal genes such as OTX2, NRL, PDC and DCT. The transfected cells also were immunocytochemically examined for retina-specific neuronal markers, including Rhodopsin, red opsin, CRX, Thy1, CD73, recoverin and PKCα, to determine the cellular fate of the transfected hRPE cells. Data showed that upon miR-183/-96/-182 overexpression in hRPE cultures, the expression of neuronal genes including OTX2, NRL, PDC and DCT was also upregulated. Moreover, miR-183 cluster-treated hRPE cells were immunoreactive for neuronal markers such as Rhodopsin, red opsin, CRX and Thy1. Both transcriptional and translational upregulation of neuronal genes in miR-183 cluster-treated hRPE cells suggests that in vitro overexpression of miR-183 cluster could trigger reprogramming of hRPE cells to retinal neuron fate. Copyright © 2016 Elsevier Inc. All rights reserved.

Escherichia coli rpiA gene encoding ribose phosphate isomerase A

DEFF Research Database (Denmark)

Hove-Jensen, Bjarne; Maigaard, Marianne

1993-01-01

The rpiA gene encoding ribose phosphate isomerase A was cloned from phage 1A2(471) of the Kohara gene library. Subcloning, restriction, and complementation analyses revealed an 1,800-bp SspI-generated DNA fragment that contained the entire control and coding sequences. This DNA fragment was seque......The rpiA gene encoding ribose phosphate isomerase A was cloned from phage 1A2(471) of the Kohara gene library. Subcloning, restriction, and complementation analyses revealed an 1,800-bp SspI-generated DNA fragment that contained the entire control and coding sequences. This DNA fragment...

Interaction of p53 with prolyl isomerases: Healthy and unhealthy relationships.

Science.gov (United States)

Mantovani, Fiamma; Zannini, Alessandro; Rustighi, Alessandra; Del Sal, Giannino

2015-10-01

The p53 protein family, comprising p53, p63 and p73, is primarily involved in preserving genome integrity and preventing tumor onset, and also affects a range of physiological processes. Signal-dependent modifications of its members and of other pathway components provide cells with a sophisticated code to transduce a variety of stress signaling into appropriate responses. TP53 mutations are highly frequent in cancer and lead to the expression of mutant p53 proteins that are endowed with oncogenic activities and sensitive to stress signaling. p53 family proteins have unique structural and functional plasticity, and here we discuss the relevance of prolyl-isomerization to actively shape these features. The anti-proliferative functions of the p53 family are carefully activated upon severe stress and this involves the interaction with prolyl-isomerases. In particular, stress-induced stabilization of p53, activation of its transcriptional control over arrest- and cell death-related target genes and of its mitochondrial apoptotic function, as well as certain p63 and p73 functions, all require phosphorylation of specific S/T-P motifs and their subsequent isomerization by the prolyl-isomerase Pin1. While these functions of p53 counteract tumorigenesis, under some circumstances their activation by prolyl-isomerases may have negative repercussions (e.g. tissue damage induced by anticancer therapies and ischemia-reperfusion, neurodegeneration). Moreover, elevated Pin1 levels in tumor cells may transduce deregulated phosphorylation signaling into activation of mutant p53 oncogenic functions. The complex repertoire of biological outcomes induced by p53 finds mechanistic explanations, at least in part, in the association between prolyl-isomerases and the p53 pathway. This article is part of a Special Issue entitled Proline-directed foldases: Cell signaling catalysts and drug targets. Copyright © 2015 Elsevier B.V. All rights reserved.

Exogenous NAD(+) decreases oxidative stress and protects H2O2-treated RPE cells against necrotic death through the up-regulation of autophagy.

Science.gov (United States)

Zhu, Ying; Zhao, Ke-Ke; Tong, Yao; Zhou, Ya-Li; Wang, Yi-Xiao; Zhao, Pei-Quan; Wang, Zhao-Yang

2016-05-31

Increased oxidative stress, which can lead to the retinal pigment epithelium (RPE) cell death by inducing ATP depletion and DNA repair, is believed to be a prominent pathology in age-related macular degeneration (AMD). In the present study, we showed that and 0.1 mM nicotinamide adenine dinucleotide (NAD(+)) administration significantly blocked RPE cell death induced by 300 μM H2O2. Further investigation showed that H2O2 resulted in increased intracellular ROS level, activation of PARP-1 and subsequently necrotic death of RPE cells. Exogenous NAD(+) administration significantly decreased intracellular and intranuclear ROS levels in H2O2-treated RPE cells. In addition, NAD(+) administration to H2O2-treated RPE cells inhibited the activation of PARP-1 and protected the RPE cells against necrotic death. Moreover, exogenous NAD(+) administration up-regulated autophagy in the H2O2-treated RPE cells. Inhibition of autophagy by LY294002 blocked the decrease of intracellular and intranuclear ROS level. Besides, inhibition of autophagy by LY294002 abolished the protection of exogenous NAD(+) against H2O2-induced cell necrotic death. Taken together, our findings indicate that that exogenous NAD(+) administration suppresses H2O2-induced oxidative stress and protects RPE cells against PARP-1 mediated necrotic death through the up-regulation of autophagy. The results suggest that exogenous NAD(+) administration might be potential value for the treatment of AMD.

Exogenous NAD+ decreases oxidative stress and protects H2O2-treated RPE cells against necrotic death through the up-regulation of autophagy

Science.gov (United States)

Zhu, Ying; Zhao, Ke-ke; Tong, Yao; Zhou, Ya-li; Wang, Yi-xiao; Zhao, Pei-quan; Wang, Zhao-yang

2016-01-01

Increased oxidative stress, which can lead to the retinal pigment epithelium (RPE) cell death by inducing ATP depletion and DNA repair, is believed to be a prominent pathology in age-related macular degeneration (AMD). In the present study, we showed that and 0.1 mM nicotinamide adenine dinucleotide (NAD+) administration significantly blocked RPE cell death induced by 300 μM H2O2. Further investigation showed that H2O2 resulted in increased intracellular ROS level, activation of PARP-1 and subsequently necrotic death of RPE cells. Exogenous NAD+ administration significantly decreased intracellular and intranuclear ROS levels in H2O2-treated RPE cells. In addition, NAD+ administration to H2O2-treated RPE cells inhibited the activation of PARP-1 and protected the RPE cells against necrotic death. Moreover, exogenous NAD+ administration up-regulated autophagy in the H2O2-treated RPE cells. Inhibition of autophagy by LY294002 blocked the decrease of intracellular and intranuclear ROS level. Besides, inhibition of autophagy by LY294002 abolished the protection of exogenous NAD+ against H2O2-induced cell necrotic death. Taken together, our findings indicate that that exogenous NAD+ administration suppresses H2O2-induced oxidative stress and protects RPE cells against PARP-1 mediated necrotic death through the up-regulation of autophagy. The results suggest that exogenous NAD+ administration might be potential value for the treatment of AMD. PMID:27240523

Validity and reliability of the session-RPE method for quantifying training in Australian football: a comparison of the CR10 and CR100 scales.

Science.gov (United States)

Scott, Tannath J; Black, Cameron R; Quinn, John; Coutts, Aaron J

2013-01-01

The purpose of this study was to examine and compare the criterion validity and test-retest reliability of the CR10 and CR100 rating of perceived exertion (RPE) scales for team sport athletes that undertake high-intensity, intermittent exercise. Twenty-one male Australian football (AF) players (age: 19.0 ± 1.8 years, body mass: 83.92 ± 7.88 kg) participated the first part (part A) of this study, which examined the construct validity of the session-RPE (sRPE) method for quantifying training load in AF. Ten male athletes (age: 16.1 ± 0.5 years) participated in the second part of the study (part B), which compared the test-retest reliability of the CR10 and CR100 RPE scales. In part A, the validity of the sRPE method was assessed by examining the relationships between sRPE, and objective measures of internal (i.e., heart rate) and external training load (i.e., distance traveled), collected from AF training sessions. Part B of the study assessed the reliability of sRPE through examining the test-retest reliability of sRPE during 3 different intensities of controlled intermittent running (10, 11.5, and 13 km·h(-1)). Results from part A demonstrated strong correlations for CR10- and CR100-derived sRPE with measures of internal training load (Banisters TRIMP and Edwards TRIMP) (CR10: r = 0.83 and 0.83, and CR100: r = 0.80 and 0.81, p training load (distance, higher speed running and player load) for both the CR10 (r = 0.81, 0.71, and 0.83) and CR100 (r = 0.78, 0.69, and 0.80) were significant (p reliability for both the CR10 (31.9% CV) and CR100 (38.6% CV) RPE scales after short bouts of intermittent running. Collectively, these results suggest both CR10- and CR100-derived sRPE methods have good construct validity for assessing training load in AF. The poor levels of reliability revealed under field testing indicate that the sRPE method may not be sensible to detecting small changes in exercise intensity during brief intermittent running bouts. Despite this limitation

Kinase-dead ATM protein is highly oncogenic and can be preferentially targeted by Topo-isomerase I inhibitors.

Science.gov (United States)

Yamamoto, Kenta; Wang, Jiguang; Sprinzen, Lisa; Xu, Jun; Haddock, Christopher J; Li, Chen; Lee, Brian J; Loredan, Denis G; Jiang, Wenxia; Vindigni, Alessandro; Wang, Dong; Rabadan, Raul; Zha, Shan

2016-06-15

Missense mutations in ATM kinase, a master regulator of DNA damage responses, are found in many cancers, but their impact on ATM function and implications for cancer therapy are largely unknown. Here we report that 72% of cancer-associated ATM mutations are missense mutations that are enriched around the kinase domain. Expression of kinase-dead ATM (Atm(KD/-)) is more oncogenic than loss of ATM (Atm(-/-)) in mouse models, leading to earlier and more frequent lymphomas with Pten deletions. Kinase-dead ATM protein (Atm-KD), but not loss of ATM (Atm-null), prevents replication-dependent removal of Topo-isomerase I-DNA adducts at the step of strand cleavage, leading to severe genomic instability and hypersensitivity to Topo-isomerase I inhibitors. Correspondingly, Topo-isomerase I inhibitors effectively and preferentially eliminate Atm(KD/-), but not Atm-proficientor Atm(-/-) leukemia in animal models. These findings identify ATM kinase-domain missense mutations as a potent oncogenic event and a biomarker for Topo-isomerase I inhibitor based therapy.

Cloning and characterization of peptidylprolyl isomerase B in the ...

African Journals Online (AJOL)

Peptidylprolyl isomerases (PPIases) play essential roles in protein folding and are implicated in immune response and cell cycle control. Our previous proteomic analysis indicated that Bombyx mori PPIases may be involved in anti- Bombyx mori nucleopolyhedrovirus (BmNPV) response. To help investigate this mechanism, ...

Crystallization and preliminary X-ray characterization of phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv

International Nuclear Information System (INIS)

Mathur, Divya; Anand, Kanchan; Mathur, Deepika; Jagadish, Nirmala; Suri, Anil; Garg, Lalit C.

2007-01-01

The phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv was crystallized and diffraction data were collected to 2.8 Å resolution. Phosphoglucose isomerase is a ubiquitous enzyme that catalyzes the isomerization of d-glucopyranose-6-phosphate to d-fructofuranose-6-phosphate. The present investigation reports the expression, purification, crystallization and preliminary crystallographic studies of the phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv, which shares 46% sequence identity with that of its human host. The recombinant protein, which was prepared using an Escherichia coli expression system, was crystallized by the hanging-drop vapour-diffusion method. The crystals diffracted to a resolution of 2.8 Å and belonged to the orthorhombic space group I2 1 2 1 2 1 , with unit-cell parameters a = 109.0, b = 119.8, c = 138.9 Å

Functional recovery after experimental RPE debridement, mfERG studies in a porcine model

DEFF Research Database (Denmark)

Sørensen, Nina Buus; Lassota, Nathan; Kyhn, Maria Voss

2013-01-01

BACKGROUND: The correlation between histologically identified regeneration of retinal pigment epithelium (RPE) and functional outcome measured by multifocal electroretinography (mfERG) following surgical debridement is examined in a porcine model. In humans, visual acuity is reduced in diseases......, and by brushing the Bruch's membrane with a silicone catheter. Immediately following surgery (baseline) and after 2 and 6 weeks respectively, the animals were examined by mfERG, fundus photographs (FPs), fluorescein angiograms (FAs), and histopathology. RESULTS: The mfERG P1 amplitude was decreased 2 weeks (T2....... CONCLUSION: This is the first study to show that the histological regeneration of hypopigmented RPE correlates to a return of the retinal function, measured by mfERG....

Transcriptomic analysis across nasal, temporal, and macular regions of human neural retina and RPE/choroid by RNA-Seq

Science.gov (United States)

Whitmore, S. Scott; Wagner, Alex H.; DeLuca, Adam P.; Drack, Arlene V.; Stone, Edwin M.; Tucker, Budd A.; Zeng, Shemin; Braun, Terry A.; Mullins, Robert F.; Scheetz, Todd E.

2014-01-01

Proper spatial differentiation of retinal cell types is necessary for normal human vision. Many retinal diseases, such as Best disease and male germ cell associated kinase (MAK)-associated retinitis pigmentosa, preferentially affect distinct topographic regions of the retina. While much is known about the distribution of cell-types in the retina, the distribution of molecular components across the posterior pole of the eye has not been well-studied. To investigate regional difference in molecular composition of ocular tissues, we assessed differential gene expression across the temporal, macular, and nasal retina and retinal pigment epithelium (RPE)/choroid of human eyes using RNA-Seq. RNA from temporal, macular, and nasal retina and RPE/choroid from four human donor eyes was extracted, poly-A selected, fragmented, and sequenced as 100 bp read pairs. Digital read files were mapped to the human genome and analyzed for differential expression using the Tuxedo software suite. Retina and RPE/choroid samples were clearly distinguishable at the transcriptome level. Numerous transcription factors were differentially expressed between regions of the retina and RPE/choroid. Photoreceptor-specific genes were enriched in the peripheral samples, while ganglion cell and amacrine cell genes were enriched in the macula. Within the RPE/choroid, RPE-specific genes were upregulated at the periphery while endothelium associated genes were upregulated in the macula. Consistent with previous studies, BEST1 expression was lower in macular than extramacular regions. The MAK gene was expressed at lower levels in macula than in extramacular regions, but did not exhibit a significant difference between nasal and temporal retina. The regional molecular distinction is greatest between macula and periphery and decreases between different peripheral regions within a tissue. Datasets such as these can be used to prioritize candidate genes for possible involvement in retinal diseases with

Transcriptomic analysis across nasal, temporal, and macular regions of human neural retina and RPE/choroid by RNA-Seq.

Science.gov (United States)

Whitmore, S Scott; Wagner, Alex H; DeLuca, Adam P; Drack, Arlene V; Stone, Edwin M; Tucker, Budd A; Zeng, Shemin; Braun, Terry A; Mullins, Robert F; Scheetz, Todd E

2014-12-01

Proper spatial differentiation of retinal cell types is necessary for normal human vision. Many retinal diseases, such as Best disease and male germ cell associated kinase (MAK)-associated retinitis pigmentosa, preferentially affect distinct topographic regions of the retina. While much is known about the distribution of cell types in the retina, the distribution of molecular components across the posterior pole of the eye has not been well-studied. To investigate regional difference in molecular composition of ocular tissues, we assessed differential gene expression across the temporal, macular, and nasal retina and retinal pigment epithelium (RPE)/choroid of human eyes using RNA-Seq. RNA from temporal, macular, and nasal retina and RPE/choroid from four human donor eyes was extracted, poly-A selected, fragmented, and sequenced as 100 bp read pairs. Digital read files were mapped to the human genome and analyzed for differential expression using the Tuxedo software suite. Retina and RPE/choroid samples were clearly distinguishable at the transcriptome level. Numerous transcription factors were differentially expressed between regions of the retina and RPE/choroid. Photoreceptor-specific genes were enriched in the peripheral samples, while ganglion cell and amacrine cell genes were enriched in the macula. Within the RPE/choroid, RPE-specific genes were upregulated at the periphery while endothelium associated genes were upregulated in the macula. Consistent with previous studies, BEST1 expression was lower in macular than extramacular regions. The MAK gene was expressed at lower levels in macula than in extramacular regions, but did not exhibit a significant difference between nasal and temporal retina. The regional molecular distinction is greatest between macula and periphery and decreases between different peripheral regions within a tissue. Datasets such as these can be used to prioritize candidate genes for possible involvement in retinal diseases with

Arabidopsis Phosphomannose Isomerase 1, but Not Phosphomannose Isomerase 2, Is Essential for Ascorbic Acid Biosynthesis*S⃞

OpenAIRE

Maruta, Takanori; Yonemitsu, Miki; Yabuta, Yukinori; Tamoi, Masahiro; Ishikawa, Takahiro; Shigeoka, Shigeru

2008-01-01

We studied molecular and functional properties of Arabidopsis phosphomannose isomerase isoenzymes (PMI1 and PMI2) that catalyze reversible isomerization between d-fructose 6-phosphate and d-mannose 6-phosphate (Man-6P). The apparent Km and Vmax values for Man-6P of purified recombinant PMI1 were 41.3 ± 4.2 μm and 1.89 μmol/min/mg protein, respectively, whereas those of purified recombinant PMI2 were 372 ± 13 μm and 22.5 μmol/min/mg protein, respectively. Both PMI1 ...

RPE vs. Percentage 1RM Loading in Periodized Programs Matched for Sets and Repetitions

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Helms, Eric R.; Byrnes, Ryan K.; Cooke, Daniel M.; Haischer, Michael H.; Carzoli, Joseph P.; Johnson, Trevor K.; Cross, Matthew R.; Cronin, John B.; Storey, Adam G.; Zourdos, Michael C.

2018-01-01

Purpose: To investigate differences between rating of perceived exertion (RPE) and percentage one-repetition maximum (1RM) load assignment in resistance-trained males (19–35 years) performing protocols with matched sets and repetitions differentiated by load-assignment. Methods: Participants performed squats then bench press 3x/weeks in a daily undulating format over 8-weeks. Participants were counterbalanced by pre-test 1RM then assigned to percentage 1RM (1RMG, n = 11); load-assignment via percentage 1RMs, or RPE groups (RPEG, n = 10); participant-selected loads to reach target RPE ranges. Ultrasonography determined pre and post-test pectoralis (PMT), and vastus lateralis muscle thickness at 50 (VLMT50) and 70% (VLMT70) femur-length. Results: Bench press (1RMG +9.64 ± 5.36; RPEG + 10.70 ± 3.30 kg), squat (1RMG + 13.91 ± 5.89; RPEG + 17.05 ± 5.44 kg) and their combined-total 1RMs (1RMG + 23.55 ± 10.38; RPEG + 27.75 ± 7.94 kg) increased (p 0.05). Magnitude-based inferences revealed 79, 57, and 72% chances of mean small effect size (ES) advantages for squat; ES 90% confidence limits (CL) = 0.50 ± 0.63, bench press; ES 90% CL = 0.28 ± 0.73, and combined-total; ES 90% CL = 0.48 ± 0.68 respectively, in RPEG. There were 4, 14, and 6% chances 1RMG had a strength advantage of the same magnitude, and 18, 29, and 22% chances, respectively of trivial differences between groups. Conclusions: Both loading-types are effective. However, RPE-based loading may provide a small 1RM strength advantage in a majority of individuals. PMID:29628895

A2E Suppresses Regulatory Function of RPE Cells in Th1 Cell Differentiation Via Production of IL-1β and Inhibition of PGE2.

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Shi, Qian; Wang, Qiu; Li, Jing; Zhou, Xiaohui; Fan, Huimin; Wang, Fenghua; Liu, Haiyun; Sun, Xiangjun; Sun, Xiaodong

2015-12-01

Inflammatory status of RPE cells induced by A2E is essential in the development of AMD. Recent research indicated T-cell immunity was involved in the pathological progression of AMD. This study was designed to investigate how A2E suppresses immunoregulatory function of RPE cells in T-cell immunity in vitro. Mouse RPE cells or human ARPE19 cells were stimulated with A2E, and co-cultured with naïve T cells under Th1, Th2, Th17, and regulatory T cell (Treg) polarization conditions. The intracellular cytokines or transcript factors of the induced T-cells subset were detected with flow cytometer and qRT-PCR. The ROS levels were detected, and the factors and possible pathways involved in the A2E-laden RPE cells were analyzed through neutralization antibody of IL-1β and inhibitors of related pathways. The A2E reduced regulatory function of RPE cells in Treg differentiation. The A2E-laden RPE cells promoted polarization of Th1 cells in vitro, but not Th2 or Th17 differentiation. The A2E induced RPE cells to release inflammatory cytokines and ROS, but PGE2 production was inhibited. Through neutralization of IL-1β or inhibition of COX2-PGE2 pathways, A2E-laden RPE cells expressed reduced effect in inducing Th1 cells. The A2E inhibited regulatory function of RPE cells in suppressing Th1 cell immunity in vitro through production of IL-1β and inhibition of PGE2. Our data indicate that A2E could suppress immunoregulatory function of RPE cells and adaptive immunity might play a role in the immune pathogenesis of AMD.

Prevention of UV irradiation induced suppression of monocyte functions by retinoids and carotenoids in vitro

International Nuclear Information System (INIS)

Schoen, D.J.; Watson, R.R.

1988-01-01

The effects of stimulation of human peripheral blood monocytes in vitro with retinoids and carotenoids, and subsequent exposure to ultraviolet light of the B wavelength were measured. The compounds were applied to the monocytes in culture for 24 h, and the washed cells were then exposed to UVB light up to 220 J/m 2 . The compounds tested protected the monocyte from UVB induced damage to phagocytic activity. This protection may be due to the antioxidant or UVB energy-quenching properties of these compounds. Monocyte cytotoxicity against a melanoma cell line was stimulated by exposure to the retinoids or carotenoids, but a protective effect in vitro against UVB damage was not seen for this cell function. (author)

Quantitative Fundus Autofluorescence in Best Vitelliform Macular Dystrophy: RPE Lipofuscin is not Increased in Non-Lesion Areas of Retina.

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Sparrow, Janet R; Duncker, Tobias; Woods, Russell; Delori, François C

2016-01-01

Since the lipofuscin of retinal pigment epithelial (RPE) cells has been implicated in the pathogenesis of Best vitelliform macular dystrophy, we quantified fundus autofluorescence (quantitative fundus autofluorescence, qAF) as an indirect measure of RPE lipofuscin levels. Mean non-lesion qAF was found to be within normal limits for age. By spectral domain optical coherence tomography (SD-OCT) vitelliform lesions presented as fluid-filled subretinal detachments containing reflective material. We discuss photoreceptor outer segment debris as the source of the intense fluorescence of these lesions and loss of anion channel functioning as an explanation for the bullous photoreceptor-RPE detachment. Unexplained is the propensity of the disease for central retina.

Zeaxanthin Inhibits Hypoxia-Induced VEGF Secretion by RPE Cells through Decreased Protein Levels of Hypoxia-Inducible Factors-1α

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Richard Rosen

2015-01-01

Full Text Available Hypoxia is the most important stimulus leading to upregulation of VEGF in the retina and this is caused by accumulation of hypoxia-inducible factors-1α (HIF-1α protein. The effects of zeaxanthin, a natural phytochemical, on the VEGF and HIF-1α expression in the primary culture of human retinal pigment epithelial (RPE cells were studied. An in vitro RPE cell hypoxia model was established by placing cells under 1% oxygen pressure or by adding cobalt chloride (CoCl2 to the culture medium. RPE cells and conditioned media were collected from cultures treated with and without zeaxanthin under normoxic and hypoxic conditions. VEGF and HIF-1α protein and RNA levels were measured by ELISA kits and RT-PCR, respectively. Hypoxia caused a significant increase of VEGF expression and accumulation of HIF-1α in RPE cells. Zeaxanthin at 50–150 μM significantly inhibited the expression of VEGF and accumulation of HIF-1α protein caused by hypoxia but did not affect expression of VEGF and HIF-1α under normoxic conditions. This is the first report on the effect of zeaxanthin on VEGF and HIF-1α levels in cultured RPE cells and suggests that zeaxanthin may have potential value in the prevention and treatment of various retinal diseases associated with vascular leakage and neovascularization.

Obtaining mutants of Streptomyces griseoflavus strain 1339, producers of glucose isomerase, following gamma irradiation

International Nuclear Information System (INIS)

Dzhedzheva, G.; Stoeva, N.; Stojchev, M.

1990-01-01

A water suspension of Streptomyces griseoflavus strain 1339 spores of a density of 8.7.10 6 spores/cm 3 is gamma irradiated ( 60 Co, RHM-γ-20, 30.3 Gy/min). The survival of Streptomyces griseoflavus strain 1339 spores was determined depending on radiation doses, exposure times and incubation temperature. Five major morphological types of colonies were isolated, characterized by different levels of glucose isomerase activity. Maximum specific glucose isomerase activity (GIU/g) was attained after the third gamma irradiation step using a dose of 3000 Gy. 2 tabs., 3 figs., 7 refs

Gait and posture analysis in patients with maxillary transverse discrepancy, before and after RPE.

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Mason, Martina; Spolaor, Fabiola; Guiotto, Annamaria; De Stefani, Alberto; Gracco, Antonio; Sawacha, Zimi

2018-03-01

The purpose of this study was to evaluate the effects of the rapid palatal expansion (RPE) on posture and gait analysis in subjects with maxillary transverse discrepancies. Forty-one patients between 6 and 12 years were divided into 3 groups: 10 control subjects (Cs), 16 patients with unilateral posterior crossbite (CbMono), 15 patients with maxillary transverse discrepancy and no crossbite (Nocb). Every subject underwent gait analysis and posturographic examination in order to evaluate the presence of balance alterations before (T0) and after (T4) RPE application. The examinations were performed through a six-cameras stereophotogrammetric system (60-120Hz, BTS S.p.A.) synchronized with two force plates (FP4060, Bertec Corp.). Romberg test was performed on a force plate, and the statokinesiogram and joint kinematics were evaluated. One-way Anova was performed among the variables after evidence of normal distribution (Levene's test for equality of variances) and Kruskal-Wallis test (Ptest was performed, or Kruskal-Wallis test, instead when comparing pre- and post-RPE application within the same group of subjects (P<0.05). Tamane T2 or Bonferroni correction was applied where needed. The posturographic analysis reveal significant differences across the 3 population: 95% power frequency in medio-lateral and antero-posterior direction in T0, median frequency in medio-lateral direction in T0, mean power frequency in medio-lateral direction in T0. Significant differences were also registered in the three-dimensional joints kinematics variables, mainly between Cs and Cbmono in T0 and T4 and between Cbmono and Nocb in T4. A detectable correlation between dental occlusion and body posture is shown in this study that confirms another benefit of the RPE. This was mainly revealed in the dynamic posture where modifications at the mandibular level affect the whole body. Copyright © 2018. Published by Elsevier Masson SAS.

Hepatic retinoid levels in seven fish species (teleosts) from a tropical coastal lagoon receiving effluents from iron-ore mining and processing.

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Pereira, Adriana A; van Hattum, Bert; Brouwer, Abraham

2012-02-01

The present study was undertaken to investigate the possible effects of Fe and trace element exposure on hepatic levels of retinoids in seven fish species. Concentrations of retinoids were measured in fish collected from a coastal lagoon in Brazil that receives effluents from an iron-ore mining and processing plant. Fish from nearby coastal lagoons were also included to assess possible differences related to chemical exposure. Results indicated considerable differences in hepatic retinoid composition among the various species investigated. The most striking differences were in retinol and derivative-specific profiles and in didehydro retinol and derivative-specific profiles. The Perciformes species Geophagus brasiliensis, Tilapia rendalli, Mugil liza, and Cichla ocellaris and the Characiforme Hoplias malabaricus were characterized as retinol and derivative-specific, while the Siluriformes species Hoplosternum littorale and Rhamdia quelen were didehydro retinol and derivative-specific fish species. A negative association was observed between Al, Pb, As, and Cd and hepatic didehydro retinoid levels. Fish with higher levels of hepatic Fe, Cu, and Zn showed unexpectedly significant positive correlations with increased hepatic retinol levels. This finding, associated with the positive relationships between retinol and retinyl palmitate with lipid peroxidation, may suggest that vitamin A is mobilized from other tissues to increase hepatic antioxidant levels for protection against oxidative damage. These data show significant but dissimilar associations between trace element exposure and hepatic retinoid levels in fish species exposed to iron-ore mining and processing effluents, without apparent major impacts on fish health and condition. Copyright © 2011 SETAC.

Synergistic Effects of PPARγ Ligands and Retinoids in Cancer Treatment

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Masahito Shimizu

2008-01-01

Full Text Available Peroxisome proliferator-activated receptors (PPARs are members of the nuclear receptor superfamily. The activation of PPARs by their specific ligands is regarded as one of the promising strategies to inhibit cancer cell growth. However, recent clinical trials targeting several common cancers showed no beneficial effect when PPAR ligands are used as a monotherapy. Retinoid X receptors (RXRs, which play a critical role in normal cell proliferation as a master regulator for nuclear receptors, preferentially form heterodimers with PPARs. A malfunction of RXRα due to phosphorylation by the Ras/MAPK signaling pathway is associated with the development of certain types of human malignancies. The activation of PPARγ/RXR heterodimer by their respective ligands synergistically inhibits cell growth, while inducing apoptosis in human colon cancer cells when the phosphorylation of RXRα was inhibited. We herein review the synergistic antitumor effects produced by the combination of the PPAR, especially PPARγ, ligands plus other agents, especially retinoids, in a variety of human cancers. We also focus on the phosphorylation of RXRα because the inhibition of RXRα phosphorylation and the restoration of its physiological function may activate PPAR/RXR heterodimer and, therefore, be a potentially effective and critical strategy for the inhibition of cancer cell growth.

Human triose-phosphate isomerase deficiency: a single amino acid substitution results in a thermolabile enzyme.

OpenAIRE

Daar, I O; Artymiuk, P J; Phillips, D C; Maquat, L E

1986-01-01

Triose-phosphate isomerase (TPI; D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1) deficiency is a recessive disorder that results in hemolytic anemia and neuromuscular dysfunction. To determine the molecular basis of this disorder, a TPI allele from two unrelated patients homozygous for TPI deficiency was compared with an allele from a normal individual. Each disease-associated sequence harbors a G X C----C X G transversion in the codon for amino acid-104 and specifies a structurally...

Genomic analysis of a xylose operon and characterization of novel xylose isomerase and xylulokinase from Bacillus coagulans NL01.

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Zheng, Zhaojuan; Lin, Xi; Jiang, Ting; Ye, Weihua; Ouyang, Jia

2016-08-01

To investigate the xylose operon and properties of xylose isomerase and xylulokinase in Bacillus coagulans that can effectively ferment xylose to lactic acid. The xylose operon is widely present in B. coagulans. It is composed of four putative ORFs. Novel xylA and xylB from B. coagulans NL01 were cloned and expressed in Escherichia coli. Sequence of xylose isomerase was more conserved than that of xylulokinase. Both the enzymes exhibited maximum activities at pH 7-8 but with a high temperature maximum of 80-85 °C, divalent metal ion was prerequisite for their activation. Xylose isomerase and xylulokinase were most effectively activated by Ni(2+) and Co(2+), respectively. Genomic analysis of xylose operon has contributed to understanding xylose metabolism in B. coagulans and the novel xylose isomerase and xylulokinase might provide new alternatives for metabolic engineering of other strains to improve their fermentation performance on xylose.

N-retinylidene-phosphatidylethanolamine is the preferred retinoid substrate for the photoreceptor-specific ABC transporter ABCA4 (ABCR).

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Beharry, Seelochan; Zhong, Ming; Molday, Robert S

2004-12-24

ABCA4, a member of the family of ATP binding cassette (ABC) proteins found in rod and cone photoreceptors, has been implicated in the transport of retinoid compounds across the outer segment disk membrane following the photoactivation of rhodopsin. Mutations in the ABCA4 gene are responsible for Stargardt macular dystrophy and related retinal degenerative diseases that cause a loss in vision. To identify the retinoid substrate that interacts with ABCA4, we have isolated ABCA4 from rod outer segment disk membranes on an immunoaffinity matrix and analyzed retinoid compounds that bind to ABCA4 using high performance liquid chromatography and radiolabeling methods. When all-trans-retinal was added to ABCA4 in the presence of phosphatidylethanolamine, approximately 0.9 mol of N-retinylidene-phosphatidylethanolamine and 0.3 mol of all-trans-retinal were bound per mol of ABCA4 with an apparent K(d) of 2-5 microm. ATP and GTP released these retinoids from ABCA4, whereas ADP, GDP, and nonhydrolyzable derivatives, adenosine 5'-(beta,gamma-imido)triphosphate and guanosine 5'-(beta,gamma-imido)triphosphate, were ineffective. One mole of N-retinyl-phosphatidylethanolamine, the reduced form of N-retinylidene-phosphatidylethanolamine, bound per mol of ABCA4, whereas 0.3 mol of all-trans-retinal were bound in the absence of phosphatidylethanolamine. No binding of all-trans-retinol to ABCA4 was observed. Our results indicate that ABCA4 preferentially binds N-retinylidene-phosphatidylethanolamine with high affinity in the absence of ATP. Our studies further suggest that ATP binding and hydrolysis induces a protein conformational change that causes N-retinylidene-phosphatidylethanolamine to dissociate from ABCA4.

Distinct populations of hepatic stellate cells in the mouse liver have different capacities for retinoid and lipid storage.

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Diana N D'Ambrosio

Full Text Available Hepatic stellate cell (HSC lipid droplets are specialized organelles for the storage of retinoid, accounting for 50-60% of all retinoid present in the body. When HSCs activate, retinyl ester levels progressively decrease and the lipid droplets are lost. The objective of this study was to determine if the HSC population in a healthy, uninjured liver demonstrates heterogeneity in its capacity for retinoid and lipid storage in lipid droplets. To this end, we utilized two methods of HSC isolation, which leverage distinct properties of these cells, including their vitamin A content and collagen expression. HSCs were isolated either from wild type (WT mice in the C57BL/6 genetic background by flotation in a Nycodenz density gradient, followed by fluorescence activated cell sorting (FACS based on vitamin A autofluorescence, or from collagen-green fluorescent protein (GFP mice by FACS based on GFP expression from a GFP transgene driven by the collagen I promoter. We show that GFP-HSCs have: (i increased expression of typical markers of HSC activation; (ii decreased retinyl ester levels, accompanied by reduced expression of the enzyme needed for hepatic retinyl ester synthesis (LRAT; (iii decreased triglyceride levels; (iv increased expression of genes associated with lipid catabolism; and (v an increase in expression of the retinoid-catabolizing cytochrome, CYP2S1.Our observations suggest that the HSC population in a healthy, uninjured liver is heterogeneous. One subset of the total HSC population, which expresses early markers of HSC activation, may be "primed" and ready for rapid response to acute liver injury.

Dose-dependent pharmacokinetics and teratogenic activity of topical retinoids

International Nuclear Information System (INIS)

Sharma, R.P.; Willhite, C.C.; Berry, D.L.; Allen, P.V.

1990-01-01

Oral retinoid treatment can be teratogenic and topical applications are used to treat acne and smooth wrinkles. A single topical trace (2.5 μg; 191 μCi/kg) or high (1.3 mg; 195 μCi/kg) dose of all-trans-[10, 11- 3 H 2 ] retinoic acid (RA) dissolved in acetone was applied to 4 cm 2 shaved dorsal hamster skin. Peak plasma radioactivity (C max ) occurred at 12 and 36 hr and mean t1/2 values for parent PA absorption were 48 min and 2.8 hr, for trace and high dose, respectively. The dermal RA C max values were only 2% of that after an equivalent oral dose, but plasma AUC after dermal treatment was 63% of the oral value. The mean t1/2 for rapid elimination was shorter for the high (57 min) than for the trace (6.9 hr) dose, but t1/2 values for slow elimination were comparable (t1/2 high = 51.2 hr; t1/2 trace = 36.8 hr). Single topical application of 10-30 mg/kg RA or 5 mg/kg etretinate (Ro 10-9359) to pregnant hamsters (day 8) caused local hyperkeratosis, but failed to induce terata. Similar application of 10-1000 μg/kg arotinoid Ro 13-6298 caused dose-dependent terata, being twice as embryolethal by parenteral as enteric dosing. Skin toxicity and attenuated maternal blood levels limit the amount of retinoids that can reach the embryo

Gremlin promotes retinal pigmentation epithelial (RPE) cell proliferation, migration and VEGF production via activating VEGFR2-Akt-mTORC2 signaling.

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Liu, Yuan; Chen, Zhijun; Cheng, Haixia; Chen, Juan; Qian, Jing

2017-01-03

Retinopathy of prematurity (ROP) is characterized by late-phase pathologic retinal vasoproliferation. Gremlin is a novel vascular endothelial growth factors (VEGF) receptor 2 (VEGFR2) agonist and promotes angiogenic response. We demonstrated that gremlin expression was significantly increased in retinas of ROP model mice, which was correlated with VEGF upregulation. In retinal pigmentation epithelial (RPE) cells, gremlin activated VEGFR2-Akt-mTORC2 (mammalian target of rapamycin complex 2) signaling, and promoted cell proliferation, migration and VEGF production. VEGFR inhibition (by SU5416) or shRNA knockdown almost abolished gremlin-mediated pleiotropic functions in RPE cells. Further, pharmacological inhibition of Akt-mTOR, or shRNA knockdown of key mTORC2 component (Rictor or Sin1) also attenuated gremlin-exerted activities in RPE cells. We conclude that gremlin promotes RPE cell proliferation, migration and VEGF production possibly via activating VEGFR2-Akt-mTORC2 signaling. Gremlin could be a novel therapeutic target of ROP or other retinal vasoproliferation diseases.

[Deficiency of triosephosphate isomerase. Apropos of 2 new cases].

Science.gov (United States)

Delso Martínez, M C; Uriel Miñana, P; Pérez Lugmus, G; Giménez Mas, J A; Baldellou Vázquez, A

1983-08-01

Two siblings, born of a no consanguineous couple, a female and a male, affected by a severe and progressive neurological disease and chronic hemolytic anemia are presented. Their clinical, hematological, biochemical and pathological studies are discussed. One of the patients showed a triosephosphate isomerase deficiency and the carrier condition of their parents was tested. Commentaries about physiopathology of this disease are made.

RETINOID RECEPTORS IN BONE AND THEIR ROLE IN BONE REMODELING

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Petra eHenning

2015-03-01

Full Text Available Vitamin A (retinol is a necessary and important constituent of the body which is provided by food intake of retinyl esters and carotenoids. Vitamin A is known best for being important for vision, but in addition to the eye, vitamin A is necessary in numerous other organs in the body, including the skeleton. Vitamin A is converted to an active compound, all-trans-retinoic acid (ATRA, which is responsible for most of its biological actions. ATRA binds to intracellular nuclear receptors called retinoic acid receptors (RAR, RAR, RAR. RARs and closely related retinoid X receptors (RXR, RXR, RXR form heterodimers which bind to DNA and function as ligand activated transcription factors. It has been known for many years that hypervitaminosis A promotes skeleton fragility by increasing osteoclast formation and decreasing cortical bone mass. Some epidemiological studies have suggested that increased intake of vitamin A and increased serum levels of retinoids may decrease bone mineral density and increase fracture rate, but the literature on this is not conclusive. The current review summarizes how vitamin A is taken up by the intestine, metabolized, stored in the liver and processed to ATRA. ATRA’s effects on formation and activity of osteoclasts and osteoblasts are outlined, and a summary of clinical data pertaining to vitamin A and bone is presented.

RPE vs. Percentage 1RM Loading in Periodized Programs Matched for Sets and Repetitions

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Eric R. Helms

2018-03-01

Full Text Available Purpose: To investigate differences between rating of perceived exertion (RPE and percentage one-repetition maximum (1RM load assignment in resistance-trained males (19–35 years performing protocols with matched sets and repetitions differentiated by load-assignment.Methods: Participants performed squats then bench press 3x/weeks in a daily undulating format over 8-weeks. Participants were counterbalanced by pre-test 1RM then assigned to percentage 1RM (1RMG, n = 11; load-assignment via percentage 1RMs, or RPE groups (RPEG, n = 10; participant-selected loads to reach target RPE ranges. Ultrasonography determined pre and post-test pectoralis (PMT, and vastus lateralis muscle thickness at 50 (VLMT50 and 70% (VLMT70 femur-length.Results: Bench press (1RMG +9.64 ± 5.36; RPEG + 10.70 ± 3.30 kg, squat (1RMG + 13.91 ± 5.89; RPEG + 17.05 ± 5.44 kg and their combined-total 1RMs (1RMG + 23.55 ± 10.38; RPEG + 27.75 ± 7.94 kg increased (p < 0.05 in both groups as did PMT (1RMG + 1.59 ± 1.33; RPEG +1.90 ± 1.91 mm, VLMT50 (1RMG +2.13 ± 1.95; RPEG + 1.85 ± 1.97 mm and VLMT70 (1RMG + 2.40 ± 2.22; RPEG + 2.31 ± 2.27 mm. Between-group differences were non-significant (p > 0.05. Magnitude-based inferences revealed 79, 57, and 72% chances of mean small effect size (ES advantages for squat; ES 90% confidence limits (CL = 0.50 ± 0.63, bench press; ES 90% CL = 0.28 ± 0.73, and combined-total; ES 90% CL = 0.48 ± 0.68 respectively, in RPEG. There were 4, 14, and 6% chances 1RMG had a strength advantage of the same magnitude, and 18, 29, and 22% chances, respectively of trivial differences between groups.Conclusions: Both loading-types are effective. However, RPE-based loading may provide a small 1RM strength advantage in a majority of individuals.

Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS/TEN): could retinoids play a causative role?

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Mawson, Anthony R; Eriator, Ike; Karre, Sridhar

2015-01-12

Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) are overlapping manifestations on a spectrum of acute drug-induced conditions associated with severe blistering, skin peeling, and multi-organ damage. TEN is an eruption resembling severe scalding, with ≥30% skin detachment. SJS is a mild form of TEN, characterized histologically by epidermal keratinocyte apoptosis with dermo-epidermal separation and extensive small blisters with <10% body surface skin detachment. The syndrome can be induced by numerous medications and typically occurs 1-4 weeks after the initiation of therapy. Granulysin is found in the lesions of patients with SJS/TEN and plays a significant pathogenic role in the condition, but the overall mechanisms linking medications, granulysin, and disease manifestations remain obscure. This paper reviews evidence suggesting that the different medications implicated in SJS/TEN have the common property of interacting and synergizing with endogenous retinoids (vitamin A and its congeners), in many instances causing the latter to accumulate in and damage the liver, the main storage organ for vitamin A. It is hypothesized that liver damage leads to the spillage of toxic retinoid compounds into the circulation, resulting in an endogenous form of hypervitaminosis A and cytotoxicity with widespread apoptosis, mediated by granulysin and recognized as SJS/TEN. Subject to testing, the model suggests that symptom worsening could be arrested at onset by lowering the concentration of circulating retinoids and/or granulysin via phlebotomy or plasmapheresis or by pharmacological measures to limit their expression.

Peretinoin, an Acyclic Retinoid, Inhibits Hepatitis B Virus Replication by Suppressing Sphingosine Metabolic Pathway In Vitro

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Kazuhisa Murai

2018-01-01

Full Text Available Hepatocellular carcinoma (HCC frequently develops from hepatitis C virus (HCV and hepatitis B virus (HBV infection. We previously reported that peretinoin, an acyclic retinoid, inhibits HCV replication. This study aimed to examine the influence of peretinoin on the HBV lifecycle. HBV-DNA and covalently closed circular DNA (cccDNA were evaluated by a qPCR method in HepG2.2.15 cells. Peretinoin significantly reduced the levels of intracellular HBV-DNA, nuclear cccDNA, and HBV transcript at a concentration that did not induce cytotoxicity. Conversely, other retinoids, such as 9-cis, 13-cis retinoic acid (RA, and all-trans-retinoic acid (ATRA, had no effect or rather increased HBV replication. Mechanistically, although peretinoin increased the expression of HBV-related transcription factors, as observed for other retinoids, peretinoin enhanced the binding of histone deacetylase 1 (HDAC1 to cccDNA in the nucleus and negatively regulated HBV transcription. Moreover, peretinoin significantly inhibited the expression of SPHK1, a potential inhibitor of HDAC activity, and might be involved in hepatic inflammation, fibrosis, and HCC. SPHK1 overexpression in cells cancelled the inhibition of HBV replication induced by peretinoin. This indicates that peretinoin activates HDAC1 and thereby suppresses HBV replication by inhibiting the sphingosine metabolic pathway. Therefore, peretinoin may be a novel therapeutic agent for HBV replication and chemoprevention against HCC.

Spontaneous correction of anterior crossbite by RPE anchored on deciduous teeth in the early mixed dentition.

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Rosa, M; Lucchi, P; Mariani, L; Caprioglio, A

2012-09-01

The purpose of this study was to evaluate the effectiveness of Haas RPE anchored on deciduous teeth in the early mixed dentition, for inducing the spontaneous correction of permanent incisor's crossbite, without compliance, without post bite-plane and no involvement of the permanent teeth. The sample group comprised 50 consecutive patients (mean age 8y 5m, SD 2y 1m), 31 males, 19 females. They showed a cross-bite affecting one or more permanent incisors, for a total of 70 teeth. The patients were treated with Haas RPE appliance anchored on second deciduous molars and bonded on deciduous canines. No direct forces were applied on the permanent teeth. Anterior crossbite self-corrected 'spontaneously' in 84% of the cases. Lateral incisors had a higher rate of self-correction than central incisors. All hyper-divergent subjects showed a spontaneous crossbite self-correction. The early maxillary expansion by Haas RPE anchored on deciduous teeth is an efficient and effective procedure to induce the anterior crossbite self-correction in the early mixed dentition without the need of a bite-plane, no involvement of the permanent teeth and without compliance.

Evaluation of retinoids for induction of the redundant gene ABCD2 as an alternative treatment option in X-linked adrenoleukodystrophy.

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Franziska D Weber

Full Text Available X-linked adrenoleukodystrophy (X-ALD, the most common peroxisomal disorder, is a clinically heterogeneous disease that can manifest as devastating inflammatory cerebral demyelination (CALD leading to death of affected males. Currently, the only curative treatment is allogeneic hematopoietic stem cell transplantation (HSCT. However, HSCT is only effective when performed at an early stage because the inflammation may progress for eighteen months after HSCT. Thus, alternative treatment options able to immediately halt the progression are urgently needed. X-ALD is caused by mutations in the ABCD1 gene, encoding the peroxisomal membrane protein ABCD1, resulting in impaired very long-chain fatty acid metabolism. The related ABCD2 protein is able to functionally compensate for ABCD1-deficiency both in vitro and in vivo. Recently, we demonstrated that of the cell types derived from CD34+ stem cells, predominantly monocytes but not lymphocytes are metabolically impaired in X-ALD. As ABCD2 is virtually not expressed in these cells, we hypothesize that a pharmacological up-regulation of ABCD2 should compensate metabolically and halt the inflammation in CALD. Retinoids are anti-inflammatory compounds known to act on ABCD2. Here, we investigated the capacity of selected retinoids for ABCD2 induction in human monocytes/macrophages. In THP-1 cells, 13-cis-retinoic acid reached the highest, fivefold, increase in ABCD2 expression. To test the efficacy of retinoids in vivo, we analyzed ABCD2 mRNA levels in blood cells isolated from acne patients receiving 13-cis-retinoic acid therapy. In treated acne patients, ABCD2 mRNA levels were comparable to pre-treatment levels in monocytes and lymphocytes. Nevertheless, when primary monocytes were in vitro differentiated into macrophages and treated with 13-cis-retinoic acid, we observed a fourfold induction of ABCD2. However, the level of ABCD2 induction obtained by retinoids alone is probably not of therapeutic relevance

TNF-α promotes human retinal pigment epithelial (RPE) cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression through activation of Akt/mTORC1 signaling

International Nuclear Information System (INIS)

Wang, Cheng-hu; Cao, Guo-Fan; Jiang, Qin; Yao, Jin

2012-01-01

Highlights: ► TNF-α induces MMP-9 expression and secretion to promote RPE cell migration. ► MAPK activation is not critical for TNF-α-induced MMP-9 expression. ► Akt and mTORC1 signaling mediate TNF-α-induced MMP-9 expression. ► SIN1 knockdown showed no significant effect on MMP-9 expression by TNF-α. -- Abstract: Tumor necrosis factor-alpha (TNF-α) promotes in vitro retinal pigment epithelial (RPE) cell migration to initiate proliferative vitreoretinopathy (PVR). Here we report that TNF-α promotes human RPE cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression. Inhibition of MMP-9 by its inhibitor or its neutralizing antibody inhibited TNF-α-induced in vitro RPE cell migration. Reversely, exogenously-added active MMP-9 promoted RPE cell migration. Suppression Akt/mTOR complex 1(mTORC1) activation by LY 294002 and rapamycin inhibited TNF-α-mediated MMP-9 expression. To introduce a constitutively active Akt (CA-Akt) in cultured RPE cells increased MMP-9 expression, and to block mTORC1 activation by rapamycin inhibited its effect. RNA interference (RNAi)-mediated silencing of SIN1, a key component of mTOR complex 2 (mTORC2), had no effect on MMP-9 expression or secretion. In conclusion, this study suggest that TNF-α promotes RPE cell migration by inducing MMP-9 expression through activation of Akt/ mTORC1, but not mTORC2 signaling.

TNF-{alpha} promotes human retinal pigment epithelial (RPE) cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression through activation of Akt/mTORC1 signaling

Energy Technology Data Exchange (ETDEWEB)

Wang, Cheng-hu; Cao, Guo-Fan [The Affiliated Eye Hospital of Nanjing Medical University, Nanjing 210029 (China); Jiang, Qin, E-mail: Jqin710@vip.sina.com [The Affiliated Eye Hospital of Nanjing Medical University, Nanjing 210029 (China); Yao, Jin, E-mail: dryaojin@yahoo.com [The Affiliated Eye Hospital of Nanjing Medical University, Nanjing 210029 (China)

2012-08-17

Highlights: Black-Right-Pointing-Pointer TNF-{alpha} induces MMP-9 expression and secretion to promote RPE cell migration. Black-Right-Pointing-Pointer MAPK activation is not critical for TNF-{alpha}-induced MMP-9 expression. Black-Right-Pointing-Pointer Akt and mTORC1 signaling mediate TNF-{alpha}-induced MMP-9 expression. Black-Right-Pointing-Pointer SIN1 knockdown showed no significant effect on MMP-9 expression by TNF-{alpha}. -- Abstract: Tumor necrosis factor-alpha (TNF-{alpha}) promotes in vitro retinal pigment epithelial (RPE) cell migration to initiate proliferative vitreoretinopathy (PVR). Here we report that TNF-{alpha} promotes human RPE cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression. Inhibition of MMP-9 by its inhibitor or its neutralizing antibody inhibited TNF-{alpha}-induced in vitro RPE cell migration. Reversely, exogenously-added active MMP-9 promoted RPE cell migration. Suppression Akt/mTOR complex 1(mTORC1) activation by LY 294002 and rapamycin inhibited TNF-{alpha}-mediated MMP-9 expression. To introduce a constitutively active Akt (CA-Akt) in cultured RPE cells increased MMP-9 expression, and to block mTORC1 activation by rapamycin inhibited its effect. RNA interference (RNAi)-mediated silencing of SIN1, a key component of mTOR complex 2 (mTORC2), had no effect on MMP-9 expression or secretion. In conclusion, this study suggest that TNF-{alpha} promotes RPE cell migration by inducing MMP-9 expression through activation of Akt/ mTORC1, but not mTORC2 signaling.

SAXS-WAXS studies of the low-resolution structure in solution of xylose/glucose isomerase from Streptomyces rubiginosus

Science.gov (United States)

Kozak, Maciej; Taube, Michał

2009-10-01

The structure and conformation of molecule of xylose/glucose isomerase from Streptomyces rubiginosus in solution (at pH 6 and 7.6; with and without the substrate) has been studied by small- and wide-angle scattering of synchrotron radiation (SAXS-WAXS). On the basis of the SAXS-WAXS data, the low-resolution structure in solution has been reconstructed using ab inito methods. A comparison of the models of glucose isomerase shows only small differences between the model in solution and the crystal structure.

Assessment of the efficacy and safety of a combination of 2 topical retinoids (RetinSphere) in maintaining post-treatment response of acne to oral isotretinoin.

Science.gov (United States)

Truchuelo, M T; Jiménez, N; Mavura, D; Jaén, P

2015-03-01

The high rate of relapse of acne lesions following oral isotretinoin treatment is a common problem which remains unsolved. To avoid or minimize relapses, topical retinoids have been used for many years as maintenance treatment. However, adverse effects frequently occur. To determine the efficacy and safety of a new retinoid combination (Retinsphere technology) in maintaining post-treatment response to oral isotretinoin. Prospective, randomized, double-blind and vehicle-controlled study of 30 patients with acne previously treated with isotretinoin. Treatment with the retinoid combination was applied to one side of the face and vehicle was applied to the other, once daily, for 3 months. Standardized photographs were taken using RBX technology at baseline, 1.5 months and 3 months. The primary efficacy endpoint was the appearance of relapse on the treated side compared to the vehicle-treated side. Other endpoints included lesion count, investigator-reported improvement, patient-reported improvement, impact on quality-of-life, and side effects. Although the majority of patients did not reach the total target dose of oral isotretinoin, the relapse rate was significantly lower on the retinoid-treated side compared to the vehicle-treated side. Likewise, improved lesion count and excellent tolerance were observed. This new retinoid combination (Retinsphere technology) were effective and safe as maintenance therapy after post-treatment response to oral isotretinoin in patients with acne. Copyright © 2014 Elsevier España, S.L.U. and AEDV. All rights reserved.

Pathogenesis and prophylaxis of AMD: focus on oxidative stress and antioxidants

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Anna Wiktorowska-Owczarek

2010-07-01

Full Text Available Age-related macular degeneration (AMD is the leading cause of severe visual loss and blindness in people over 55. Its pathogenesis – likely multifactorial, involving a complex interaction of metabolic, functional, genetic and environmental factors – remains poorly understood. Among molecular links in pathogenesis of AMD is the oxidative stress in the retina, a structure that is particularly susceptible to damage by reactive oxygen species (ROS since photoreceptor outer segment (POS membranes are rich in polyunsaturated fatty acids which can be readily oxidized and can initiate a cytotoxic chain reaction. Occurring in the neighborhood of photoreceptors, the retinal pigment epithelial cells (RPE actively contribute to both the retinoid cycle and catabolism of constantly shed and phagocytized parts of photoreceptor outer segments. Enzymatic degradation of photoreceptor fragments occurring in RPE phagolysosomes is not complete and undigested material in the form of insoluble aggregates, called lipofuscin, is deposited in lysosomes of RPE cells. Lipofuscin contains a mixture of diverse molecular components including retinoid-derived compounds, some of which displaying potent photoinducible properties, contributing to an enhancement and propagation of the oxidative stress. The retina possesses defense mechanisms against the oxidative stress that effectively neutralize the consequences of reactive oxygen species actions under normal conditions. A key role in the antioxidant defense plays an array of substances, including: xanthophylls (lutein and zeaxanthin, vitamin C and E, and glutathione. This paper surveys the current concepts on the role of the oxidative stress in pathophysiology of AMD, and describes major components of the antioxidant defense system, including their use in AMD prophylaxis and therapy.

The action of a dietary retinoid on gene expression and cancer induction in electron-irradiated rat skin

International Nuclear Information System (INIS)

Burns, F.J.; Chen, S.; Xu, G.; Wu, F.; Tang, M.S.

2002-01-01

Current models of radiation carcinogenesis generally assume that the DNA is damaged in a variety of ways by the radiation and that subsequent cell divisions contribute to the conversion of the damage to heritable mutations. Cancer may seem complex and intractable, but its complexity provides multiple opportunities for preventive interventions. Mitotic inhibitors are among the strongest cancer preventive agents, not only slowing the growth rate of preneoplasias but also increasing the fidelity of DNA repair processes. Ionizing radiation, including electrons, is a strong inducer of cancer in rat skin, and dietary retinoids have shown potent cancer preventive activity in the same system. A non-toxic dietary dose of retinyl acetate altered gene expression levels 24 hours after electron irradiation of rat skin. Of the 8740 genes on an Affymetrix rat expression array, the radiation significantly (5 fold or higher) altered 188, while the retinoid altered 231, including 16 radiation-altered genes that were reversely altered. While radiation strongly affected the expression of stress response, immune/inflammation and nucleic acid metabolism genes, the retinoid most strongly affected proliferation-related genes, including some significant reversals, such as, keratin 14, retinol binding protein, and calcium binding proteins. These results point to reversal of proliferation-relevant genes as a likely basis for the anti-radiogenic effects of dietary retinyl acetate. (author)

Affinity labeling and characterization of the active site histidine of glucosephosphate isomerase

International Nuclear Information System (INIS)

Gibson, D.R.; Gracy, R.W.; Hartman, F.C.

1980-01-01

N-bromoacetylethanolamine phosphate was found to act as a specific affinity label for the active center of glucosephosphate isomerase. The inactivation process followed pseudo-first order kinetics, was irreversible, and exhibited rate saturation kinetics with minimal half-lives of inactivation of 4.5 and 6.3 min for the enzyme isolated from human placenta and rabbit muscle, respectively. The pH dependence of the inactivation process closely paralleled the pH dependence of the overall catalytic process with pK/sub a/ values at pH 6.4 and 9.0. The stoichiometry of labeling of either enzyme, as determined with N-bromo[ 14 C 2 ]acetylethanolamine phosphate, was 1 eq of the affinity label/subunit of enzyme. After acid hydrolysis and amino acid analysis of the radioactive affinity-labeled human enzyme, only radioactive 3-carboxymethyl histidine was found. In the case of the rabbit enzyme, the only radioactive derivative obtained was 1-carboxymethyl histidine. Active site tryptic peptides were isolated by solvent extraction, thin layer peptide fingerprinting, and ion exchange chromatography before and after removal of the phosphate from the active site peptide. Amino acid analysis of the labeled peptides from the two species were very similar. Using high sensitivity methods for sequence analysis, the primary structure of the active site was established as Val-Leu-His-Ala-Glu-Asn-Val-Asp (Gly,Thr,Ser) Glu-Ile (Thr-Gly-His-Lys-Glx)-Tyr-Phe. Apparent sequence homology between the catalytic center of glucosephosphate isomerase and triosephosphate isomerase suggest that the two enzymes may have evolved from a common ancestral gene

Identification of a class of nonlinear state-space models using RPE techniques

DEFF Research Database (Denmark)

Zhou, W. W.; Blanke, Mogens

1986-01-01

The recursive prediction error methods in state-space form have been efficiently used as parameter identifiers for linear systems, and especially Ljung's innovations filter using a Newton search direction has proved to be quite ideal. In this paper, the RPE method in state-space form is developed...... a quite convincing performance of the filter as combined parameter and state estimator....

Studies on the production of glucose isomerase by Bacillus licheniformis

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Nwokoro Ogbonnaya

2015-09-01

Full Text Available This work reports the effects of some culture conditions on the production of glucose isomerase by Bacillus licheniformis. The bacterium was selected based on the release of 3.62 mg/mL fructose from the fermentation of glucose. Enzyme was produced using a variety of carbon substrates but the highest enzyme activity was detected in a medium containing 0.5% xylose and 1% glycerol (specific activity = 6.88 U/mg protein. Media containing only xylose or glucose gave lower enzyme productivies (specific activities= 4.60 and 2.35 U/mg protein respectively. The effects of nitrogen substrates on glucose isomerase production showed that yeast extract supported maximum enzyme activity (specific activity = 5.24 U/mg protein. Lowest enzyme activity was observed with sodium trioxonitrate (specific activity = 2.44 U/mg protein. In general, organic nitrogen substrates supported higher enzyme productivity than inorganic nitrogen substrates. Best enzyme activity was observed in the presence of Mg2+ (specific activity = 6.85 U/mg protein while Hg2+ was inhibitory (specific activity = 1.02 U/mg protein. The optimum pH for best enzyme activity was 6.0 while optimum temperature for enzyme production was 50ºC.

Structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC118.

Science.gov (United States)

Lobley, Carina M C; Aller, Pierre; Douangamath, Alice; Reddivari, Yamini; Bumann, Mario; Bird, Louise E; Nettleship, Joanne E; Brandao-Neto, Jose; Owens, Raymond J; O'Toole, Paul W; Walsh, Martin A

2012-12-01

The structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC188 has been determined at 1.72 Å resolution. The structure was solved by molecular replacement, which identified the functional homodimer in the asymmetric unit. Despite only showing 57% sequence identity to its closest homologue, the structure adopted the typical α and β D-ribose 5-phosphate isomerase fold. Comparison to other related structures revealed high homology in the active site, allowing a model of the substrate-bound protein to be proposed. The determination of the structure was expedited by the use of in situ crystallization-plate screening on beamline I04-1 at Diamond Light Source to identify well diffracting protein crystals prior to routine cryocrystallography.

Reversal of blindness in animal models of leber congenital amaurosis using optimized AAV2-mediated gene transfer.

Science.gov (United States)

Bennicelli, Jeannette; Wright, John Fraser; Komaromy, Andras; Jacobs, Jonathan B; Hauck, Bernd; Zelenaia, Olga; Mingozzi, Federico; Hui, Daniel; Chung, Daniel; Rex, Tonia S; Wei, Zhangyong; Qu, Guang; Zhou, Shangzhen; Zeiss, Caroline; Arruda, Valder R; Acland, Gregory M; Dell'Osso, Lou F; High, Katherine A; Maguire, Albert M; Bennett, Jean

2008-03-01

We evaluated the safety and efficacy of an optimized adeno-associated virus (AAV; AAV2.RPE65) in animal models of the RPE65 form of Leber congenital amaurosis (LCA). Protein expression was optimized by addition of a modified Kozak sequence at the translational start site of hRPE65. Modifications in AAV production and delivery included use of a long stuffer sequence to prevent reverse packaging from the AAV inverted-terminal repeats, and co-injection with a surfactant. The latter allows consistent and predictable delivery of a given dose of vector. We observed improved electroretinograms (ERGs) and visual acuity in Rpe65 mutant mice. This has not been reported previously using AAV2 vectors. Subretinal delivery of 8.25 x 10(10) vector genomes in affected dogs was well tolerated both locally and systemically, and treated animals showed improved visual behavior and pupillary responses, and reduced nystagmus within 2 weeks of injection. ERG responses confirmed the reversal of visual deficit. Immunohistochemistry confirmed transduction of retinal pigment epithelium cells and there was minimal toxicity to the retina as judged by histopathologic analysis. The data demonstrate that AAV2.RPE65 delivers the RPE65 transgene efficiently and quickly to the appropriate target cells in vivo in animal models. This vector holds great promise for treatment of LCA due to RPE65 mutations.

Glucose(xylose isomerase production by Streptomyces sp. CH7 grown on agricultural residues

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Kankiya Chanitnun

2012-09-01

Full Text Available Streptomyces sp. CH7 was found to efficiently produce glucose(xylose isomerase when grown on either xylan or agricultural residues. This strain produced a glucose(xylose isomerase activity of roughly 1.8 U/mg of protein when it was grown in medium containing 1% xylose as a carbon source. Maximal enzymatic activities of about 5 and 3 U/mg were obtained when 1% xylan and 2.5% corn husks were used, respectively. The enzyme was purified from a mycelial extract to 16-fold purity with only two consecutive column chromatography steps using Macro-prep DEAE and Sephacryl-300, respectively. The approximate molecular weight of the purified enzyme is 170 kDa, and it has four identical subunits of 43.6 kDa as estimated by SDS-PAGE. Its Km values for glucose and xylose were found to be 258.96 and 82.77 mM, respectively, and its Vmax values are 32.42 and 63.64 μM/min/mg, respectively. The purified enzyme is optimally active at 85ºC and pH 7.0. It is stable at pH 5.5-8.5 and at temperatures up to 60ºC after 30 min. These findings indicate that glucose(xylose isomerase from Streptomyces sp. CH7 has the potential for industrial applications, especially for high-fructose syrup production and bioethanol fermentation from hemicellulosic hydrolysates by Saccharomyces cerevisiae.

Liver X receptors balance lipid stores in hepatic stellate cells through Rab18, a retinoid responsive lipid droplet protein.

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O'Mahony, Fiona; Wroblewski, Kevin; O'Byrne, Sheila M; Jiang, Hongfeng; Clerkin, Kara; Benhammou, Jihane; Blaner, William S; Beaven, Simon W

2015-08-01

Liver X receptors (LXRs) are determinants of hepatic stellate cell (HSC) activation and liver fibrosis. Freshly isolated HSCs from Lxrαβ(-/-) mice have increased lipid droplet (LD) size, but the functional consequences of this are unknown. Our aim was to determine whether LXRs link cholesterol to retinoid storage in HSCs and how this impacts activation. Primary HSCs from Lxrαβ(-/-) and wild-type mice were profiled by gene array during in vitro activation. Lipid content was quantified by high-performance liquid chromatography and mass spectroscopy. Primary HSCs were treated with nuclear receptor ligands, transfected with small interfering RNA and plasmid constructs, and analyzed by immunocytochemistry. Lxrαβ(-/-) HSCs have increased cholesterol and retinyl esters. The retinoid increase drives intrinsic retinoic acid receptor signaling, and activation occurs more rapidly in Lxrαβ(-/-) HSCs. We identify Rab18 as a novel retinoic acid-responsive, LD-associated protein that helps mediate stellate cell activation. Rab18 mRNA, protein, and membrane insertion increase during activation. Both Rab18 guanosine triphosphatase activity and isoprenylation are required for stellate cell LD loss and induction of activation markers. These phenomena are accelerated in Lxrαβ(-/-) HSCs, where there is greater retinoic acid flux. Conversely, Rab18 knockdown retards LD loss in culture and blocks activation, just like the functional mutants. Rab18 is also induced with acute liver injury in vivo. Retinoid and cholesterol metabolism are linked in stellate cells by the LD-associated protein Rab18. Retinoid overload helps explain the profibrotic phenotype of Lxrαβ(-/-) mice, and we establish a pivotal role for Rab18 GTPase activity and membrane insertion in wild-type stellate cell activation. Interference with Rab18 may have significant therapeutic benefit in ameliorating liver fibrosis. © 2015 by the American Association for the Study of Liver Diseases.

Liver X Receptors Balance Lipid Stores in Hepatic Stellate Cells via Rab18, a Retinoid Responsive Lipid Droplet Protein

Science.gov (United States)

O’Mahony, Fiona; Wroblewski, Kevin; O’Byrne, Sheila M.; Jiang, Hongfeng; Clerkin, Kara; Benhammou, Jihane; Blaner, William S.; Beaven, Simon W.

2014-01-01

Liver X receptors (LXRs) are determinants of hepatic stellate cell (HSC) activation and liver fibrosis. Freshly isolated HSCs from Lxrαβ−/− mice have increased lipid droplet (LD) size but the functional consequences of this are unknown. Our aim was to determine whether LXRs link cholesterol to retinoid storage in HSCs and how this impacts activation. Primary HSCs from Lxrαβ−/− and wild-type (WT) mice were profiled by gene array during in vitro activation. Lipid content was quantified by HPLC and mass spectroscopy. Primary HSCs were treated with nuclear receptor ligands, transfected with siRNA and plasmid constructs, and analyzed by immunocytochemistry. Lxrαβ−/− HSCs have increased cholesterol and retinyl esters (CEs & REs). The retinoid increase drives intrinsic retinoic acid receptor (RAR) signaling and activation occurs more rapidly in Lxrαβ−/− HSCs. We identify Rab18 as a novel retinoic acid responsive, lipid droplet associated protein that helps mediate stellate cell activation. Rab18 mRNA, protein, and membrane insertion increase during activation. Both Rab18 GTPase activity and isoprenylation are required for stellate cell lipid droplet loss and induction of activation markers. These phenomena are accelerated in the Lxrαβ−/− HSCs, where there is greater retinoic acid flux. Conversely, Rab18 knockdown retards lipid droplet loss in culture and blocks activation, just like the functional mutants. Rab18 is also induced with acute liver injury in vivo. Conclusion Retinoid and cholesterol metabolism are linked in stellate cells by the LD associated protein, Rab18. Retinoid overload helps explain the pro-fibrotic phenotype of Lxrαβ−/− mice and we establish a pivotal role for Rab18 GTPase activity and membrane insertion in wild-type stellate cell activation. Interference with Rab18 may have significant therapeutic benefit in ameliorating liver fibrosis. PMID:25482505

Carotenoids and retinoids in Finnish foods: dairy products and eggs.

Science.gov (United States)

Ollilainen, V; Heinonen, M; Linkola, E; Varo, P; Koivistoinen, P

1989-09-01

As part of an overall composition study of Finnish foods, the carotenoid and retinoid content of 20 dairy product samples and eggs were determined by HPLC. The total beta-carotene (all-trans beta-carotene plus 15-cis beta-carotene) was quantitated for dairy products. For egg and egg yolk, lutein content was also determined. Only traces of lycopene, cryptoxanthin, and alpha-carotene were present. All-trans retinol and 13-cis retinol were the major retinoids in dairy products. Small amounts of 9-cis, 11-cis, and 9,11-cis retinols were found. High values of both retinol and beta-carotene were found in full fat cheeses and whipping cream: from 179.0 (cheese, Edam-type) to 318.7 micrograms/100 g (whipping cream) and from 86.7 (cheese, Edam-type) to 186.5 micrograms/100 g (whipping cream) for all-trans retinol and total beta-carotene, respectively. The retinol content averaged 16.3, 32.6, and 52.2 and that of beta-carotene 9.6, 16.7, and 3.0 micrograms/100 g in milk (1.9% fat), milk (3.9% fat), and human milk, respectively. The major pigment in eggs and egg yolk was lutein, 619.5 micrograms/100 g in eggs and 1575.8 micrograms/100 g in egg yolk. According to this study, at the present level of consumption in Finland, milk, milk products (excluding butter), and eggs result in a daily intake of about 350 retinol equivalents, and consequently, are a major source of vitamin A.

Direct production of D-arabinose from D-xylose by a coupling reaction using D-xylose isomerase, D-tagatose 3-epimerase and D-arabinose isomerase.

Science.gov (United States)

Sultana, Ishrat; Mizanur, Rahman Md; Takeshita, Kei; Takada, Goro; Izumori, Ken

2003-01-01

Klebsiella pneumoniae 40bXX, a mutant strain that constitutively produces D-arabinose isomerase (D-AI), was isolated through a series of repeated subcultures from the parent strain on a mineral salt medium supplemented with L-Xylose as the sole carbon source. D-AI could be efficiently immobilized on chitopearl beads. The optimum temperature for the activity of the immobilized enzyme was 40 degrees C and the enzyme was stable up to 50 degrees C. The D-Al was active at pH 10.0 and was stable in the range of pH 6.0-11.0. The enzyme required manganese ions for maximum activity. Three immobilized enzymes, D-xylose isomerase (D-XI), D-tagatose 3-epimerase (D-TE and D-AI were used for the preparation of D-arabinose from D-xylose in a coupling reaction. After completion of the reaction, degradation of D-xylulose was carried out by Saccharomyces cerevisiae. The reaction mixture containing D-Xylose, D-ribulose and the product was then separated by ion exchange column chromatography. After crystallization, the product was checked by HPLC, IR spectroscopy, NMR spectroscopy and optical rotation measurements. Finally, 2.0 g of D-arabinose could be obtained from 5 g of the substrate.

Cell-mediated immunity against human retinal extract, S-antigen, and interphotoreceptor retinoid binding protein in onchocercal chorioretinopathy

NARCIS (Netherlands)

van der Lelij, A.; Rothova, A.; Stilma, J. S.; Hoekzema, R.; Kijlstra, A.

1990-01-01

Autoimmune mechanisms are thought to be involved in the pathogenesis of onchocercal chorioretinopathy. Cell-mediated immune responses to human retinal S-antigen, interphotoreceptor retinoid binding protein (IRBP), and crude retinal extract were investigated in patients with onchocerciasis from

Retinoid Expression in Onchocercal Skin Disease: Pilot Study.

Science.gov (United States)

Mawson, Anthony R; Makunde, Williams H; Penman, Alan D; Hernandez Morales, Veronica de Los Angeles; Kalinga, Akili K; Francis, Filbert; Rubinchik, Semyon; Kibweja, Addow

2017-01-01

Based on the observation that the parasite Onchocerca volvulus selectively absorbs vitamin A from the host, and the known toxicity of vitamin A in higher concentration, it was hypothesized that dying microfilariae (mf) release their stores of vitamin A (retinoids) into the host circulation in toxic concentrations, inducing the signs and symptoms of onchocerciasis. We conducted a pilot study to test the hypothesis in Songea communities in Southern Tanzania, where mass drug administration with ivermectin had not been implemented by the time of the survey. The specific aim was to evaluate the correlation between the diagnosis of onchocerciasis and increased levels of retinoic acid at infection sites. The analysis was performed by determining copy numbers of a genome of O volvulus present in skin snip samples of persons with onchocerciacis, and correlating these numbers with expression levels of retinoic acid receptor-α (RAR-α), which is inducible by retinoic acid. Total DNA and RNA were extracted from each of 25 mf-positive and 25 mf-negative skin samples and evaluated using quantitative polymerase chain reaction with appropriate negative controls. Analysis of the samples, adjusted with glyceraldehyde 3-phosphate dehydrogenase gene levels, revealed that most samples with detectable RAR-α transcripts had higher levels of RAR-α expression than the assay control. However, the quality and number of samples were insufficient for statistical analysis. Fold data on the expression levels of both O volvulus DNA and RAR RNA suggested a possible trend toward higher relative RAR-α expression in samples with higher levels of O volvulus DNA ( r 2  = 0.25, P  = .079). Evidence of a contribution of vitamin A to the pathology of onchocerciasis thus remains elusive. Future studies on the role of retinoids in onchocerciasis will require larger groups of participants as well as careful monitoring of the cold chain and tissue storage procedures in view of the sensitivity of

Cystic fibrosis transmembrane conductance regulator contributes to reacidification of alkalinized lysosomes in RPE cells.

Science.gov (United States)

Liu, Ji; Lu, Wennan; Guha, Sonia; Baltazar, Gabriel C; Coffey, Erin E; Laties, Alan M; Rubenstein, Ronald C; Reenstra, William W; Mitchell, Claire H

2012-07-15

The role of the cystic fibrosis transmembrane conductance regulator (CFTR) in lysosomal acidification has been difficult to determine. We demonstrate here that CFTR contributes more to the reacidification of lysosomes from an elevated pH than to baseline pH maintenance. Lysosomal alkalinization is increasingly recognized as a factor in diseases of accumulation, and we previously showed that cAMP reacidified alkalinized lysosomes in retinal pigmented epithelial (RPE) cells. As the influx of anions to electrically balance proton accumulation may enhance lysosomal acidification, the contribution of the cAMP-activated anion channel CFTR to lysosomal reacidification was probed. The antagonist CFTR(inh)-172 had little effect on baseline levels of lysosomal pH in cultured human RPE cells but substantially reduced the reacidification of compromised lysosomes by cAMP. Likewise, CFTR activators had a bigger impact on cells whose lysosomes had been alkalinized. Knockdown of CFTR with small interfering RNA had a larger effect on alkalinized lysosomes than on baseline levels. Inhibition of CFTR in isolated lysosomes altered pH. While CFTR and Lamp1 were colocalized, treatment with cAMP did not increase targeting of CFTR to the lysosome. The inhibition of CFTR slowed lysosomal degradation of photoreceptor outer segments while activation of CFTR enhanced their clearance from compromised lysosomes. Activation of CFTR acidified RPE lysosomes from the ABCA4(-/-) mouse model of recessive Stargardt's disease, whose lysosomes are considerably alkalinized. In summary, CFTR contributes more to reducing lysosomal pH from alkalinized levels than to maintaining baseline pH. Treatment to activate CFTR may thus be of benefit in disorders of accumulation associated with lysosomal alkalinization.

Bioconversion of D-galactose into D-tagatose by expression of L-arabinose isomerase.

Science.gov (United States)

Roh, H J; Kim, P; Park, Y C; Choi, J H

2000-02-01

D-Tagatose is a potential bulking agent in food as a non-calorific sweetener. To produce D-tagatose from cheaper resources, plasmids harbouring the L-arabinose isomerase gene (araA) from Escherichia coli, Bacillus subtilis and Salmonella typhimurium were constructed because L-arabinose isomerase was suggested previously as an enzyme that mediates the bioconversion of galactose into tagatose as well as that of arabinose to ribulose. The constructed plasmids were named pTC101, pTC105 and pTC106, containing araA from E. coli, B. subtilis and S. typhimurium respectively. In the cultures of recombinant E. coli with pTC101, pTC105 and pTC106, tagatose was produced from galactose in 9.9, 7.1 and 6.9% yields respectively. The enzyme extract of E. coli with the plasmid pTC101 also converted galactose into tagatose with a 96.4% yield.

Duration of oral tetracycline-class antibiotic therapy and use of topical retinoids for the treatment of acne among general practitioners (GP): A retrospective cohort study.

Science.gov (United States)

Barbieri, John S; Hoffstad, Ole; Margolis, David J

2016-12-01

Guidelines recommend limiting the duration of oral antibiotic therapy in acne to 3 to 6 months and prescribing concomitant topical retinoids for all patients. We sought to evaluate the duration of therapy with oral tetracyclines and the use of topical retinoids among patients with acne treated primarily by general practitioners in the United Kingdom. We conducted a retrospective cohort study using the Health Improvement Network database. The mean duration of therapy was 175.1 days. Of antibiotic courses, 62% were not associated with a topical retinoid; 29% exceeded 6 months in duration. If all regions were to achieve uses similar to the region with the shortest mean duration of therapy, approximately 3.3 million antibiotic days per year could be avoided in the United Kingdom. The Health Improvement Network does not include information on acne severity and clinical outcomes. Prescribing behavior for oral antibiotics in the treatment of acne among general practitioners is not aligned with current guideline recommendations. Increasing the use of topical retinoids and considering alternative agents to oral antibiotics when appropriate represent opportunities to reduce antibiotic exposure and associated complications such as antibiotic resistance and to improve outcomes in patients treated for acne. Copyright © 2016 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.

Ultrastructure and 3H-TdR autoradiography after topical application of aromatic retinoid in guinea pigs

International Nuclear Information System (INIS)

Ueda, Keiichi; Maruo, Mitsuru; Inoue, Yuji; Wakabayashi, Shunji

1981-01-01

Histological, autoradiographical and electron microscopical investigations were performed on the epidermis of guinea pigs after topical application of 0.1% aromatic retinoid ointment for 7 days. Histologically, the epidermis exhibited a thickening of granular layer and acanthosis, without a prolonged rete ridge. Labelling indices were 3 to 4 times higher than in the control epidermis. Electron microscopically, the nuclei in the spinous and the basal layer were large in size and nucleoli were increased in number. In the cytoplasm, tonofilaments were reduced in number and lots of vacuoles containing less dense amorphous material appeared. Amorphous material was present in the intercellular spaces of the epidermis. Differentiation and proliferation of the epidermis in guinea pigs after topical application of aromatic retinoid showed almost the same response in quantity to the epidermis after topical application of retinoic acid, while the epidermal alternation was slight in degree than in the epidermis after topical application of retinoic acid. (author)

Mechanism of RPE cell death in α-crystallin deficient mice: a novel and critical role for MRP1-mediated GSH efflux.

Directory of Open Access Journals (Sweden)

Parameswaran G Sreekumar

Full Text Available Absence of α-crystallins (αA and αB in retinal pigment epithelial (RPE cells renders them susceptible to oxidant-induced cell death. We tested the hypothesis that the protective effect of α-crystallin is mediated by changes in cellular glutathione (GSH and elucidated the mechanism of GSH efflux. In α-crystallin overexpressing cells resistant to cell death, cellular GSH was >2 fold higher than vector control cells and this increase was seen particularly in mitochondria. The high GSH levels associated with α-crystallin overexpression were due to increased GSH biosynthesis. On the other hand, cellular GSH was decreased by 50% in murine retina lacking αA or αB crystallin. Multiple multidrug resistance protein (MRP family isoforms were expressed in RPE, among which MRP1 was the most abundant. MRP1 was localized to the plasma membrane and inhibition of MRP1 markedly decreased GSH efflux. MRP1-suppressed cells were resistant to cell death and contained elevated intracellular GSH and GSSG. Increased GSH in MRP1-supressed cells resulted from a higher conversion of GSSG to GSH by glutathione reductase. In contrast, GSH efflux was significantly higher in MRP1 overexpressing RPE cells which also contained lower levels of cellular GSH and GSSG. Oxidative stress further increased GSH efflux with a decrease in cellular GSH and rendered cells apoptosis-prone. In conclusion, our data reveal for the first time that 1 MRP1 mediates GSH and GSSG efflux in RPE cells; 2 MRP1 inhibition renders RPE cells resistant to oxidative stress-induced cell death while MRP1 overexpression makes them susceptible and 3 the antiapoptotic function of α-crystallin in oxidatively stressed cells is mediated in part by GSH and MRP1. Our findings suggest that MRP1 and α crystallin are potential therapeutic targets in pathological retinal degenerative disorders linked to oxidative stress.

Open reading frame 176 in the photosynthesis gene cluster of Rhodobacter capsulatus encodes idi, a gene for isopentenyl diphosphate isomerase.

OpenAIRE

Hahn, F M; Baker, J A; Poulter, C D

1996-01-01

Isopentenyl diphosphate (IPP) isomerase catalyzes an essential activation step in the isoprenoid biosynthetic pathway. A database search based on probes from the highly conserved regions in three eukaryotic IPP isomerases revealed substantial similarity with ORF176 in the photosynthesis gene cluster in Rhodobacter capsulatus. The open reading frame was cloned into an Escherichia coli expression vector. The encoded 20-kDa protein, which was purified in two steps by ion exchange and hydrophobic...

Crystal structure of Pyrococcus furiosus phosphoglucose isomerase: Implications for substrate binding and catalysis

NARCIS (Netherlands)

Berrisford, J.M.; Akerboom, A.P.; Turnbull, A.P.; Geus, de D.; Sedelnikova, S.E.; Staton, I.; McLeod, C.W.; Verhees, C.H.; Oost, van der J.; Rice, D.W.; Baker, P.J.

2003-01-01

Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization between D-fructose 6-phosphate and D-glucose 6-phosphate as part of the glycolytic pathway. PGI from the Archaea Pyrococcus furiosus (Pfu) was crystallized, and its structure was determined by x-ray diffraction to a 2-Angstrom

Allergen-Induced Dermatitis Causes Alterations in Cutaneous Retinoid-Mediated Signaling in Mice

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Gericke, Janine; Ittensohn, Jan; Mihály, Johanna; Dubrac, Sandrine; Rühl, Ralph

2013-01-01

Nuclear receptor-mediated signaling via RARs and PPARδ is involved in the regulation of skin homeostasis. Moreover, activation of both RAR and PPARδ was shown to alter skin inflammation. Endogenous all-trans retinoic acid (ATRA) can activate both receptors depending on specific transport proteins: Fabp5 initiates PPARδ signaling whereas Crabp2 promotes RAR signaling. Repetitive topical applications of ovalbumin (OVA) in combination with intraperitoneal injections of OVA or only intraperitoneal OVA applications were used to induce allergic dermatitis. In our mouse model, expression of IL-4, and Hbegf increased whereas expression of involucrin, Abca12 and Spink5 decreased in inflamed skin, demonstrating altered immune response and epidermal barrier homeostasis. Comprehensive gene expression analysis showed alterations of the cutaneous retinoid metabolism and retinoid-mediated signaling in allergic skin immune response. Notably, ATRA synthesis was increased as indicated by the elevated expression of retinaldehyde dehydrogenases and increased levels of ATRA. Consequently, the expression pattern of genes downstream to RAR was altered. Furthermore, the increased ratio of Fabp5 vs. Crabp2 may indicate an up-regulation of the PPARδ pathway in allergen-induced dermatitis in addition to the altered RAR signaling. Thus, our findings suggest that ATRA levels, RAR-mediated signaling and signaling involved in PPARδ pathways are mainly increased in allergen-induced dermatitis and may contribute to the development and/or maintenance of allergic skin diseases. PMID:23977003

Gestational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin alters retinoid homeostasis in maternal and perinatal tissues of the Holtzman rat

International Nuclear Information System (INIS)

Kransler, Kevin M.; Tonucci, David A.; McGarrigle, Barbara P.; Napoli, Joseph L.; Olson, James R.

2007-01-01

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), one of the most widely studied environmental contaminants, causes a variety of adverse health effects including teratogenesis and altered development which may be related to disruptions in retinoid homeostasis. The purpose of this study was to determine the effect that gestational administration of TCDD has on retinoid homeostasis in both pregnant Holtzman rats and developing fetuses and neonates. A single oral dose of TCDD (0, 1.5, 3, or 6 μg/kg) was administered to pregnant rats on gestation day 10, with fetuses analyzed on gestation days 17 and 20, and neonates analyzed on post natal day 7. Exposure to TCDD generally produced decreases in the concentrations of retinyl esters, such as retinyl palmitate, and retinol in maternal and perinatal liver and lung, while increasing levels in the maternal kidney. Additionally, perinatal hepatic retinol binding protein 1-dependent retinyl ester hydrolysis was also decrease by TCDD. Sensitivity of the developing perinates to TCDD appeared to have an age-related component demonstrated by an increased rate of mortality and significant alterations to body weight and length on post natal day 7 relative to that observed at gestation day 20. A unique observation made in this study was a significant decrease in lung weight observed in the perinates exposed to TCDD. Taken together, these data demonstrate that TCDD significantly alters retinoid homeostasis in tissues of the developing fetus and neonate, suggesting that their unique sensitivity to TCDD may at least be in part the result of altered retinoid homeostasis

Maternal retinoids control type 3 innate lymphoid cells and set the offspring immunity

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van de Pavert, Serge A.; Ferreira, Manuela; Domingues, Rita G.; Ribeiro, Hélder; Molenaar, Rosalie; Moreira-Santos, Lara; Almeida, Francisca F.; Ibiza, Sales; Barbosa, Inês; Goverse, Gera; Labão-Almeida, Carlos; Godinho-Silva, Cristina; Konijn, Tanja; Schooneman, Dennis; O'Toole, Tom; Mizee, Mark R.; Habani, Yasmin; Haak, Esther; Santori, Fabio R.; Littman, Dan R.; Schulte-Merker, Stefan; Dzierzak, Elaine; Simas, J. Pedro; Mebius, Reina E.; Veiga-Fernandes, Henrique

2014-04-01

The impact of nutritional status during fetal life on the overall health of adults has been recognized; however, dietary effects on the developing immune system are largely unknown. Development of secondary lymphoid organs occurs during embryogenesis and is considered to be developmentally programmed. Secondary lymphoid organ formation depends on a subset of type 3 innate lymphoid cells (ILC3) named lymphoid tissue inducer (LTi) cells. Here we show that mouse fetal ILC3s are controlled by cell-autonomous retinoic acid (RA) signalling in utero, which pre-sets the immune fitness in adulthood. We found that embryonic lymphoid organs contain ILC progenitors that differentiate locally into mature LTi cells. Local LTi cell differentiation was controlled by maternal retinoid intake and fetal RA signalling acting in a haematopoietic cell-autonomous manner. RA controlled LTi cell maturation upstream of the transcription factor RORγt. Accordingly, enforced expression of Rorgt restored maturation of LTi cells with impaired RA signalling, whereas RA receptors directly regulated the Rorgt locus. Finally, we established that maternal levels of dietary retinoids control the size of secondary lymphoid organs and the efficiency of immune responses in the adult offspring. Our results reveal a molecular link between maternal nutrients and the formation of immune structures required for resistance to infection in the offspring.

Monomethylfumarate induces γ-globin expression and fetal hemoglobin production in cultured human retinal pigment epithelial (RPE) and erythroid cells, and in intact retina.

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Promsote, Wanwisa; Makala, Levi; Li, Biaoru; Smith, Sylvia B; Singh, Nagendra; Ganapathy, Vadivel; Pace, Betty S; Martin, Pamela M

2014-05-13

Sickle retinopathy (SR) is a major cause of vision loss in sickle cell disease (SCD). There are no strategies to prevent SR and treatments are extremely limited. The present study evaluated (1) the retinal pigment epithelial (RPE) cell as a hemoglobin producer and novel cellular target for fetal hemoglobin (HbF) induction, and (2) monomethylfumarate (MMF) as an HbF-inducing therapy and abrogator of oxidative stress and inflammation in SCD retina. Human globin gene expression was evaluated by RT-quantitative (q)PCR in the human RPE cell line ARPE-19 and in primary RPE cells isolated from Townes humanized SCD mice. γ-Globin promoter activity was monitored in KU812 stable dual luciferase reporter expressing cells treated with 0 to 1000 μM dimethylfumarate, MMF, or hydroxyurea (HU; positive control) by dual luciferase assay. Reverse transcriptase-qPCR, fluorescence-activated cell sorting (FACS), immunofluorescence, and Western blot techniques were used to evaluate γ-globin expression and HbF production in primary human erythroid progenitors, ARPE-19, and normal hemoglobin producing (HbAA) and homozygous β(s) mutation (HbSS) RPE that were treated similarly, and in MMF-injected (1000 μM) HbAA and HbSS retinas. Dihydroethidium labeling and nuclear factor (erythroid-derived 2)-like 2 (Nrf2), IL-1β, and VEGF expression were also analyzed. Retinal pigment epithelial cells express globin genes and synthesize adult and fetal hemoglobin MMF stimulated γ-globin expression and HbF production in cultured RPE and erythroid cells, and in HbSS mouse retina where it also reduced oxidative stress and inflammation. The production of hemoglobin by RPE suggests the potential involvement of this cell type in the etiology of SR. Monomethylfumarate influences multiple parameters consistent with improved retinal health in SCD and may therefore be of therapeutic potential in SR treatment. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

Two dietary polyphenols, fisetin and luteolin, reduce inflammation but augment DNA damage-induced toxicity in human RPE cells.

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Hytti, Maria; Szabó, Dora; Piippo, Niina; Korhonen, Eveliina; Honkakoski, Paavo; Kaarniranta, Kai; Petrovski, Goran; Kauppinen, Anu

2017-04-01

Plant-derived polyphenols are known to possess anti-inflammatory and antioxidant effects. In recent years, several studies have investigated their potential benefits for treating chronic diseases associated with prolonged inflammation and excessive oxidative stress, such as age-related macular degeneration (AMD). Previously, two polyphenols, fisetin and luteolin, have been reported to increase the survival of retinal pigment epithelial (RPE) cells suffering from oxidative stress as well as decreasing inflammation but the benefits of polyphenol therapy seem to depend on the model system used. Our aim was to analyze the effects of fisetin and luteolin on inflammation and cellular viability in a model of nonoxidative DNA damage-induced cell death in human RPE (hRPE) cells. Pretreatment of ARPE-19 or primary hRPE cells with the polyphenols augmented etoposide-induced cell death as measured by the lactate dehydrogenase and 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. However, the treatment was able to reduce the release of two proinflammatory cytokines, IL-6 and IL-8, which were determined by enzyme-linked Immunosorbent assay. Analyses of caspase 3 activity, p53 acetylation and SIRT1 protein levels revealed the apoptotic nature of etoposide-evoked cell death and that fisetin and luteolin augmented the etoposide-induced acetylation of p53 and decreased SIRT1 levels. Taken together, our findings suggest that the cytoprotective effects of fisetin and luteolin depend on the stressor they need to combat, whereas their anti-inflammatory potential is sustained over a variety of model systems. Careful consideration of disease pathways will be necessary before fisetin or luteolin can be recommended as therapeutic agents for inflammatory diseases in general and specifically AMD. Copyright © 2017 Elsevier Inc. All rights reserved.

A human apoB100 transgenic mouse expresses human apoB100 in the RPE and develops features of early AMD

DEFF Research Database (Denmark)

Fujihara, Masashi; Bartels, Emil; Nielsen, Lars B

2009-01-01

changes consistent with early human AMD including loss of basal infoldings and accumulation of cytoplasmic vacuoles in the RPE, and basal laminar deposits containing long-spacing collagen and heterogeneous debris in Bruch membrane of apoB100 mice. In apoB100 mice given a high-fat diet, basal linear...... transgenic for a human genomic fragment encoding the full length human apoB ("apoB100" mice) and litter-mate control mice were given a normal chow or high-fat diet for 12 months. Mice were evaluated for human apoB mRNA expression in the RPE/choroid and liver by RT-qPCR. Phenotypic changes associated......-like deposits were identified in 12-month-old mice. Linear regression analysis showed that the genotype (human apoB transgene) was a stronger influencing factor than high-fat diet in producing AMD-like lesions used in this study. Human apoB100 transgenic mice overexpress apoB in RPE and, with time, develop...

Patient-specific mutations impair BESTROPHIN1’s essential role in mediating Ca2+-dependent Cl- currents in human RPE

Energy Technology Data Exchange (ETDEWEB)

Li, Yao [Jonas Children’s Vision Care, and Bernard and Shirlee Brown Glaucoma Laboratory, Department of Ophthalmology and Pathology & Cell Biology, Edward S. Harkness Eye Institute, New York Presbyterian Hospital/Columbia University, New York, United States; Zhang, Yu [Department of Pharmacology and Physiology, School of Medicine and Dentistry, University of Rochester, Rochester, United States; Xu, Yu [Jonas Children’s Vision Care, and Bernard and Shirlee Brown Glaucoma Laboratory, Department of Ophthalmology and Pathology & Cell Biology, Edward S. Harkness Eye Institute, New York Presbyterian Hospital/Columbia University, New York, United States; Department of Ophthalmology, Xinhua Hospital affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China; Kittredge, Alec [Department of Pharmacology and Physiology, School of Medicine and Dentistry, University of Rochester, Rochester, United States; Ward, Nancy [Department of Pharmacology and Physiology, School of Medicine and Dentistry, University of Rochester, Rochester, United States; Chen, Shoudeng [Molecular Imaging Center, Department of Experimental Medicine, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, China; Tsang, Stephen H. [Jonas Children’s Vision Care, and Bernard and Shirlee Brown Glaucoma Laboratory, Department of Ophthalmology and Pathology & Cell Biology, Edward S. Harkness Eye Institute, New York Presbyterian Hospital/Columbia University, New York, United States; Yang, Tingting [Department of Pharmacology and Physiology, School of Medicine and Dentistry, University of Rochester, Rochester, United States

2017-10-24

Mutations in the human BEST1 gene lead to retinal degenerative diseases displaying progressive vision loss and even blindness. BESTROPHIN1, encoded by BEST1, is predominantly expressed in retinal pigment epithelium (RPE), but its physiological role has been a mystery for the last two decades. Using a patient-specific iPSC-based disease model and interdisciplinary approaches, we comprehensively analyzed two distinct BEST1 patient mutations, and discovered mechanistic correlations between patient clinical phenotypes, electrophysiology in their RPEs, and the structure and function of BESTROPHIN1 mutant channels. Our results revealed that the disease-causing mechanism of BEST1 mutations is centered on the indispensable role of BESTROPHIN1 in mediating the long speculated Ca2+-dependent Cl- current in RPE, and demonstrate that the pathological potential of BEST1 mutations can be evaluated and predicted with our iPSC-based ‘disease-in-a-dish’ approach. Moreover, we demonstrated that patient RPE is rescuable with viral gene supplementation, providing a proof-of-concept for curing BEST1-associated diseases.

A library of fluorescent peptides for exploring the substrate specificities of prolyl isomerases

NARCIS (Netherlands)

Zoldak, G.; Aumuller, T.; Lucke, C.; Hritz, J.; Oostenbrink, C.; Fischer, G.; Schmid, F.X.

2009-01-01

To fully explore the substrate specificities of prolyl isomerases, we synthesized a library of 20 tetrapeptides that are labeled with a 2-aminobenzoyl (Abz) group at the amino terminus and a p-nitroanilide (pNA) group at the carboxy terminus. In this peptide library of the general formula

Proline substitutions in a Mip-like peptidyl-prolyl cis-trans isomerase severely affect its structure, stability, shape and activity

Directory of Open Access Journals (Sweden)

Soumitra Polley

2015-01-01

Full Text Available FKBP22, an Escherichia coli-specific peptidyl-prolyl cis-trans isomerase, shows substantial homology with the Mip-like virulence factors. Mip-like proteins are homodimeric and possess a V-shaped conformation. Their N-terminal domains form dimers, whereas their C-terminal domains bind protein/peptide substrates and distinct inhibitors such as rapamycin and FK506. Interestingly, the two domains of the Mip-like proteins are separated by a lengthy, protease-susceptible α-helix. To delineate the structural requirement of this domain-connecting region in Mip-like proteins, we have investigated a recombinant FKBP22 (rFKBP22 and its three point mutants I65P, V72P and A82P using different probes. Each mutant harbors a Pro substitution mutation at a distinct location in the hinge region. We report that the three mutants are not only different from each other but also different from rFKBP22 in structure and activity. Unlike rFKBP22, the three mutants were unfolded by a non-two state mechanism in the presence of urea. In addition, the stabilities of the mutants, particularly I65P and V72P, differed considerably from that of rFKBP22. Conversely, the rapamycin binding affinity of no mutant was different from that of rFKBP22. Of the mutants, I65P showed the highest levels of structural/functional loss and dissociated partly in solution. Our computational study indicated a severe collapse of the V-shape in I65P due to the anomalous movement of its C-terminal domains. The α-helical nature of the domain-connecting region is, therefore, critical for the Mip-like proteins.

Administration of Mycobacterium leprae rHsp65 aggravates experimental autoimmune uveitis in mice.

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Eliana B Marengo

Full Text Available The 60 kDa heat shock protein family, Hsp60, constitutes an abundant and highly conserved class of molecules that are highly expressed in chronic-inflammatory and autoimmune processes. Experimental autoimmune uveitis [EAU] is a T cell mediated intraocular inflammatory disease that resembles human uveitis. Mycobacterial and homologous Hsp60 peptides induces uveitis in rats, however their participation in aggravating the disease is poorly known. We here evaluate the effects of the Mycobacterium leprae Hsp65 in the development/progression of EAU and the autoimmune response against the eye through the induction of the endogenous disequilibrium by enhancing the entropy of the immunobiological system with the addition of homologous Hsp. B10.RIII mice were immunized subcutaneously with interphotoreceptor retinoid-binding protein [IRBP], followed by intraperitoneally inoculation of M. leprae recombinant Hsp65 [rHsp65]. We evaluated the proliferative response, cytokine production and the percentage of CD4(+IL-17(+, CD4(+IFN-gamma(+ and CD4(+Foxp3(+ cells ex vivo, by flow cytometry. Disease severity was determined by eye histological examination and serum levels of anti-IRBP and anti-Hsp60/65 measured by ELISA. EAU scores increased in the Hsp65 group and were associated with an expansion of CD4(+IFN-gamma(+ and CD4(+IL-17(+ T cells, corroborating with higher levels of IFN-gamma. Our data indicate that rHsp65 is one of the managers with a significant impact over the immune response during autoimmunity, skewing it to a pathogenic state, promoting both Th1 and Th17 commitment. It seems comprehensible that the specificity and primary function of Hsp60 molecules can be considered as a potential pathogenic factor acting as a whistleblower announcing chronic-inflammatory diseases progression.

Crystallization and preliminary X-ray diffraction studies of l-rhamnose isomerase from Pseudomonas stutzeri

International Nuclear Information System (INIS)

Yoshida, Hiromi; Wayoon, Poonperm; Takada, Goro; Izumori, Ken; Kamitori, Shigehiro

2006-01-01

Recombinant l-rhamnose isomerase from P. stutzeri has been crystallized. Diffraction data have been collected to 2.0 Å resolution. l-Rhamnose isomerase from Pseudomonas stutzeri (P. stutzeril-RhI) catalyzes not only the reversible isomerization of l-rhamnose to l-rhamnulose, but also isomerization between various rare aldoses and ketoses. Purified His-tagged P. stutzeril-RhI was crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the monoclinic space group P2 1 , with unit-cell parameters a = 74.3, b = 104.0, c = 107.0 Å, β = 106.8°. Diffraction data have been collected to 2.0 Å resolution. The molecular weight of the purified P. stutzeril-RhI with a His tag at the C-terminus was confirmed to be 47.7 kDa by MALDI–TOF mass-spectrometric analysis and the asymmetric unit is expected to contain four molecules

The retinoids. A review of their clinical pharmacology and therapeutic use.

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Orfanos, C E; Ehlert, R; Gollnick, H

1987-10-01

With the introduction of the synthetic retinoids, oral therapy with an acceptable risk/benefit ratio became possible for a variety of skin diseases including severe acne, psoriasis and numerous genodermatoses. This article reviews the clinical pharmacology, mechanisms of action and therapeutic use of the retinoids, particularly isotretinoin (13-cis-retinoic acid) and etretinate. The free aromatic acid of etretinate, etretin, and the new polyaromatic retinoid compounds (arotinoids) are also discussed. Isotretinoin is used clinically for oral therapy of severe acne, but is also recommended for severe Gram-negative folliculitis and rosacea not responding to traditional therapy. The results of several studies have established that acne therapy should be started with 1.0 mg/kg/day for 2 to 3 months after which the daily dosage should be lowered to 0.2 to 0.5 mg/kg/day for another 2 to 3 months. This therapeutic regimen of isotretinoin has proven to be the most successful in preventing relapses. Etretinate is particularly useful for oral therapy of widespread plaque-like, pustular and erythrodermic psoriasis, and of generalised lichen planus, Darier's disease and severe congenital ichthyoses. Whereas pustular forms of psoriasis require a high daily dosage of 1.0 mg/kg/day, erythrodermic psoriasis should be treated with a lower dosage of 0.25 to 0.35 mg/kg/day. In chronic plaque-like psoriasis, a mean daily dosage of 0.5 mg/kg/day over several weeks to months, usually combined with photo(chemo)therapy, tar or dithranol, is recommended. Other indications for oral etretinate therapy are adequately treated with a moderate dosage of 0.4 to 0.75 mg/kg/day. Etretin differs from etretinate in having a much shorter elimination half-life of 2 to 3 days, in contrast to 80 to 100 days after long term administration of etretinate. Moreover, it has not been shown to increase serum cholesterol levels. However, its clinical efficacy is not yet clearly established. Among the arotinoids

Ultrastructure and /sup 3/H-TdR autoradiography after topical application of aromatic retinoid in guinea pigs

Energy Technology Data Exchange (ETDEWEB)

Ueda, K.; Maruo, M.; Inoue, Y.; Wakabayashi, S. (Kyoto Prefectural Univ. of Medicine (Japan))

1981-12-01

Histological, autoradiographical and electron microscopical investigations were performed on the epidermis of guinea pigs after topical application of 0.1% aromatic retinoid ointment for 7 days. Histologically, the epidermis exhibited a thickening of granular layer and acanthosis, without a prolonged rete ridge. Labelling indices were 3 to 4 times higher than in the control epidermis. Electron microscopically, the nuclei in the spinous and the basal layer were large in size and nucleoli were increased in number. In the cytoplasm, tonofilaments were reduced in number and lots of vacuoles containing less dense amorphous material appeared. Amorphous material was present in the intercellular spaces of the epidermis. Differentiation and proliferation of the epidermis in guinea pigs after topical application of aromatic retinoid showed almost the same response in quantity to the epidermis after topical application of retinoic acid, while the epidermal alternation was slight in degree than in the epidermis after topical application of retinoic acid.

Putrescine treatment reverses α-tocopherol-induced desynchronization of polyamine and retinoid metabolism during rat liver regeneration

Directory of Open Access Journals (Sweden)

Lourdes Sánchez-Sevilla

2016-10-01

Full Text Available Abstract Background The pre-treatment with α-tocopherol inhibits progression of rat liver proliferation induced by partial hepatectomy (PH, by decreasing and/or desynchronizing cyclin D1 expression and activation into the nucleus, activation and nuclear translocation of STAT-1 and -3 proteins and altering retinoid metabolism. Interactions between retinoic acid and polyamines have been reported in the PH-induced rat liver regeneration. Therefore, we evaluated the effect of low dosage of α-tocopherol on PH-induced changes in polyamine metabolism. Methods This study evaluated the participation of polyamine synthesis and metabolism during α-tocopherol-induced inhibition of rat liver regeneration. In PH-rats (Wistar treated with α-tocopherol and putrescine, parameters indicative of cell proliferation, lipid peroxidation, ornithine decarboxylase expression (ODC, and polyamine levels, were determined. Results Pre-treatment with α-tocopherol to PH-animals exerted an antioxidant effect, shifting earlier the increased ODC activity and expression, temporally affecting polyamine synthesis and ornithine metabolism. Whereas administration of putrescine induced minor changes in PH-rats, the concomitant treatment actually counteracted most of adverse actions exerted by α-tocopherol on the remnant liver, restituting its proliferative potential, without changing its antioxidant effect. Putrescine administration to these rats was also associated with lower ODC expression and activity in the proliferating liver, but the temporally shifting in the amount of liver polyamines induced by α-tocopherol, was also “synchronized” by the putrescine administration. The latter is supported by the fact that a close relationship was observed between fluctuations of polyamines and retinoids. Conclusions Putrescine counteracted most adverse actions exerted by α-tocopherol on rat liver regeneration, restoring liver proliferative potential and restituting the decreased

Heterologous expression and characterization of Bacillus coagulans L-arabinose isomerase.

Science.gov (United States)

Zhou, Xingding; Wu, Jin Chuan

2012-05-01

Bacillus coagulans has been of great commercial interest over the past decade owing to its strong ability of producing optical pure L: -lactic acid from both hexose and pentose sugars including L: -arabinose with high yield, titer and productivity under thermophilic conditions. The L: -arabinose isomerase (L-AI) from Bacillus coagulans was heterologously over-expressed in Escherichia coli. The open reading frame of the L-AI has 1,422 nucleotides encoding a protein with 474 amino acid residues. The recombinant L-AI was purified to homogeneity by one-step His-tag affinity chromatography. The molecular mass of the enzyme was estimated to be 56 kDa by SDS-PAGE. The enzyme was most active at 70°C and pH 7.0. The metal ion Mn(2+) was shown to be the best activator for enzymatic activity and thermostability. The enzyme showed higher activity at acidic pH than at alkaline pH. The kinetic studies showed that the K (m), V (max) and k (cat)/K (m) for the conversion of L: -arabinose were 106 mM, 84 U/mg and 34.5 mM(-1)min(-1), respectively. The equilibrium ratio of L: -arabinose to L: -ribulose was 78:22 under optimal conditions. L: -ribulose (97 g/L) was obtained from 500 g/l of L: -arabinose catalyzed by the enzyme (8.3 U/mL) under the optimal conditions within 1.5 h, giving at a substrate conversion of 19.4% and a production rate of 65 g L(-1) h(-1).

Molecular identification, immunolocalization, and characterization of Clonorchis sinensis triosephosphate isomerase.

Science.gov (United States)

Zhou, Juanjuan; Liao, Hua; Li, Shan; Zhou, Chenhui; Huang, Yan; Li, Xuerong; Liang, Chi; Yu, Xinbing

2015-08-01

Clonorchis sinensis triosephosphate isomerase (CsTIM) is a key regulatory enzyme of glycolysis and gluconeogenesis, which catalyzes the interconversion of glyceraldehyde 3-phosphate to dihydroxyacetone phosphate. In this study, the biochemical characterizations of CsTIM have been examined. A full-length complementary DNA (cDNA; Cs105350) sequence encoding CsTIM was obtained from our C. sinensis cDNA library. The open reading frame of CsTIM contains 759 bp which encodes 252 amino acids. The amino acid sequence of CsTIM shares 60-65% identity with other species. Western blot analysis displayed that recombinant CsTIM (rCsTIM) can be probed by anti-rCsTIM rat serum and anti-C. sinensis excretory/secretory products (anti-CsESPs) rat serum. Quantitative reverse transcription (RT)-PCR and western blotting analysis revealed that CsTIM messenger RNA (mRNA) and protein were differentially expressed in development cycle stages of the parasite, including adult worm, metacercaria, excysted metacercaria, and egg. In addition, immunolocalization assay showed that CsTIM was located in the seminal vesicle, eggs, and testicle. Moreover, rCsTIM exhibited active enzyme activity in catalytic reactions. The Michaelis constant (K m) of rCsTIM was 0.33 mM, when using glyceraldehyde 3-phosphate as the substrate. The optimal temperature and pH of CsTIM were 37 °C and 7.5-9.5, respectively. Collectively, these results suggest that CsTIM is an important protein involved in glycometabolism, and CsTIM possibly take part in many biological functions in the growth and development of C. sinensis.

Thermostable L-arabinose isomerase from Bacillus stearothermophilus IAM 11001 for D-tagatose production: gene cloning, purification and characterisation.

Science.gov (United States)

Cheng, Lifang; Mu, Wanmeng; Jiang, Bo

2010-06-01

D-Tagatose, as one of the rare sugars, has been found to be a natural and safe low-calorie sweetener in food products and is classified as a GRAS substance. L-Arabinose isomerase (L-AI, EC 5.3.1.4), catalysing the isomerisations of L-arabinose and D-galactose to L-ribulose and D-tagatose respectively, is considered to be the most promising enzyme for the production of D-tagatose. The araA gene encoding an L-AI from Bacillus stearothermophilus IAM 11001 was cloned, sequenced and overexpressed in Escherichia coli. The gene is composed of 1491 bp nucleotides and codes for a protein of 496 amino acid residues. The recombinant L-AI was purified to electrophoretical homogeneity by affinity chromatography. The purified enzyme was optimally active at 65 degrees C and pH 7.5 and had an absolute requirement for the divalent metal ion Mn(2+) for both catalytic activity and thermostability. The enzyme was relatively active and stable at acidic pH of 6. The bioconversion yield of D-galactose to D-tagatose by the purified L-AI after 12 h at 65 degrees C reached 36%. The purified L-AI from B. stearothermophilus IAM 11001 was characterised and shown to be a good candidate for potential application in D-tagatose production. Copyright (c) 2010 Society of Chemical Industry.

Reversal of Blindness in Animal Models of Leber Congenital Amaurosis Using Optimized AAV2-mediated Gene Transfer

OpenAIRE

Bennicelli, Jeannette; Wright, John Fraser; Komaromy, Andras; Jacobs, Jonathan B; Hauck, Bernd; Zelenaia, Olga; Mingozzi, Federico; Hui, Daniel; Chung, Daniel; Rex, Tonia S; Wei, Zhangyong; Qu, Guang; Zhou, Shangzhen; Zeiss, Caroline; Arruda, Valder R

2008-01-01

We evaluated the safety and efficacy of an optimized adeno-associated virus (AAV; AAV2.RPE65) in animal models of the RPE65 form of Leber congenital amaurosis (LCA). Protein expression was optimized by addition of a modified Kozak sequence at the translational start site of hRPE65. Modifications in AAV production and delivery included use of a long stuffer sequence to prevent reverse packaging from the AAV inverted-terminal repeats, and co-injection with a surfactant. The latter allows consis...

The acid-tolerant L-arabinose isomerase from the mesophilic Shewanella sp. ANA-3 is highly active at low temperatures

Science.gov (United States)

2011-01-01

Background L-arabinose isomerases catalyse the isomerization of L-arabinose into L-ribulose at insight biological systems. At industrial scale of this enzyme is used for the bioconversion of D-galactose into D-tagatose which has many applications in pharmaceutical and agro-food industries. The isomerization reaction is thermodynamically equilibrated, and therefore the bioconversion rates is shifted towards tagatose when the temperature is increased. Moreover, to prevent secondary reactions it will be of interest to operate at low pH. The profitability of this D-tagatose production process is mainly related to the use of lactose as cheaper raw material. In many dairy products it will be interesting to produce D-tagatose during storage. This requires an efficient L-arabinose isomerase acting at low temperature and pH values. Results The gene encoding the L-arabinose isomerase from Shewanella sp. ANA-3 was cloned and overexpressed in Escherichia coli. The purified protein has a tetrameric arrangement composed by four identical 55 kDa subunits. The biochemical characterization of this enzyme showed that it was distinguishable by its maximal activity at low temperatures comprised between 15-35°C. Interestingly, this biocatalyst preserves more than 85% of its activity in a broad range of temperatures from 4.0 to 45°C. Shewanella sp. ANA-3 L-arabinose isomerase was also optimally active at pH 5.5-6.5 and maintained over 80% of its activity at large pH values from 4.0 to 8.5. Furthermore, this enzyme exhibited a weak requirement for metallic ions for its activity evaluated at 0.6 mM Mn2+. Stability studies showed that this protein is highly stable mainly at low temperature and pH values. Remarkably, T268K mutation clearly enhances the enzyme stability at low pH values. Use of this L-arabinose isomerase for D-tagatose production allows the achievement of attractive bioconversion rates of 16% at 4°C and 34% at 35°C. Conclusions Here we reported the purification and the

The acid-tolerant L-arabinose isomerase from the mesophilic Shewanella sp. ANA-3 is highly active at low temperatures

Directory of Open Access Journals (Sweden)

Rhimi Moez

2011-11-01

Full Text Available Abstract Background L-arabinose isomerases catalyse the isomerization of L-arabinose into L-ribulose at insight biological systems. At industrial scale of this enzyme is used for the bioconversion of D-galactose into D-tagatose which has many applications in pharmaceutical and agro-food industries. The isomerization reaction is thermodynamically equilibrated, and therefore the bioconversion rates is shifted towards tagatose when the temperature is increased. Moreover, to prevent secondary reactions it will be of interest to operate at low pH. The profitability of this D-tagatose production process is mainly related to the use of lactose as cheaper raw material. In many dairy products it will be interesting to produce D-tagatose during storage. This requires an efficient L-arabinose isomerase acting at low temperature and pH values. Results The gene encoding the L-arabinose isomerase from Shewanella sp. ANA-3 was cloned and overexpressed in Escherichia coli. The purified protein has a tetrameric arrangement composed by four identical 55 kDa subunits. The biochemical characterization of this enzyme showed that it was distinguishable by its maximal activity at low temperatures comprised between 15-35°C. Interestingly, this biocatalyst preserves more than 85% of its activity in a broad range of temperatures from 4.0 to 45°C. Shewanella sp. ANA-3 L-arabinose isomerase was also optimally active at pH 5.5-6.5 and maintained over 80% of its activity at large pH values from 4.0 to 8.5. Furthermore, this enzyme exhibited a weak requirement for metallic ions for its activity evaluated at 0.6 mM Mn2+. Stability studies showed that this protein is highly stable mainly at low temperature and pH values. Remarkably, T268K mutation clearly enhances the enzyme stability at low pH values. Use of this L-arabinose isomerase for D-tagatose production allows the achievement of attractive bioconversion rates of 16% at 4°C and 34% at 35°C. Conclusions Here we

Identification of a Class of Non-linear State Space Models using RPE Techniques

DEFF Research Database (Denmark)

Zhou, Wei-Wu; Blanke, Mogens

1989-01-01

The RPE (recursive prediction error) method in state-space form is developed in the nonlinear systems and extended to include the exact form of a nonlinearity, thus enabling structure preservation for certain classes of nonlinear systems. Both the discrete and the continuous-discrete versions...... of the algorithm in an innovations model are investigated, and a nonlinear simulation example shows a quite convincing performance of the filter as combined parameter and state estimator...

Effects of polybrominated diphenyl ethers (PBDEs) and their derivatives on protein disulfide isomerase activity and growth hormone release of GH3 cells.

Science.gov (United States)

Hashimoto, Shoko; Yoshimura, Hiromi; Okada, Kazushi; Uramaru, Naoto; Sugihara, Kazumi; Kitamura, Shigeyuki; Imaoka, Susumu

2012-03-19

Polybrominated diphenyl ethers (PBDEs) have been used in a variety of consumer products such as flame retardants and recently have been known to be widespread environmental pollutants, which probably affect biological functions of mammalian cells. However, the risk posed by PBDE metabolites has not been clarified. Our previous study suggested that bisphenol A (BPA), an endocrine-disrupting chemical, binds to protein disulfide isomerase (PDI) and inhibits its activity. PDI is an isomerase enzyme in the endoplasmic reticulum and facilitates the formation or cleavage of disulfide bonds. PDI consists of a, b, b', and a' domains and the c region, with the a and a' domains having isomerase active sites. In the present study, we tested the effects of 10 kinds of PBDE compounds and their metabolites on PDI. OH-PBDEs specifically inhibited the isomerase activity of PDI, with 4'-OH-PBDE more effective than 2' (or 2)-OH-PBDEs. 4'-OH-PBDE inhibited the isomerase activity of the b'a'c fragment but not that of ab and a'c, suggesting that the b' domain of PDI is essential for the inhibition by 4'-OH-PBDE. We also investigated the effects of these chemicals on the production of growth hormone (GH) in GH3 cells. In GH3 cells, levels of mRNA and protein of GH stimulated by T(3) were reduced by 4'-OH-PBDE and 4'-MeO-PBDE. The reduction in GH expression caused by these compounds was not changed by the overexpression or knockdown of PDI in GH3 cells, while these manipulations of PDI levels significantly suppressed the expression of GH. These results suggest that the biological effects of PBDEs differed depending on their brominated and hydroxylated positions. © 2011 American Chemical Society

Phospho-carboxyl-terminal domain binding and the role of a prolyl isomerase in pre-mRNA 3'-End formation.

Science.gov (United States)

Morris, D P; Phatnani, H P; Greenleaf, A L

1999-10-29

A phospho-carboxyl-terminal domain (CTD) affinity column created with yeast CTD kinase I and the CTD of RNA polymerase II was used to identify Ess1/Pin1 as a phospho-CTD-binding protein. Ess1/Pin1 is a peptidyl prolyl isomerase involved in both mitotic regulation and pre-mRNA 3'-end formation. Like native Ess1, a GSTEss1 fusion protein associates specifically with the phosphorylated but not with the unphosphorylated CTD. Further, hyperphosphorylated RNA polymerase II appears to be the dominant Ess1 binding protein in total yeast extracts. We demonstrate that phospho-CTD binding is mediated by the small WW domain of Ess1 rather than the isomerase domain. These findings suggest a mechanism in which the WW domain binds the phosphorylated CTD of elongating RNA polymerase II and the isomerase domain reconfigures the CTD though isomerization of proline residues perhaps by a processive mechanism. This process may be linked to a variety of pre-mRNA maturation events that use the phosphorylated CTD, including the coupled processes of pre-mRNA 3'-end formation and transcription termination.

Nucleotide sequence of the triosephosphate isomerase gene from Macaca mulatta

Energy Technology Data Exchange (ETDEWEB)

Old, S.E.; Mohrenweiser, H.W. (Univ. of Michigan, Ann Arbor (USA))

1988-09-26

The triosephosphate isomerase gene from a rhesus monkey, Macaca mulatta, charon 34 library was sequenced. The human and chimpanzee enzymes differ from the rhesus enzyme at ASN 20 and GLU 198. The nucleotide sequence identity between rhesus and human is 97% in the coding region and >94% in the flanking regions. Comparison of the rhesus and chimp genes, including the intron and flanking sequences, does not suggest a mechanism for generating the two TPI peptides of proliferating cells from hominoids and a single peptide from the rhesus gene.

Biochemical properties of L-arabinose isomerase from Clostridium hylemonae to produce D-tagatose as a functional sweetener.

Science.gov (United States)

Nguyen, Tien-Kieu; Hong, Moon-Gi; Chang, Pahn-Shick; Lee, Byung-Hoo; Yoo, Sang-Ho

2018-01-01

d-Tagatose has gained substantial interest due to its potential functionalities as a sucrose substitute. In this study, the gene araA, encoding l-arabinose isomerase (l-AI) from Clostridium hylemonae (DSM 15053), was cloned and expressed in Escherichia coli BL21 (DE3). This gene consists of 1,506 nucleotides and encodes a protein of 501 amino acid residues with a calculated molecular mass of 56,554 Da. Since l-AI was expressed as an intracellular inclusion body, this enzyme was solubilized with guanidine hydrochloride, refolded, and activated with a descending concentration gradient of urea. The purified enzyme exhibited the greatest activity at 50°C, pH 7-7.5, and required 1 mM of Mg2+ as a cofactor. Notably, the catalytic efficiency (3.69 mM-1sec-1) of l-AI from C. hylemonae on galactose was significantly greater than that of other previously reported enzymes. The bioconversion yield of d-tagatose using the C. hylemonae l-arabinose isomerase at 60°C reached approximately 46% from 10 mM of d-galactose after 2 h. From these results, it is suggested that the l-arabinose isomerase from C. hylemonae could be utilized as a potential enzyme for d-tagatose production due to its high conversion yield at an industrially competitive temperature.

Theileria parasites secrete a prolyl isomerase to maintain host leukocyte transformation.

Science.gov (United States)

Marsolier, J; Perichon, M; DeBarry, J D; Villoutreix, B O; Chluba, J; Lopez, T; Garrido, C; Zhou, X Z; Lu, K P; Fritsch, L; Ait-Si-Ali, S; Mhadhbi, M; Medjkane, S; Weitzman, J B

2015-04-16

Infectious agents develop intricate mechanisms to interact with host cell pathways and hijack their genetic and epigenetic machinery to change host cell phenotypic states. Among the Apicomplexa phylum of obligate intracellular parasites, which cause veterinary and human diseases, Theileria is the only genus that transforms its mammalian host cells. Theileria infection of bovine leukocytes induces proliferative and invasive phenotypes associated with activated signalling pathways, notably JNK and AP-1 (ref. 2). The transformed phenotypes are reversed by treatment with the theilericidal drug buparvaquone. We used comparative genomics to identify a homologue of the peptidyl-prolyl isomerase PIN1 in T. annulata (TaPIN1) that is secreted into the host cell and modulates oncogenic signalling pathways. Here we show that TaPIN1 is a bona fide prolyl isomerase and that it interacts with the host ubiquitin ligase FBW7, leading to its degradation and subsequent stabilization of c-JUN, which promotes transformation. We performed in vitro and in silico analysis and in vivo zebrafish xenograft experiments to demonstrate that TaPIN1 is directly inhibited by the anti-parasite drug buparvaquone (and other known PIN1 inhibitors) and is mutated in a drug-resistant strain. Prolyl isomerization is thus a conserved mechanism that is important in cancer and is used by Theileria parasites to manipulate host oncogenic signalling.

Inhibiting prolyl isomerase activity by hybrid organic-inorganic molecules containing rhodium(II) fragments.

Science.gov (United States)

Coughlin, Jane M; Kundu, Rituparna; Cooper, Julian C; Ball, Zachary T

2014-11-15

A small molecule containing a rhodium(II) tetracarboxylate fragment is shown to be a potent inhibitor of the prolyl isomerase FKBP12. The use of small molecules conjugates of rhodium(II) is presented as a general strategy for developing new protein inhibitors based on distinct structural and sequence features of the enzyme active site. Copyright © 2014 Elsevier Ltd. All rights reserved.

17-AAG, a Hsp90 inhibitor, attenuates the hypoxia-induced expression of SDF-1alpha and ILK in mouse RPE cells.

Science.gov (United States)

Wang, Ye Qing; Zhang, Xiao Mei; Wang, Xiao Dan; Wang, Bin Jie; Wang, Wei

2010-03-01

The aim of this study was to investigate the changes of SDF-1alpha and ILK expression in mouse retinal pigment epithelium (RPE) cells in response to hypoxia, and the effect of 17-Allylamino-17-demethoxygeldanamycin (17-AAG), a heat shock protein 90 (Hsp90) inhibitor, on the hypoxia-induced expression of SDF-1alpha and ILK. RPE cells were cultured with 200 micromol/L cobalt chloride (CoCl(2)) for different times (1, 3, 6, 12, 24, 72 h) to imitate chemical hypoxia. Pretreatment of 17-AAG was 1 h prior to hypoxic insult. Cellular viability after 17-AAG treatment was assessed by MTT assay, and the changes of SDF-1alpha and ILK expression were examined by RT-PCR and Western blot. Up-regulation of SDF-1alpha and ILK expression in response to hypoxia was observed. One hour pretreatment of 17-AAG could remarkably decreased the hypoxia-induced SDF-1alpha and ILK expression in vitro. Our results indicated that SDF-1alpha and ILK involved in the hypoxic response of RPE cells, and 1 h pretreatment of 17-AAG had an inhibitive effect on the hypoxia-induced SDF-1alpha and ILK expression.

Hyperglycaemia exacerbates choroidal neovascularisation in mice via the oxidative stress-induced activation of STAT3 signalling in RPE cells.

Directory of Open Access Journals (Sweden)

Xia Li

Full Text Available Choroidal neovascularisation (CNV that occurs as a result of age-related macular degeneration (AMD causes severe vision loss among elderly patients. The relationship between diabetes and CNV remains controversial. However, oxidative stress plays a critical role in the pathogenesis of both AMD and diabetes. In the present study, we investigated the influence of diabetes on experimentally induced CNV and on the underlying molecular mechanisms of CNV. CNV was induced via photocoagulation in the ocular fundi of mice with streptozotocin-induced diabetes. The effect of diabetes on the severity of CNV was measured. An immunofluorescence technique was used to determine the levels of oxidative DNA damage by anti-8-hydroxy-2-deoxyguanosine (8-OHdG antibody, the protein expression of phosphorylated signal transducer and activator of transcription 3 (p-STAT3 and vascular endothelial growth factor (VEGF, in mice with CNV. The production of reactive oxygen species (ROS in retinal pigment epithelial (RPE cells that had been cultured under high glucose was quantitated using the 2',7'-dichlorofluorescein diacetate (DCFH-DA method. p-STAT3 expression was examined using Western blot analysis. RT-PCR and ELISA processes were used to detect VEGF expression. Hyperglycaemia exacerbated the development of CNV in mice. Oxidative stress levels and the expression of p-STAT3 and VEGF were highly elevated both in mice and in cultured RPE cells. Treatment with the antioxidant compound N-acetyl-cysteine (NAC rescued the severity of CNV in diabetic mice. NAC also inhibited the overexpression of p-STAT3 and VEGF in CNV and in RPE cells. The JAK-2/STAT3 pathway inhibitor AG490 blocked VEGF expression but had no effect on the production of ROS in vitro. These results suggest that hyperglycaemia promotes the development of CNV by inducing oxidative stress, which in turn activates STAT3 signalling in RPE cells. Antioxidant supplementation helped attenuate the development of CNV

Ultrastructure and /sup 3/H-TdR autoradiogram in the skin of guinea pigs after topical application of aromatic retinoid (Ro 10-9359)

Energy Technology Data Exchange (ETDEWEB)

Ueda, K.; Inoue, Y.; Wakabayashi, S.; Kitamura, T. (Kyoto Prefectural Univ. of Medicine (Japan))

1982-01-01

The effect of topical application with 0.1% aromatic retinoid ointment for 7 days on quinea pig epidermis were investigated histologically, electron microscopically and autoradiographically. 1. Histological changes of the epidermis after topical application of aromatic retinoid were characterized by acanthosis and thickening of epidermal layer without prolonged rete ridge. Epidermal cells were stained slightly positive with alcian blue. 2. Labelling index in epidermis was 18% high in average rate, and labelled cells distributed in basal layer and 2 to 3 upper basal layers. Labelling index was 3 to 4 times higher than in control skin. 3. Ultrastructural changes were observed in nuclei and cytoplasm of epidermal cells. Nuclei were large in size and irregular in shape, and nucleoli were large in size and increased in number. In the cytoplasm, tonofilament was reduced in number and lots of vacuoles containing less dense material appeared. Mitochondria, endoplasmic reticulum and ribosomes were rich in number. In the intercellular spaces, amorphous less dense materials were seen. Desmosomes were kept in figure and were floating in the wide intercellular spaces. 4. Differentiation and proliferation of the epidermis in guinea pig after topical application of aromatic retinoid and retinoic acid were almost same in quality of the reaction, but the reaction was different in quantity between both agents.

[Immunosuppresive agents, retinoids and new trends in the therapy of psoriasis].

Science.gov (United States)

Svozil, M

2002-11-01

Some psoriasis forms can be successfully treated with topical medicaments; serious and extensive forms cannot be cured without systemic treatment with retinoids (etretinate, acitretin, tazarotene) or with immunosuppressives (methotrexate, cyclosporine A, tacrolimus, SDZ 281-240). Immunotherapy and nucleotide analogues are being newly introduced, a number of potentially effective substances interfering with pathogenetic mechanisms participating in the emergence and self-prolongation of psoriasis are in the stage of research and clinical trials, and gene therapy possibilities are being investigated. The development and testing of drugs serving therapy for psoriasis correlates with the development of knowledge about the origins of the disease and the mechanisms of how antipsoriatics in current use as well as the new potential ones work.

Ethanol production from lignocellulosic hydrolysates using engineered Saccharomyces cerevisiae harboring xylose isomerase-based pathway.

Science.gov (United States)

Ko, Ja Kyong; Um, Youngsoon; Woo, Han Min; Kim, Kyoung Heon; Lee, Sun-Mi

2016-06-01

The efficient co-fermentation of glucose and xylose is necessary for the economically feasible bioethanol production from lignocellulosic biomass. Even with xylose utilizing Saccharomyces cerevisiae, the efficiency of the lignocellulosic ethanol production remains suboptimal mainly due to the low conversion yield of xylose to ethanol. In this study, we evaluated the co-fermentation performances of SXA-R2P-E, a recently engineered isomerase-based xylose utilizing strain, in mixed sugars and in lignocellulosic hydrolysates. In a high-sugar fermentation with 70g/L of glucose and 40g/L of xylose, SXA-R2P-E produced 50g/L of ethanol with an yield of 0.43gethanol/gsugars at 72h. From dilute acid-pretreated hydrolysates of rice straw and hardwood (oak), the strain produced 18-21g/L of ethanol with among the highest yield of 0.43-0.46gethanol/gsugars ever reported. This study shows a highly promising potential of a xylose isomerase-expressing strain as an industrially relevant ethanol producer from lignocellulosic hydrolysates. Copyright © 2016 Elsevier Ltd. All rights reserved.

In-house SIRAS phasing of the polyunsaturated fatty-acid isomerase from Propionibacterium acnes

International Nuclear Information System (INIS)

Liavonchanka, Alena; Hornung, Ellen; Feussner, Ivo; Rudolph, Markus

2006-01-01

Low iodide concentrations were sufficient to allow SAD and SIRAS phasing of cubic crystals of a novel fatty acid isomerase using Cu Kα radiation. The polyenoic fatty-acid isomerase from Propionibacterium acnes (PAI) catalyzes the double-bond isomerization of linoleic acid to conjugated linoleic acid, which is a dairy- or meat-derived fatty acid in the human diet. PAI was overproduced in Escherichia coli and purified to homogeneity as a yellow-coloured protein. The nature of the bound cofactor was analyzed by absorption and fluorescence spectroscopy. Single crystals of PAI were obtained in two crystal forms. Cubic shaped crystals belong to space group I2 1 3, with a unit-cell parameter of 160.4 Å, and plate-like crystals belong to the monoclinic space group C2, with unit-cell parameters a = 133.7, b = 60.8, c = 72.2 Å, β = 115.8°. Both crystal forms contain one molecule per asymmetric unit and diffract to a resolution of better than 2.0 Å. Initial phases were obtained by SIRAS from in-house data from a cubic crystal that was soaked with an unusually low KI concentration of 0.25 M

Combination Superficial Peels With Salicylic Acid and Post-Peel Retinoids.

Science.gov (United States)

Kligman, Douglas E; Draelos, Zoe D

2016-04-01

Salicylic acid (SA) and retinoids, tretinoin (all-trans retinoic acid [ATRA]), and retinol (all-trans retinol) are widely used as topical agents for the improvement of photodamage and acne vulgaris. They can be used in daily take-home products or as part of an in-office procedure, combining the benefits of a keratolytic (SA) and a retinoid. The objective of this research was to compare the efficacy for ameliorating photodamage of topical tretinoin (0.25%) and retinol (0.25%) to baseline and with each other when applied after a 30% salicylic acid peel on human facial skin. Twenty female subjects received a full face 30% SA peel followed by the overnight application of tretinoin to a 1 randomized half-face and retinol to the opposite side (split-face study). The identical procedure was repeated at week 2. Double-blinded subject and investigator assessments of the results were captured at weeks 2 and 4. By investigator evaluation, both peeling regimens were effective in improving photodamage parameters compared to baseline. (ATRA P-values at week 4 were: P=.00008 texture, P=.00013 roughness, P=.00221 pores, P=.00098 dryness, P=.02770 erythema, and P=.00008 overall appearance. Retinol P-values at week 4 were: P=.00019 texture, P=.00053 roughness, P=.00221 pores, P=.00147 dryness, P=.02770 erythema, and P=.0043 overall appearance.) By subject self-assessment compared with baseline, both tretinoin and retinol were effective in improving overall appearance (ATRA P=.0229 and retinol P=.0190). By investigator evaluation comparing tretinoin with retinol, tretinoin was slightly better than retinol at week 4 in improving texture P=.00506, roughness P=.01171, and overall appearance P=.00506. By subject self-assessment comparing tretinoin with retinol, there was no difference in overall appearance (ATRA P=.2367 and retinol P=.3613). Either topical tretinoin (0.25%) or retinol (0.25%) can be used safely and effectively when applied in office immediately after SA peeling to

Triosephosphate isomerase: energetics of the reaction catalyzed by the yeast enzyme expressed in Escherichia coli

International Nuclear Information System (INIS)

Nickbarg, E.B.; Knowles, J.R.

1988-01-01

Triosephosphate isomerase from bakers' yeast, expressed in Escherichia coli strain DF502(p12), has been purified to homogeneity. The kinetics of the reaction in each direction have been determined at pH 7.5 and 30 degrees C. Deuterium substitution at the C-2 position of substrate (R)-glyceraldehyde phosphate and at the 1-pro-R position of substrate dihydroxyacetone phosphate results in kinetic isotope effects on kcat of 1.6 and 3.4, respectively. The extent of transfer of tritium from [1(R)- 3 H]dihydroxyacetone phosphate to product (R)-glyceraldehyde phosphate during the catalyzed reaction is only 3% after 66% conversion to product, indicating that the enzymic base that mediates proton transfer is in rapid exchange with solvent protons. When the isomerase-catalyzed reaction is run in tritiated water in each direction, radioactivity is incorporated both into the remaining substrate and into the product. In the exchange-conversion experiment with dihydroxyacetone phosphate as substrate, the specific radioactivity of remaining dihydroxyacetone phosphate rises as a function of the extent of reaction with a slope of about 0.3, while the specific radioactivity of the products is 54% that of the solvent. In the reverse direction with (R)-glyceraldehyde phosphate as substrate, the specific radioactivity of the product formed is only 11% that of the solvent, while the radioactivity incorporated into the remaining substrate (R)-glyceraldehyde phosphate also rises as a function of the extent of reaction with a slope of 0.3. These results have been analyzed according to the protocol described earlier to yield the free energy profile of the reaction catalyzed by the yeast isomerase

A Protein Disulfide Isomerase Gene Fusion Expression System That Increases the Extracellular Productivity of Bacillus brevis

Science.gov (United States)

Kajino, Tsutomu; Ohto, Chikara; Muramatsu, Masayoshi; Obata, Shusei; Udaka, Shigezo; Yamada, Yukio; Takahashi, Haruo

2000-01-01

We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system. PMID:10653729

The basic tilted helix bundle domain of the prolyl isomerase FKBP25 is a novel double-stranded RNA binding module

Science.gov (United States)

Dilworth, David; Bonnafous, Pierre; Edoo, Amiirah Bibi; Bourbigot, Sarah; Pesek-Jardim, Francy; Gudavicius, Geoff; Serpa, Jason J.; Petrotchenko, Evgeniy V.; Borchers, Christoph H.

2017-01-01

Abstract Prolyl isomerases are defined by a catalytic domain that facilitates the cis–trans interconversion of proline residues. In most cases, additional domains in these enzymes add important biological function, including recruitment to a set of protein substrates. Here, we report that the N-terminal basic tilted helix bundle (BTHB) domain of the human prolyl isomerase FKBP25 confers specific binding to double-stranded RNA (dsRNA). This binding is selective over DNA as well as single-stranded oligonucleotides. We find that FKBP25 RNA-association is required for its nucleolar localization and for the vast majority of its protein interactions, including those with 60S pre-ribosome and early ribosome biogenesis factors. An independent mobility of the BTHB and FKBP catalytic domains supports a model by which the N-terminus of FKBP25 is anchored to regions of dsRNA, whereas the FKBP domain is free to interact with neighboring proteins. Apart from the identification of the BTHB as a new dsRNA-binding module, this domain adds to the growing list of auxiliary functions used by prolyl isomerases to define their primary cellular targets. PMID:29036638

RPE y velocidad como marcadores de intensidad en el press de banca

OpenAIRE

Bautista, I. J.; Chirosa, I. J.; Chirosa, L. J.; Martín, I.; Revilla, J.

2016-01-01

El propósito de esta investigación fue analizar la relación entre la evolución de la velocidad media (Velmedia) y los valores de la escala OMNI-RES, en 3 intensidades (carga Inicial [CI], máxima potencia [MP] y repetición máxima [RM]). 38 sujetos divididos en dos grupos: Entrenados (G1, n = 19) y No Entrenados (G2, n = 19) realizaron un test incremental de cargas en el ejercicio del press de banca. En la percepción subjetiva del esfuerzo (RPE) no se encontraron diferencias significativas entr...

Quantification of training load using session RPE method and performance in futsal. DOI: 10.5007/1980-0037.2012v14n1p73

Directory of Open Access Journals (Sweden)

Victor Hugo Freitas

2011-12-01

Full Text Available There is a need of studies about training load (TL quantification using session rating of perceived exertion (session-RPE and its relation to athlete’s performance in futsal. The objective of this study was to describe training load behavior using session-RPE method and to analyze futsal athlete´s performance during a preparatory period and its relation to TL. Twelve male futsal players (age 24,92 ± 5,20 year, weight 73,42 ± 5,70 kg, height 175,83 ± 5,05 cm were submitted to 14 weeks of training quantified by the session-RPE method. It was measured maximum volume of oxygen (VO₂max, velocity, agility and vertical jump (VJ before and after the training period and also the VJ every week. Using the Wilcoxon test, it was observed an increase in VO₂max (p=0,004, VJ (p=0,003, velocity (p=0,003 and agility (p=0,002 after the training period. Using the ANOVA to repeated measures (Tukey post-hoc, it was observed a greater training load (TL in the first mesocycle compared to the others and a greater CT in the second compared to the third mesocycle. It was not found a relation between VJ and TL. It was concluded that the session RPE allowed an effective TL quantification through the training period and the athlete’s performance increased along with the progressive TL reduction description. There wasn’t correlation between the TL and performance.

Quantification of training load using session RPE method and performance in futsal. DOI: 10.5007/1980-0037.2012v14n1p73

Directory of Open Access Journals (Sweden)

Maurício Gattas Bara Filho

2012-01-01

Full Text Available There is a need of studies about training load (TL quantification using session rating of perceived exertion (session-RPE and its relation to athlete’s performance in futsal. The objective of this study was to describe training load behavior using session-RPE method and to analyze futsal athlete´s performance during a preparatory period and its relation to TL. Twelve male futsal players (age 24,92 ± 5,20 year, weight 73,42 ± 5,70 kg, height 175,83 ± 5,05 cm were submitted to 14 weeks of training quantified by the session-RPE method. It was measured maximum volume of oxygen (VO₂max, velocity, agility and vertical jump (VJ before and after the training period and also the VJ every week. Using the Wilcoxon test, it was observed an increase in VO₂max (p=0,004, VJ (p=0,003, velocity (p=0,003 and agility (p=0,002 after the training period. Using the ANOVA to repeated measures (Tukey post-hoc, it was observed a greater training load (TL in the first mesocycle compared to the others and a greater CT in the second compared to the third mesocycle. It was not found a relation between VJ and TL. It was concluded that the session RPE allowed an effective TL quantification through the training period and the athlete’s performance increased along with the progressive TL reduction description. There wasn’t correlation between the TL and performance.

Structural insights from a novel invertebrate triosephosphate isomerase from Litopenaeus vannamei

Science.gov (United States)

Lopez-Zavala, Alonso A.; Carrasco-Miranda, Jesus S.; Ramirez-Aguirre, Claudia D.; López-Hidalgo, Marisol; Benitez-Cardoza, Claudia G.; Ochoa-Leyva, Adrian; Cardona-Felix, Cesar S.; Diaz-Quezada, Corina; Rudiño-Piñera, Enrique; Sotelo-Mundo, Rogerio R.; Brieba, Luis G.

2016-01-01

Triosephosphate isomerase (TIM; EC 5.3.1.1) is a key enzyme involved in glycolysis and gluconeogenesis. Glycolysis is one of the most regulated metabolic pathways, however little is known about the structural mechanisms for its regulation in non-model organisms, like crustaceans. To understand the structure and function of this enzyme in invertebrates, we obtained the crystal structure of triosephosphate isomerase from the marine Pacific whiteleg shrimp (Litopenaeus vannamei, LvTIM) in complex with its inhibitor 2-phosphogyceric acid (2-PG) at 1.7 Å resolution. LvTIM assembles as a homodimer with residues 166-176 covering the active site and residue Glu166 interacting with the inhibitor. We found that LvTIM is the least stable TIM characterized to date, with the lowest range of melting temperatures, and with the lowest activation enthalpy associated with the thermal unfolding process reported. In TIMs dimer stabilization is maintained by an interaction of loop 3 by a set of hydrophobic contacts between subunits. Within these contacts, the side chain of a hydrophobic residue of one subunit fits into a cavity created by a set of hydrophobic residues in the neighboring subunit, via a "ball and socket" interaction. LvTIM presents a Cys47 at the "ball" inter-subunit contact indicating that the character of this residue is responsible for the decrease in dimer stability. Mutational studies show that this residue plays a role in dimer stability but is not a solely determinant for dimer formation. PMID:27614148

Retinol Dehydrogenases Regulate Vitamin A Metabolism for Visual Function

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Bhubanananda Sahu

2016-11-01

Full Text Available The visual system produces visual chromophore, 11-cis-retinal from dietary vitamin A, all-trans-retinol making this vitamin essential for retinal health and function. These metabolic events are mediated by a sequential biochemical process called the visual cycle. Retinol dehydrogenases (RDHs are responsible for two reactions in the visual cycle performed in retinal pigmented epithelial (RPE cells, photoreceptor cells and Müller cells in the retina. RDHs in the RPE function as 11-cis-RDHs, which oxidize 11-cis-retinol to 11-cis-retinal in vivo. RDHs in rod photoreceptor cells in the retina work as all-trans-RDHs, which reduce all-trans-retinal to all-trans-retinol. Dysfunction of RDHs can cause inherited retinal diseases in humans. To facilitate further understanding of human diseases, mouse models of RDHs-related diseases have been carefully examined and have revealed the physiological contribution of specific RDHs to visual cycle function and overall retinal health. Herein we describe the function of RDHs in the RPE and the retina, particularly in rod photoreceptor cells, their regulatory properties for retinoid homeostasis and future therapeutic strategy for treatment of retinal diseases.

Effect of gamma irradiation on whole-cell glucose isomerase. Pt.1

International Nuclear Information System (INIS)

Bachman, S.; Gebicka, L.

1984-01-01

Gamma-rays induced inactivation of Actinoplanes missouriensis and Streptomyces olivaceus glucose isomerase has been studied. This enzyme exhibits high resistance against ionizing radiation. The D 37 value was found to be equal to 131 kGy for Actinoplanes missouriensis cells and 88 kGy for Streptomyces olivaceus cells when irradiated in the dry state in the presence of air. Mg 2+ ions do not affect the radiosensitivity of the enzyme in cells, while the addition of Co 2+ ions to the cell suspension increases its stability against ionizing radiation. (orig.) [de

Production of D-tagatose, a low caloric sweetener during milk fermentation using L-arabinose isomerase.

Science.gov (United States)

Rhimi, Moez; Chouayekh, Hichem; Gouillouard, Isabelle; Maguin, Emmanuelle; Bejar, Samir

2011-02-01

Lactobacillusdelbrueckii subsp. bulgaricus and Streptococcus thermophilus are used for the biotransformation of milk in yoghurt. During milk fermentation, these lactic acid bacteria (LAB) hydrolyze lactose producing a glucose moiety that is further metabolized and a galactose moiety that they are enable to metabolize. We investigated the ability of L. bulgaricus and S. thermophilus strains expressing a heterologous L-arabinose isomerase to convert residual D-galactose to D-tagatose. The Bacillus stearothermophilus US100l-arabinose isomerase (US100l-AI) was expressed in both LAB, using a new shuttle vector where the araA US100 gene is under the control of the strong and constitutive promoter of the L. bulgaricus ATCC 11842 hlbA gene. The production of L-AI by these LAB allowed the bioconversion of D-galactose to D-tagatose during fermentation in laboratory media and milk. We also established that the addition of L-AI to milk also allowed the conversion of D-galactose into D-tagatose during the fermentation process. Copyright © 2010 Elsevier Ltd. All rights reserved.

A preliminary X-ray study of sedoheptulose-7-phosphate isomerase from Burkholderia pseudomallei

International Nuclear Information System (INIS)

Kim, Mi-Sun; Shin, Dong Hae

2009-01-01

Sedoheptulose-7-phosphate isomerase (GmhA) from B. pseudomallei is one of the targets of antibiotic adjuvants for melioidosis. In this study, GmhA has been cloned, expressed, purified and crystallized. Sedoheptulose-7-phosphate isomerase (GmhA) converts d-sedoheptulose 7-phosphate to d,d-heptose 7-phosphate. This is the first step in the biosynthesis pathway of NDP-heptose, which is responsible for the pleiotropic phenotype. This biosynthesis pathway is the target of inhibitors to increase the membrane permeability of Gram-negative pathogens or of adjuvants working synergistically with known antibiotics. Burkholderia pseudomallei is the causative agent of melioidosis, a seriously invasive disease in animals and humans in tropical and subtropical areas. GmhA from B. pseudomallei is one of the targets of antibiotic adjuvants for melioidosis. In this study, GmhA has been cloned, expressed, purified and crystallized. Synchrotron X-ray data were also collected to 1.9 Å resolution. The crystal belonged to the primitive orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 61.3, b = 84.2, c = 142.3 Å. A full structural determination is under way in order to provide insights into the structure–function relationships of this protein

Compact conformations of human protein disulfide isomerase.

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Shang Yang

Full Text Available Protein disulfide isomerase (PDI composed of four thioredoxin-like domains a, b, b', and a', is a key enzyme catalyzing oxidative protein folding in the endoplasmic reticulum. Large scale molecular dynamics simulations starting from the crystal structures of human PDI (hPDI in the oxidized and reduced states were performed. The results indicate that hPDI adopts more compact conformations in solution than in the crystal structures, which are stabilized primarily by inter-domain interactions, including the salt bridges between domains a and b' observed for the first time. A prominent feature of the compact conformations is that the two catalytic domains a and a' can locate close enough for intra-molecular electron transfer, which was confirmed by the characterization of an intermediate with a disulfide between the two domains. Mutations, which disrupt the inter-domain interactions, lead to decreased reductase activity of hPDI. Our molecular dynamics simulations and biochemical experiments reveal the intrinsic conformational dynamics of hPDI and its biological impact.

The crystal structure of a multifunctional protein: Phosphoglucose isomerase/autocrine motility factor/neuroleukin

OpenAIRE

Sun, Yuh-Ju; Chou, Chia-Cheng; Chen, Wei-Shone; Wu, Rong-Tsun; Meng, Menghsiao; Hsiao, Chwan-Deng

1999-01-01

Phosphoglucose isomerase (PGI) plays a central role in both the glycolysis and the gluconeogenesis pathways. We present here the complete crystal structure of PGI from Bacillus stearothermophilus at 2.3-Å resolution. We show that PGI has cell-motility-stimulating activity on mouse colon cancer cells similar to that of endogenous autocrine motility factor (AMF). PGI can also enhance neurite outgrowth on neuronal progenitor cells similar to that observed for neuroleukin. The results confirm tha...

Crystallization and preliminary X-ray crystallographic analysis of l-rhamnose isomerase with a novel high thermostability from Bacillus halodurans

International Nuclear Information System (INIS)

Doan, Thi-Ngoc-Thanh; Prabhu, Ponnandy; Kim, Jin-Kwang; Ahn, Yeh-Jin; Natarajan, Sampath; Kang, Lin-Woo; Park, Geon Tae; Lim, Sang-Boem; Lee, Jung-Kul

2010-01-01

l-Rhamnose isomerase (l-RhI) from B. halodurans has been purified and crystallized. The crystals of l-RhI belonged to the monoclinic space group P2 1 , with unit-cell parameters a = 83.2, b = 164.9, c = 92.0 Å, β = 116.0°, and diffracted to 2.5 Å resolution. l-Rhamnose isomerases catalyze isomerization between l-rhamnose (6-deoxy-l-mannose) and l-rhamnulose (6-deoxy-l-fructose), which is the first step in rhamnose catabolism. l-Rhamnose isomerase from Bacillus halodurans ATCC BAA-125 (BHRI) exhibits interesting characteristics such as high thermostability and selective substrate specificity. BHRI fused with an HHHHHH sequence was purified and crystallized in order to elucidate the molecular basis of its unique enzymatic properties. The crystals were grown by the hanging-drop vapour-diffusion method and belonged to the monoclinic space group P2 1 , with unit-cell parameters a = 83.2, b = 164.9, c = 92.0 Å, β = 116.0°. Diffraction data were collected to 2.5 Å resolution. According to a Matthews coefficient calculation, there are four monomers in the asymmetric unit with a V M of 3.0 Å 3 Da −1 and a solvent content of 59.3%. The initial structure of BHRI has been determined by the molecular-replacement method

The peptidyl prolyl cis/trans isomerase Pin1/Ess1 inhibits phosphorylation and toxicity of tau in a yeast model for Alzheimer’s disease

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Ann De Vos

2015-04-01

Full Text Available Since hyperphosphorylation of protein tau is a crucial event in Alzheimer’s disease, additional mechanisms besides the interplay of kinase and phosphatase activities are investigated, such as the effect of the peptidyl prolyl cis/trans isomerase Pin1. This isomerase was shown to bind and isomerize phosphorylated protein tau, thereby restoring the microtubule associated protein function of tau as well as promoting the dephosphorylation of the protein by the trans-dependent phosphatase PP2A. In this study we used models based on Saccharomyces cerevisiae to further elucidate the influence of Pin1 and its yeast ortholog Ess1 on tau phosphorylation and self-assembly. We could demonstrate that in yeast, a lack of Pin1 isomerase activity leads to an increase in phosphorylation of tau at Thr231, comparable to AD brain and consistent with earlier findings in other model organisms. However, we could also distinguish an effect by Pin1 on other residues of tau, i.e. Ser235 and Ser198/199/202. Furthermore, depletion of Pin1 isomerase activity results in reduced growth of the yeast cells, which is enhanced upon expression of tau. This suggests that the accumulation of hyperphosphorylated and aggregation-prone tau causes cytotoxicity in yeast. This study introduces yeast as a valuable model organism to characterize in detail the effect of Pin1 on the biochemical characteristics of protein tau, more specifically its phosphorylation and aggregation.

Loss of function of the retinoid-related nuclear receptor (RORB) gene and epilepsy

DEFF Research Database (Denmark)

Rudolf, Gabrielle; Lesca, Gaetan; Mehrjouy, Mana M

2016-01-01

nuclear receptor (RORβ), in four affected family members. In addition, two de novo variants (c.218T>C/p.(Leu73Pro); c.1249_1251delACG/p.(Thr417del)) were identified in sporadic patients by trio-based exome sequencing. We also found two de novo deletions in patients with behavioral and cognitive impairment...... in various types of epilepsies in the past few years. In the present study, we performed whole-exome sequencing in a family with GGE consistent with the diagnosis of eyelid myoclonia with absences. We found a nonsense variant (c.196C>T/p.(Arg66*)) in RORB, which encodes the beta retinoid-related orphan...

Mechanism of ultraviolet light induced catabolite repression of L-arabinose isomerase

Energy Technology Data Exchange (ETDEWEB)

Bhatnagar, D; Bhattacharya, A K [Banaras Hindu Univ. (India). Inst. of Medical Sciences

1982-12-01

An attempt has been made to find out how U.V. irradiation of E.coli B/r cells causes catabolite repression to inhibit L-arabinose isomerase synthesis. The results presented show that U.V. irradiation leads to a lowering of the cellular cyclic AMP level and of the cyclic AMP binding activity. Unlike catabolite repression by glucose, no small molecular weight compound is involved in U.V. light induced inhibition of the binding activity. It is therefore concluded that the mechanism of catabolite repression induced by U.V. appears to be different from that of the catabolite repression by glucose.

40 CFR 65.65 - Monitoring.

Science.gov (United States)

2010-07-01

... 40 Protection of Environment 15 2010-07-01 2010-07-01 false Monitoring. 65.65 Section 65.65 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) CONSOLIDATED FEDERAL AIR RULE Process Vents § 65.65 Monitoring. (a) An owner or operator of a Group 2A process vent...

Preliminary crystallographic analysis of two hypothetical ribose-5-phosphate isomerases from Streptococcus mutans

International Nuclear Information System (INIS)

Wang, Chen; Fan, Xuexin; Cao, Xiaofang; Liu, Xiang; Li, Lanfen; Su, Xiaodong

2012-01-01

Two hypothetical ribose-5-phosphate isomerases from S. mutans have been produced in E. coli and crystallized. The crystals diffracted to high resolutions suitable for crystallographic analyses. Study of the enzymes from sugar metabolic pathways may provide a better understanding of the pathogenesis of the human oral pathogen Streptococcus mutans. Bioinformatics, biochemical and crystallization methods were used to characterize and understand the function of two putative ribose-5-phosphate isomerases: SMU1234 and SMU2142. The proteins were cloned and constructed with N-terminal His tags. Protein purification was performed by Ni 2+ -chelating and size-exclusion chromatography. The crystals of SUM1234 diffracted to 1.9 Å resolution and belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 48.97, b = 98.27, c = 101.09 Å, α = β = γ = 90°. The optimized SMU2142 crystals diffracted to 2.7 Å resolution and belonged to space group P1, with unit-cell parameters a = 53.7, b = 54.1, c = 86.5 Å, α = 74.2, β = 73.5, γ = 83.7°. Initial phasing of both proteins was attempted by molecular replacement; the structure of SMU1234 could easily be solved, but no useful results were obtained for SMU2142. Therefore, SeMet-labelled SMU2142 will be prepared for phasing

Long-term effect of gene therapy on Leber's congenital amaurosis.

Science.gov (United States)

Bainbridge, James W B; Mehat, Manjit S; Sundaram, Venki; Robbie, Scott J; Barker, Susie E; Ripamonti, Caterina; Georgiadis, Anastasios; Mowat, Freya M; Beattie, Stuart G; Gardner, Peter J; Feathers, Kecia L; Luong, Vy A; Yzer, Suzanne; Balaggan, Kamaljit; Viswanathan, Ananth; de Ravel, Thomy J L; Casteels, Ingele; Holder, Graham E; Tyler, Nick; Fitzke, Fred W; Weleber, Richard G; Nardini, Marko; Moore, Anthony T; Thompson, Debra A; Petersen-Jones, Simon M; Michaelides, Michel; van den Born, L Ingeborgh; Stockman, Andrew; Smith, Alexander J; Rubin, Gary; Ali, Robin R

2015-05-14

Mutations in RPE65 cause Leber's congenital amaurosis, a progressive retinal degenerative disease that severely impairs sight in children. Gene therapy can result in modest improvements in night vision, but knowledge of its efficacy in humans is limited. We performed a phase 1-2 open-label trial involving 12 participants to evaluate the safety and efficacy of gene therapy with a recombinant adeno-associated virus 2/2 (rAAV2/2) vector carrying the RPE65 complementary DNA, and measured visual function over the course of 3 years. Four participants were administered a lower dose of the vector, and 8 were administered a higher dose. In a parallel study in dogs, we investigated the relationship among vector dose, visual function, and electroretinography (ERG) findings. Improvements in retinal sensitivity were evident, to varying extents, in six participants for up to 3 years, peaking at 6 to 12 months after treatment and then declining. No associated improvement in retinal function was detected by means of ERG. Three participants had intraocular inflammation, and two had clinically significant deterioration of visual acuity. The reduction in central retinal thickness varied among participants. In dogs, RPE65 gene therapy with the same vector at lower doses improved vision-guided behavior, but only higher doses resulted in improvements in retinal function that were detectable with the use of ERG. Gene therapy with rAAV2/2 RPE65 vector improved retinal sensitivity, albeit modestly and temporarily. Comparison with the results obtained in the dog model indicates that there is a species difference in the amount of RPE65 required to drive the visual cycle and that the demand for RPE65 in affected persons was not met to the extent required for a durable, robust effect. (Funded by the National Institute for Health Research and others; ClinicalTrials.gov number, NCT00643747.).

Characterization of a monoclonal antibody that specifically inhibits triosephosphate isomerase activity of Taenia solium.

Science.gov (United States)

Víctor, Sanabria-Ayala; Yolanda, Medina-Flores; Araceli, Zavala-Carballo; Lucía, Jiménez; Abraham, Landa

2013-08-01

In the present study, we obtained and characterized partially a monoclonal antibody (4H11D10B11 mAb) against triosephosphate isomerase from Taenia solium (TTPI). This antibody recognized the enzyme by both ELISA and western blot and was able to inhibit its enzymatic activity in 74%. Moreover, the antigen-binding fragments (Fabs), products of digestion of the monoclonal antibody with papain, retained almost the same inhibitory effect. We determined the binding site by ELISA; synthetic peptides containing sequences from different non-conserved regions of the TTPI were confronted to the 4H11D10B11 mAb. The epitope recognized by the monoclonal antibody was located on peptide TTPI-56 (ATPAQAQEVHKVVRDWIRKHVDAGIADKARI), and an analysis of mimotopes, obtained with the 4H11D10B11 mAb, suggests that the epitope spans the sequence WIRKHVDAGIAD, residues 193-204 of the enzyme. This epitope is located within helix 6, next to loop 6, an essential active loop during catalysis. The antibody did not recognize triosephosphate isomerase from man and pig, definitive and intermediary hosts of T. solium, respectively. Furthermore, it did not bind to the catalytic site, since kinetic analysis demonstrated that inhibition had a non-competitive profile. Copyright © 2013 Elsevier Inc. All rights reserved.

A dual inhibitor against prolyl isomerase Pin1 and cyclophilin discovered by a novel real-time fluorescence detection method

International Nuclear Information System (INIS)

Mori, Tadashi; Hidaka, Masafumi; Lin, Yi-Chin; Yoshizawa, Ibuki; Okabe, Takayoshi; Egashira, Shinichiro; Kojima, Hirotatsu; Nagano, Tetsuo; Koketsu, Mamoru; Takamiya, Mari; Uchida, Takafumi

2011-01-01

Research highlights: → A Pin1 (prolyl isomerase) inhibitor, TME-001, has been discovered by using a new established high-throughput screening method. → The TME-001 showed a cell-active inhibition with lower cytotoxic effect than known Pin1 inhibitors. → Kinetic analyses revealed that the TME-001 is the first compound that exhibits dual inhibition of Pin1 and another type of prolyl isomerase, cyclophilin. → Thus, similarities of structure and reaction mechanism between Pin1 and cyclophilin are proposed. -- Abstract: Pin1, a peptidyl prolyl cis/trans isomerase (PPIase), is a potential target molecule for cancer, infectious disease, and Alzheimer's disease. We established a high-throughput screening method for Pin1 inhibitors, which employs a real-time fluorescence detector. This screening method identified 66 compounds that inhibit Pin1 out of 9756 compounds from structurally diverse chemical libraries. Further evaluations of surface plasmon resonance methods and a cell proliferation assay were performed. We discovered a cell-active inhibitor, TME-001 (2-(3-chloro-4-fluoro-phenyl)-isothiazol-3-one). Surprisingly, kinetic analyses revealed that TME-001 is the first compound that exhibits dual inhibition of Pin1 (IC 50 = 6.1 μM) and cyclophilin, another type of PPIase, (IC 50 = 13.7 μM). This compound does not inhibit FKBP. This finding suggests the existence of similarities of structure and reaction mechanism between Pin1 and cyclophilin, and may lead to a more complete understanding of the active sites of PPIases.

Vitamin A Derivatives as Treatment Options for Retinal Degenerative Diseases

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Tadao Maeda

2013-07-01

Full Text Available The visual cycle is a sequential enzymatic reaction for vitamin A, all-trans-retinol, occurring in the outer layer of the human retina and is essential for the maintenance of vision. The central source of retinol is derived from dietary intake of both retinol and pro-vitamin A carotenoids. A series of enzymatic reactions, located in both the photoreceptor outer segment and the retinal pigment epithelium, transform retinol into the visual chromophore 11-cis-retinal, regenerating visual pigments. Retina specific proteins carry out the majority of the visual cycle, and any significant interruption in this sequence of reactions is capable of causing varying degrees of blindness. Among these important proteins are Lecithin:retinol acyltransferase (LRAT and retinal pigment epithelium-specific 65-kDa protein (RPE65 known to be responsible for esterification of retinol to all-trans-retinyl esters and isomerization of these esters to 11-cis-retinal, respectively. Deleterious mutations in these genes are identified in human retinal diseases that cause blindness, such as Leber congenital amaurosis (LCA and retinitis pigmentosa (RP. Herein, we discuss the pathology of 11-cis-retinal deficiency caused by these mutations in both animal disease models and human patients. We also review novel therapeutic strategies employing artificial visual chromophore 9-cis-retinoids which have been employed in clinical trials involving LCA patients.

Alanine/RPE dosimetry in the process of blood irradiation; Dosimetria alanina/RPE en el proceso de irradiacion de sangre

Energy Technology Data Exchange (ETDEWEB)

Chen, F.; Covas, D.T.; Baffa, O. [Departamento de Fisica e Matematica, FFCLRP, Universidade de Sao Paulo, 14040-901 Ribeirao Preto-SP (Brazil)

2000-07-01

A set of eighty dosemeters approximately of Dl- alanine was proportioned to the Hemo center of Hospital das Clinicas of Medicine Faculty at Ribeirao Preto (HC-FRMP) in the Sao Paulo University, with the purpose to realize a quality control of the radiation dose supplied to blood bags. These dosemeters were divided in eight groups and irradiated for a two months period.The dosemeters were adhered to the bags and arranged inside of an acrylic box filled water with dimensions: (40x40x20) cm{sup 3}. The box irradiation was made using two fields parallel-opposed of (40x40) cm{sup 2} at 80 cm distance source-surface, in the Radiotherapy Service of HC-FMRP, with a {sup 60} Co teletherapy unit. The irradiation time was sufficient to supply a dose of 20 Gy approximately in the box center. The RPE measures were realized in a Varian E-4 spectrometer operating in X-band. For the total of dosemeters and for the irradiation volume, the minimum and maximum doses were of 14 Gy and 23 Gy respectively. The mean dose was (18{+-}2) Gy (1 {sigma}), and the variability coefficient 11 %. Around 5 % of the bags received a dose under of the recommended limit by the Brazilian legislation and as consequence, the exposure time was increased. (Author)

Inhibition of estrogen-responsive gene activation by the retinoid X receptor beta: evidence for multiple inhibitory pathways.

OpenAIRE

Segars, J H; Marks, M S; Hirschfeld, S; Driggers, P H; Martinez, E; Grippo, J F; Brown, M; Wahli, W; Ozato, K

1993-01-01

The retinoid X receptor beta (RXR beta; H-2RIIBP) forms heterodimers with various nuclear hormone receptors and binds multiple hormone response elements, including the estrogen response element (ERE). In this report, we show that endogenous RXR beta contributes to ERE binding activity in nuclear extracts of the human breast cancer cell line MCF-7. To define a possible regulatory role of RXR beta regarding estrogen-responsive transcription in breast cancer cells, RXR beta and a reporter gene d...

Alginate as a cell culture substrate for growth and differentiation of human retinal pigment epithelial cells.

Science.gov (United States)

Heidari, Razeih; Soheili, Zahra-Soheila; Samiei, Shahram; Ahmadieh, Hamid; Davari, Maliheh; Nazemroaya, Fatemeh; Bagheri, Abouzar; Deezagi, Abdolkhalegh

2015-03-01

The purpose of this study was to evaluate retinal pigment epithelium (RPE) cells' behavior in alginate beads that establish 3D environment for cellular growth and mimic extracellular matrix versus the conventional 2D monolayer culture. RPE cells were encapsulated in alginate beads by dripping alginate cell suspension into CaCl2 solution. Beads were suspended in three different media including Dulbecco's modified Eagle's medium (DMEM)/F12 alone, DMEM/F12 supplemented with 10 % fetal bovine serum (FBS), and DMEM/F12 supplemented with 30 % human amniotic fluid (HAF). RPE cells were cultivated on polystyrene under the same conditions as controls. Cell phenotype, cell proliferation, cell death, and MTT assay, immunocytochemistry, and real-time RT-PCR were performed to evaluate the effect of alginate on RPE cells characteristics and integrity. RPE cells can survive and proliferate in alginate matrixes. Immunocytochemistry analysis exhibited Nestin, RPE65, and cytokeratin expressions in a reasonable number of cultured cells in alginate beads. Real-time PCR data demonstrated high levels of Nestin, CHX10, RPE65, and tyrosinase gene expressions in RPE cells immobilized in alginate when compared to 2D monolayer culture systems. The results suggest that alginate can be used as a reliable scaffold for maintenance of RPE cells' integrity and in vitro propagation of human retinal progenitor cells for cell replacement therapies in retinal diseases.

The multidrug resistance IncA/C transferable plasmid encodes a novel domain-swapped dimeric protein-disulfide isomerase.

Science.gov (United States)

Premkumar, Lakshmanane; Kurth, Fabian; Neyer, Simon; Schembri, Mark A; Martin, Jennifer L

2014-01-31

The multidrug resistance-encoding IncA/C conjugative plasmids disseminate antibiotic resistance genes among clinically relevant enteric bacteria. A plasmid-encoded disulfide isomerase is associated with conjugation. Sequence analysis of several IncA/C plasmids and IncA/C-related integrative and conjugative elements (ICE) from commensal and pathogenic bacteria identified a conserved DsbC/DsbG homolog (DsbP). The crystal structure of DsbP reveals an N-terminal domain, a linker region, and a C-terminal catalytic domain. A DsbP homodimer is formed through domain swapping of two DsbP N-terminal domains. The catalytic domain incorporates a thioredoxin-fold with characteristic CXXC and cis-Pro motifs. Overall, the structure and redox properties of DsbP diverge from the Escherichia coli DsbC and DsbG disulfide isomerases. Specifically, the V-shaped dimer of DsbP is inverted compared with EcDsbC and EcDsbG. In addition, the redox potential of DsbP (-161 mV) is more reducing than EcDsbC (-130 mV) and EcDsbG (-126 mV). Other catalytic properties of DsbP more closely resemble those of EcDsbG than EcDsbC. These catalytic differences are in part a consequence of the unusual active site motif of DsbP (CAVC); substitution to the EcDsbC-like (CGYC) motif converts the catalytic properties to those of EcDsbC. Structural comparison of the 12 independent subunit structures of DsbP that we determined revealed that conformational changes in the linker region contribute to mobility of the catalytic domain, providing mechanistic insight into DsbP function. In summary, our data reveal that the conserved plasmid-encoded DsbP protein is a bona fide disulfide isomerase and suggest that a dedicated oxidative folding enzyme is important for conjugative plasmid transfer.

Genome sequence of carboxylesterase, carboxylase and xylose isomerase producing alkaliphilic haloarchaeon Haloterrigena turkmenica WANU15

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Samy Selim

2016-03-01

Full Text Available We report draft genome sequence of Haloterrigena turkmenica strain WANU15, isolated from Soda Lake. The draft genome size is 2,950,899 bp with a G + C content of 64% and contains 49 RNA sequence. The genome sequence can be accessed at DDBJ/EMBL/GenBank under the accession no. LKCV00000000. Keywords: Soda Lake, Haloterrigena turkmenica, Carboxylesterase, Carboxylase, Xylose isomerase, Whole genome sequencing

Revisiting the mechanistic basis of the French Paradox: red wine inhibits the activity of protein disulfide isomerase in vitro

Science.gov (United States)

Galinski, Christine N.; Zwicker, Jeffrey I.; Kennedy, Daniel R.

2015-01-01

Introduction Although epidemiologic evidence points to cardioprotective activity of red wine, the mechanistic basis for antithrombotic activity has not been established. Quercetin and related flavonoids are present in high concentrations in red but not white wine. Quercetin-glycosides were recently shown to prevent thrombosis in animal models through the inhibition of extracellular protein disulfide isomerase (PDI). We evaluated whether red or white wine inhibited PDI activity in vitro. Methods Quercetin levels in red and white wines were measured by HPLC analysis. Inhibition of PDI activity by red and white wines was assessed by an insulin reduction turbidity assay at various concentrations of wine. PDI inhibition was confirmed using a reduced peptide that contained a disulfide containing peptide as a substrate. The inhibition of PDI related thiol isomerases ERp5 and ERp57 was also assessed. Results We observed a dose-dependent decrease of PDI activity for a variety of red but not white wines. Red wine diluted to 3% final concentration resulted in over 80% inhibition of PDI activity by insulin reductase assay for all varieties tested. This inhibition was also observed in the peptide based assay. Red grape juice yielded similar results but ethanol alone did not affect PDI activity. Interestingly, red wine also inhibited the PDI related thiol isomerases ERp5 and ERp57, albeit to a lesser degree than PDI. Conclusions PDI activity is inhibited by red wine and grape juice, identifying a potentially novel mechanism underlying the cardiovascular benefits attributed to wine consumption. PMID:26585763

PlGF gene knockdown in human retinal pigment epithelial cells.

Science.gov (United States)

Akrami, Hassan; Soheili, Zahra-Soheila; Sadeghizadeh, Majid; Ahmadieh, Hamid; Rezaeikanavi, Mozhgan; Samiei, Shahram; Khalooghi, Keynoush

2011-04-01

To evaluate the knockdown of placental growth factor (PlGF) gene expression in human retinal pigment epithelium (RPE) cells and its effect on cell proliferation, apoptosis and angiogenic potential of RPE cells. Human RPE cells were isolated by dispase I solution and cultured in DMEM/F12 supplemented with 10% fetal calf serum (FCS). A small interfering RNA (siRNA) corresponding to PlGF mRNA and a scrambled siRNA (scRNA) were introduced into the cells. Cell proliferation and cell death were examined by ELISA. PlGF mRNA and protein were quantified by real-time polymerase chain reaction (PCR) and western blot. The levels of gene expression for human retinal pigment epithelium-specific protein 65 kDa (RPE65), cellular retinaldehyde-binding protein (CRALBP) and tyrosinase were examined by real-time PCR. The angiogenic activity of RPE cell-derived conditioned media was assayed by a tube formation assay using human umbilical vein endothelial cells (HUVECs). At a final siRNA concentration of 20 pmol/ml, the transfection efficiency was about 80%. The amount of PlGF transcripts was reduced to 10% after 36 h of incubation, and the amount of PlGF protein in culture supernatant was significantly decreased. Suppression of PlGF gene had no effect on RPE cell proliferation and survival, and there were no notable changes in the transcript levels of RPE65, CRALBP or tyrosinase for the cultures treated by siRNA cognate to PlGF. Vascular tube formation was efficiently reduced in HUVECs. Our findings present PlGF as a key modulator of angiogenic potential in RPE cells of the human retina.

Crystal Structure of Escherichia coli L-Arabinose Isomerase (ECAI), The Putative Target of Biological Tagatose Production

Energy Technology Data Exchange (ETDEWEB)

Manjasetty,B.; Chance, M.

2006-01-01

Escherichia coli L-arabinose isomerase (ECAI; EC 5.3.1.4) catalyzes the isomerization of L-arabinose to L-ribulose in vivo. This enzyme is also of commercial interest as it catalyzes the conversion of D-galactose to D-tagatose in vitro. The crystal structure of ECAI was solved and refined at 2.6 Angstroms resolution. The subunit structure of ECAI is organized into three domains: an N-terminal, a central and a C-terminal domain. It forms a crystallographic trimeric architecture in the asymmetric unit. Packing within the crystal suggests the idea that ECAI can form a hexameric assembly. Previous electron microscopic and biochemical studies supports that ECAI is hexameric in solution. A comparison with other known structures reveals that ECAI adopts a protein fold most similar to E. coli fucose isomerase (ECFI) despite very low sequence identity 9.7%. The structural similarity between ECAI and ECFI with regard to number of domains, overall fold, biological assembly, and active site architecture strongly suggests that the enzymes have functional similarities. Further, the crystal structure of ECAI forms a basis for identifying molecular determinants responsible for isomerization of arabinose to ribulose in vivo and galactose to tagatose in vitro.

Isolation and characterization of a spontaneously immortalized bovine retinal pigmented epithelial cell line

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Griffiths T Daniel

2009-05-01

Full Text Available Abstract Background The Retinal Pigmented Epithelium (RPE is juxtaposed with the photoreceptor outer segments of the eye. The proximity of the photoreceptor cells is a prerequisite for their survival, as they depend on the RPE to remove the outer segments and are also influenced by RPE cell paracrine factors. RPE cell death can cause a progressive loss of photoreceptor function, which can diminish vision and, over time, blindness ensues. Degeneration of the retina has been shown to induce a variety of retinopathies, such as Stargardt's disease, Cone-Rod Dystrophy (CRD, Retinitis Pigmentosa (RP, Fundus Flavimaculatus (FFM, Best's disease and Age-related Macular Degeneration (AMD. We have cultured primary bovine RPE cells to gain a further understanding of the mechanisms of RPE cell death. One of the cultures, named tRPE, surpassed senescence and was further characterized to determine its viability as a model for retinal diseases. Results The tRPE cell line has been passaged up to 150 population doublings and was shown to be morphologically similar to primary cells. They have been characterized to be of RPE origin by reverse transcriptase PCR and immunocytochemistry using the RPE-specific genes RPE65 and CRALBP and RPE-specific proteins RPE65 and Bestrophin. The tRPE cells are also immunoreactive to vimentin, cytokeratin and zonula occludens-1 antibodies. Chromosome analysis indicates a normal diploid number. The tRPE cells do not grow in suspension or in soft agar. After 3H thymidine incorporation, the cells do not appear to divide appreciably after confluency. Conclusion The tRPE cells are immortal, but still exhibit contact inhibition, serum dependence, monolayer growth and secrete an extra-cellular matrix. They retain the in-vivo morphology, gene expression and cell polarity. Additionally, the cells endocytose exogenous melanin, A2E and purified lipofuscin granules. This cell line may be a useful in-vitro research model for retinal

Domain architecture of protein-disulfide isomerase facilitates its dual role as an oxidase and an isomerase in Ero1p-mediated disulfide formation

DEFF Research Database (Denmark)

Kulp, M. S.; Frickel, E. M.; Ellgaard, Lars

2006-01-01

reduction/rearrangement of non-native disulfides is poorly understood. We analyzed the role of individual PDI domains in disulfide bond formation in a reaction driven by their natural oxidant, Ero1p. We found that Ero1p oxidizes the isolated PDI catalytic thioredoxin domains, A and A' at the same rate......Native disulfide bond formation in eukaryotes is dependent on protein-disulfide isomerase (PDI) and its homologs, which contain varying combinations of catalytically active and inactive thioredoxin domains. However, the specific contribution of PDI to the formation of new disulfides versus...... catalytic (A) domain. The specific order of thioredoxin domains in PDI is important in establishing the asymmetry in the rate of oxidation of the two active sites thus allowing A and A', two thioredoxin domains that are similar in sequence and structure, to serve opposing functional roles as a disulfide...

Overexpression, purification, crystallization and preliminary diffraction studies of the Protaminobacter rubrum sucrose isomerase SmuA

International Nuclear Information System (INIS)

Ravaud, Stéphanie; Watzlawick, Hildegard; Haser, Richard; Mattes, Ralf; Aghajari, Nushin

2005-01-01

The P. rubrum sucrose isomerase SmuA, a key enzyme in the industrial production of isomaltulose, was crystallized and diffraction data were collected to 1.95 Å resolution. Palatinose (isomaltulose, α-d-glucosylpyranosyl-1,6-d-fructofuranose), a nutritional and acariogenic reducing sugar, is industrially obtained from sucrose by using immobilized cells of Protaminobacter rubrum that produce the sucrose isomerase SmuA. The isomerization of sucrose catalyzed by this enzyme also results in the formation of trehalulose (α-d-glucosylpyranosyl-1,1-d-fructofuranose) in smaller amounts and glucose, fructose and eventually isomaltose as by-products, which lower the yield of the reaction and complicate the recovery of palatinose. The determination of the three-dimensional structure of SmuA will provide a basis for rational protein-engineering studies in order to optimize the industrial production of palatinose. A recombinant form of the 67.3 kDa SmuA enzyme has been crystallized in the native state by the vapour-diffusion method. Crystals belong to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 61.6, b = 81.4, c = 135.6 Å, and diffract to 1.95 Å resolution on a synchrotron-radiation source

Human triose-phosphate isomerase deficiency: a single amino acid substitution results in a thermolabile enzyme.

Science.gov (United States)

Daar, I O; Artymiuk, P J; Phillips, D C; Maquat, L E

1986-10-01

Triose-phosphate isomerase (TPI; D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1) deficiency is a recessive disorder that results in hemolytic anemia and neuromuscular dysfunction. To determine the molecular basis of this disorder, a TPI allele from two unrelated patients homozygous for TPI deficiency was compared with an allele from a normal individual. Each disease-associated sequence harbors a G X C----C X G transversion in the codon for amino acid-104 and specifies a structurally altered protein in which a glutamate residue is replaced by an aspartate residue. The importance of glutamate-104 to enzyme structure and function is implicated by its conservation in the TPI protein of all species that have been characterized to date. The glutamate-to-aspartate substitution results in a thermolabile enzyme as demonstrated by assays of TPI activity in cultured fibroblasts of each patient and cultured Chinese hamster ovary (CHO) cells that were stably transformed with the mutant alleles. Although this substitution conserves the overall charge of amino acid-104, the x-ray crystal structure of chicken TPI indicates that the loss of a side-chain methylene group (-CH2CH2COO- ---- -CH2COO-) is sufficient to disrupt the counterbalancing of charges that normally exists within a hydrophobic pocket of the native enzyme.

Characterization of an L-arabinose isomerase from Bacillus thermoglucosidasius for D-tagatose production.

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Seo, Myung-Ji

2013-01-01

L-Arabinose isomerase from Bacillus thermoglucosidasius KCTC 1828 (BTAI) was expressed in Escherichia coli. The optimal temperature and pH for the activity of the purified BTAI were 40 °C and pH 7.0. The Mn(2+) ion was an activator of BTAI activity. The kinetic parameters of BTAI for D-galactose were a K(m) of 175 mM and a k(cat)/K(m) of 2.8 mM(-1)min(-1). The conversion ratio by BTAI to D-tagatose reached 45.6% at 40 °C.

A comparison of carotenoids, retinoids, and tocopherols in the serum and buccal mucosa of chronic cigarette smokers versus nonsmokers.

OpenAIRE

Gabriel, Helen E.; Liu, Zhenhua; Crott, Jimmy W.; Choi, Sang-Woon; Song, Byeng Chun; Mason, Joel B.; Johnson, Elizabeth J.

2006-01-01

Biomarker: micronucleiDiet, food or substance: carotenoids, retinoids, and tocopherols. Study type: humans Study design: case-control studyStudy size: 35 smokers and 21 nonsmokers Tissue/biological material/sample size: Buccal Mucosa Cells (BMC); blood. Method of analysis: freshly prepared BMC suspension were smeared on a microscope slide and scored for the presence of micronuclei. Impact on outcome (including dose-response): General linear regression with adjustments for dietary intake showe...

Polimorfisme Enzim Glucose-6-Phosphate Isomerase pada Tiga Populasi Tuna Sirip Kuning (Thunnus albacares)

OpenAIRE

Permana, Gusti Ngurah; Hutapea, Jhon H.; Moria, Sari Budi; Haryanti, Haryanti

2006-01-01

Samples of yellowfin tuna (Thunnus albacares) were taken from three locations Bali, North Sulawesi and North Maluku. The glucose-6-phosphate isomerase (GPI) was analyzed from liver using allozyme electrophoresis method. Polymorphism of GPI enzyme was observed and four alleles (A, B ,C, D) were found in Bali population, three alleles (A,B,C) were found in North Maluku and North Sulawesi populations. Heterozygosity values, from Bali, North Maluku and North Sulawesi were 0.419; 0.417; 0.143 resp...

Selective ligand activity at Nur/retinoid X receptor complexes revealed by dimer-specific bioluminescence resonance energy transfer-based sensors

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Giner, Xavier C; Cotnoir-White, David; Mader, Sylvie; Lévesque, Daniel

2017-01-01

Retinoid X receptors (RXR) play a role as master regulators due to their capacity to form heterodimers with other nuclear receptors. Accordingly, retinoid signaling is involved in multiple biological processes, including development, cell differentiation, metabolism and cell death. However, the role and functions of RXR in different heterodimer complexes remain unsolved, mainly because most RXR drugs (called rexinoids) are not selective to specific heterodimer complexes. This also strongly limits the use of rexinoids for specific therapeutic approaches. In order to better characterize rexinoids at specific nuclear receptor complexes, we have developed and optimized luciferase protein complementation-based Bioluminescence Resonance Energy Transfer (BRET) assays, which can directly measure recruitment of a co-activator motif fused to yellow fluorescent protein (YFP) by specific nuclear receptor dimers. To validate the assays, we compared rexinoid modulation of co-activator recruitment by RXR homodimer, and heterodimers Nur77/RXR and Nurr1/RXR. Results reveal that some rexinoids display selective co-activator recruitment activities with homo- or hetero-dimer complexes. In particular, SR11237 (BMS649) has increased potency for recruitment of co-activator motif and transcriptional activity with the Nur77/RXR heterodimer compared to other complexes. This technology should prove useful to identify new compounds with specificity for individual dimeric species formed by nuclear receptors. PMID:26148973

Protein Disulfide Isomerase and Host-Pathogen Interaction

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Beatriz S. Stolf

2011-01-01

Full Text Available Reactive oxygen species (ROS production by immunological cells is known to cause damage to pathogens. Increasing evidence accumulated in the last decade has shown, however, that ROS (and redox signals functionally regulate different cellular pathways in the host-pathogen interaction. These especially affect (i pathogen entry through protein redox switches and redox modification (i.e., intra- and interdisulfide and cysteine oxidation and (ii phagocytic ROS production via Nox family NADPH oxidase enzyme and the control of phagolysosome function with key implications for antigen processing. The protein disulfide isomerase (PDI family of redox chaperones is closely involved in both processes and is also implicated in protein unfolding and trafficking across the endoplasmic reticulum (ER and towards the cytosol, a thiol-based redox locus for antigen processing. Here, we summarise examples of the cellular association of host PDI with different pathogens and explore the possible roles of pathogen PDIs in infection. A better understanding of these complex regulatory steps will provide insightful information on the redox role and coevolutional biological process, and assist the development of more specific therapeutic strategies in pathogen-mediated infections.

Developmentally Regulated Production of meso-Zeaxanthin in Chicken Retinal Pigment Epithelium/Choroid and Retina.

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Gorusupudi, Aruna; Shyam, Rajalekshmy; Li, Binxing; Vachali, Preejith; Subhani, Yumna K; Nelson, Kelly; Bernstein, Paul S

2016-04-01

meso-Zeaxanthin is a carotenoid that is rarely encountered in nature outside of the vertebrate eye. It is not a constituent of a normal human diet, yet this carotenoid comprises one-third of the primate macular pigment. In the current study, we undertook a systematic approach to biochemically characterize the production of meso-zeaxanthin in the vertebrate eye. Fertilized White Leghorn chicken eggs were analyzed for the presence of carotenoids during development. Yolk, liver, brain, serum, retina, and RPE/choroid were isolated, and carotenoids were extracted. The samples were analyzed on C-30 or chiral HPLC columns to determine the carotenoid composition. Lutein and zeaxanthin were found in all studied nonocular tissues, but no meso-zeaxanthin was ever detected. Among the ocular tissues, the presence of meso-zeaxanthin was consistently observed starting at embryonic day 17 (E17) in the RPE/choroid, several days before its consistent detection in the retina. If RPE/choroid of an embryo was devoid of meso-zeaxanthin, the corresponding retina was always negative as well. This is the first report of developmentally regulated synthesis of meso-zeaxanthin in a vertebrate system. Our observations suggest that the RPE/choroid is the primary site of meso-zeaxanthin synthesis. Identification of meso-zeaxanthin isomerase enzyme in the developing chicken embryo will facilitate our ability to determine the biochemical mechanisms responsible for production of this unique carotenoid in other higher vertebrates, such as humans.

Enhanced pest resistance and increased phenolic production in maize callus transgenically expressing a maize chalcone isomerase -3 like gene

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Significant losses in maize production are due to damage by insects and ear rot fungi. A gene designated as chalcone-isomerase-like, located in a quantitative trait locus for resistance to Fusarium ear rot fungi, was cloned from a Fusarium ear rot resistant inbred and transgenically expressed in mai...

L-Rhamnose isomerase and its use for biotechnological production of rare sugars.

Science.gov (United States)

Xu, Wei; Zhang, Wenli; Zhang, Tao; Jiang, Bo; Mu, Wanmeng

2016-04-01

L-Rhamnose isomerase (L-RI, EC 5.3.1.14), catalyzing the isomerization between L-rhamnose and L-rhamnulose, plays an important role in microbial L-rhamnose metabolism and thus occurs in a wide range of microorganisms. It attracts more and more attention because of its broad substrate specificity and its great potential in enzymatic production of various rare sugars. In this article, the enzymatic properties of various reported L-RIs were compared in detail, and their applications in the production of L-rhamnulose and various rare sugars including D-allose, D-gulose, L-lyxose, L-mannose, L-talose, and L-galactose were also reviewed.

Co-expression of D-glucose isomerase and D-psicose 3-epimerase: development of an efficient one-step production of D-psicose.

Science.gov (United States)

Men, Yan; Zhu, Yueming; Zeng, Yan; Izumori, Ken; Sun, Yuanxia; Ma, Yanhe

2014-10-01

D-Psicose has been attracting attention in recent years because of its alimentary activities and is used as an ingredient in a range of foods and dietary supplements. To develop a one-step enzymatic process of D-psicose production, thermoactive D-glucose isomerase and the D-psicose 3-epimerase obtained from Bacillus sp. and Ruminococcus sp., respectively, were successfully co-expressed in Escherichia coli BL21 strain. The substrate of one-step enzymatic process was D-glucose. The co-expression system exhibited maximum activity at 65 °C and pH 7.0. Mg(2+) could enhance the output of D-psicose by 2.32 fold to 1.6 g/L from 10 g/L of D-glucose. When using high-fructose corn syrup (HFCS) as substrate, 135 g/L D-psicose was produced under optimum conditions. The mass ratio of D-glucose, D-fructose, and D-psicose was almost 3.0:2.7:1.0, when the reaction reached equilibrium after an 8h incubation time. This co-expression system approaching to produce D-psicose has potential application in food and beverage products, especially softdrinks. Copyright © 2014 Elsevier Inc. All rights reserved.

Molecular Characterization and Analysis of a Novel Protein Disulfide Isomerase-Like Protein of Eimeria tenella

OpenAIRE

Han, Hongyu; Dong, Hui; Zhu, Shunhai; Zhao, Qiping; Jiang, Lianlian; Wang, Yange; Li, Liujia; Wu, Youlin; Huang, Bing

2014-01-01

Protein disulfide isomerase (PDI) and PDI-like proteins are members of the thioredoxin superfamily. They contain thioredoxin-like domains and catalyze the physiological oxidation, reduction and isomerization of protein disulfide bonds, which are involved in cell function and development in prokaryotes and eukaryotes. In this study, EtPDIL, a novel PDI-like gene of Eimeria tenella, was cloned using rapid amplification of cDNA ends (RACE) according to the expressed sequence tag (EST). The EtPDI...

Thermoinactivation Mechanism of Glucose Isomerase

Science.gov (United States)

Lim, Leng Hong; Saville, Bradley A.

In this article, the mechanisms of thermoinactivation of glucose isomerase (GI) from Streptomyces rubiginosus (in soluble and immobilized forms) were investigated, particularly the contributions of thiol oxidation of the enzyme's cysteine residue and a "Maillard-like" reaction between the enzyme and sugars in high fructose corn syrup (HFCS). Soluble GI (SGI) was successfully immobilized on silica gel (13.5 μm particle size), with an activity yield between 20 and 40%. The immobilized GI (IGI) has high enzyme retention on the support during the glucose isomerization process. In batch reactors, SGI (half-life =145 h) was more stable than IGI (half-life=27 h) at 60°C in HFCS, whereas at 80°C, IGI (half-life=12 h) was more stable than SGI (half-life=5.2 h). IGI was subject to thiol oxidation at 60°C, which contributed to the enzyme's deactivation. IGI was subject to thiol oxidation at 80°C, but this did not contribute to the deactivation of the enzyme. SGI did not undergo thiol oxidation at 60°C, but at 80°C SGI underwent severe precipitation and thiol oxidation, which caused the enzyme to deactivate. Experimental results show that immobilization suppresses the destablizing effect of thiol oxidation on GI. A "Maillard-like" reaction between SGI and the sugars also caused SGI thermoinactivation at 60, 70, and 80°C, but had minimal effect on IGI. At 60 and 80°C, IGI had higher thermostability in continuous reactors than in batch reactors, possibily because of reduced contact with deleterious compounds in HFCS.

Characterization of the triple-component linoleic acid isomerase in Lactobacillus plantarum ZS2058 by genetic manipulation.

Science.gov (United States)

Yang, B; Qi, H; Gu, Z; Zhang, H; Chen, W; Chen, H; Chen, Y Q

2017-11-01

To assess the mechanism for conjugated linoleic acid (CLA) production in Lactobacillus plantarum ZS2058. CLA has attracted great interests for decades due to its health-associated benefits including anticancer, anti-atherogenic, anti-obesity and modulation of the immune system. A number of microbial CLA producers were widely reported including lactic acid bacteria. Lactobacillus plantarum ZS2058, an isolate from Chinese traditional fermented food, could convert LA to CLA with various intermediates. To characterize the genetic determinants for generating CLA, a cre-lox-based system was utilized to delete the genes encoding myosin cross-reactive antigen (MCRA), short-chain dehydrogenase/oxidoreductase (DH) and acetoacetate decarboxylase (DC) in Lact. plantarum ZS2058, respectively. Neither intermediate was detected in the corresponding gene deletion mutant. Meanwhile all those mutants could recover the ability to convert linoleic acid to CLA when the corresponding gene was completed. The results indicated that CLA production was a multiple-step reaction catalysed by triple-component linoleate isomerase system encoded by mcra, dh and dc. Multicomponent linoleic acid isomerase provided important results for illustration unique mechanism for CLA production in Lact. plantarum ZS2058. Lactobacilli with CLA production ability offer novel opportunities for functional food development. © 2017 The Society for Applied Microbiology.

Transmutation of human glutathione transferase A2-2 with peroxidase activity into an efficient steroid isomerase.

Science.gov (United States)

Pettersson, Par L; Johansson, Ann-Sofie; Mannervik, Bengt

2002-08-16

A major goal in protein engineering is the tailor-making of enzymes for specified chemical reactions. Successful attempts have frequently been based on directed molecular evolution involving libraries of random mutants in which variants with desired properties were identified. For the engineering of enzymes with novel functions, it would be of great value if the necessary changes of the active site could be predicted and implemented. Such attempts based on the comparison of similar structures with different substrate selectivities have previously met with limited success. However, the present work shows that the knowledge-based redesign restricted to substrate-binding residues in human glutathione transferase A2-2 can introduce high steroid double-bond isomerase activity into the enzyme originally characterized by glutathione peroxidase activity. Both the catalytic center activity (k(cat)) and catalytic efficiency (k(cat)/K(m)) match the values of the naturally evolved glutathione transferase A3-3, the most active steroid isomerase known in human tissues. The substrate selectivity of the mutated glutathione transferase was changed 7000-fold by five point mutations. This example demonstrates the functional plasticity of the glutathione transferase scaffold as well as the potential of rational active-site directed mutagenesis as a complement to DNA shuffling and other stochastic methods for the redesign of proteins with novel functions.

Cloning retinoid and peroxisome proliferator-activated nuclear receptors of the Pacific oyster and in silico binding to environmental chemicals.

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Susanne Vogeler

Full Text Available Disruption of nuclear receptors, a transcription factor superfamily regulating gene expression in animals, is one proposed mechanism through which pollution causes effects in aquatic invertebrates. Environmental pollutants have the ability to interfere with the receptor's functions through direct binding and inducing incorrect signals. Limited knowledge of invertebrate endocrinology and molecular regulatory mechanisms, however, impede the understanding of endocrine disruptive effects in many aquatic invertebrate species. Here, we isolated three nuclear receptors of the Pacific oyster, Crassostrea gigas: two isoforms of the retinoid X receptor, CgRXR-1 and CgRXR-2, a retinoic acid receptor ortholog CgRAR, and a peroxisome proliferator-activated receptor ortholog CgPPAR. Computer modelling of the receptors based on 3D crystal structures of human proteins was used to predict each receptor's ability to bind to different ligands in silico. CgRXR showed high potential to bind and be activated by 9-cis retinoic acid and the organotin tributyltin (TBT. Computer modelling of CgRAR revealed six residues in the ligand binding domain, which prevent the successful interaction with natural and synthetic retinoid ligands. This supports an existing theory of loss of retinoid binding in molluscan RARs. Modelling of CgPPAR was less reliable due to high discrepancies in sequence to its human ortholog. Yet, there are suggestions of binding to TBT, but not to rosiglitazone. The effect of potential receptor ligands on early oyster development was assessed after 24h of chemical exposure. TBT oxide (0.2μg/l, all-trans retinoic acid (ATRA (0.06 mg/L and perfluorooctanoic acid (20 mg/L showed high effects on development (>74% abnormal developed D-shelled larvae, while rosiglitazone (40 mg/L showed no effect. The results are discussed in relation to a putative direct (TBT disruption effect on nuclear receptors. The inability of direct binding of ATRA to CgRAR suggests

The susceptibility of the retina to photochemical damage from visible light

Science.gov (United States)

Hunter, Jennifer J; Morgan, Jessica I W; Merigan, William H; Sliney, David H; Sparrow, Janet R; Williams, David R

2011-01-01

The photoreceptor/RPE complex must maintain a delicate balance between maximizing the absorption of photons for vision and retinal image quality while simultaneously minimizing the risk of photodamage when exposed to bright light. We review the recent discovery of two new effects of light exposure on the photoreceptor/RPE complex in the context of current thinking about the causes of retinal phototoxicity. These effects are autofluorescence photobleaching in which exposure to bright light reduces lipofuscin autofluorescence and, at higher light levels, RPE disruption in which the pattern of autofluorescence is permanently altered following light exposure. Both effects occur following exposure to visible light at irradiances that were previously thought to be safe. Photopigment, retinoids involved in the visual cycle, and bisretinoids in lipofuscin have been implicated as possible photosensitizers for photochemical damage. The mechanism of RPE disruption may follow either of these paths. On the other hand, autofluorescence photobleaching is likely an indicator of photooxidation of lipofuscin. The permanent changes inherent in RPE disruption might require modification of the light safety standards. AF photobleaching recovers after several hours although the mechanisms by which this occurs are not yet clear. Understanding the mechanisms of phototoxicity is all the more important given the potential for increased susceptibility in the presence of ocular diseases that affect either the visual cycle and/or lipofuscin accumulation. In addition, knowledge of photochemical mechanisms can improve our understanding of some disease processes that may be influenced by light exposure, such as some forms of Leber’s congenital amaurosis, and aid in the development of new therapies. Such treatment prior to intentional light exposures, as in ophthalmic examinations or surgeries, could provide an effective preventative strategy. PMID:22085795

Effect of Am-80, A Novel Retinoid Derivative, On Contact Hypersensitivity Caused by Repeated Applications of Hapten in Mice

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Satoru Niwa

2000-01-01

Full Text Available Some retinoids show an anti-inflammatory action through regulation of transcription of various genes. In the present study, the inhibitory effect of 4-((5,6,7,8- tetrahydro-5,5,8,8-tetramethyl-2-naphthyl carbamoyl benzoic acid (Am-80, a synthetic retinoid, on mouse contact hypersensitivity provoked by repeated applications of 2,4-dinitrofluorobenzene (DNFB to the ear was investigated. Five-fold applications of DNFB on ears once per week elicited severe contact dermatitis with marked infiltration of inflammatory cells and elevation of anti-dinitrophenyl (DNP-IgE antibody in the serum. The Am-80 significantly inhibited ear swelling in a dose-dependent manner. In the histopathologic study, infiltration of inflammatory cells was clearly decreased by Am-80. However, Am-80 did not affect the production of DNP-specific IgE antibody both at the transcriptional and post-transcriptional levels. The effects of Am-80 on the transcriptional level of cytokines, interferon (IFN-γ, interleukin (IL-1 and IL-4 in cervical lymph nodes were investigated. Marked elevation of mRNA for all cytokines was observed and Am-80 potently inhibited the expression of IFN-γ mRNA, but not IL-1 and IL-4 mRNA. These findings indicated that Am-80 may inhibit the contact dermatitis at the post-sensitization phase by inhibiting IFN-γ production at the transcriptional level in mice.

Helicobacter pylori Peptidyl Prolyl Isomerase Expression Is Associated with the Severity of Gastritis.

Science.gov (United States)

Oghalaie, Akbar; Saberi, Samaneh; Esmaeili, Maryam; Ebrahimzadeh, Fatemeh; Barkhordari, Farzaneh; Ghamarian, Abdolreza; Tashakoripoor, Mohammad; Abdirad, Afshin; Eshagh Hosseini, Mahmoud; Khalaj, Vahid; Mohammadi, Marjan

2016-12-01

Helicobacter pylori secretory peptidyl prolyl isomerase, HP0175, is progressively identified as a pro-inflammatory and pro-carcinogenic protein, which serves to link H. pylori infection to its more severe clinical outcomes. Here, we have analyzed host HP0175-specific antibody responses in relation to the severity of gastritis. The HP0175 gene fragment was PCR-amplified, cloned, expressed and purified by Ni-NTA affinity chromatography. Serum antigen-specific antibody responses of non-ulcer dyspeptic patients (N = 176) against recombinant HP0175 were detected by western blotting. The infection status of these subjects was determined by rapid urease test, culture, histology, and serology. The grade of inflammation and stage of atrophy were scored blindly according to the OLGA staging system. The recombinant HP0175 (rHP0175) was expressed as a ~35 kDa protein and its identity was confirmed by western blotting using anti-6X His tag antibody and pooled H. pylori-positive sera. Serum IgG antibodies against rHP0175 segregated our patients into two similar-sized groups of sero-positives (90/176, 51.1 %) and sero-negatives (86/176, 48.9 %). The former presented with higher grades of gastric inflammation (OR = 4.4, 95 % CI = 1.9-9.9, P = 0.001) and stages of gastric atrophy (OR = 18.3, 95 %CI = 1.4-246.6, P = 0.028). Our findings lend further support to the pro-inflammatory nature of H. pylori peptidyl prolyl isomerase (HP0175) and recommends this antigen as a non-invasive serum biomarker of the severity of H. pylori-associated gastritis.

Kinetic analysis of the mechanism and specificity of protein-disulfide isomerase using fluorescence-quenched peptides

DEFF Research Database (Denmark)

Westphal, V; Spetzler, J C; Meldal, M

1998-01-01

Protein-disulfide isomerase (PDI) is an abundant folding catalyst in the endoplasmic reticulum of eukaryotic cells. PDI introduces disulfide bonds into newly synthesized proteins and catalyzes disulfide bond isomerizations. We have synthesized a library of disulfide-linked fluorescence......-quenched peptides, individually linked to resin beads, for two purposes: 1) to probe PDI specificity, and 2) to identify simple, sensitive peptide substrates of PDI. Using this library, beads that became rapidly fluorescent by reduction by human PDI were selected. Amino acid sequencing of the bead-linked peptides...

Cheilitis in acne vulgaris patients with no previous use of systemic retinoid products.

Science.gov (United States)

Balighi, Kamran; Daneshpazhooh, Maryam; Lajevardi, Vahideh; Talebi, Shahin; Azizpour, Arghavan

2017-08-01

Isotretinoin is commonly used in the treatment of acne vulgaris. While one of the more common side-effects is cheilitis, we have observed an increased incidence of cheilitis prior to the commencement of systemic isotretinoin. The aim of this study was to assess the prevalence of cheilitis among acne vulgaris patients. A non-interventional cross-sectional study of patients with acne vulgaris. Patients with previous use of systemic retinoids were excluded. The patients were examined for signs and symptoms of cheilitis. Of a total of 400 patients, 134 (34%) had evidence of cheilitis at initial presentation. Two-thirds (63%) were female (P acne excorie, compared with only 8% of patients with no signs of cheilitis. Our findings suggest that cheilitis is quite common among acne vulgaris patients even before treatment with isotretinoin. © 2016 The Australasian College of Dermatologists.

Vitamin D receptor–retinoid X receptor heterodimer signaling regulates oligodendrocyte progenitor cell differentiation

Science.gov (United States)

de la Fuente, Alerie Guzman; Errea, Oihana; van Wijngaarden, Peter; Gonzalez, Ginez A.; Kerninon, Christophe; Jarjour, Andrew A.; Lewis, Hilary J.; Jones, Clare A.; Nait-Oumesmar, Brahim; Zhao, Chao; Huang, Jeffrey K.; ffrench-Constant, Charles

2015-01-01

The mechanisms regulating differentiation of oligodendrocyte (OLG) progenitor cells (OPCs) into mature OLGs are key to understanding myelination and remyelination. Signaling via the retinoid X receptor γ (RXR-γ) has been shown to be a positive regulator of OPC differentiation. However, the nuclear receptor (NR) binding partner of RXR-γ has not been established. In this study we show that RXR-γ binds to several NRs in OPCs and OLGs, one of which is vitamin D receptor (VDR). Using pharmacological and knockdown approaches we show that RXR–VDR signaling induces OPC differentiation and that VDR agonist vitamin D enhances OPC differentiation. We also show expression of VDR in OLG lineage cells in multiple sclerosis. Our data reveal a role for vitamin D in the regenerative component of demyelinating disease and identify a new target for remyelination medicines. PMID:26644513

Glucose isomerization in simulated moving bed reactor by Glucose isomerase

Directory of Open Access Journals (Sweden)

Eduardo Alberto Borges da Silva

2006-05-01

Full Text Available Studies were carried out on the production of high-fructose syrup by Simulated Moving Bed (SMB technology. A mathematical model and numerical methodology were used to predict the behavior and performance of the simulated moving bed reactors and to verify some important aspects for application of this technology in the isomerization process. The developed algorithm used the strategy that considered equivalences between simulated moving bed reactors and true moving bed reactors. The kinetic parameters of the enzymatic reaction were obtained experimentally using discontinuous reactors by the Lineweaver-Burk technique. Mass transfer effects in the reaction conversion using the immobilized enzyme glucose isomerase were investigated. In the SMB reactive system, the operational variable flow rate of feed stream was evaluated to determine its influence on system performance. Results showed that there were some flow rate values at which greater purities could be obtained.Neste trabalho a tecnologia de Leito Móvel Simulado (LMS reativo é aplicada no processo de isomerização da glicose visando à produção de xarope concentrado de frutose. É apresentada a modelagem matemática e uma metodologia numérica para predizer o comportamento e o desempenho de unidades reativas de leito móvel simulado para verificar alguns aspectos importantes para o emprego desta tecnologia no processo de isomerização. O algoritmo desenvolvido utiliza a abordagem que considera as equivalências entre as unidades reativas de leito móvel simulado e leito móvel verdadeiro. Parâmetros cinéticos da reação enzimática são obtidos experimentalmente usando reatores em batelada pela técnica Lineweaver-Burk. Efeitos da transferência de massa na conversão de reação usando a enzima imobilizada glicose isomerase são verificados. No sistema reativo de LMS, a variável operacional vazão da corrente de alimentação é avaliada para conhecer o efeito de sua influência no

Mechanisms of Neuroprotection by Protein Disulphide Isomerase in Amyotrophic Lateral Sclerosis

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Adam K. Walker

2011-01-01

Full Text Available Amyotrophic lateral sclerosis (ALS is a devastating neurodegenerative disease characterised by the progressive loss of motor neurons, leading to paralysis and death within several years of onset. Although protein misfolding is a key feature of ALS, the upstream triggers of disease remain elusive. Recently, endoplasmic reticulum (ER stress was identified as an early and central feature in ALS disease models as well as in human patient tissues, indicating that ER stress could be an important process in disease pathogenesis. One important chaperone induced by ER stress is protein disulphide isomerase (PDI, which is both upregulated and posttranslationally inhibited by S-nitrosylation in ALS. In this paper, we present evidence from studies of genetics, model organisms, and patient tissues which indicate an active role for PDI and ER stress in ALS disease processes.

NADPH-d activity in rat thymus after the application of retinoid acid

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F. Dorko

2012-02-01

Full Text Available The aim of this work was to determine the localization of nicotinamide-adenine dinucleotide phosphate-diaphorase (NADPH-d activity as the marker for synthesis of nitric oxide synthase (NOS in the rat thymus after the application of retinoid acid (RA on 1st, 7th, 14th and 21st days of gestation. The given results can build the basis for understanding of the role of NOS in rat thymus. NADPH-d positive cells were represented with dark-blue color and were localized on corticomedullar junction of the thymus. These cells were of different intensity of coloring and were shaped in oval, circle or irregular forms. NADPH-d positive nerve fibers were observed in perivascular topography. They were marked more strongly in the case of control group. The result of application of RA to gravid rats was that the birth weights of newborn rats and their thymuses were smaller, but without statistically significance.

Identification and characterization of a novel L-arabinose isomerase from Anoxybacillus flavithermus useful in D-tagatose production.

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Li, Yanjun; Zhu, Yueming; Liu, Anjun; Sun, Yuanxia

2011-05-01

D-Tagatose is a highly functional rare ketohexose and many attempts have been made to convert D-galactose into the valuable D-tagatose using L-arabinose isomerase (L-AI). In this study, a thermophilic strain possessing L-AI gene was isolated from hot spring sludge and identified as Anoxybacillus flavithermus based on its physio-biochemical characterization and phylogenetic analysis of its 16s rRNA gene. Furthermore, the gene encoding L-AI from A. flavithermus (AFAI) was cloned and expressed at a high level in E. coli BL21(DE3). L-AI had a molecular weight of 55,876 Da, an optimum pH of 10.5 and temperature of 95°C. The results showed that the conversion equilibrium shifted to more D-tagatose from D-galactose by raising the reaction temperatures and adding borate. A 60% conversion of D-galactose to D-tagatose was observed at an isomerization temperature of 95°C with borate. The catalytic efficiency (k (cat) /K (m)) for D-galactose with borate was 9.47 mM(-1) min(-1), twice as much as that without borate. Our results indicate that AFAI is a novel hyperthermophilic and alkaliphilic isomerase with a higher catalytic efficiency for D-galactose, suggesting its great potential for producing D-tagatose.

Human cyclophilin B: A second cyclophilin gene encodes a peptidyl-prolyl isomerase with a signal sequence

International Nuclear Information System (INIS)

Price, E.R.; Zydowsky, L.D.; Jin, Mingjie; Baker, C.H.; McKeon, F.D.; Walsh, C.T.

1991-01-01

The authors report the cloning and characterization of a cDNA encoding a second human cyclosporin A-binding protein (hCyPB). Homology analyses reveal that hCyPB is a member of the cyclophilin B (CyPB) family, which includes yeast CyPB, Drosophila nina A, and rat cyclophilin-like protein. This family is distinguished from the cyclophilin A (CyPA) family by the presence of endoplasmic reticulum (ER)-directed signal sequences. hCyPB has a hydrophobic leader sequence not found in hCyPA, and its first 25 amino acids are removed upon expression in Escherichia coli. Moreover, they show that hCyPB is a peptidyl-prolyl cis-trans isomerase which can be inhibited by cyclosporin A. These observations suggest that other members of the CyPB family will have similar enzymatic properties. Sequence comparisons of the CyPB proteins show a central, 165-amino acid peptidyl-prolyl isomerase and cyclosprorin A-binding domain, flanked by variable N-terminal and C-terminal domains. These two variable regions may impart compartmental specificity and regulation to this family of cyclophilin proteins containing the conserved core domain. Northern blot analyses show that hCyPB mRNA is expressed in the Jurkat T-cell line, consistent with its possible target role in cyclosporin A-mediated immunosuppression

Control analysis of the role of triosephosphate isomerase in glucose metabolism in Lactococcus lactis

DEFF Research Database (Denmark)

Solem, Christian; Købmann, Brian Jensen; Jensen, Peter Ruhdal

2008-01-01

Triosephosphate isomerase (TPI), which catalyses the conversion of dihydroxyacetone phosphate (DHAP) to glyceraldehyde-3-phosphate (G3P), was studied for its control on glycolysis and mixed acid production in L. lactis subspecies lactis IL1403 and L. lactis subspecies cremoris MG1363. Strains...... metabolites glucose-6-phosphate, fructose-1,6-bisphosphate and DHAP in the IL1403 derivatives were essentially unchanged for TPI activities from 26% to 225%. At a TPI activity of 3%, the level of DHAP increased four times. The finding that an increased level of DHAP coincides with an increase in formate...

Probable Chemical Hypoxia Effects on Progress of CNV Through Induction of Promoter CpG Demethylation and Overexpression of IL17RC in Human RPE Cells.

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Alivand, Mohammad Reza; Sabouni, Farzaneh; Soheili, Zahra-Soheila

2016-09-01

To survey the changes of promoter CpG methylation status and mRNA expression of IL17RC (interleukin 17 receptor C) gene in retinal pigment epithelium (RPE) cells under chemical hypoxia condition for choroidal neovascularization (CNV) modeling in vitro. RPE cells were cultured in both untreated as a control group and treated by cobalt chloride media as a hypoxia group for various concentrations (100-150μM) and times (24-36 hrs.) To confirm chemical hypoxia condition, mRNA expression of HIF (Hypoxia Inducible Factor) -1α, -2α, and Vascular Endothelial Growth Factor (VEGF) was compared between two groups by Real-time PCR. Also, in normoxia and hypoxia conditions, IL17RC expression changes and promoter CpG methylation status were evaluated by Real-time PCR and methylation-specific PCR (MSP) techniques, respectively. Overexpression of HIF-1α, HIF-2α, and VEGF was significant in hypoxia versus normoxia conditions. Our data showed overexpression of IL17RC (2.1- to 6.3-fold) and decreasing of its promoter methylation in comparison with hypoxia and normoxia conditions. It was found that there are significant association between promoter methylation status and expression of IL17RC in chemical hypoxia condition. Therefore, methylation of IL17RC could play as a marker in CNV and degeneration of RPE cells in vitro. Additionally, HIF-α and methylation phenomena may be considered as critical targets for blocking in angiogenesis of age-related degeneration in future studies.

Rapid expansion of the protein disulfide isomerase gene family facilitates the folding of venom peptides

DEFF Research Database (Denmark)

Safavi-Hemami, Helena; Li, Qing; Jackson, Ronneshia L.

2016-01-01

Formation of correct disulfide bonds in the endoplasmic reticulum is a crucial step for folding proteins destined for secretion. Protein disulfide isomerases (PDIs) play a central role in this process. We report a previously unidentified, hypervariable family of PDIs that represents the most...... diverse gene family of oxidoreductases described in a single genus to date. These enzymes are highly expressed specifically in the venom glands of predatory cone snails, animals that synthesize a remarkably diverse set of cysteine-rich peptide toxins (conotoxins). Enzymes in this PDI family, termed...

The Expression of Millettia pinnata Chalcone Isomerase in Saccharomyces cerevisiae Salt-Sensitive Mutants Enhances Salt-Tolerance

OpenAIRE

Wang, Hui; Hu, Tangjin; Huang, Jianzi; Lu, Xiang; Huang, Baiqu; Zheng, Yizhi

2013-01-01

The present study demonstrates a new Millettia pinnata chalcone isomerase (MpCHI) whose transcription level in leaf was confirmed to be enhanced after being treated by seawater or NaCl (500 mM) via transcriptome sequencing and Real-Time Quantitative Reverse Transcription PCR (QRT-PCR) analyses. Its full length cDNA (666 bp) was obtained by 3'-end and 5'-end Rapid Amplification of cDNA Ends (RACE). The analysis via NCBI BLAST indicates that both aminoacid sequence and nucleotide sequ...

Production of D-tagatose at high temperatures using immobilized Escherichia coli cells expressing L-arabinose isomerase from Thermotoga neapolitana.

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Hong, Young-Ho; Lee, Dong-Woo; Lee, Sang-Jae; Choe, Eun-Ah; Kim, Seong-Bo; Lee, Yoon-Hee; Cheigh, Chan-Ick; Pyun, Yu-Ryang

2007-04-01

Escherichia coli cells expressing L-arabinose isomerase from Thermotoga neapolitana (TNAI) were immobilized in calcium alginate beads. The resulting cell reactor (2.4 U, t (1/2) = 43 days at 70 degrees C) in a continuous recycling mode at 70 degrees C produced 49 and 38 g D-tagatose/l from 180 and 90 g D-galactose/l, respectively, within 12 h.

Effects of a synthetic retinoid on skin structure, matrix metalloproteinases, and procollagen in healthy and high-risk subjects with diabetes.

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Zeng, Wei; Tahrani, Abd; Shakher, Jayadave; Varani, James; Hughes, Sharon; Dubb, Kiran; Stevens, Martin J

2011-01-01

In diabetes, foot ulceration may result from increased skin fragility. Retinoids can reverse some diabetes-induced deficits of skin structure and function, but their clinical utility is limited by skin irritation. The effects of diabetes and MDI 301, a nonirritating synthetic retinoid, and retinoic acid have been evaluated on matrix metalloproteinases (MMPs), procollagen expression, and skin structure in skin biopsies from nondiabetic volunteers and diabetic subjects at risk of foot ulceration using organ culture techniques. Zymography and enzyme-linked immunosorbent assay were utilized for analysis of MMP-1, -2, and -9 and tissue inhibitor of metalloproteinase-1 (TIMP-1) and immunohistochemistry for type I procollagen protein abundance. Collagen structure parameters were assessed in formalin-fixed, paraffin-embedded tissue sections. The % of active MMP-1 and -9 was higher and TIMP-1 abundance was lower in subjects with diabetes. Type 1 procollagen abundance was reduced and skin structural deficits were increased in diabetes. Three μM MDI 301 reduced active MMP-1 and -9 abundance by 29% (P structural deficit scores. Two μM retinoic acid reduced MMP-1 but did not significantly affect skin structure. These data indicate that diabetic patients at risk of foot ulceration have deficits of skin structure and function. MDI 301 offers potential for repairing this skin damage complicating diabetes. Copyright © 2011 Elsevier Inc. All rights reserved.

Alanine/RPE dosimetry in the process of blood irradiation

International Nuclear Information System (INIS)

Chen, F.; Covas, D.T.; Baffa, O.

2000-01-01

A set of eighty dosemeters approximately of Dl- alanine was proportioned to the Hemo center of Hospital das Clinicas of Medicine Faculty at Ribeirao Preto (HC-FRMP) in the Sao Paulo University, with the purpose to realize a quality control of the radiation dose supplied to blood bags. These dosemeters were divided in eight groups and irradiated for a two months period.The dosemeters were adhered to the bags and arranged inside of an acrylic box filled water with dimensions: (40x40x20) cm 3 . The box irradiation was made using two fields parallel-opposed of (40x40) cm 2 at 80 cm distance source-surface, in the Radiotherapy Service of HC-FMRP, with a 60 Co teletherapy unit. The irradiation time was sufficient to supply a dose of 20 Gy approximately in the box center. The RPE measures were realized in a Varian E-4 spectrometer operating in X-band. For the total of dosemeters and for the irradiation volume, the minimum and maximum doses were of 14 Gy and 23 Gy respectively. The mean dose was (18±2) Gy (1 σ), and the variability coefficient 11 %. Around 5 % of the bags received a dose under of the recommended limit by the Brazilian legislation and as consequence, the exposure time was increased. (Author)

The human protein disulfide isomerase gene family

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Galligan James J

2012-07-01

Full Text Available Abstract Enzyme-mediated disulfide bond formation is a highly conserved process affecting over one-third of all eukaryotic proteins. The enzymes primarily responsible for facilitating thiol-disulfide exchange are members of an expanding family of proteins known as protein disulfide isomerases (PDIs. These proteins are part of a larger superfamily of proteins known as the thioredoxin protein family (TRX. As members of the PDI family of proteins, all proteins contain a TRX-like structural domain and are predominantly expressed in the endoplasmic reticulum. Subcellular localization and the presence of a TRX domain, however, comprise the short list of distinguishing features required for gene family classification. To date, the PDI gene family contains 21 members, varying in domain composition, molecular weight, tissue expression, and cellular processing. Given their vital role in protein-folding, loss of PDI activity has been associated with the pathogenesis of numerous disease states, most commonly related to the unfolded protein response (UPR. Over the past decade, UPR has become a very attractive therapeutic target for multiple pathologies including Alzheimer disease, Parkinson disease, alcoholic and non-alcoholic liver disease, and type-2 diabetes. Understanding the mechanisms of protein-folding, specifically thiol-disulfide exchange, may lead to development of a novel class of therapeutics that would help alleviate a wide range of diseases by targeting the UPR.

Effects of peptidyl-prolyl isomerase 1 depletion in animal models of prion diseases.

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Legname, Giuseppe; Virgilio, Tommaso; Bistaffa, Edoardo; De Luca, Chiara Maria Giulia; Catania, Marcella; Zago, Paola; Isopi, Elisa; Campagnani, Ilaria; Tagliavini, Fabrizio; Giaccone, Giorgio; Moda, Fabio

2018-04-20

Pin1 is a peptidyl-prolyl isomerase that induces the cis-trans conversion of specific Ser/Thr-Pro peptide bonds in phosphorylated proteins, leading to conformational changes through which Pin1 regulates protein stability and activity. Since down-regulation of Pin1 has been described in several neurodegenerative disorders, including Alzheimer's Disease (AD), Parkinson's Disease (PD) and Huntington's Disease (HD), we investigated its potential role in prion diseases. Animals generated on wild-type (Pin1 +/+ ), hemizygous (Pin1 +/- ) or knock-out (Pin1 -/- ) background for Pin1 were experimentally infected with RML prions. The study indicates that, neither the total depletion nor reduced levels of Pin1 significantly altered the clinical and neuropathological features of the disease.

A method for the production of D-tagatose using a recombinant Pichia pastoris strain secreting β-D-galactosidase from Arthrobacter chlorophenolicus and a recombinant L-arabinose isomerase from Arthrobacter sp. 22c

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Wanarska Marta

2012-08-01

Full Text Available Abstract Background D-Tagatose is a natural monosaccharide which can be used as a low-calorie sugar substitute in food, beverages and pharmaceutical products. It is also currently being tested as an anti-diabetic and obesity control drug. D-Tagatose is a rare sugar, but it can be manufactured by the chemical or enzymatic isomerization of D-galactose obtained by a β-D-galactosidase-catalyzed hydrolysis of milk sugar lactose and the separation of D-glucose and D-galactose. L-Arabinose isomerases catalyze in vitro the conversion of D-galactose to D-tagatose and are the most promising enzymes for the large-scale production of D-tagatose. Results In this study, the araA gene from psychrotolerant Antarctic bacterium Arthrobacter sp. 22c was isolated, cloned and expressed in Escherichia coli. The active form of recombinant Arthrobacter sp. 22c L-arabinose isomerase consists of six subunits with a combined molecular weight of approximately 335 kDa. The maximum activity of this enzyme towards D-galactose was determined as occurring at 52°C; however, it exhibited over 60% of maximum activity at 30°C. The recombinant Arthrobacter sp. 22c L-arabinose isomerase was optimally active at a broad pH range of 5 to 9. This enzyme is not dependent on divalent metal ions, since it was only marginally activated by Mg2+, Mn2+ or Ca2+ and slightly inhibited by Co2+ or Ni2+. The bioconversion yield of D-galactose to D-tagatose by the purified L-arabinose isomerase reached 30% after 36 h at 50°C. In this study, a recombinant Pichia pastoris yeast strain secreting β-D-galactosidase Arthrobacter chlorophenolicus was also constructed. During cultivation of this strain in a whey permeate, lactose was hydrolyzed and D-glucose was metabolized, whereas D-galactose was accumulated in the medium. Moreover, cultivation of the P. pastoris strain secreting β-D-galactosidase in a whey permeate supplemented with Arthrobacter sp. 22c L-arabinose isomerase resulted in a 90% yield

A method for the production of D-tagatose using a recombinant Pichia pastoris strain secreting β-D-galactosidase from Arthrobacter chlorophenolicus and a recombinant L-arabinose isomerase from Arthrobacter sp. 22c.

Science.gov (United States)

Wanarska, Marta; Kur, Józef

2012-08-23

D-Tagatose is a natural monosaccharide which can be used as a low-calorie sugar substitute in food, beverages and pharmaceutical products. It is also currently being tested as an anti-diabetic and obesity control drug. D-Tagatose is a rare sugar, but it can be manufactured by the chemical or enzymatic isomerization of D-galactose obtained by a β-D-galactosidase-catalyzed hydrolysis of milk sugar lactose and the separation of D-glucose and D-galactose. L-Arabinose isomerases catalyze in vitro the conversion of D-galactose to D-tagatose and are the most promising enzymes for the large-scale production of D-tagatose. In this study, the araA gene from psychrotolerant Antarctic bacterium Arthrobacter sp. 22c was isolated, cloned and expressed in Escherichia coli. The active form of recombinant Arthrobacter sp. 22c L-arabinose isomerase consists of six subunits with a combined molecular weight of approximately 335 kDa. The maximum activity of this enzyme towards D-galactose was determined as occurring at 52°C; however, it exhibited over 60% of maximum activity at 30°C. The recombinant Arthrobacter sp. 22c L-arabinose isomerase was optimally active at a broad pH range of 5 to 9. This enzyme is not dependent on divalent metal ions, since it was only marginally activated by Mg2+, Mn2+ or Ca2+ and slightly inhibited by Co2+ or Ni2+. The bioconversion yield of D-galactose to D-tagatose by the purified L-arabinose isomerase reached 30% after 36 h at 50°C. In this study, a recombinant Pichia pastoris yeast strain secreting β-D-galactosidase Arthrobacter chlorophenolicus was also constructed. During cultivation of this strain in a whey permeate, lactose was hydrolyzed and D-glucose was metabolized, whereas D-galactose was accumulated in the medium. Moreover, cultivation of the P. pastoris strain secreting β-D-galactosidase in a whey permeate supplemented with Arthrobacter sp. 22c L-arabinose isomerase resulted in a 90% yield of lactose hydrolysis, the complete utilization

Long-Term Effect of Gene Therapy on Leber's Congenital Amaurosis

OpenAIRE

Bainbridge, James W B; Mehat, Manjit S; Sundaram, Venki; Robbie, Scott J; Barker, Susie E; Ripamonti, Caterina; Georgiadis, Anastasios; Mowat, Freya M; Beattie, Stuart G; Gardner, Peter J; Feathers, Kecia L; Luong, Vy A; Yzer, Suzanne; Balaggan, Kamaljit; Viswanathan, Ananth

2015-01-01

Background Mutations in RPE65 cause Leber's congenital amaurosis, a progressive retinal degenerative disease that severely impairs sight in children. Gene therapy can result in modest improvements in night vision, but knowledge of its efficacy in humans is limited. Methods We performed a phase 1-2 open-label trial involving 12 participants to evaluate the safety and efficacy of gene therapy with a recombinant adeno-associated virus 2/2 (rAAV2/2) vector carrying the RPE65 complementary DNA, an...

The clinical impact of vehicle technology using a patented formulation of benzoyl peroxide 5%/clindamycin 1% gel: comparative assessments of skin tolerability and evaluation of combination use with a topical retinoid.

Science.gov (United States)

Del Rosso, James Q; Tanghetti, Emil

2006-02-01

A major challenge encountered in clinical practice in patients with acne vulgaris is irritation related to topical medications used for treatment. Advances in vehicle technology have improved formulations containing active ingredients known to produce irritation in some patients, such as benzoyl peroxide (BP) and topical retinoids. Clinical studies, including combination therapy studies have demonstrated that certain additives, such as silicates and specific humectants, reduce irritation by maintaining barrier integrity. A patented gel formulation of BP 5%/clindamycin phosphate 1% (clindamycin) containing dimethicone and glycerin has been studied both as a monotherapy and in combination with topical retinoid use. This article evaluates specific vehicle additives included in this gel formulation and explains their role in reducing irritation. Data from clinical trials utilizing this technology in acne management are also reviewed.

Purification and characterization of an L-arabinose isomerase from an isolated strain of Geobacillus thermodenitrificans producing D-tagatose.

Science.gov (United States)

Kim, Hye-Jung; Oh, Deok-Kun

2005-11-04

The araA gene, encoding l-arabinose isomerase (AI), from the thermophilic bacterium Geobacillus thermodenitrificans was cloned and expressed in Escherichia coli. Recombinant AI was isolated with a final purity of about 97% and a final specific activity of 2.10 U/mg. The molecular mass of the purified AI was estimated to be about 230 kDa to be a tetramer composed of identical subunits. The AI exhibited maximum activity at 70 degrees C and pH 8.5 in the presence of Mn2+. The enzyme was stable at temperatures below 60 degrees C and within the pH range 7.5-8.0. d-Galactose and l-arabinose as substrate were isomerized with high activities. Ribitol was the strongest competitive inhibitor of AI with a Ki of 5.5mM. The apparent Km and Vmax for L-arabinose were 142 mM and 86 U/mg, respectively, whereas those for d-galactose were 408 mM and 6.9 U/mg, respectively. The catalytic efficiency (kcat/Km) was 48 mM(-1)min(-1) for L-arabinose and 0.5mM(-1)min(-1) for D-galactose. Mn2+ was a competitive activator and increased the thermal stability of the AI. The D-tagatose yield produced by AI from d-galactose was 46% without the addition of Mn2+ and 48% with Mn2+ after 300 min at 65 degrees C.

A feasible enzymatic process for D-tagatose production by an immobilized thermostable L-arabinose isomerase in a packed-bed bioreactor.

Science.gov (United States)

Kim, Hye-Jung; Ryu, Se-Ah; Kim, Pil; Oh, Deok-Kun

2003-01-01

To develop a feasible enzymatic process for d-tagatose production, a thermostable l-arabinose isomerase, Gali152, was immobilized in alginate, and the galactose isomerization reaction conditions were optimized. The pH and temperature for the maximal galactose isomerization reaction were pH 8.0 and 65 degrees C in the immobilized enzyme system and pH 7.5 and 60 degrees C in the free enzyme system. The presence of manganese ion enhanced galactose isomerization to tagatose in both the free and immobilized enzyme systems. The immobilized enzyme was more stable than the free enzyme at the same pH and temperature. Under stable conditions of pH 8.0 and 60 degrees C, the immobilized enzyme produced 58 g/L of tagatose from 100 g/L galactose in 90 h by batch reaction, whereas the free enzyme produced 37 g/L tagatose due to its lower stability. A packed-bed bioreactor with immobilized Gali152 in alginate beads produced 50 g/L tagatose from 100 g/L galactose in 168 h, with a productivity of 13.3 (g of tagatose)/(L-reactor.h) in continuous mode. The bioreactor produced 230 g/L tagatose from 500 g/L galactose in continuous recycling mode, with a productivity of 9.6 g/(L.h) and a conversion yield of 46%.

Combination of retinal pigment epithelium cell-conditioned medium and photoreceptor outer segments stimulate mesenchymal stem cell differentiation toward a functional retinal pigment epithelium cell phenotype.

Science.gov (United States)

Huang, Chen; Zhang, Jing; Ao, Mingxin; Li, Ying; Zhang, Chun; Xu, Yonggen; Li, Xuemin; Wang, Wei

2012-02-01

Recent studies have suggested that bone marrow-derived mesenchymal stem cells (BMMSCs) are capable of retinal tissue-specific differentiation but not retinal pigment epithelium (RPE) cell-specific differentiation. Photoreceptor outer segments (POS) contribute to RPE development and maturation. However, there has been no standard culture system that fosters the differentiation of BMMSCs into mature RPE cells in vitro. In this study, we investigated if the soluble factors from RPE cells and POS could differentiate BMMSCs into cells having a phenotype characteristic of RPE cells. Rat BMMSCs were separately co-cultured with RPE cells, or they were exposed to either control medium, RPE cell-conditioned medium (RPECM), POS, or a combination of RPECM and POS (RPECM-POS). After 7 days, the cells were analyzed for morphology and the expression of RPE markers (cytokeratin 8, CRALBP, and RPE65) to assess the RPE differentiation. Significantly higher pigment accumulation and increased protein expression of the three markers were seen in cells cultured in RPECM-POS than in other treated cultures. Furthermore, the RPECM-POS-treated cultures displayed ultrastructural features typical of RPE cells, expressed RPE cell functional proteins, and had the capability to phagocytose POS. Together, theses results suggest the combination of RPECM and POS stimulate BMMSCs differentiation toward a functional RPE phenotype. Our results provide the foundation for a new route to RPE regenerative therapy involving BMMSCs. Future work isolating the active agent in RPECM and POS would be useful in therapies for RPE diseases or in developing appropriately pre-differentiated BMMSCs for tissue-engineered RPE reconstruction. Copyright © 2011 Wiley Periodicals, Inc.

Retinoid X Receptor α-Dependent HBV Minichromosome Remodeling and Viral Replication.

Science.gov (United States)

Zhang, Yan; He, Song; Guo, Jin-Jun; Peng, Hong; Fan, Jia-Hao; Li, Qing-Ling

2017-01-01

The HBV covalently closed circular DNA (cccDNA) is organized into a minichromosome in the nuclei of infected hepatocytes through interactions with histone and nonhistone proteins. Retinoid X receptor α (RXRα), a liver-enriched nuclear receptor, participates in regulation of HBV replication and transcription through modulation of HBV enhancer 1 and core promoter activity. This study investigated RXRα involvement in HBV cccDNA epigenetic modifications. Quantitative cccDNA chromatin immunoprecipitation (ChIP) was applied to study the recruitment of RXRα, histones, and chromatin-modifying enzymes to HBV minichromosome in HepG2 cells after transfection of the linear HBV genome. RXRα Was found to directly bind to HBV cccDNA; recruitment of RXRα to HBV mini-chromosome paralleled HBV replication, histone recruitment, and histone acetylation in HBVcccDNA. Moreover, RXRα overexpression or knock-down significantly increased or impaired the recruitment of the p300 acetyltransferase to cccDNAminichromosome. Our results confirmed the regulation of RXRα on HBV replication in vitro and demonstrated the modulation of RXRα on HBV cccDNA epigenetics. These findings provide a profound theoretical and experimental basis for late-model antiviral treatment acting on the HBV cccDNA and minichromosome.

Waterborne cadmium and nickel impact oxidative stress responses and retinoid metabolism in yellow perch

International Nuclear Information System (INIS)

Defo, Michel A.; Bernatchez, Louis; Campbell, Peter G.C.; Couture, Patrice

2014-01-01

Highlights: • Cd and Ni affected indicators of retinoid metabolism and oxidative stress in fish. • Liver rdh-2 transcription levels increase in fish exposed to waterborne Cd. • Liver REH and LdRAT activities increase with increasing kidney Cd concentration. • Changes at molecular levels do not always mean changes at the functional levels. • Multi-level biological approaches are needed when assessing fish metal toxicology. - Abstract: In this experiment, we studied the transcriptional and functional (enzymatic) responses of yellow perch (Perca flavescens) to metal stress, with a focus on oxidative stress and vitamin A metabolism. Juvenile yellow perch were exposed to two environmentally relevant concentrations of waterborne cadmium (Cd) and nickel (Ni) for a period of 6 weeks. Kidney Cd and Ni bioaccumulation significantly increased with increasing metal exposure. The major retinoid metabolites analyzed in liver and muscle decreased with metal exposure except at high Cd exposure where no variation was reported in liver. A decrease in free plasma dehydroretinol was also observed with metal exposure. In the liver of Cd-exposed fish, both epidermal retinol dehydrogenase 2 transcription level and corresponding enzyme activities retinyl ester hydrolase and lecithin dehydroretinyl acyl transferase increased. In contrast, muscle epidermal retinol dehydrogenase 2 transcription level decreased with Cd exposure. Among antioxidant defences, liver transcription levels of catalase, microsomal glutathione-S-transferase-3 and glucose-6-phosphate dehydrogenase were generally enhanced in Cd-exposed fish and this up-regulation was accompanied by an increase in the activities of corresponding enzymes, except for microsomal glutathione-S-transferase. No consistent pattern in antioxidant defence responses was observed between molecular and biochemical response when fish were exposed to Ni, suggesting a non-synchronous response of antioxidant defence in fish exposed to

Waterborne cadmium and nickel impact oxidative stress responses and retinoid metabolism in yellow perch

Energy Technology Data Exchange (ETDEWEB)

Defo, Michel A. [Institut national de la recherche scientifique (INRS), Centre Eau Terre Environnement, 490 de la Couronne, Québec, Québec G1K 9A9 (Canada); Bernatchez, Louis [Institut de Biologie Intégrative et des Systèmes (IBIS), Université Laval, Québec, Québec G1V 0A6 (Canada); Campbell, Peter G.C. [Institut national de la recherche scientifique (INRS), Centre Eau Terre Environnement, 490 de la Couronne, Québec, Québec G1K 9A9 (Canada); Couture, Patrice, E-mail: patrice.couture@ete.inrs.ca [Institut national de la recherche scientifique (INRS), Centre Eau Terre Environnement, 490 de la Couronne, Québec, Québec G1K 9A9 (Canada)

2014-09-15

Highlights: • Cd and Ni affected indicators of retinoid metabolism and oxidative stress in fish. • Liver rdh-2 transcription levels increase in fish exposed to waterborne Cd. • Liver REH and LdRAT activities increase with increasing kidney Cd concentration. • Changes at molecular levels do not always mean changes at the functional levels. • Multi-level biological approaches are needed when assessing fish metal toxicology. - Abstract: In this experiment, we studied the transcriptional and functional (enzymatic) responses of yellow perch (Perca flavescens) to metal stress, with a focus on oxidative stress and vitamin A metabolism. Juvenile yellow perch were exposed to two environmentally relevant concentrations of waterborne cadmium (Cd) and nickel (Ni) for a period of 6 weeks. Kidney Cd and Ni bioaccumulation significantly increased with increasing metal exposure. The major retinoid metabolites analyzed in liver and muscle decreased with metal exposure except at high Cd exposure where no variation was reported in liver. A decrease in free plasma dehydroretinol was also observed with metal exposure. In the liver of Cd-exposed fish, both epidermal retinol dehydrogenase 2 transcription level and corresponding enzyme activities retinyl ester hydrolase and lecithin dehydroretinyl acyl transferase increased. In contrast, muscle epidermal retinol dehydrogenase 2 transcription level decreased with Cd exposure. Among antioxidant defences, liver transcription levels of catalase, microsomal glutathione-S-transferase-3 and glucose-6-phosphate dehydrogenase were generally enhanced in Cd-exposed fish and this up-regulation was accompanied by an increase in the activities of corresponding enzymes, except for microsomal glutathione-S-transferase. No consistent pattern in antioxidant defence responses was observed between molecular and biochemical response when fish were exposed to Ni, suggesting a non-synchronous response of antioxidant defence in fish exposed to

Focal epithelial hyperplasia by human papillomavirus (HPV)-32 misdiagnosed as HPV-16 and treated with combination of retinoids, imiquimod and quadrivalent HPV vaccine.

Science.gov (United States)

Gemigniani, Franco; Hernández-Losa, Javier; Ferrer, Berta; García-Patos, Vicente

2015-12-01

Focal epithelial hyperplasia (FEH) or Heck's disease is a rare, benign and asymptomatic mucosal proliferation associated with human papillomavirus (HPV) infection, mainly with genotypes 13 and 32. We report a florid case of FEH in an 11-year-old Haitian girl with systemic lupus erythematosus receiving immunosuppressive therapy. Cryotherapy was previously performed on numerous occasions with no results. We decided to prescribe a non-invasive and more comfortable treatment. A combination of topical retinoid and imiquimod cream was well tolerated and led to an important improvement. The evidence of infection by HPV-16 detected by polymerase chain reaction (PCR) technique, prompted us to prescribe the quadrivalent HPV vaccine (types 6, 11,16 and 18). Subsequent PCR sequencing with generic primers GP5-GP6 and further BLAST comparative analysis confirmed that genomic viral sequence in our case truly corresponded with HPV-32. This molecular misdiagnosis can be explained by the similarity between genomic sequences of both HPV-16 and -32 genotypes. At the 1-year follow up, we observed total clinical improvement and no recurrences of the disease. Complete healing in this case may correspond to a potential action of topical retinoid, imiquimod and the cross-protection mechanism of the quadrivalent HPV vaccine. © 2015 Japanese Dermatological Association.

Independent Activation of Hepatitis B Virus Biosynthesis by Retinoids, Peroxisome Proliferators, and Bile Acids

Science.gov (United States)

Reese, Vanessa C.; Oropeza, Claudia E.

2013-01-01

In the human hepatoma cell line HepG2, retinoic acid, clofibric acid, and bile acid treatment can only modestly increase hepatitis B virus (HBV) biosynthesis. Utilizing the human embryonic kidney cell line 293T, it was possible to demonstrate that the retinoid X receptor α (RXRα) plus its ligand can support viral biosynthesis independently of additional nuclear receptors. In addition, RXRα/peroxisome proliferator-activated receptor α (PPARα) and RXRα/farnesoid X receptor α (FXRα) heterodimeric nuclear receptors can also mediate ligand-dependent HBV transcription and replication when activated by clofibric acid and bile acid, respectively, independently of a requirement for the ligand-dependent activation of RXRα. These observations indicate that there are at least three possible modes of ligand-mediated activation of HBV transcription and replication existing within hepatocytes, suggesting that multiple independent mechanisms control viral production in the livers of infected individuals. PMID:23135717

The Expansion of RPE Atrophy after the Inverted ILM Flap Technique for a Chronic Large Macular Hole

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Hisanori Imai

2014-03-01

Full Text Available Purpose: To report a case of the expansion of submacular retinal pigment epithelium (RPE atrophy after using the inverted internal limiting membrane (ILM flap technique for a persisting, large, stage IV macular hole (MH. Case Report: A 79-year-old woman presented with a chronic large MH that remained open despite pars plana vitrectomy (PPV. The surgery was performed twice for the MH closure 14 years earlier. ILM peeling was not performed during the previous surgeries. The best-corrected visual acuity (BCVA with the Landolt ring chart was 0.08 at her visit. The minimum MH diameter was 1,240 μm. Inverted ILM flap technique with 20% SF6 gas tamponade was performed for the MH closure. For the inverted ILM flap technique, 25-gauge PPV and ILM staining with indocyanine green were used. The ILM was peeled off for 2 disc diameters around the MH, but the ILM was not removed completely. The ILM was then inverted and covered the MH. Results: One month after surgery, the MH was closed, accompanied by glial cell proliferation spreading from the inverted ILM flap (as reported before. On the other hand, the area of the submacular RPE atrophy, which was already observed 1 week after surgery, gradually increased in size. BCVA improved to 0.3 six months after the surgery. Conclusions: The inverted ILM flap technique may be promising even for persisting large MH which were not closed in previous surgeries, but long-term observation is needed because the detailed behavior of the inverted ILM and the Müller cells after surgery is not yet known.

Cyclophilin40 isomerase activity is regulated by a temperature-dependent allosteric interaction with Hsp90.

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Blackburn, Elizabeth A; Wear, Martin A; Landré, Vivian; Narayan, Vikram; Ning, Jia; Erman, Burak; Ball, Kathryn L; Walkinshaw, Malcolm D

2015-09-01

Cyclophilin 40 (Cyp40) comprises an N-terminal cyclophilin domain with peptidyl-prolyl isomerase (PPIase) activity and a C-terminal tetratricopeptide repeat (TPR) domain that binds to the C-terminal-EEVD sequence common to both heat shock protein 70 (Hsp70) and Hsp90. We show in the present study that binding of peptides containing the MEEVD motif reduces the PPIase activity by ∼30%. CD and fluorescence assays show that the TPR domain is less stable than the cyclophilin domain and is stabilized by peptide binding. Isothermal titration calorimetry (ITC) shows that the affinity for the-MEEVD peptide is temperature sensitive in the physiological temperature range. Results from these biophysical studies fit with the MD simulations of the apo and holo (peptide-bound) structures which show a significant reduction in root mean square (RMS) fluctuation in both TPR and cyclophilin domains when-MEEVD is bound. The MD simulations of the apo-protein also highlight strong anti-correlated motions between residues around the PPIase-active site and a band of residues running across four of the seven helices in the TPR domain. Peptide binding leads to a distortion in the shape of the active site and a significant reduction in these strongly anti-correlated motions, providing an explanation for the allosteric effect of ligand binding and loss of PPIase activity. Together the experimental and MD results suggest that on heat shock, dissociation of Cyp40 from complexes mediated by the TPR domain leads to an increased pool of free Cyp40 capable of acting as an isomerase/chaperone in conditions of cellular stress. © 2015 Authors.

PBN (Phenyl-N-Tert-Butylnitrone-Derivatives Are Effective in Slowing the Visual Cycle and Rhodopsin Regeneration and in Protecting the Retina from Light-Induced Damage.

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Megan Stiles

Full Text Available A2E and related toxic molecules are part of lipofuscin found in the retinal pigment epithelial (RPE cells in eyes affected by Stargardt's disease, age-related macular degeneration (AMD, and other retinal degenerations. A novel therapeutic approach for treating such degenerations involves slowing down the visual cycle, which could reduce the amount of A2E in the RPE. This can be accomplished by inhibiting RPE65, which produces 11-cis-retinol from all-trans-retinyl esters. We recently showed that phenyl-N-tert-butylnitrone (PBN inhibits RPE65 enzyme activity in RPE cells. In this study we show that like PBN, certain PBN-derivatives (PBNDs such as 4-F-PBN, 4-CF3-PBN, 3,4-di-F-PBN, and 4-CH3-PBN can inhibit RPE65 and synthesis of 11-cis-retinol in in vitro assays using bovine RPE microsomes. We further demonstrate that systemic (intraperitoneal, IP administration of these PBNDs protect the rat retina from light damage. Electroretinography (ERG and histological analysis showed that rats treated with PBNDs retained ~90% of their photoreceptor cells compared to a complete loss of function and 90% loss of photoreceptors in the central retina in rats treated with vehicle/control injections. Topically applied PBN and PBNDs also significantly slowed the rate of the visual cycle in mouse and baboon eyes. One hour dark adaptation resulted in 75-80% recovery of bleachable rhodopsin in control/vehicle treated mice. Eye drops of 5% 4-CH3-PBN were most effective, inhibiting the regeneration of bleachable rhodopsin significantly (60% compared to vehicle control. In addition, a 10% concentration of PBN and 5% concentration of 4-CH3-PBN in baboon eyes inhibited the visual cycle by 60% and by 30%, respectively. We have identified a group of PBN related nitrones that can reach the target tissue (RPE by systemic and topical application and slow the rate of rhodopsin regeneration and therefore the visual cycle in mouse and baboon eyes. PBNDs can also protect the rat

PBN (Phenyl-N-Tert-Butylnitrone)-Derivatives Are Effective in Slowing the Visual Cycle and Rhodopsin Regeneration and in Protecting the Retina from Light-Induced Damage.

Science.gov (United States)

Stiles, Megan; Moiseyev, Gennadiy P; Budda, Madeline L; Linens, Annette; Brush, Richard S; Qi, Hui; White, Gary L; Wolf, Roman F; Ma, Jian-Xing; Floyd, Robert; Anderson, Robert E; Mandal, Nawajes A

2015-01-01

A2E and related toxic molecules are part of lipofuscin found in the retinal pigment epithelial (RPE) cells in eyes affected by Stargardt's disease, age-related macular degeneration (AMD), and other retinal degenerations. A novel therapeutic approach for treating such degenerations involves slowing down the visual cycle, which could reduce the amount of A2E in the RPE. This can be accomplished by inhibiting RPE65, which produces 11-cis-retinol from all-trans-retinyl esters. We recently showed that phenyl-N-tert-butylnitrone (PBN) inhibits RPE65 enzyme activity in RPE cells. In this study we show that like PBN, certain PBN-derivatives (PBNDs) such as 4-F-PBN, 4-CF3-PBN, 3,4-di-F-PBN, and 4-CH3-PBN can inhibit RPE65 and synthesis of 11-cis-retinol in in vitro assays using bovine RPE microsomes. We further demonstrate that systemic (intraperitoneal, IP) administration of these PBNDs protect the rat retina from light damage. Electroretinography (ERG) and histological analysis showed that rats treated with PBNDs retained ~90% of their photoreceptor cells compared to a complete loss of function and 90% loss of photoreceptors in the central retina in rats treated with vehicle/control injections. Topically applied PBN and PBNDs also significantly slowed the rate of the visual cycle in mouse and baboon eyes. One hour dark adaptation resulted in 75-80% recovery of bleachable rhodopsin in control/vehicle treated mice. Eye drops of 5% 4-CH3-PBN were most effective, inhibiting the regeneration of bleachable rhodopsin significantly (60% compared to vehicle control). In addition, a 10% concentration of PBN and 5% concentration of 4-CH3-PBN in baboon eyes inhibited the visual cycle by 60% and by 30%, respectively. We have identified a group of PBN related nitrones that can reach the target tissue (RPE) by systemic and topical application and slow the rate of rhodopsin regeneration and therefore the visual cycle in mouse and baboon eyes. PBNDs can also protect the rat retina from

Disposition and biotransformation of the acetylenic retinoid tazarotene in humans.

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Attar, Mayssa; Yu, Dale; Ni, Jinsong; Yu, Zhiling; Ling, Kah-Hiing John; Tang-Liu, Diane D-S

2005-10-01

Oral tazarotene, an acetylenic retinoid, is in clinical development for the treatment of psoriasis. The disposition and biotransformation of tazarotene were investigated in six healthy male volunteers, following a single oral administration of a 6 mg (100 microCi) dose of [14C]tazarotene, in a gelatin capsule. Blood levels of radioactivity peaked 2 h postdose and then rapidly declined. Total recovery of radioactivity was 89.2+/-8.0% of the administered dose, with 26.1+/-4.2% in urine and 63.0+/-7.0% in feces, within 7 days of dosing. Only tazarotenic acid, the principle active metabolite formed via esterase hydrolysis of tazarotene, was detected in blood. One major urinary oxidative metabolite, tazarotenic acid sulfoxide, accounted for 19.2+/-3.0% of the dose. The majority of radioactivity recovered in the feces was attributed to tazarotenic acid representing 46.9+/-9.9% of the dose and only 5.82+/-3.84% of dose was excreted as unchanged tazarotene. Thus following oral administration, tazarotene was rapidly absorbed and underwent extensive hydrolysis to tazarotenic acid, the major circulating species in the blood that was then excreted unchanged in feces. A smaller fraction of tazarotenic acid was further metabolized to an inactive sulfoxide that was excreted in the urine. Copyright (c) 2005 Wiley-Liss, Inc. and the American Pharmacists Association

Retinoides (RARβ y CRBP1 en carcinoma de pulmón a células no pequeñas Retinoid expression (RARβ and CRBP1 in non-small-cell lung carcinoma

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Laura V. Mauro

2008-06-01

Full Text Available Aunque los pacientes con cáncer de pulmón a células no pequeñas en estadios tempranos (NSCLC tienen buen pronóstico, el 20-30% recae, siendo relevante la identificación de biomarcadores pronósticos. Los retinoides regulan crecimiento y diferenciación, y pueden antagonizar la progresión tumoral. Su efecto depende del transporte citosólico mediado por moléculas como CRBP1, y de la unión a receptores específicos (RARβ. Alteraciones en esta vía se asociaron con cáncer. Nuestro objetivo fue estudiar la expresión, mediante inmunohistoquímica, de RARβ y CRBP1 en el tejido tumoral de 49 pacientes NSCLC Estadio I/II, obtenido durante la cirugía. La supervivencia se analizó mediante los test Log Rank y multivariado de Cox. El 44.9% de los tumores fueron positivos para RARβ con expresión a nivel citoplasmático, mientras que el 34.7% lo expresó a nivel nuclear. La tinción para CRBP1 se observó en el 61.2% de los tumores. No se encontró asociación entre la expresión de ambas moléculas y las características clinicopatológicas (sexo, tamaño tumoral, nódulos línfáticos comprometidos, histopatología y p53. Tampoco se encontró asociación con el hábito de fu-mar. La presencia de células tumorales en el lavado pleural se asoció significativamente con la expresión de CRBP1. Por otro lado, se demostró asociación entre la expresión elevada de RARβ citoplasmático y menor supervivencia global (LR 4.17, p=0.0412. El análisis multivariado no mostró asociación con otras variables de pronóstico en NSCLC. En conclusión, en este grupo de pacientes NSCLC Estadio I/II, RARβ pareciera predecir la supervivencia global en forma independiente.Although early-stage non-small-cell lung carcinoma (NSCLC patients have a relative by favorable prognosis, the risk of a bad outcome remains substantial. Identification of reliable prognostic markers for disease recurrence and death has meaningful clinical application. Retinoids are

Integrating protein structures and precomputed genealogies in the Magnum database: Examples with cellular retinoid binding proteins

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Bradley Michael E

2006-02-01

Full Text Available Abstract Background When accurate models for the divergent evolution of protein sequences are integrated with complementary biological information, such as folded protein structures, analyses of the combined data often lead to new hypotheses about molecular physiology. This represents an excellent example of how bioinformatics can be used to guide experimental research. However, progress in this direction has been slowed by the lack of a publicly available resource suitable for general use. Results The precomputed Magnum database offers a solution to this problem for ca. 1,800 full-length protein families with at least one crystal structure. The Magnum deliverables include 1 multiple sequence alignments, 2 mapping of alignment sites to crystal structure sites, 3 phylogenetic trees, 4 inferred ancestral sequences at internal tree nodes, and 5 amino acid replacements along tree branches. Comprehensive evaluations revealed that the automated procedures used to construct Magnum produced accurate models of how proteins divergently evolve, or genealogies, and correctly integrated these with the structural data. To demonstrate Magnum's capabilities, we asked for amino acid replacements requiring three nucleotide substitutions, located at internal protein structure sites, and occurring on short phylogenetic tree branches. In the cellular retinoid binding protein family a site that potentially modulates ligand binding affinity was discovered. Recruitment of cellular retinol binding protein to function as a lens crystallin in the diurnal gecko afforded another opportunity to showcase the predictive value of a browsable database containing branch replacement patterns integrated with protein structures. Conclusion We integrated two areas of protein science, evolution and structure, on a large scale and created a precomputed database, known as Magnum, which is the first freely available resource of its kind. Magnum provides evolutionary and structural

Mannose Phosphate Isomerase Isoenzymes in Plutella xylostella Support Common Genetic Bases of Resistance to Bacillus thuringiensis Toxins in Lepidopteran Species

OpenAIRE

Herrero, Salvador; Ferré, Juan; Escriche, Baltasar

2001-01-01

A strong correlation between two mannose phosphate isomerase (MPI) isoenzymes and resistance to Cry1A toxins from Bacillus thuringiensis has been found in a Plutella xylostella population. MPI linkage to Cry1A resistance had previously been reported for a Heliothis virescens population. The fact that the two populations share similar biochemical, genetic, and cross-resistance profiles of resistance suggests the occurrence of homologous resistance loci in both species.

Characterization of a spontaneously generated murine retinal pigmented epithelium cell line; a model for in vitro experiments.

Science.gov (United States)

Ranaei Pirmardan, Ehsan; Soheili, Zahra-Soheila; Samiei, Shahram; Ahmadieh, Hamid; Mowla, Seyed Javad; Ezzati, Razie; Naseri, Marzieh

2016-10-01

Retinal pigmented epithelium (RPE), the outermost layer of the retina, has a key role in maintaining retinal cells' functions. Severity of the culture of RPE cells has exerted many limitations to both in vitro and in vivo studies and its therapeutic applications. Therefore, establishment of RPE cell lines with high proliferative potential can considerably improve study of RPE cell biology. Here we report generation of a spontaneously immortalized murine RPE cell line in primary mouse RPE cell culture. Founded colonized cells were picked up and expression of RPE and retinal progenitor cells' (RPC) markers were studied using immunocytochemistry (ICC). Emerged cells cultured over 35 passages and population doubling times in different serum concentrations were calculated. We also investigated the ability of cells for becoming transfected by calcium-phosphate method and for becoming infected by adeno-associated virus serotype 2 (AAV2) using flow cytometry. Data showed that the cobblestone constituent cells expressed RPE65, cytokeratin and ZO1 and moreover several progenitor markers such as Pax6, Sox2, Nestin and Chx10. It revealed that, despite primary RPE cells, the newly emerged cells were easily transfectable and were highly infectable when compared with HEK293T cells. Our data indicated that the emerged mouse RPE cell line pretended RPC-like phenotype and also simultaneously expressed RPE markers. It would be a promising model for leading studies on RPE and RPC cells and substantially confirmed the great RPE plasticity and its invaluable potential in research studies. Copyright © 2016 Elsevier Inc. All rights reserved.

Roles of Retinoids and Retinoic Acid Receptors in the Regulation of Hematopoietic Stem Cell Self-Renewal and Differentiation

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Louise E. Purton

2007-01-01

Full Text Available Multipotent hematopoietic stem cells (HSCs sustain blood cell production throughout an individual's lifespan through complex processes ultimately leading to fates of self-renewal, differentiation or cell death decisions. A fine balance between these decisions in vivo allows for the size of the HSC pool to be maintained. While many key factors involved in regulating HSC/progenitor cell differentiation and cell death are known, the critical regulators of HSC self-renewal are largely unknown. In recent years, however, a number of studies describing methods of increasing or decreasing the numbers of HSCs in a given population have emerged. Of major interest here are the emerging roles of retinoids in the regulation of HSCs.

Subretinal Implantation of Retinal Pigment Epithelial Cells Derived From Human Embryonic Stem Cells: Improved Survival When Implanted as a Monolayer

Science.gov (United States)

Diniz, Bruno; Thomas, Padmaja; Thomas, Biju; Ribeiro, Ramiro; Hu, Yuntao; Brant, Rodrigo; Ahuja, Ashish; Zhu, Danhong; Liu, Laura; Koss, Michael; Maia, Mauricio; Chader, Gerald; Hinton, David R.; Humayun, Mark S.

2013-01-01

Purpose. To evaluate cell survival and tumorigenicity of human embryonic stem cell–derived retinal pigment epithelium (hESC-RPE) transplantation in immunocompromised nude rats. Cells were transplanted as a cell suspension (CS) or as a polarized monolayer plated on a parylene membrane (PM). Methods. Sixty-nine rats (38 male, 31 female) were surgically implanted with CS (n = 33) or PM (n = 36). Cohort subsets were killed at 1, 6, and 12 months after surgery. Both ocular tissues and systemic organs (brain, liver, kidneys, spleen, heart, and lungs) were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Every fifth section was stained with hematoxylin and eosin and analyzed histologically. Adjacent sections were processed for immunohistochemical analysis (as needed) using the following antibodies: anti-RPE65 (RPE-specific marker), anti-TRA-1-85 (human cell marker), anti-Ki67 (proliferation marker), anti-CD68 (macrophage), and anti-cytokeratin (epithelial marker). Results. The implanted cells were immunopositive for the RPE65 and TRA-1-85. Cell survival (P = 0.006) and the presence of a monolayer (P < 0.001) of hESC-RPE were significantly higher in eyes that received the PM. Gross morphological and histological analysis of the eye and the systemic organs after the surgery revealed no evidence of tumor or ectopic tissue formation in either group. Conclusions. hESC-RPE can survive for at least 12 months in an immunocompromised animal model. Polarized monolayers of hESC-RPE show improved survival compared to cell suspensions. The lack of teratoma or any ectopic tissue formation in the implanted rats bodes well for similar results with respect to safety in human subjects. PMID:23833067

FabQ, a Dual-Function Dehydratase/Isomerase, Circumvents the Last Step of the Classical Fatty Acid Synthesis Cycle

OpenAIRE

Bi, Hongkai; Wang, Haihong; Cronan, John E.

2013-01-01

In the classical anaerobic pathway of unsaturated fatty acid biosynthesis, that of Escherichia coli, the double bond is introduced into the growing acyl chain by the FabA dehydratase/isomerase. Another dehydratase, FabZ, functions in the chain elongation cycle. In contrast, Aerococcus viridans has only a single FabA/FabZ homolog we designate FabQ. FabQ can not only replace the function of E. coli FabZ in vivo, but it also catalyzes the isomerization required for unsaturated fatty acid biosynt...

Inhibition of d-xylose isomerase by polyols: atomic details by joint X-ray/neutron crystallography

Energy Technology Data Exchange (ETDEWEB)

Kovalevsky, Andrey, E-mail: ayk@lanl.gov [Los Alamos National Laboratory, PO Box 1663, MS M888, Los Alamos, NM 87545 (United States); Hanson, B. Leif [University of Toledo, 2801 West Bancroft Street, Toledo, OH 43606 (United States); Mason, Sax A. [Institut Laue–Langevin, 6 Rue Jules Horowitz, 38042 Grenoble (France); Forsyth, V. Trevor [Institut Laue–Langevin, 6 Rue Jules Horowitz, 38042 Grenoble (France); Keele University, Staffordshire (United Kingdom); Fisher, Zoe [Los Alamos National Laboratory, PO Box 1663, MS M888, Los Alamos, NM 87545 (United States); Mustyakimov, Marat [Los Alamos National Laboratory, PO Box 1663, MS M888, Los Alamos, NM 87545 (United States); Oak Ridge National Laboratory, PO Box 2008, MS 6475, Oak Ridge, TN 37831 (United States); Blakeley, Matthew P. [Institut Laue–Langevin, 6 Rue Jules Horowitz, 38042 Grenoble (France); Keen, David A. [Harwell Science and Innovation Campus, Didcot, Oxon OX11 0QX (United Kingdom); Langan, Paul [Oak Ridge National Laboratory, PO Box 2008, MS 6475, Oak Ridge, TN 37831 (United States); Los Alamos National Laboratory, PO Box 1663, MS M888, Los Alamos, NM 87545 (United States)

2012-09-01

A joint X-ray/neutron structure of d-xylose isomerase in complex with the inhibitor sorbitol was determined at room temperature at an acidic pH of 5.9. Protonation of the O5 O atom of the sugar was directly observed in the nuclear density maps. Under acidic conditions sorbitol gains a water-mediated interaction with the enzyme active site, which may explain the increased potency of the inhibitor at low pH. d-Xylose isomerase (XI) converts the aldo-sugars xylose and glucose to their keto analogs xylulose and fructose, but is strongly inhibited by the polyols xylitol and sorbitol, especially at acidic pH. In order to understand the atomic details of polyol binding to the XI active site, a 2.0 Å resolution room-temperature joint X-ray/neutron structure of XI in complex with Ni{sup 2+} cofactors and sorbitol inhibitor at pH 5.9 and a room-temperature X-ray structure of XI containing Mg{sup 2+} ions and xylitol at the physiological pH of 7.7 were obtained. The protonation of oxygen O5 of the inhibitor, which was found to be deprotonated and negatively charged in previous structures of XI complexed with linear glucose and xylulose, was directly observed. The Ni{sup 2+} ions occupying the catalytic metal site (M2) were found at two locations, while Mg{sup 2+} in M2 is very mobile and has a high B factor. Under acidic conditions sorbitol gains a water-mediated interaction that connects its O1 hydroxyl to Asp257. This contact is not found in structures at basic pH. The new interaction that is formed may improve the binding of the inhibitor, providing an explanation for the increased affinity of the polyols for XI at low pH.

Construction of phosphomannose isomerase (PMI) transformation vectors and evaluation of the effectiveness of vectors in tobacco (Nicotiana tabacum L).

Science.gov (United States)

Bahariah, Bohari; Parveez, Ghulam Kadir Ahmad; Masani, Mat Yunus Abdul; Khalid, Norzulaani

2012-01-01

Phosphomannose isomerase (pmi) gene isolated from Escherichia coli allows transgenic plants carrying it to convert mannose-6- phosphate (from mannose), a carbon source that could not be naturally utilized by plants into fructose-6-phosphate which can be utilized by plants as a carbon source. This conversion ability provides energy source to allow the transformed cells to survive on the medium containing mannose. In this study, four transformation vectors carrying the pmi gene alone or in combination with the β-glucuronidase (gusA) gene were constructed and driven by either the maize ubiquitin (Ubi1) or the cauliflower mosaic virus (CaMV35S) promoter. Restriction digestion, PCR amplification and sequencing were carried out to ensure sequence integrity and orientation. Tobacco was used as a model system to study the effectiveness of the constructs and selection system. PMI11G and pMI3G, which carry gusA gene, were used to study the gene transient expression in tobacco. PMI3 construct, which only carries the pmi gene driven by CaMV35S promoter, was stably transformed into tobacco using biolistics after selection on 30 g 1(-1) mannose without sucrose. Transgenic plants were verified using PCR analysis. PMI/pmi - Phosphomannose isomerase, Ubi1 - Maize ubiquitin promoter, CaMV35S - Cauliflower mosaic virus 35S promoter, gusA - β-glucuronidase GUS reporter gene.

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of the mannose 6-phosphate isomerase from Salmonella typhimurium

Energy Technology Data Exchange (ETDEWEB)

Gowda, Giri; Sagurthi, Someswar Rao [Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012 (India); Savithri, H. S. [Department of Biochemistry, Indian Institute of Science, Bangalore 560 012 (India); Murthy, M. R. N., E-mail: mrn@mbu.iisc.ernet.in [Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012 (India)

2008-02-01

The cloning, expression, purification, crystallization and preliminary X-ray crystallographic studies of mannose 6-phosphate isomerase from S. typhimurium are reported. Mannose 6-phosphate isomerase (MPI; EC 5.3.1.8) catalyzes the reversible isomerization of d-mannose 6-phosphate (M6P) and d-fructose 6-phosphate (F6P). In the eukaryotes and prokaryotes investigated to date, the enzyme has been reported to play a crucial role in d-mannose metabolism and supply of the activated mannose donor guanosine diphosphate d-mannose (GDP-d-mannose). In the present study, MPI was cloned from Salmonella typhimurium, overexpressed in Escherichia coli and purified using Ni–NTA affinity column chromatography. Purified MPI crystallized in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 36.03, b = 92.2, c = 111.01 Å. A data set extending to 1.66 Å resolution was collected with 98.8% completeness using an image-plate detector system mounted on a rotating-anode X-ray generator. The asymmetric unit of the crystal cell was compatible with the presence of a monomer of MPI. A preliminary structure solution of the enzyme has been obtained by molecular replacement using Candida albicans MPI as the phasing model and the program Phaser. Further refinement and model building are in progress.

Characterization of a spontaneously generated murine retinal pigmented epithelium cell line; a model for in vitro experiments

International Nuclear Information System (INIS)

Ranaei Pirmardan, Ehsan; Soheili, Zahra-Soheila; Samiei, Shahram; Ahmadieh, Hamid; Mowla, Seyed Javad; Ezzati, Razie; Naseri, Marzieh

2016-01-01

Retinal pigmented epithelium (RPE), the outermost layer of the retina, has a key role in maintaining retinal cells’ functions. Severity of the culture of RPE cells has exerted many limitations to both in vitro and in vivo studies and its therapeutic applications. Therefore, establishment of RPE cell lines with high proliferative potential can considerably improve study of RPE cell biology. Here we report generation of a spontaneously immortalized murine RPE cell line in primary mouse RPE cell culture. Founded colonized cells were picked up and expression of RPE and retinal progenitor cells’ (RPC) markers were studied using immunocytochemistry (ICC). Emerged cells cultured over 35 passages and population doubling times in different serum concentrations were calculated. We also investigated the ability of cells for becoming transfected by calcium-phosphate method and for becoming infected by adeno-associated virus serotype 2 (AAV2) using flow cytometry. Data showed that the cobblestone constituent cells expressed RPE65, cytokeratin and ZO1 and moreover several progenitor markers such as Pax6, Sox2, Nestin and Chx10. It revealed that, despite primary RPE cells, the newly emerged cells were easily transfectable and were highly infectable when compared with HEK293T cells. Our data indicated that the emerged mouse RPE cell line pretended RPC-like phenotype and also simultaneously expressed RPE markers. It would be a promising model for leading studies on RPE and RPC cells and substantially confirmed the great RPE plasticity and its invaluable potential in research studies. - Highlights: • Isolation of a spontaneously generated retinal pigmented epithelium cell line is reported. • The cells express some of the retinal progenitor cell markers in addition to the RPE markers. • The aforesaid cell line is highly transfecable and considerably infectable by AAV2. • These results confirm the great RPE plasticity and its invaluable potential in research studies.

Characterization of a spontaneously generated murine retinal pigmented epithelium cell line; a model for in vitro experiments

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Ranaei Pirmardan, Ehsan [Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Soheili, Zahra-Soheila [Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology, Tehran (Iran, Islamic Republic of); Samiei, Shahram [Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran (Iran, Islamic Republic of); Ahmadieh, Hamid [Ophthalmic Research Center, Shahid Beheshti University of Medical Sciences, Tehran (Iran, Islamic Republic of); Mowla, Seyed Javad [Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Ezzati, Razie [Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology, Tehran (Iran, Islamic Republic of); Naseri, Marzieh [Department of Molecular Medicine, Faculty of Advanced Technology, Iran University of Medical Sciences, Tehran (Iran, Islamic Republic of)

2016-10-01

Retinal pigmented epithelium (RPE), the outermost layer of the retina, has a key role in maintaining retinal cells’ functions. Severity of the culture of RPE cells has exerted many limitations to both in vitro and in vivo studies and its therapeutic applications. Therefore, establishment of RPE cell lines with high proliferative potential can considerably improve study of RPE cell biology. Here we report generation of a spontaneously immortalized murine RPE cell line in primary mouse RPE cell culture. Founded colonized cells were picked up and expression of RPE and retinal progenitor cells’ (RPC) markers were studied using immunocytochemistry (ICC). Emerged cells cultured over 35 passages and population doubling times in different serum concentrations were calculated. We also investigated the ability of cells for becoming transfected by calcium-phosphate method and for becoming infected by adeno-associated virus serotype 2 (AAV2) using flow cytometry. Data showed that the cobblestone constituent cells expressed RPE65, cytokeratin and ZO1 and moreover several progenitor markers such as Pax6, Sox2, Nestin and Chx10. It revealed that, despite primary RPE cells, the newly emerged cells were easily transfectable and were highly infectable when compared with HEK293T cells. Our data indicated that the emerged mouse RPE cell line pretended RPC-like phenotype and also simultaneously expressed RPE markers. It would be a promising model for leading studies on RPE and RPC cells and substantially confirmed the great RPE plasticity and its invaluable potential in research studies. - Highlights: • Isolation of a spontaneously generated retinal pigmented epithelium cell line is reported. • The cells express some of the retinal progenitor cell markers in addition to the RPE markers. • The aforesaid cell line is highly transfecable and considerably infectable by AAV2. • These results confirm the great RPE plasticity and its invaluable potential in research studies.

Prevalence of choroidal nevus and retinal pigment epithelial alterations in vitiligo patients.

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Fleissig, Efrat; Pavlovksy, Mor; Loewenstein, Anat; Zur, Dinah; Newman, Hadas; Keren, Shay; Goldenberg, Dafna; Bar-Ilan, Efrat; Goldstein, Michaella

2018-05-01

To investigate ocular manifestations in patients with vitiligo by multimodal imaging, including optical coherence tomography (OCT), color fundus photography, and fundus autofluorescence (FAF). In this prospective, observational clinical study, vitiligo patients underwent ophthalmologic and dermatologic clinical assessment and imaging by spectral-domain OCT, FAF, and color fundus imaging. Ocular echography was performed as indicated. Statistical analysis was performed using paired T test and Pearson correlation. A total of 61 eyes of 31 vitiligo patients were examined. Ocular findings consisted of choroidal nevi (n = 10, 32%), of which four (40%) were bilateral; two patients (6.5%) had a prominent choroidal pattern, two (6.5%) had hypopigmentary retinal pigment epithelium (RPE) lesions, and one (3.2%) had peripapillary atrophy of the RPE. Choroidal nevi were demonstrated only in eyes of patients with generalized vitiligo and were more common with upper body involvement (p = 0.02) and more prevalent in women (p = 0.02). Hypopigmentary lesions were detected in two patients and demonstrated on OCT as RPE atrophy and as photoreceptor/RPE changes. In this case series, vitiligo patients had a higher rate of choroidal nevi than previously reported. The hypopigmentary vitiliginous fundus lesions were depicted on OCT as photoreceptor and RPE atrophy. These findings may suggest the advisability of regular ocular monitoring for vitiligo patients.

Peretinoin, an acyclic retinoid, improves the hepatic gene signature of chronic hepatitis C following curative therapy of hepatocellular carcinoma

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Honda, Masao; Yamashita, Taro; Yamashita, Tatsuya; Arai, Kuniaki; Sakai, Yoshio; Sakai, Akito; Nakamura, Mikiko; Mizukoshi, Eishiro; Kaneko, Shuichi

2013-01-01

The acyclic retinoid, peretinoin, has been shown to be effective for suppressing hepatocellular carcinoma (HCC) recurrence after definitive treatment in a small-scale randomized clinical trial. However, little has been documented about the mechanism by which peretinoin exerts its inhibitory effects against recurrent HCC in humans in vivo. Twelve hepatitis C virus-positive patients whose HCC had been eradicated through curative resection or ablation underwent liver biopsy at baseline and week 8 of treatment with either a daily dose of 300 or 600 mg peretinoin. RNA isolated from biopsy samples was subjected to gene expression profile analysis. Peretinoin treatment elevated the expression levels of IGFBP6, RBP1, PRB4, CEBPA, G0S2, TGM2, GPRC5A, CYP26B1, and many other retinoid target genes. Elevated expression was also observed for interferon-, Wnt-, and tumor suppressor-related genes. By contrast, decreased expression levels were found for mTOR- and tumor progression-related genes. Interestingly, gene expression profiles for week 8 of peretinoin treatment could be classified into two groups of recurrence and non-recurrence with a prediction accuracy rate of 79.6% (P<0.05). In the liver of patients with non-recurrence, expression of PDGFC and other angiogenesis genes, cancer stem cell marker genes, and genes related to tumor progression was down-regulated, while expression of genes related to hepatocyte differentiation, tumor suppression genes, and other genes related to apoptosis induction was up-regulated. Gene expression profiling at week 8 of peretinoin treatment could successfully predict HCC recurrence within 2 years. This study is the first to show the effect of peretinoin in suppressing HCC recurrence in vivo based on gene expression profiles and provides a molecular basis for understanding the efficacy of peretinoin

Cloning, expression and characterization of L-arabinose isomerase from Thermotoga neapolitana: bioconversion of D-galactose to D-tagatose using the enzyme.

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Kim, Byoung-Chan; Lee, Yoon-Hee; Lee, Han-Seung; Lee, Dong-Woo; Choe, Eun-Ah; Pyun, Yu-Ryang

2002-06-18

Gene araA encoding an L-arabinose isomerase (AraA) from the hyperthermophile, Thermotoga neapolitana 5068 was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a polypeptide of 496 residues with a calculated molecular mass of 56677 Da. The deduced amino acid sequence has 94.8% identical amino acids compared with the residues in a putative L-arabinose isomerase of Thermotoga maritima. The recombinant enzyme expressed in E. coli was purified to homogeneity by heat treatment, ion exchange chromatography and gel filtration. The thermophilic enzyme had a maximum activity of L-arabinose isomerization and D-galactose isomerization at 85 degrees C, and required divalent cations such as Co(2+) and Mn(2+) for its activity and thermostability. The apparent K(m) values of the enzyme for L-arabinose and D-galactose were 116 mM (v(max), 119 micromol min(-1) mg(-1)) and 250 mM (v(max), 14.3 micromol min(-1) mg(-1)), respectively, that were determined in the presence of both 1 mM Co(2+) and 1 mM Mn(2+). A 68% conversion of D-galactose to D-tagatose was obtained using the recombinant enzyme at the isomerization temperature of 80 degrees C.

On the structure and function of the phytoene desaturase CRTI from Pantoea ananatis, a membrane-peripheral and FAD-dependent oxidase/isomerase.

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Patrick Schaub

Full Text Available CRTI-type phytoene desaturases prevailing in bacteria and fungi can form lycopene directly from phytoene while plants employ two distinct desaturases and two cis-tans isomerases for the same purpose. This property renders CRTI a valuable gene to engineer provitamin A-formation to help combat vitamin A malnutrition, such as with Golden Rice. To understand the biochemical processes involved, recombinant CRTI was produced and obtained in homogeneous form that shows high enzymatic activity with the lipophilic substrate phytoene contained in phosphatidyl-choline (PC liposome membranes. The first crystal structure of apo-CRTI reveals that CRTI belongs to the flavoprotein superfamily comprising protoporphyrinogen IX oxidoreductase and monoamine oxidase. CRTI is a membrane-peripheral oxidoreductase which utilizes FAD as the sole redox-active cofactor. Oxygen, replaceable by quinones in its absence, is needed as the terminal electron acceptor. FAD, besides its catalytic role also displays a structural function by enabling the formation of enzymatically active CRTI membrane associates. Under anaerobic conditions the enzyme can act as a carotene cis-trans isomerase. In silico-docking experiments yielded information on substrate binding sites, potential catalytic residues and is in favor of single half-site recognition of the symmetrical C(40 hydrocarbon substrate.

Retinal pigment epithelial atrophy following indocyanine green dye-assisted surgery for serous macular detachment

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Hussain Nazimul

2008-01-01

Full Text Available To report subretinal migration of indocyanine green dye (ICG and subsequent retinal pigment epithelial (RPE atrophy during macular surgery for serous macular detachment. A 65-year-old woman presented with residual epiretinal membrane and serous detachment of the macula following vitreoretinal surgery for epiretinal membrane. She underwent resurgery with ICG-assisted internal limiting membrane peeling and intraocular tamponade. Intraoperatively a large area of subretinal ICG was seen with subsequent RPE mottling and atrophy of the macula in the area involved during follow-up. This case demonstrates that subretinal migration of ICG is possible and can be toxic to RPE.

Fe65-PTB2 Dimerization Mimics Fe65-APP Interaction

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Lukas P. Feilen

2017-05-01

Full Text Available Physiological function and pathology of the Alzheimer’s disease causing amyloid precursor protein (APP are correlated with its cytosolic adaptor Fe65 encompassing a WW and two phosphotyrosine-binding domains (PTBs. The C-terminal Fe65-PTB2 binds a large portion of the APP intracellular domain (AICD including the GYENPTY internalization sequence fingerprint. AICD binding to Fe65-PTB2 opens an intra-molecular interaction causing a structural change and altering Fe65 activity. Here we show that in the absence of the AICD, Fe65-PTB2 forms a homodimer in solution and determine its crystal structure at 2.6 Å resolution. Dimerization involves the unwinding of a C-terminal α-helix that mimics binding of the AICD internalization sequence, thus shielding the hydrophobic binding pocket. Specific dimer formation is validated by nuclear magnetic resonance (NMR techniques and cell-based analyses reveal that Fe65-PTB2 together with the WW domain are necessary and sufficient for dimerization. Together, our data demonstrate that Fe65 dimerizes via its APP interaction site, suggesting that besides intra- also intermolecular interactions between Fe65 molecules contribute to homeostatic regulation of APP mediated signaling.

Fe65-PTB2 Dimerization Mimics Fe65-APP Interaction.

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Feilen, Lukas P; Haubrich, Kevin; Strecker, Paul; Probst, Sabine; Eggert, Simone; Stier, Gunter; Sinning, Irmgard; Konietzko, Uwe; Kins, Stefan; Simon, Bernd; Wild, Klemens

2017-01-01

Physiological function and pathology of the Alzheimer's disease causing amyloid precursor protein (APP) are correlated with its cytosolic adaptor Fe65 encompassing a WW and two phosphotyrosine-binding domains (PTBs). The C-terminal Fe65-PTB2 binds a large portion of the APP intracellular domain (AICD) including the GYENPTY internalization sequence fingerprint. AICD binding to Fe65-PTB2 opens an intra-molecular interaction causing a structural change and altering Fe65 activity. Here we show that in the absence of the AICD, Fe65-PTB2 forms a homodimer in solution and determine its crystal structure at 2.6 Å resolution. Dimerization involves the unwinding of a C-terminal α-helix that mimics binding of the AICD internalization sequence, thus shielding the hydrophobic binding pocket. Specific dimer formation is validated by nuclear magnetic resonance (NMR) techniques and cell-based analyses reveal that Fe65-PTB2 together with the WW domain are necessary and sufficient for dimerization. Together, our data demonstrate that Fe65 dimerizes via its APP interaction site, suggesting that besides intra- also intermolecular interactions between Fe65 molecules contribute to homeostatic regulation of APP mediated signaling.

Module structure of interphotoreceptor retinoid-binding protein (IRBP may provide bases for its complex role in the visual cycle – structure/function study of Xenopus IRBP

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Ghosh Debashis

2007-08-01

Full Text Available Abstract Background Interphotoreceptor retinoid-binding protein's (IRBP remarkable module structure may be critical to its role in mediating the transport of all-trans and 11-cis retinol, and 11-cis retinal between rods, cones, RPE and Müller cells during the visual cycle. We isolated cDNAs for Xenopus IRBP, and expressed and purified its individual modules, module combinations, and the full-length polypeptide. Binding of all-trans retinol, 11-cis retinal and 9-(9-anthroyloxy stearic acid were characterized by fluorescence spectroscopy monitoring ligand-fluorescence enhancement, quenching of endogenous protein fluorescence, and energy transfer. Finally, the X-ray crystal structure of module-2 was used to predict the location of the ligand-binding sites, and compare their structures among modules using homology modeling. Results The full-length Xenopus IRBP cDNA codes for a polypeptide of 1,197 amino acid residues beginning with a signal peptide followed by four homologous modules each ~300 amino acid residues in length. Modules 1 and 3 are more closely related to each other than either is to modules 2 and 4. Modules 1 and 4 are most similar to the N- and C-terminal modules of the two module IRBP of teleosts. Our data are consistent with the model that vertebrate IRBPs arose through two genetic duplication events, but that the middle two modules were lost during the evolution of the ray finned fish. The sequence of the expressed full-length IRBP was confirmed by liquid chromatography-tandem mass spectrometry. The recombinant full-length Xenopus IRBP bound all-trans retinol and 11-cis retinaldehyde at 3 to 4 sites with Kd's of 0.2 to 0.3 μM, and was active in protecting all-trans retinol from degradation. Module 2 showed selectivity for all-trans retinol over 11-cis retinaldehyde. The binding data are correlated to the results of docking of all-trans-retinol to the crystal structure of Xenopus module 2 suggesting two ligand-binding sites

Roles of Prolyl Isomerases in RNA-Mediated Gene Expression

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Roopa Thapar

2015-05-01

Full Text Available The peptidyl-prolyl cis-trans isomerases (PPIases that include immunophilins (cyclophilins and FKBPs and parvulins (Pin1, Par14, Par17 participate in cell signaling, transcription, pre-mRNA processing and mRNA decay. The human genome encodes 19 cyclophilins, 18 FKBPs and three parvulins. Immunophilins are receptors for the immunosuppressive drugs cyclosporin A, FK506, and rapamycin that are used in organ transplantation. Pin1 has also been targeted in the treatment of Alzheimer’s disease, asthma, and a number of cancers. While these PPIases are characterized as molecular chaperones, they also act in a nonchaperone manner to promote protein-protein interactions using surfaces outside their active sites. The immunosuppressive drugs act by a gain-of-function mechanism by promoting protein-protein interactions in vivo. Several immunophilins have been identified as components of the spliceosome and are essential for alternative splicing. Pin1 plays roles in transcription and RNA processing by catalyzing conformational changes in the RNA Pol II C-terminal domain. Pin1 also binds several RNA binding proteins such as AUF1, KSRP, HuR, and SLBP that regulate mRNA decay by remodeling mRNP complexes. The functions of ribonucleoprotein associated PPIases are largely unknown. This review highlights PPIases that play roles in RNA-mediated gene expression, providing insight into their structures, functions and mechanisms of action in mRNP remodeling in vivo.

Cloning of araA Gene Encoding L-Arabinose Isomerase from Marine Geobacillus stearothermophilus Isolated from Tanjung Api, Poso, Indonesia

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DEWI FITRIANI

2010-06-01

Full Text Available L-arabinose isomerase is an enzyme converting D-galactose to D-tagatose. D-tagatose is a potential sweetener-sucrose substitute which has low calorie. This research was to clone and sequence araA gene from marine bacterial strain Geobacillus stearothermophilus isolated from Tanjung Api Poso Indonesia. The amplified araA gene consisted of 1494 bp nucleotides encoding 497 amino acids. DNA alignment analysis showed that the gene had high homology with that of G. stearothermophilus T6. The enzyme had optimum activity at high temperature and alkalin condition.

Spatial organization of lipids in the human retina and optic nerve by MALDI imaging mass spectrometry.

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Zemski Berry, Karin A; Gordon, William C; Murphy, Robert C; Bazan, Nicolas G

2014-03-01

MALDI imaging mass spectrometry (IMS) was used to characterize lipid species within sections of human eyes. Common phospholipids that are abundant in most tissues were not highly localized and observed throughout the accessory tissue, optic nerve, and retina. Triacylglycerols were highly localized in accessory tissue, whereas sulfatide and plasmalogen glycerophosphoethanolamine (PE) lipids with a monounsaturated fatty acid were found enriched in the optic nerve. Additionally, several lipids were associated solely with the inner retina, photoreceptors, or retinal pigment epithelium (RPE); a plasmalogen PE lipid containing DHA (22:6), PE(P-18:0/22:6), was present exclusively in the inner retina, and DHA-containing glycerophosphatidylcholine (PC) and PE lipids were found solely in photoreceptors. PC lipids containing very long chain (VLC)-PUFAs were detected in photoreceptors despite their low abundance in the retina. Ceramide lipids and the bis-retinoid, N-retinylidene-N-retinylethanolamine, was tentatively identified and found only in the RPE. This MALDI IMS study readily revealed the location of many lipids that have been associated with degenerative retinal diseases. Complex lipid localization within retinal tissue provides a global view of lipid organization and initial evidence for specific functions in localized regions, offering opportunities to assess their significance in retinal diseases, such as macular degeneration, where lipids have been implicated in the disease process.

Enhanced activity and stability of L-arabinose isomerase by immobilization on aminopropyl glass.

Science.gov (United States)

Zhang, Ye-Wang; Jeya, Marimuthu; Lee, Jung-Kul

2011-03-01

Immobilization of Bacillus licheniformis L: -arabinose isomerase (BLAI) on aminopropyl glass modified with glutaraldehyde (4 mg protein g support⁻¹) was found to enhance the enzyme activity. The immobilization yield of BLAI was proportional to the quantity of amino groups on the surface of support. Reducing particle size increased the adsorption capacity (q(m)) and affinity (k(a)). The pH and temperature for immobilization were optimized to be pH 7.1 and 33 °C using response surface methodology (RSM). The immobilized enzyme was characterized and compared to the free enzyme. There is no change in optimal pH and temperature before and after immobilization. However, the immobilized BLAI enzyme achieved 145% of the activity of the free enzyme. Correspondingly, the catalytic efficiency (k(cat)/K(m)) was improved 1.47-fold after immobilization compared to the free enzyme. The thermal stability was improved 138-fold (t₁/₂) increased from 2 to 275 h) at 50 °C following immobilization.

The crystal structure of a multifunctional protein: phosphoglucose isomerase/autocrine motility factor/neuroleukin.

Science.gov (United States)

Sun, Y J; Chou, C C; Chen, W S; Wu, R T; Meng, M; Hsiao, C D

1999-05-11

Phosphoglucose isomerase (PGI) plays a central role in both the glycolysis and the gluconeogenesis pathways. We present here the complete crystal structure of PGI from Bacillus stearothermophilus at 2.3-A resolution. We show that PGI has cell-motility-stimulating activity on mouse colon cancer cells similar to that of endogenous autocrine motility factor (AMF). PGI can also enhance neurite outgrowth on neuronal progenitor cells similar to that observed for neuroleukin. The results confirm that PGI is neuroleukin and AMF. PGI has an open twisted alpha/beta structural motif consisting of two globular domains and two protruding parts. Based on this substrate-free structure, together with the previously published biological, biochemical, and modeling results, we postulate a possible substrate-binding site that is located within the domains' interface for PGI and AMF. In addition, the structure provides evidence suggesting that the top part of the large domain together with one of the protruding loops might participate in inducing the neurotrophic activity.

Identification of interacting proteins of retinoid-related orphan nuclear receptor gamma in HepG2 cells

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Ze-Min Huang1,#, Jun Wu2,#, Zheng-Cai Jia1, Yi Tian1, Jun Tang3, Yan Tang1, Ying Wang2, Yu-Zhang Wu1,* & Bing Ni1,*

2012-06-01

Full Text Available The retinoid-related orphan nuclear receptor gamma (RORγplays critical roles in regulation of development, immunity andmetabolism. As transcription factor usually forms a proteincomplex to function, thus capturing and dissecting of theRORγ protein complex will be helpful for exploring themechanisms underlying those functions. After construction ofthe recombinant tandem affinity purification (TAP plasmid,pMSCVpuro RORγ-CTAP(SG, the nuclear localization ofRORγ-CTAP(SG fusion protein was verified. Followingisolation of RORγ protein complex by TAP strategy, sevencandidate interacting proteins were identified. Finally, the heatshock protein 90 (HSP90 and receptor-interacting protein 140(RIP140 were confirmed to interplay with RORγ byco-immunoprecipitation. Interference of HSP90 or/and RIP140genes resulted in dramatically decreased expression ofCYP2C8 gene, the RORγ target gene. Data from this studydemonstrate that HSP90 and RIP140 proteins interact withRORγ protein in a complex format and function asco-activators in the RORγ-mediated regulatory processes ofHepG2 cells.

L-Arabinose isomerase and its use for biotechnological production of rare sugars.

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Xu, Zheng; Li, Sha; Feng, Xiaohai; Liang, Jinfeng; Xu, Hong

2014-11-01

L-Arabinose isomerase (AI), a key enzyme in the microbial pentose phosphate pathway, has been regarded as an important biological catalyst in rare sugar production. This enzyme could isomerize L-arabinose into L-ribulose, as well as D-galactose into D-tagatose. Both the two monosaccharides show excellent commercial values in food and pharmaceutical industries. With the identification of novel AI family members, some of them have exhibited remarkable potential in industrial applications. The biological production processes for D-tagatose and L-ribose (or L-ribulose) using AI have been developed and improved in recent years. Meanwhile, protein engineering techniques involving rational design has effectively enhanced the catalytic properties of various AIs. Moreover, the crystal structure of AI has been disclosed, which sheds light on the understanding of AI structure and catalytic mechanism at molecular levels. This article reports recent developments in (i) novel AI screening, (ii) AI-mediated rare sugar production processes, (iii) molecular modification of AI, and (iv) structural biology study of AI. Based on previous reports, an analysis of the future development has also been initiated.

A Case of AML Characterized by a Novel t(4;15(q31;q22 Translocation That Confers a Growth-Stimulatory Response to Retinoid-Based Therapy

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Justin M. Watts

2017-07-01

Full Text Available Here we report the case of a 30-year-old woman with relapsed acute myeloid leukemia (AML who was treated with all-trans retinoic acid (ATRA as part of investigational therapy (NCT02273102. The patient died from rapid disease progression following eight days of continuous treatment with ATRA. Karyotype analysis and RNA-Seq revealed the presence of a novel t(4;15(q31;q22 reciprocal translocation involving the TMEM154 and RASGRF1 genes. Analysis of primary cells from the patient revealed the expression of TMEM154-RASGRF1 mRNA and the resulting fusion protein, but no expression of the reciprocal RASGRF1-TMEM154 fusion. Consistent with the response of the patient to ATRA therapy, we observed a rapid proliferation of t(4;15 primary cells following ATRA treatment ex vivo. Preliminary characterization of the retinoid response of t(4;15 AML revealed that in stark contrast to non-t(4;15 AML, these cells proliferate in response to specific agonists of RARα and RARγ. Furthermore, we observed an increase in the levels of nuclear RARγ upon ATRA treatment. In summary, the identification of the novel t(4;15(q31;q22 reciprocal translocation opens new avenues in the study of retinoid resistance and provides potential for a new biomarker for therapy of AML.

Periodización inversa y control de la carga mediante sRPE en Triatlon Ironman 70.3: estudio de caso

OpenAIRE

Rodríguez Reche, Sergio

2016-01-01

El propósito de este estudio de caso era evaluar y analizar la eficacia de la periodización inversa en triatlón, con una distribución de las intensidades de manera polarizada y llevando a cabo un control de la carga mediante escala de percepción de fatiga (sRPE). Para ello, se llevó a cabo un periodo de entrenamiento con el objetivo de preparar a un triatleta para el Ironman 70.3 de Pucón (Chile). Durante 63 días, el deportista realizó 124 sesiones (43 de natación, 28 de ciclismo, 36 de carre...

Whole cell immobilization of refractory glucose isomerase using tris(hydroxymethyl)phosphine as crosslinker for preparation of high fructose corn syrup at elevated temperature.

Science.gov (United States)

Jia, Dong-Xu; Wang, Teng; Liu, Zi-Jian; Jin, Li-Qun; Li, Jia-Jia; Liao, Cheng-Jun; Chen, De-Shui; Zheng, Yu-Guo

2018-04-04

Glucose isomerase (GI) responsible for catalyzing the isomerization from d-glucose to d-fructose, was an important enzyme for producing high fructose corn syrup (HFCS). In a quest to prepare HFCS at elevated temperature and facilitate enzymatic recovery, an effective procedure for whole cell immobilization of refractory Thermus oshimai glucose isomerase (ToGI) onto Celite 545 using tris(hydroxymethyl)phosphine (THP) as crosslinker was established. The immobilized biocatalyst showed an activity of approximate 127.3 U/(g·immobilized product) via optimization in terms of cells loading, crosslinker concentration and crosslinking time. The pH optimum of the immobilized biocatalyst was displaced from pH 8.0 of native enzyme to neutral pH 7.0. Compared with conventional glutaraldehyde (GLU)-immobilized cells, it possessed the enhanced thermostability with 70.1% residual activity retaining after incubation at 90°C for 72 h. Moreover, the THP-immobilized biocatalyst exhibited superior operational stability, in which it retained 85.8% of initial activity after 15 batches of bioconversion at 85°C. This study paved a way for reducing catalysis cost for upscale preparation of HFCS with higher d-fructose concentration. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

An efficient xylose-fermenting recombinant Saccharomyces cerevisiae strain obtained through adaptive evolution and its global transcription profile

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Shen, Yu; Chen, Xiao; Peng, Bingyin; Chen, Liyuan; Hou, Jin; Bao, Xiaoming [Shandong Univ., Jinan (China). State Key Lab. of Microbial Technology

2012-11-15

Factors related to ethanol production from xylose in engineered Saccharomyces cerevisiae that contain an exogenous initial metabolic pathway are still to be elucidated. In the present study, a strain that expresses the xylose isomerase gene of Piromyces sp. Pi-xylA and overexpresses XKS1, RPE1, RKI1, TAL1, and TKL1, with deleted GRE3 and COX4 genes was constructed. The xylose utilization capacity of the respiratory deficiency strain was poor but improved via adaptive evolution in xylose. The {mu}{sub max} of the evolved strain in 20 gl{sup -1} xylose is 0.11 {+-} 0.00 h{sup -1}, and the evolved strain consumed 17.83 gl{sup -1} xylose within 72 h, with an ethanol yield of 0.43 gg{sup -1} total consumed sugars during glucose-xylose cofermentation. Global transcriptional changes and effect of several specific genes were studied. The result revealed that the increased xylose isomerase activity, the upregulation of enzymes involved in glycolysis and glutamate synthesis, and the downregulation of trehalose and glycogen synthesis, may have contributed to the improved xylose utilization of the strain. Furthermore, the deletion of PHO13 decreased the xylose growth in the respiration deficiency strain although deleting PHO13 can improve the xylose metabolism in other strains. (orig.)

Retinoids repress Ah receptor CYP1A1 induction pathway through the SMRT corepressor

International Nuclear Information System (INIS)

Fallone, Frederique; Villard, Pierre-Henri; Seree, Eric; Rimet, Odile; Nguyen, Quock Binh; Bourgarel-Rey, Veronique; Fouchier, Francis; Barra, Yves; Durand, Alain; Lacarelle, Bruno

2004-01-01

CYP1A1 isoform is mainly regulated by the transcription factor AhR and to a lesser extent by the nuclear receptor RAR. The effect of a coexposure with 3MC, a AhR ligand, and RA, a RAR ligand, which are, respectively, strong and weak CYP1A1 inducers, is poorly known. We showed in Caco-2 cells that addition of RA significantly decreased 3MC-induced CYP1A1 expression by -55% for mRNA level and -30% for promoter and enzymatic activities. We further showed that RA decreased AhR protein level. Moreover, a physical interaction between AhR and the RAR-corepressor SMRT has been described in vitro. Using the corepressor inhibitor TSA, transfected-cells with SMRT cDNA, and coimmunoprecipitation experiments, we demonstrated that RA addition repressed AhR function through a marked AhR/SMRT physical interaction. This interaction explains the decrease of 3MC-induced CYP1A1 expression. This new mechanism involving the repression of AhR-induced CYP1A1 expression by retinoids allows better knowledge of the CYP1A1 regulation

Genome-Wide Profiling of Liver X Receptor, Retinoid X Receptor, and Peroxisome Proliferator-Activated Receptor α in Mouse Liver Reveals Extensive Sharing of Binding Sites

DEFF Research Database (Denmark)

Boergesen, Michael; Pedersen, Thomas Åskov; Gross, Barbara

2012-01-01

and correlate with an LXR-dependent hepatic induction of lipogenic genes. To further investigate the roles of RXR and LXR in the regulation of hepatic gene expression, we have mapped the ligand-regulated genome-wide binding of these factors in mouse liver. We find that the RXR agonist bexarotene primarily......The liver X receptors (LXRs) are nuclear receptors that form permissive heterodimers with retinoid X receptor (RXR) and are important regulators of lipid metabolism in the liver. We have recently shown that RXR agonist-induced hypertriglyceridemia and hepatic steatosis in mice are dependent on LXRs...

Functional Role of the Disulfide Isomerase ERp57 in Axonal Regeneration.

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Valentina Castillo

Full Text Available ERp57 (also known as grp58 and PDIA3 is a protein disulfide isomerase that catalyzes disulfide bonds formation of glycoproteins as part of the calnexin and calreticulin cycle. ERp57 is markedly upregulated in most common neurodegenerative diseases downstream of the endoplasmic reticulum (ER stress response. Despite accumulating correlative evidence supporting a neuroprotective role of ERp57, the contribution of this foldase to the physiology of the nervous system remains unknown. Here we developed a transgenic mouse model that overexpresses ERp57 in the nervous system under the control of the prion promoter. We analyzed the susceptibility of ERp57 transgenic mice to undergo neurodegeneration. Unexpectedly, ERp57 overexpression did not affect dopaminergic neuron loss and striatal denervation after injection of a Parkinson's disease-inducing neurotoxin. In sharp contrast, ERp57 transgenic animals presented enhanced locomotor recovery after mechanical injury to the sciatic nerve. These protective effects were associated with enhanced myelin removal, macrophage infiltration and axonal regeneration. Our results suggest that ERp57 specifically contributes to peripheral nerve regeneration, whereas its activity is dispensable for the survival of a specific neuronal population of the central nervous system. These results demonstrate for the first time a functional role of a component of the ER proteostasis network in peripheral nerve regeneration.

A Role of a Newly Identified Isomerase From Yarrowia lipolytica in Erythritol Catabolism

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Aleksandra M. Mirończuk

2018-05-01

Full Text Available Erythritol is a natural sweetener produced by microorganisms as an osmoprotectant. It belongs to the group of polyols and it can be utilized by the oleaginous yeast Yarrowia lipolytica. Despite the recent identification of the transcription factor of erythritol utilization (EUF1, the metabolic pathway of erythritol catabolism remains unknown. In this study we identified a new gene, YALI0F01628g, involved in erythritol assimilation. In silico analysis showed that YALI0F01628g is a putative isomerase and it is localized in the same region as EUF1. qRT-PCR analysis of Y. lipolytica showed a significant increase in YALI0F01628g expression during growth on erythritol and after overexpression of EUF1. Moreover, the deletion strain ΔF01628 showed significantly impaired erythritol assimilation, whereas synthesis of erythritol remained unchanged. The results showed that YALI0F1628g is involved in erythritol assimilation; thus we named the gene EYI1. Moreover, we suggest the metabolic pathway of erythritol assimilation in yeast Y. lipolytica.

Ser46 phosphorylation and prolyl-isomerase Pin1-mediated isomerization of p53 are key events in p53-dependent apoptosis induced by mutant huntingtin.

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Grison, Alice; Mantovani, Fiamma; Comel, Anna; Agostoni, Elena; Gustincich, Stefano; Persichetti, Francesca; Del Sal, Giannino

2011-11-01

Huntington disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the gene coding for huntingtin protein. Several mechanisms have been proposed by which mutant huntingtin (mHtt) may trigger striatal neurodegeneration, including mitochondrial dysfunction, oxidative stress, and apoptosis. Furthermore, mHtt induces DNA damage and activates a stress response. In this context, p53 plays a crucial role in mediating mHtt toxic effects. Here we have dissected the pathway of p53 activation by mHtt in human neuronal cells and in HD mice, with the aim of highlighting critical nodes that may be pharmacologically manipulated for therapeutic intervention. We demonstrate that expression of mHtt causes increased phosphorylation of p53 on Ser46, leading to its interaction with phosphorylation-dependent prolyl isomerase Pin1 and consequent dissociation from the apoptosis inhibitor iASPP, thereby inducing the expression of apoptotic target genes. Inhibition of Ser46 phosphorylation by targeting homeodomain-interacting protein kinase 2 (HIPK2), PKCδ, or ataxia telangiectasia mutated kinase, as well as inhibition of the prolyl isomerase Pin1, prevents mHtt-dependent apoptosis of neuronal cells. These results provide a rationale for the use of small-molecule inhibitors of stress-responsive protein kinases and Pin1 as a potential therapeutic strategy for HD treatment.

Investor Outlook: Significance of the Positive LCA2 Gene Therapy Phase III Results.

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Schimmer, Joshua; Breazzano, Steven

2015-12-01

Spark Therapeutics recently reported positive phase III results for SPK-RPE65 targeting the treatment of visual impairment caused by RPE65 gene mutations (often referred to as Leber congenital amaurosis type 2, or LCA2, but may include other retinal disorders), marking an important inflection point for the field of gene therapy. The results highlight the ability to successfully design and execute a randomized trial of a gene therapy and also reinforce the potentially predictive nature of early preclinical and clinical data. The results are expected to pave the way for the first approved gene therapy product in the United States and should sustain investor interest and confidence in gene therapy for many approaches, including retina targeting and beyond.

Novel Epigenetic Controlling of Hypoxia Pathway Related to Overexpression and Promoter Hypomethylation of TET1 and TET2 in RPE Cells.

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Alivand, Mohammad Reza; Soheili, Zahra-Soheila; Pornour, Majid; Solali, Saeed; Sabouni, Farzaneh

2017-10-01

CpG methylation of DNA takes part in a specific epigenetic memory that plays crucial roles in the differentiation and abnormality of the cells. The methylation pattern aberration of genomes is affected in three ways, namely DNA methyltransferase (DNMT), ten-eleven translocation (TET), and methyl-binding domain (MBD) proteins. Of these, TET enzymes have recently been demonstrated to be master modifier enzymes in the DNA methylation process. Additionally, recent studies emphasize that not only epigenetic phenomena play a role in controlling hypoxia pathway, but the hypoxia condition also triggers hypomethylation of genomes that may help with the expression of hypoxia pathway genes. In this study, we suggested that TET1 and TET2 could play a role in the demethylation of genomes under chemical hypoxia conditions. Herein, the evaluating methylation status and mRNA expression of mentioned genes were utilized through real-time PCR and methylation-specific PCR (MSP), respectively. Our results showed that TET1 and TET2 genes were overexpressed (P < 0.05) under chemical hypoxia conditions in Retinal Pigment Epithelial (RPE) cells, whereas the promoter methylation status of them were hypomethylated in the same condition. Therefore, chemical hypoxia not only causes overexpression of TET1 and TET2 but also could gradually do promoter demethylation of same genes. This is the first study to show the relationship between epigenetics and the expression of mentioned genes related to hypoxia pathways. Furthermore, it seems that these associations in RPE cells are subjected to chemical hypoxia as a mechanism that could play a crucial role in methylation pattern changes of hypoxia-related diseases such as cancer and ischemia. J. Cell. Biochem. 118: 3193-3204, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Synthesis and modifications of heterocyclic derivatives of D-arabinose: potential inhibitors of glucose-6-phosphate isomerase and glucosamine-6-phosphate synthase

International Nuclear Information System (INIS)

Viana, Renato Marcio Ribeiro; Prado, Maria Auxiliadora Fontes; Alves, Ricardo Jose

2008-01-01

The synthesis of -5-(D-arabino-1,2,3,4-tetrahydroxybutyl)tetrazole and -2-(d-arabino-1,2,3,4-tetra-acetoxybutyl)-5-methyl-1,3,4-oxadiazole from d-arabinose is described. Attempts at removing the protecting groups of the oxadiazole derivative were unsuccessful, leading to products resulting from the opening of the oxadiazole ring. The unprotected tetrazole derivative was selectively phosphorylated at the primary hydroxyl group with diethyl phosphoryl chloride. The resulting 5-[d-arabino-4-(diethylphosphoryloxy)-1,2,3-trihydroxybutyl]tetrazole is a protected form of a potential inhibitor of the enzymes glucose-6-phosphate isomerase and glucosamine synthase. (author)

PDILT, a divergent testis-specific protein disulfide isomerase with a non-classical SXXC motif that engages in disulfide-dependent interactions in the endoplasmic reticulum.

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van Lith, Marcel; Hartigan, Nichola; Hatch, Jennifer; Benham, Adam M

2005-01-14

Protein disulfide isomerase (PDI) is the archetypal enzyme involved in the formation and reshuffling of disulfide bonds in the endoplasmic reticulum (ER). PDI achieves its redox function through two highly conserved thioredoxin domains, and PDI can also operate as an ER chaperone. The substrate specificities and the exact functions of most other PDI family proteins remain important unsolved questions in biology. Here, we characterize a new and striking member of the PDI family, which we have named protein disulfide isomerase-like protein of the testis (PDILT). PDILT is the first eukaryotic SXXC protein to be characterized in the ER. Our experiments have unveiled a novel, glycosylated PDI-like protein whose tissue-specific expression and unusual motifs have implications for the evolution, catalytic function, and substrate selection of thioredoxin family proteins. We show that PDILT is an ER resident glycoprotein that liaises with partner proteins in disulfide-dependent complexes within the testis. PDILT interacts with the oxidoreductase Ero1alpha, demonstrating that the N-terminal cysteine of the CXXC sequence is not required for binding of PDI family proteins to ER oxidoreductases. The expression of PDILT, in addition to PDI in the testis, suggests that PDILT performs a specialized chaperone function in testicular cells. PDILT is an unusual PDI relative that highlights the adaptability of chaperone and redox function in enzymes of the endoplasmic reticulum.

Space, self, and the theater of consciousness.

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Trehub, Arnold

2007-06-01

Over a decade ago, I introduced a large-scale theory of the cognitive brain which explained for the first time how the human brain is able to create internal models of its intimate world and invent models of a wider universe. An essential part of the theoretical model is an organization of neuronal mechanisms which I have named the Retinoid Model [Trehub, A. (1977). Neuronal models for cognitive processes: Networks for learning, perception and imagination. Journal of Theoretical Biology, 65, 141-169; Trehub, A. (1991). The Cognitive Brain: MIT Press]. This hypothesized brain system has structural and dynamic properties enabling it to register and appropriately integrate disparate foveal stimuli into a perspectival, egocentric representation of an extended 3D world scene including a neuronally tokened locus of the self which, in this theory, is the neuronal origin of retinoid space. As an integral part of the larger neuro-cognitive model, the retinoid system is able to perform many other useful perceptual and higher cognitive functions. In this paper, I draw on the hypothesized properties of this system to argue that neuronal activity within the retinoid structure constitutes the phenomenal content of consciousness and the unique sense of self that each of us experiences.

In silico cloning and B/T cell epitope prediction of triosephosphate isomerase from Echinococcus granulosus.

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Wang, Fen; Ye, Bin

2016-10-01

Cystic echinococcosis is a worldwide zoonosis caused by Echinococcus granulosus. Because the methods of diagnosis and treatment for cystic echinococcosis were limited, it is still necessary to screen target proteins for the development of new anti-hydatidosis vaccine. In this study, the triosephosphate isomerase gene of E. granulosus was in silico cloned. The B cell and T cell epitopes were predicted by bioinformatics methods. The cDNA sequence of EgTIM was composition of 1094 base pairs, with an open reading frame of 753 base pairs. The deduced amino acid sequences were composed of 250 amino acids. Five cross-reactive epitopes, locating on 21aa-35aa, 43aa-57aa, 94aa-107aa, 115-129aa, and 164aa-183aa, could be expected to serve as candidate epitopes in the development of vaccine against E. granulosus. These results could provide bases for gene cloning, recombinant expression, and the designation of anti-hydatidosis vaccine.

Circumpolar assessment of rhizosphere priming shows limited increase in carbon loss estimates for permafrost soils but large regional variability

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Wild, B.; Keuper, F.; Kummu, M.; Beer, C.; Blume-Werry, G.; Fontaine, S.; Gavazov, K.; Gentsch, N.; Guggenberger, G.; Hugelius, G.; Jalava, M.; Koven, C.; Krab, E. J.; Kuhry, P.; Monteux, S.; Richter, A.; Shazhad, T.; Dorrepaal, E.

2017-12-01

Predictions of soil organic carbon (SOC) losses in the northern circumpolar permafrost area converge around 15% (± 3% standard error) of the initial C pool by 2100 under the RCP 8.5 warming scenario. Yet, none of these estimates consider plant-soil interactions such as the rhizosphere priming effect (RPE). While laboratory experiments have shown that the input of plant-derived compounds can stimulate SOC losses by up to 1200%, the magnitude of RPE in natural ecosystems is unknown and no methods for upscaling exist so far. We here present the first spatial and depth explicit RPE model that allows estimates of RPE on a large scale (PrimeSCale). We combine available spatial data (SOC, C/N, GPP, ALT and ecosystem type) and new ecological insights to assess the importance of the RPE at the circumpolar scale. We use a positive saturating relationship between the RPE and belowground C allocation and two ALT-dependent rooting-depth distribution functions (for tundra and boreal forest) to proportionally assign belowground C allocation and RPE to individual soil depth increments. The model permits to take into account reasonable limiting factors on additional SOC losses by RPE including interactions between spatial and/or depth variation in GPP, plant root density, SOC stocks and ALT. We estimate potential RPE-induced SOC losses at 9.7 Pg C (5 - 95% CI: 1.5 - 23.2 Pg C) by 2100 (RCP 8.5). This corresponds to an increase of the current permafrost SOC-loss estimate from 15% of the initial C pool to about 16%. If we apply an additional molar C/N threshold of 20 to account for microbial C limitation as a requirement for the RPE, SOC losses by RPE are further reduced to 6.5 Pg C (5 - 95% CI: 1.0 - 16.8 Pg C) by 2100 (RCP 8.5). Although our results show that current estimates of permafrost soil C losses are robust without taking into account the RPE, our model also highlights high-RPE risk in Siberian lowland areas and Alaska north of the Brooks Range. The small overall impact of

Natural and synthetic retinoids afford therapeutic effects on intracerebral hemorrhage in mice.

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Matsushita, Hideaki; Hijioka, Masanori; Hisatsune, Akinori; Isohama, Yoichiro; Shudo, Koichi; Katsuki, Hiroshi

2012-05-15

We have recently proposed that retinoic acid receptor (NR1B) is a promising target of neuroprotective therapy for intracerebral hemorrhage, since pretreatment of mice with an NR1B1/NR1B2 agonist Am80 attenuated various pathological and neurological abnormalities associated with the disease. In the present study we further addressed the effects of retinoids as potential therapeutic drugs, using a collagenase-induced model of intracerebral hemorrhage. Daily oral administration of all-trans retinoic acid (ATRA; 5 and 15 mg/kg), a naturally occurring NR1B agonist, from 1 day before collagenase injection significantly inhibited loss of neurons within the hematoma. ATRA in the same treatment regimen also decreased the number of activated microglia/macrophages around the hematoma but did not affect the hematoma volume. ATRA (15 mg/kg) as well as Am80 (5mg/kg) rescued neurons in the central region of hematoma, even when drug administration was started from 6h after induction of intracerebral hemorrhage. However, in this post-treatment regimen, only Am80 significantly decreased the number of activated microglia/macrophages. With regard to neurological deficits, both ATRA (15 mg/kg) and Am80 (5mg/kg) given in the post-treatment regimen improved performance of mice in the beam-walking test and the modified limb-placing test. ATRA and Am80 also significantly attenuated damage of axon tracts as revealed by amyloid precursor protein immunohistochemistry. These results underscore potential therapeutic values of NR1B agonists for intracerebral hemorrhage. Copyright © 2012 Elsevier B.V. All rights reserved.

Engineering the l-Arabinose Isomerase from Enterococcus Faecium for d-Tagatose Synthesis.

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de Sousa, Marylane; Manzo, Ricardo M; García, José L; Mammarella, Enrique J; Gonçalves, Luciana R B; Pessela, Benevides C

2017-12-06

l-Arabinose isomerase (EC 5.3.1.4) (l-AI) from Enterococcus faecium DBFIQ E36 was overproduced in Escherichia coli by designing a codon-optimized synthetic araA gene. Using this optimized gene, two N- and C-terminal His-tagged-l-AI proteins were produced. The cloning of the two chimeric genes into regulated expression vectors resulted in the production of high amounts of recombinant N -His-l-AI and C -His-l-AI in soluble and active forms. Both His-tagged enzymes were purified in a single step through metal-affinity chromatography and showed different kinetic and structural characteristics. Analytical ultracentrifugation revealed that C -His-l-AI was preferentially hexameric in solution, whereas N -His-l-AI was mainly monomeric. The specific activity of the N -His-l-AI at acidic pH was higher than that of C -His-l-AI and showed a maximum bioconversion yield of 26% at 50 °C for d-tagatose biosynthesis, with Km and Vmax parameters of 252 mM and 0.092 U mg -1 , respectively. However, C -His-l-AI was more active and stable at alkaline pH than N -His-l-AI. N -His-l-AI follows a Michaelis-Menten kinetic, whereas C -His-l-AI fitted to a sigmoidal saturation curve.

47 CFR 65.105 - Discovery.

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2010-10-01

... 47 Telecommunication 3 2010-10-01 2010-10-01 false Discovery. 65.105 Section 65.105... OF RETURN PRESCRIPTION PROCEDURES AND METHODOLOGIES Procedures § 65.105 Discovery. (a) Participants... evidence. (c) Discovery requests pursuant to § 65.105(b), including written interrogatories, shall be filed...

Bioconversion of D-galactose to D-tagatose: continuous packed bed reaction with an immobilized thermostable L-arabinose isomerase and efficient purification by selective microbial degradation.

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Liang, Min; Chen, Min; Liu, Xinying; Zhai, Yafei; Liu, Xian-wei; Zhang, Houcheng; Xiao, Min; Wang, Peng

2012-02-01

The continuous enzymatic conversion of D-galactose to D-tagatose with an immobilized thermostable L-arabinose isomerase in packed-bed reactor and a novel method for D-tagatose purification were studied. L-arabinose isomerase from Thermoanaerobacter mathranii (TMAI) was recombinantly overexpressed and immobilized in calcium alginate. The effects of pH and temperature on D-tagatose production reaction catalyzed by free and immobilized TMAI were investigated. The optimal condition for free enzyme was pH 8.0, 60°C, 5 mM MnCl(2). However, that for immobilized enzyme was pH 7.5, 75°C, 5 mM MnCl(2). In addition, the catalytic activity of immobilized enzyme at high temperature and low pH was significantly improved compared with free enzyme. The optimum reaction yield with immobilized TMAI increased by four percentage points to 43.9% compared with that of free TMAI. The highest productivity of 10 g/L h was achieved with the yield of 23.3%. Continuous production was performed at 70°C; after 168 h, the reaction yield was still above 30%. The resultant syrup was then incubated with Saccharomyces cerevisiae L1 cells. The selective degradation of D-galactose was achieved, obtaining D-tagatose with the purity above 95%. The established production and separation methods further potentiate the industrial production of D-tagatose via bioconversion and biopurification processes.

Retinoid X receptor gene expression and protein content in tissues of the rock shell Thais clavigera

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Horiguchi, Toshihiro [Research Center for Environmental Risk, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506 (Japan)], E-mail: thorigu@nies.go.jp; Nishikawa, Tomohiro [Research Center for Environmental Risk, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506 (Japan); Ohta, Yasuhiko [Department of Veterinary Science, Faculty of Agriculture, Tottori University, 4-101 Koyamacho-Minami, Tottori 680-8553 (Japan); Shiraishi, Hiroaki; Morita, Masatoshi [Research Center for Environmental Risk, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506 (Japan)

2007-10-15

To elucidate the role of retinoid X receptor (RXR) in the development of imposex caused by organotin compounds in gastropod molluscs, we investigated RXR gene expression and RXR protein content in various tissues of male and female wild rock shells (Thais clavigera). Quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry with a commercial antibody against human RXR {alpha} revealed that RXR gene expression was significantly higher in the penises of males and imposex-exhibiting females than in the penis-forming areas of normal females (P < 0.01 and P < 0.05, respectively). Western blotting demonstrated that the antibody could detect rock shell RXR and showed that the male penis had the highest content of RXR protein among the analyzed tissues of males and normal females. Immunohistochemical staining revealed nuclear localization of RXR protein in the epithelial and smooth muscle cells of the vas deferens and in the interstitial or connective tissues and epidermis of the penis in males and imposex-exhibiting females. RXR could be involved in the mechanism of induction of male-type genitalia (penis and vas deferens) by organotin compounds in female rock shells.

Vitamin D Receptor, Retinoid X Receptor, Ki-67, Survivin, and Ezrin Expression in Canine Osteosarcoma

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John Davies

2012-01-01

Full Text Available Canine osteosarcoma (OS is an aggressive malignant bone tumor. Prognosis is primarily determined by clinical parameters. Vitamin D has been postulated as a novel therapeutic option for many malignancies. Upon activation, vitamin D receptors (VDRs combine with retinoid receptor (RXR forming a heterodimer initiating a cascade of events. Vitamin D's antineoplastic activity and its mechanism of action in OS remain to be clearly established. Expression of VDR, RXR, Ki-67, survivin, and ezrin was studied in 33 archived, canine OS specimens. VDR, RXR, survivin, and ezrin were expressed in the majority of cases. There was no statistically significant difference in VDR expression in relationship with tumor grade, type, or locations or animal breed, age, and/or sex. No significant association (p=0.316 between tumor grade and Ki-67 expression was found; in particular, no difference in Ki-67 expression between grades 2 and 3 OSs was found, while a negative correlation was noted between Ki-67 and VDR expression (ρ=−0.466, a positive correlation between survivin and RXR expression was found (p=0.374. A significant relationship exists between VDR and RXR expression in OSs and proliferative/apoptosis markers. These results establish a foundation for elucidating mechanisms by which vitamin D induces antineoplastic activity in OS.

Functional and structural studies of the disulfide isomerase DsbC from the plant pathogen Xylella fastidiosa reveals a redox-dependent oligomeric modulation in vitro.

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Santos, Clelton A; Toledo, Marcelo A S; Trivella, Daniela B B; Beloti, Lilian L; Schneider, Dilaine R S; Saraiva, Antonio M; Crucello, Aline; Azzoni, Adriano R; Souza, Alessandra A; Aparicio, Ricardo; Souza, Anete P

2012-10-01

Xylella fastidiosa is a Gram-negative bacterium that grows as a biofilm inside the xylem vessels of susceptible plants and causes several economically relevant crop diseases. In the present study, we report the functional and low-resolution structural characterization of the X. fastidiosa disulfide isomerase DsbC (XfDsbC). DsbC is part of the disulfide bond reduction/isomerization pathway in the bacterial periplasm and plays an important role in oxidative protein folding. In the present study, we demonstrate the presence of XfDsbC during different stages of X. fastidiosa biofilm development. XfDsbC was not detected during X. fastidiosa planktonic growth; however, after administering a sublethal copper shock, we observed an overexpression of XfDsbC that also occurred during planktonic growth. These results suggest that X. fastidiosa can use XfDsbC in vivo under oxidative stress conditions similar to those induced by copper. In addition, using dynamic light scattering and small-angle X-ray scattering, we observed that the oligomeric state of XfDsbC in vitro may be dependent on the redox environment. Under reducing conditions, XfDsbC is present as a dimer, whereas a putative tetrameric form was observed under nonreducing conditions. Taken together, our findings demonstrate the overexpression of XfDsbC during biofilm formation and provide the first structural model of a bacterial disulfide isomerase in solution. © 2012 The Authors Journal compilation © 2012 FEBS.

Optimization of Storage Temperature for Cultured ARPE-19 Cells

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Lara Pasovic

2013-01-01

Full Text Available Purpose. The establishment of future retinal pigment epithelium (RPE replacement therapy is partly dependent on the availability of tissue-engineered RPE cells, which may be enhanced by the development of suitable storage methods for RPE. This study investigates the effect of different storage temperatures on the viability, morphology, and phenotype of cultured RPE. Methods. ARPE-19 cells were cultured under standard conditions and stored in HEPES-buffered MEM at nine temperatures (4°C, 8°C, 12°C, 16°C, 20°C, 24°C, 28°C, 32°C, and 37°C for seven days. Viability and phenotype were assessed by a microplate fluorometer and epifluorescence microscopy, while morphology was analyzed by scanning electron microscopy. Results. The percentage of viable cells preserved after storage was highest in the 16°C group (48.7%±9.8%; P<0.01 compared to 4°C, 8°C, and 24°C–37°C; P<0.05 compared to 12°C. Ultrastructure was best preserved at 12°C, 16°C, and 20°C. Expression of actin, ZO-1, PCNA, caspase-3, and RPE65 was maintained after storage at 16°C compared to control cells that were not stored. Conclusion. Out of nine temperatures tested between 4°C and 37°C, storage at 12°C, 16°C, and 20°C was optimal for maintenance of RPE cell viability, morphology, and phenotype. The preservation of RPE cells is critically dependent on storage temperature.

Mapping Soluble Guanylyl Cyclase and Protein Disulfide Isomerase Regions of Interaction.

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Erin J Heckler

Full Text Available Soluble guanylyl cyclase (sGC is a heterodimeric nitric oxide (NO receptor that produces cyclic GMP. This signaling mechanism is a key component in the cardiovascular system. NO binds to heme in the β subunit and stimulates the catalytic conversion of GTP to cGMP several hundred fold. Several endogenous factors have been identified that modulate sGC function in vitro and in vivo. In previous work, we determined that protein disulfide isomerase (PDI interacts with sGC in a redox-dependent manner in vitro and that PDI inhibited NO-stimulated activity in cells. To our knowledge, this was the first report of a physical interaction between sGC and a thiol-redox protein. To characterize this interaction between sGC and PDI, we first identified peptide linkages between sGC and PDI, using a lysine cross-linking reagent and recently developed mass spectrometry analysis. Together with Flag-immunoprecipitation using sGC domain deletions, wild-type (WT and mutated PDI, regions of sGC involved in this interaction were identified. The observed data were further explored with computational modeling to gain insight into the interaction mechanism between sGC and oxidized PDI. Our results indicate that PDI interacts preferentially with the catalytic domain of sGC, thus providing a mechanism for PDI inhibition of sGC. A model in which PDI interacts with either the α or the β catalytic domain is proposed.

Creation of metal-independent hyperthermophilic L-arabinose isomerase by homologous recombination.

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Hong, Young-Ho; Lee, Dong-Woo; Pyun, Yu-Ryang; Lee, Sung Haeng

2011-12-28

Hyperthermophilic L-arabinose isomerases (AIs) are useful in the commercial production of D-tagatose as a low-calorie bulk sweetener. Their catalysis and thermostability are highly dependent on metals, which is a major drawback in food applications. To study the role of metal ions in the thermostability and catalysis of hyperthermophilic AI, four enzyme chimeras were generated by PCR-based hybridization to replace the variable N- and C-terminal regions of hyperthermophilic Thermotoga maritima AI (TMAI) and thermophilic Geobacillus stearothermophilus AI (GSAI) with those of the homologous mesophilic Bacillus halodurans AI (BHAI). Unlike Mn(2+)-dependent TMAI, the GSAI- and TMAI-based hybrids with the 72 C-terminal residues of BHAI were not metal-dependent for catalytic activity. By contrast, the catalytic activities of the TMAI- and GSAI-based hybrids containing the N-terminus (residues 1-89) of BHAI were significantly enhanced by metals, but their thermostabilities were poor even in the presence of Mn(2+), indicating that the effects of metals on catalysis and thermostability involve different structural regions. Moreover, in contrast to the C-terminal truncate (Δ20 residues) of GSAI, the N-terminal truncate (Δ7 residues) exhibited no activity due to loss of its native structure. The data thus strongly suggest that the metal dependence of the catalysis and thermostability of hyperthermophilic AIs evolved separately to optimize their activity and thermostability at elevated temperatures. This may provide effective target regions for engineering, thereby meeting industrial demands for the production of d-tagatose.

Identification of triosephosphate isomerase as a novel allergen in Octopus fangsiao.

Science.gov (United States)

Yang, Yang; Chen, Zhong-Wei; Hurlburt, Barry K; Li, Gui-Ling; Zhang, Yong-Xia; Fei, Dan-Xia; Shen, Hai-Wang; Cao, Min-Jie; Liu, Guang-Ming

2017-05-01

Octopus is an important mollusk in human dietary for its nutritional value, however it also causes allergic reactions in humans. Major allergens from octopus have been identified, while the knowledge of novel allergens remains poor. In the present study, a novel allergen with molecular weight of 28kDa protein was purified from octopus (Octopus fangsiao) and identified as triosephosphate isomerase (TIM) by mass spectrometry. TIM aggregated beyond 45°C, and its IgE-binding activity was affected under extreme pH conditions due to the altered secondary structure. In simulated gastric fluid digestion, TIM can be degraded into small fragments, while retaining over 80% of the IgE-binding activity. The full-length cDNA of O. fangsiao TIM (1140bp) was cloned, which encodes 247 amino acid residues, and the entire recombinant TIM was successfully expressed in Escherichia coli BL21, which showed similar immunoreactivity to the native TIM. Different intensity of cross-reactivity among TIM from related species revealed the complexity of its epitopes. Eight linear epitopes of TIM were predicted following bioinformatic analysis. Furthermore, a conformational epitope (A 71 G 74 S 69 D 75 T 73 F 72 V 67 ) was confirmed by the phage display technology. The results revealed the physicochemical and immunological characteristics of TIM, which is significant in the development of hyposensitivity food and allergy diagnosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Konfirmasi spesifitas GAD65 terhadap anti-GAD65 pada tikus DM dan pasien DM tipe 1

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Aulanni’a Aulanni’a

2012-02-01

Full Text Available The use of glutamic acid decarboxylase (GAD65 from bovine brain has been studied to obtain basic knowledge and diagnosis and prediction of Type 1 Diabetes Mellitus (DM patients. The importance of GAD65 in DM diagnosis based on its patogenesis. One of the autoimmune marker that can be used to detect beta-pancreas destruction in Diabetes Type I is the antibody to glutamic acid decarboxylase (GAD65. Most of the pre-diabetic patients indicate the reactive autoantibody to GAD65. For early detection of anti-GAD65 in the serum of the patient, human recombinat GAD65 has been succeed to be used. However this is not economical, therefore, it is necessary to find the alternative source of cheaper GAD65. The aim of this research is to develop an early detection kit of Type 1 DM based on antibody- GAD65, since the longest patient suffering from DM has higher probability to be complicated, especially for uncured patients. The anti- GAD65 antibodies induced by anti-GAD65 synthetized and labelled by alkaline phosphatase can be used as reagent detection early DM patients. The ten patients of DM as samples (positive of anti-GAD65 and five rats of DM were positive with western blott technique using reagents as result of this research. It can be concluded, GAD65 enzyme isolated from bovine brain induced anti-GAD65 production and have possibilities to be packaged in a diagnostic kit for patient pre DM.

Discovery of ebselen as an inhibitor of Cryptosporidium parvum glucose-6-phosphate isomerase (CpGPI by high-throughput screening of existing drugs

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Rana Eltahan

2018-04-01

Full Text Available Cryptosporidium parvum is a water-borne and food-borne apicomplexan pathogen. It is one of the top four diarrheal-causing pathogens in children under the age of five in developing countries, and an opportunistic pathogen in immunocompromised individuals. Unlike other apicomplexans, C. parvum lacks Kreb's cycle and cytochrome-based respiration, thus relying mainly on glycolysis to produce ATP. In this study, we characterized the primary biochemical features of the C. parvum glucose-6-phosphate isomerase (CpGPI and determined its Michaelis constant towards fructose-6-phosphate (Km = 0.309 mM, Vmax = 31.72 nmol/μg/min. We also discovered that ebselen, an organoselenium drug, was a selective inhibitor of CpGPI by high-throughput screening of 1200 known drugs. Ebselen acted on CpGPI as an allosteric noncompetitive inhibitor (IC50 = 8.33 μM; Ki = 36.33 μM, while complete inhibition of CpGPI activity was not achieved. Ebselen could also inhibit the growth of C. parvum in vitro (EC50 = 165 μM at concentrations nontoxic to host cells, albeit with a relatively small in vitro safety window of 4.2 (cytotoxicity TC50 on HCT-8 cells = 700 μM. Additionally, ebselen might also target other enzymes in the parasite, leading to the parasite growth reduction. Therefore, although ebselen is useful in studying the inhibition of CpGPI enzyme activity, further proof is needed to chemically and/or genetically validate CpGPI as a drug target. Keywords: Apicomplexan, Cryptosporidium parvum, Glucose-6-phosphate isomerase (GPI, Ebselen

Engineering the l-Arabinose Isomerase from Enterococcus Faecium for d-Tagatose Synthesis

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Marylane de Sousa

2017-12-01

Full Text Available l-Arabinose isomerase (EC 5.3.1.4 (l-AI from Enterococcus faecium DBFIQ E36 was overproduced in Escherichia coli by designing a codon-optimized synthetic araA gene. Using this optimized gene, two N- and C-terminal His-tagged-l-AI proteins were produced. The cloning of the two chimeric genes into regulated expression vectors resulted in the production of high amounts of recombinant N-His-l-AI and C-His-l-AI in soluble and active forms. Both His-tagged enzymes were purified in a single step through metal-affinity chromatography and showed different kinetic and structural characteristics. Analytical ultracentrifugation revealed that C-His-l-AI was preferentially hexameric in solution, whereas N-His-l-AI was mainly monomeric. The specific activity of the N-His-l-AI at acidic pH was higher than that of C-His-l-AI and showed a maximum bioconversion yield of 26% at 50 °C for d-tagatose biosynthesis, with Km and Vmax parameters of 252 mM and 0.092 U mg−1, respectively. However, C-His-l-AI was more active and stable at alkaline pH than N-His-l-AI. N-His-l-AI follows a Michaelis-Menten kinetic, whereas C-His-l-AI fitted to a sigmoidal saturation curve.

Comparison between serum levels of carcinoembryonic antigen, sialic acid and phosphohexose isomerase in lung cancer

International Nuclear Information System (INIS)

Patel, P.S.; Raval, G.N.; Rawal, R.M.; Balar, D.B.; Patel, G.H.; Shah, P.M.; Patel, D.D.

1995-01-01

The identification and application of quantifiable tumor markers as adjuncts to clinical care is a story of both success and failure. The present study compared serum levels of carcinoembryogenic antigen (CEA) with total sialic acid/total protein (TSA/TP) ration and phosphohexose isomerase (PHI) in 192 untreated lung cancer patients as well as 80 age and sex matched controls (44 non-smokers). CEA values were significantly raised (p < 0.001) in smokers as compared to the non-smokers; whereas, TSA/TP and PHI values were comparable between the groups of the groups of the controls. All the bio-markers were significantly elevated (p < 0.00.1) in untreated lung cancer patients as compared to the controls. Receiver operating characteristic curve analysis revealed higher sensitivities of TSA/TP and PHI as compared to CEA at different specificity levels between 60% and 95%. Mean values of CEA, TSA/TP and PHI were higher in non-responders compared to the responders. The results indicate that TSA/TP and PHI are superior tumor markers than CEA for lung cancer patients. (author)

Progranulin, a glycoprotein deficient in frontotemporal dementia, is a novel substrate of several protein disulfide isomerase family proteins.

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Sandra Almeida

Full Text Available The reduced production or activity of the cysteine-rich glycoprotein progranulin is responsible for about 20% of cases of familial frontotemporal dementia. However, little is known about the molecular mechanisms that govern the level and secretion of progranulin. Here we show that progranulin is expressed in mouse cortical neurons and more prominently in mouse microglia in culture and is abundant in the endoplasmic reticulum (ER and Golgi. Using chemical crosslinking, immunoprecipitation, and mass spectrometry, we found that progranulin is bound to a network of ER Ca(2+-binding chaperones including BiP, calreticulin, GRP94, and four members of the protein disulfide isomerase (PDI family. Loss of ERp57 inhibits progranulin secretion. Thus, progranulin is a novel substrate of several PDI family proteins and modulation of the ER chaperone network may be a therapeutic target for controlling progranulin secretion.

Bioengineered Bruch's-like extracellular matrix promotes retinal pigment epithelial differentiation

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Samuel McLenachan

2017-07-01

Full Text Available In the eye, the retinal pigment epithelium (RPE adheres to a complex protein matrix known as Bruch's membrane (BrM. The aim of this study was to provide enriched conditions for RPE cell culture through the production of a BrM-like matrix. Our hypothesis was that a human RPE cell line would deposit an extracellular matrix (ECM resembling BrM. The composition and structure of ECM deposited by ARPE19 cells (ARPE19-ECM was characterized. To produce ARPE19-ECM, ARPE19 cells were cultured in the presence dextran sulphate. ARPE19-ECM was decellularized using deoxycholate and characterized by immunostaining and western blot analysis. Primary human RPE and induced pluripotent stem cells were seeded onto ARPE19-ECM or geltrex coated surfaces and examined by microscopy or RT-PCR. Culture of ARPE19 cells with dextran sulphate promoted nuclear localization of SOX2, formation of tight junctions and deposition of ECM. ARPE19 cells deposited ECM proteins found in the inner layers of BrM, including fibronectin, vitronectin, collagens IV and V as well as laminin-alpha-5, but not those found in the middle elastic layer (elastin or the outer layers (collagen VI. ARPE19-ECM promoted pigmentation in human RPE and pluripotent stem cell cultures. Expression of RPE65 was significantly increased on ARPE19-ECM compared with geltrex in differentiating pluripotent stem cell cultures. ARPE19 cells deposit ECM with a composition and structure similar to BrM in the retina. Molecular cues present in ARPE19-ECM promote the acquisition and maintenance of the RPE phenotype. Together, these results demonstrate a simple method for generating a BrM-like surface for enriched RPE cell cultures.

7 CFR 65.150 - Goat.

Science.gov (United States)

2010-01-01

..., PORK, LAMB, CHICKEN, GOAT MEAT, PERISHABLE AGRICULTURAL COMMODITIES, MACADAMIA NUTS, PECANS, PEANUTS, AND GINSENG General Provisions Definitions § 65.150 Goat. Goat means meat produced from goats. ... 7 Agriculture 3 2010-01-01 2010-01-01 false Goat. 65.150 Section 65.150 Agriculture Regulations of...

7 CFR 65.190 - Lamb.

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2010-01-01

... 7 Agriculture 3 2010-01-01 2010-01-01 false Lamb. 65.190 Section 65.190 Agriculture Regulations of..., PORK, LAMB, CHICKEN, GOAT MEAT, PERISHABLE AGRICULTURAL COMMODITIES, MACADAMIA NUTS, PECANS, PEANUTS, AND GINSENG General Provisions Definitions § 65.190 Lamb. Lamb means meat produced from sheep. ...

7 CFR 65.120 - Chicken.

Science.gov (United States)

2010-01-01

... 7 Agriculture 3 2010-01-01 2010-01-01 false Chicken. 65.120 Section 65.120 Agriculture Regulations..., PORK, LAMB, CHICKEN, GOAT MEAT, PERISHABLE AGRICULTURAL COMMODITIES, MACADAMIA NUTS, PECANS, PEANUTS, AND GINSENG General Provisions Definitions § 65.120 Chicken. Chicken has the meaning given the term in...

18 CFR 366.4 - FERC-65, notification of holding company status, FERC-65A, exemption notification, and FERC-65B...

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2010-04-01

... meet the definition of a holding company as provided by § 366.1 as of February 8, 2006 shall notify the... of holding company status, FERC-65A, exemption notification, and FERC-65B, waiver notification. 366.4... OF ENERGY REGULATIONS UNDER THE PUBLIC UTILITY HOLDING COMPANY ACT OF 2005, FEDERAL POWER ACT AND...

Abscisic-acid-induced cellular apoptosis and differentiation in glioma via the retinoid acid signaling pathway.

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Zhou, Nan; Yao, Yu; Ye, Hongxing; Zhu, Wei; Chen, Liang; Mao, Ying

2016-04-15

Retinoid acid (RA) plays critical roles in regulating differentiation and apoptosis in a variety of cancer cells. Abscisic acid (ABA) and RA are direct derivatives of carotenoids and share structural similarities. Here we proposed that ABA may also play a role in cellular differentiation and apoptosis by sharing a similar signaling pathway with RA that may be involved in glioma pathogenesis. We reported for the first time that the ABA levels were twofold higher in low-grade gliomas compared with high-grade gliomas. In glioma tissues, there was a positive correlation between the ABA levels and the transcription of cellular retinoic acid-binding protein 2 (CRABP2) and a negative correlation between the ABA levels and transcription of fatty acid-binding protein 5 (FABP5). ABA treatment induced a significant increase in the expression of CRABP2 and a decrease in the expression of peroxisome proliferator-activated receptor (PPAR) in glioblastoma cells. Remarkably, both cellular apoptosis and differentiation were increased in the glioblastoma cells after ABA treatment. ABA-induced cellular apoptosis and differentiation were significantly reduced by selectively silencing RAR-α, while RAR-α overexpression exaggerated the ABA-induced effects. These results suggest that ABA may play a role in the pathogenesis of glioma by promoting cellular apoptosis and differentiation through the RA signaling pathway. © 2015 UICC.

40 CFR 65.86 - Monitoring.

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2010-07-01

... 40 Protection of Environment 15 2010-07-01 2010-07-01 false Monitoring. 65.86 Section 65.86 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) CONSOLIDATED FEDERAL AIR RULE Transfer Racks § 65.86 Monitoring. The owner or operator of a transfer rack equipped with...

45 CFR 96.65 - Discovery.

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2010-10-01

... 45 Public Welfare 1 2010-10-01 2010-10-01 false Discovery. 96.65 Section 96.65 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION BLOCK GRANTS Hearing Procedure § 96.65 Discovery. The use of interrogatories, depositions, and other forms of discovery shall not be allowed. ...

28 CFR 90.65 - Evaluation.

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2010-07-01

... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Evaluation. 90.65 Section 90.65 Judicial... Cases § 90.65 Evaluation. (a) The National Institute of Justice will conduct evaluations and studies of... with such federally-sponsored research and evaluation studies of their projects. In addition, grant...

21 CFR 820.65 - Traceability.

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2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Traceability. 820.65 Section 820.65 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES QUALITY SYSTEM REGULATION Identification and Traceability § 820.65 Traceability. Each manufacturer of a...

12 CFR 4.65 - Certification.

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2010-01-01

... 12 Banks and Banking 1 2010-01-01 2010-01-01 false Certification. 4.65 Section 4.65 Banks and Banking COMPTROLLER OF THE CURRENCY, DEPARTMENT OF THE TREASURY ORGANIZATION AND FUNCTIONS, AVAILABILITY...; Contracting for Goods and Services § 4.65 Certification. (a) Objective. To preserve the integrity and foster...

7 CFR 65.100 - Act.

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2010-01-01

... 7 Agriculture 3 2010-01-01 2010-01-01 false Act. 65.100 Section 65.100 Agriculture Regulations of... MARKETING ACT OF 1946 AND THE EGG PRODUCTS INSPECTION ACT (CONTINUED) COUNTRY OF ORIGIN LABELING OF BEEF..., AND GINSENG General Provisions Definitions § 65.100 Act. Act means the Agricultural Marketing Act of...

Human amniotic fluid promotes retinal pigmented epithelial cells' trans-differentiation into rod photoreceptors and retinal ganglion cells.

Science.gov (United States)

Ghaderi, Shima; Soheili, Zahra-Soheila; Ahmadieh, Hamid; Davari, Maliheh; Jahromi, Fatemeh Sanie; Samie, Shahram; Rezaie-Kanavi, Mozhgan; Pakravesh, Jalil; Deezagi, Abdolkhalegh

2011-09-01

To evaluate the effect of human amniotic fluid (HAF) on retinal pigmented epithelial cells growth and trans-differentiation into retinal neurons, retinal pigmented epithelium (RPE) cells were isolated from neonatal human cadaver eye globes and cultured in Dulbecco's modified Eagle's medium-F12 supplemented with 10% fetal bovine serum (FBS). Confluent monolayer cultures were trypsinized and passaged using FBS-containing or HAF-containing media. Amniotic fluid samples were received from pregnant women in the first trimester of gestation. Cell proliferation and death enzyme-linked immunosorbent assays were performed to assess the effect of HAF on RPE cell growth. Trans-differentiation into rod photoreceptors and retinal ganglion cells was also studied using immunocytochemistry and real-time polymerase chain reaction techniques. Primary cultures of RPE cells were successfully established under FBS-containing or HAF-containing media leading to rapid cell growth and proliferation. When RPE cells were moved to in vitro culture system, they began to lose their differentiation markers such as pigmentation and RPE65 marker and trans-differentiated neural-like cells followed by spheroid colonies pertaining to stem/progenitor cells were morphologically detected. Immunocytochemistry (ICC) analysis of HAF-treated cultures showed a considerable expression of Rhodopsin gene (30% Rhodopsin-positive cells) indicating trans-differentiation of RPE cells to rod photoreceptors. Real-time polymerase chain reaction revealed an HAF-dose-dependant expression of Thy-1 gene (RGC marker) and significant promoting effect of HAF on RGCs generation. The data presented here suggest that HAF possesses invaluable stimulatory effect on RPE cells growth and trans-differentiation into retinal neurons. It can be regarded as a newly introduced enriched supplement in serum-free kinds of media used in neuro-retinal regeneration studies.

Myo-inositol uptake by cultured calf retinal pigment epithelial cells: regulation by glucose

International Nuclear Information System (INIS)

Khatami, M.; Rockey, J.H.

1986-01-01

Confluent primary (P-1) or subcultured passage 2 or 3 (P-2, P-3) calf retinal pigment epithelial cells (RPE) were incubated with [ 3 H]-myo-inositol (MI, 100-200 μM) in balanced salt solution (BSS), for 5 to 60 min at 37 0 C. MI uptake into RPE (P-2, 5 days old) was saturable with K/sub m/ of 147 μM and V/sub max/ of 5.5 pmole/min/μg DNA. P-1 or P-2 incubated with 10 μM MI for 40 min accumulated MI against a concentration gradient ([MI]in/[MI]out > 20). Replacement of 150 mM NaCl in BSS by 150 mM choline-Cl reduced the uptake of MI by 87%. MI uptake was inhibited (39%) when cells were incubated in BSS in the absence of Ca Cl 2 . Transport of MI into RPE incubated in the presence of phloridzin, ouabain or 2,4-dinitrophenol (1 mM each) for 10 min was inhibited by 65, 37 and 21%, respectively. α-D-Glucose (20 mM) in the incubation media inhibited MI uptake into primary (or P-2) cultured RPE by 30 or 43% when cells were incubated for 10 or 60 min, respectively. The ability of RPE cells, grown in the presence of 50 mM glucose for 15-25 days, to concentrate MI (40 μM) was reduced up to 41%. Cultured RPE cells accumulated myo-inositol by an active transport system, sensitive to ouabain, DNP and phloridzin. High glucose in the incubation media or in the growth media inhibited the uptake of MI into calf RPE cells

47 CFR 65.500 - Net income.

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2010-10-01

... 47 Telecommunication 3 2010-10-01 2010-10-01 false Net income. 65.500 Section 65.500... OF RETURN PRESCRIPTION PROCEDURES AND METHODOLOGIES Interexchange Carriers § 65.500 Net income. The net income methodology specified in § 65.450 shall be utilized by all interexchange carriers that are...

Health screenings for women over age 65

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Health maintenance visit - women - over age 65; Physical exam - women - over age 65; Yearly exam - women - over age 65; Checkup - women - over age 65; Women's health - over age 65; Preventive care exam - women - over ...

Health screenings for men over age 65

Science.gov (United States)

Health maintenance visit - men - over age 65; Physical exam - men - over age 65; Yearly exam - men - over age 65; Checkup - men - over age 65; Men's health - over age 65; Preventive care exam - men - over ...

Gene profiling of postnatal Mfrprd6 mutant eyes reveals differential accumulation of Prss56, visual cycle and phototransduction mRNAs.

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Ramani Soundararajan

Full Text Available Mutations in the membrane frizzled-related protein (MFRP/Mfrp gene, specifically expressed in the retinal pigment epithelium (RPE and ciliary body, cause nanophthalmia or posterior microphthalmia with retinitis pigmentosa in humans, and photoreceptor degeneration in mice. To better understand MFRP function, microarray analysis was performed on eyes of homozygous Mfrprd6 and C57BL/6J mice at postnatal days (P 0 and P14, prior to photoreceptor loss. Data analysis revealed no changes at P0 but significant differences in RPE and retina-specific transcripts at P14, suggesting a postnatal influence of the Mfrprd6 allele. A subset of these transcripts was validated by quantitative real-time PCR (qRT-PCR. In Mfrprd6 eyes, a significant 1.5- to 2.0-fold decrease was observed among transcripts of genes linked to retinal degeneration, including those involved in visual cycle (Rpe65, Lrat, Rgr, phototransduction (Pde6a, Guca1b, Rgs9, and photoreceptor disc morphogenesis (Rpgrip1 and Fscn2. Levels of RPE65 were significantly decreased by 2.0-fold. Transcripts of Prss56, a gene associated with angle-closure glaucoma, posterior microphthalmia and myopia, were increased in Mfrprd6 eyes by 17-fold. Validation by qRT-PCR indicated a 3.5-, 14- and 70-fold accumulation of Prss56 transcripts relative to controls at P7, P14 and P21, respectively. This trend was not observed in other RPE or photoreceptor mutant mouse models with similar disease progression, suggesting that Prss56 upregulation is a specific attribute of the disruption of Mfrp. Prss56 and Glul in situ hybridization directly identified Müller glia in the inner nuclear layer as the cell type expressing Prss56. In summary, the Mfrprd6 allele causes significant postnatal changes in transcript and protein levels in the retina and RPE. The link between Mfrp deficiency and Prss56 up-regulation, together with the genetic association of human MFRP or PRSS56 variants and ocular size, raises the possibility that

Journal of Genetics | Indian Academy of Sciences

Indian Academy of Sciences (India)

RPE65 gene: multiplex PCR and mutation screening in patients from India with retinal degenerative diseases ... Medical and Vision Research Foundations, Sankara Nethralaya, Chennai 600 006, India; Department of Medical Retina, Medical and Vision Research Foundations, Sankara Nethralaya, Chennai 600 006, India ...

Characterization of a thermostable recombinant l-rhamnose isomerase from Caldicellulosiruptor obsidiansis OB47 and its application for the production of l-fructose and l-rhamnulose.

Science.gov (United States)

Chen, Ziwei; Xu, Wei; Zhang, Wenli; Zhang, Tao; Jiang, Bo; Mu, Wanmeng

2018-04-01

l-Hexoses are rare sugars that are important components and precursors in the synthesis of biological compounds and pharmaceutical drugs. l-Rhamnose isomerase (L-RI, EC 5.3.1.14) is an aldose-ketose isomerase that plays a significant role in the production of l-sugars. In this study, a thermostable, l-sugar-producing L-RI from the hyperthermophile Caldicellulosiruptor obsidiansis OB47 was characterized. The recombinant L-RI displayed maximal activity at pH 8.0 and 85 °C and was significantly activated by Co 2+ . It exhibited a relatively high thermostability, with measured half-lives of 24.75, 11.55, 4.15 and 3.30 h in the presence of Co 2+ at 70, 75, 80 and 85 °C, respectively. Specific activities of 277.6, 57.9, 13.7 and 9.6 U mg -1 were measured when l-rhamnose, l-mannose, d-allose and l-fructose were used as substrates, respectively. l-Rhamnulose was produced with conversion ratios of 44.0% and 38.6% from 25 and 50 g L -1 l-rhamnose, respectively. l-Fructose was also efficiently produced by the L-RI, with conversion ratios of 67.0% and 58.4% from 25 and 50 g L -1 l-mannose, respectively. The recombinant L-RI could effectively catalyze the formation of l-rhamnulose and l-fructose, suggesting that it was a promising candidate for industrial production of l-rhamnulose and l-fructose. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Jojoba Oil Soft Colloidal Nanocarrier of a Synthetic Retinoid: Preparation, Characterization and Clinical Efficacy in Psoriatic Patients.

Science.gov (United States)

Nasr, Maha; Abdel-Hamid, Sameh; Moftah, Noha H; Fadel, Maha; Alyoussef, Abdullah A

2017-01-01

Nanotechnology has provided substantial benefits in drug delivery, especially in the treatment of dermatological diseases. Psoriasis is a chronic inflammatory skin disease in which topical delivery of antipsoriatic agents is considered the first line treatment. To investigate whether the encapsulation of the synthetic retinoid tazarotene in a nanocarrier based on jojoba oil would decrease its irritation potential and clinically improve its therapeutic outcome in psoriatic patients. A microemulsion system based on jojoba wax and labrasol/plurol isostearique was prepared and characterized. The selected formula displayed spherical morphology, particle size of 15.49±2.41 nm, polydispersity index of 0.20 ±0.08, negative charge and low viscosity. The microemulsion provided two folds increase in skin deposition of tazarotene, correlating with higher reduction in psoriatic patients PASI scores after treatment (68% reduction in PASI scores versus 8.96% reduction with the marketed gel). No irritation was encountered in patients using microemulsion, with redness and inflammation reported with the marketed gel-treated patients. Jojoba oil microemulsion proved to be advantageous in reducing the irritancy of tazarotene, enhancing its skin deposition and achieving better therapeutic outcome in psoriatic patients. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Suppressive effects of a novel compound on interphotoreceptor retinoid-binding protein-induced experimental autoimmune uveoretinitis in rats

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Jun-ichi Sakai

1999-01-01

Full Text Available The immunosuppressive effect of ethyl O-(N-(pcarboxyphenyl-carbamoyl-mycophenolate(CAM was examined in interphotoreceptor retinoid-binding protein (IRBP-induced experimental autoimmune uveoretinitis (EAU in rats. Lewis rats immunized with bovine IRBP were treated with various oral doses of CAM postimmunization. The degree of inflammation was assessed clinically each day and histologically on day 14 or day 20. Production of various cytokines and IRBP-specific antibody, as well as IRBP-specific proliferation response, was assessed. Complete inhibition of EAU in rats, both by clinical and histologic criteria, was achieved with 50 mg/kg CAM when administered daily for 14 days following IRBP immunization. Partial inhibition was observed at lesser doses of CAM. This CAM-mediated response was accompanied by diminished production of cytokines interleukin-2, interferon-γ and tumor necrosis factor-α, as well as a reduction in IRBP-specific antibody production. Furthermore, administration of CAM either in the induction phase only (days 0–7 or in the effector phase only (days 9 or 11 to day 20 was also capable of suppressing EAU, as assessed histopathologically on day 20. We conclude that CAM is effective in suppressing EAU in rats and its mechanism of action appears to involve modulation of T cell function.

7 CFR 65.255 - United States.

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2010-01-01

... 7 Agriculture 3 2010-01-01 2010-01-01 false United States. 65.255 Section 65.255 Agriculture..., PEANUTS, AND GINSENG General Provisions Definitions § 65.255 United States. United States means the 50... United States. ...

A chalcone isomerase-like protein enhances flavonoid production and flower pigmentation.

Science.gov (United States)

Morita, Yasumasa; Takagi, Kyoko; Fukuchi-Mizutani, Masako; Ishiguro, Kanako; Tanaka, Yoshikazu; Nitasaka, Eiji; Nakayama, Masayoshi; Saito, Norio; Kagami, Takashi; Hoshino, Atsushi; Iida, Shigeru

2014-04-01

Flavonoids are major pigments in plants, and their biosynthetic pathway is one of the best-studied metabolic pathways. Here we have identified three mutations within a gene that result in pale-colored flowers in the Japanese morning glory (Ipomoea nil). As the mutations lead to a reduction of the colorless flavonoid compound flavonol as well as of anthocyanins in the flower petal, the identified gene was designated enhancer of flavonoid production (EFP). EFP encodes a chalcone isomerase (CHI)-related protein classified as a type IV CHI protein. CHI is the second committed enzyme of the flavonoid biosynthetic pathway, but type IV CHI proteins are thought to lack CHI enzymatic activity, and their functions remain unknown. The spatio-temporal expression of EFP and structural genes encoding enzymes that produce flavonoids is very similar. Expression of both EFP and the structural genes is coordinately promoted by genes encoding R2R3-MYB and WD40 family proteins. The EFP gene is widely distributed in land plants, and RNAi knockdown mutants of the EFP homologs in petunia (Petunia hybrida) and torenia (Torenia hybrida) had pale-colored flowers and low amounts of anthocyanins. The flavonol and flavone contents in the knockdown petunia and torenia flowers, respectively, were also significantly decreased, suggesting that the EFP protein contributes in early step(s) of the flavonoid biosynthetic pathway to ensure production of flavonoid compounds. From these results, we conclude that EFP is an enhancer of flavonoid production and flower pigmentation, and its function is conserved among diverse land plant species. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

[Screening of food-grade microorganisms for biotransformation of D-tagatose and cloning and expression of L-arabinose isomerase].

Science.gov (United States)

Men, Yan; Zhu, Yueming; Guan, Yuping; Zhang, Tongcun; Izumori, Ken; Sun, Yuanxia

2012-05-01

L-Arabinose isomerase (L-AI) is an intracellular enzyme that catalyzes the reversible isomerization of D-galactose and D-tagatose. Given the widespread use of D-tagatose in the food industry, food-grade microorganisms and the derivation of L-AI for the production of D-tagatose is gaining increased attention. In the current study, food-grade strains from different foods that can convert D-galactose to D-tagatose were screened. According to physiological, biochemical, and 16S rDNA gene analyses, the selected strain was found to share 99% identity with Pediococcus pentosaceus, and was named as Pediococcus pentosaceus PC-5. The araA gene encoding L-AI from Pediococcus pentosaceus PC-5 was cloned and overexpressed in E. coli BL21. The yield of D-tagatose using D-galactose as the substrate catalyzed by the crude enzyme in the presence of Mn2+ was found to be 33% at 40 degrees C.

7 CFR 65.160 - Ground chicken.

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2010-01-01

... 7 Agriculture 3 2010-01-01 2010-01-01 false Ground chicken. 65.160 Section 65.160 Agriculture... OF BEEF, PORK, LAMB, CHICKEN, GOAT MEAT, PERISHABLE AGRICULTURAL COMMODITIES, MACADAMIA NUTS, PECANS, PEANUTS, AND GINSENG General Provisions Definitions § 65.160 Ground chicken. Ground chicken means...

45 CFR 2400.65 - Teaching obligation.

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2010-10-01

... FOUNDATION FELLOWSHIP PROGRAM REQUIREMENTS Special Conditions § 2400.65 Teaching obligation. Upon receiving a Master's degree, each Fellow must teach American history, American government, social studies, or... 45 Public Welfare 4 2010-10-01 2010-10-01 false Teaching obligation. 2400.65 Section 2400.65...

7 CFR 65.230 - Production step.

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2010-01-01

... 7 Agriculture 3 2010-01-01 2010-01-01 false Production step. 65.230 Section 65.230 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Standards..., PEANUTS, AND GINSENG General Provisions Definitions § 65.230 Production step. Production step means, in...

47 CFR 65.800 - Rate base.

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2010-10-01

... 47 Telecommunication 3 2010-10-01 2010-10-01 false Rate base. 65.800 Section 65.800 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES (CONTINUED) INTERSTATE RATE OF RETURN PRESCRIPTION PROCEDURES AND METHODOLOGIES Rate Base § 65.800 Rate base. The rate base shall...

42 CFR 408.65 - Payment options.

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2010-10-01

... 42 Public Health 2 2010-10-01 2010-10-01 false Payment options. 408.65 Section 408.65 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES MEDICARE PROGRAM PREMIUMS FOR SUPPLEMENTARY MEDICAL INSURANCE Direct Remittance: Individual Payment § 408.65 Payment options...

7 CFR 65.170 - Ground lamb.

Science.gov (United States)

2010-01-01

... 7 Agriculture 3 2010-01-01 2010-01-01 false Ground lamb. 65.170 Section 65.170 Agriculture... OF BEEF, PORK, LAMB, CHICKEN, GOAT MEAT, PERISHABLE AGRICULTURAL COMMODITIES, MACADAMIA NUTS, PECANS, PEANUTS, AND GINSENG General Provisions Definitions § 65.170 Ground lamb. Ground lamb means comminuted...

47 CFR 65.450 - Net income.

Science.gov (United States)

2010-10-01

... 47 Telecommunication 3 2010-10-01 2010-10-01 false Net income. 65.450 Section 65.450... OF RETURN PRESCRIPTION PROCEDURES AND METHODOLOGIES Exchange Carriers § 65.450 Net income. (a) Net income shall consist of all revenues derived from the provision of interstate telecommunications services...

7 CFR 65.165 - Ground goat.

Science.gov (United States)

2010-01-01

... 7 Agriculture 3 2010-01-01 2010-01-01 false Ground goat. 65.165 Section 65.165 Agriculture... OF BEEF, PORK, LAMB, CHICKEN, GOAT MEAT, PERISHABLE AGRICULTURAL COMMODITIES, MACADAMIA NUTS, PECANS, PEANUTS, AND GINSENG General Provisions Definitions § 65.165 Ground goat. Ground goat means comminuted...

The disulfide isomerase ERp57 is required for fibrin deposition in vivo.

Science.gov (United States)

Zhou, J; Wu, Y; Wang, L; Rauova, L; Hayes, V M; Poncz, M; Essex, D W

2014-11-01

ERp57 is required for platelet function; however, whether ERp57 contributes to fibrin generation is unknown. Using an inhibitory anti-ERp57 antibody (mAb1), Pf4-Cre/ERp57(fl/fl) mice, Tie2-Cre/ERp57(fl/fl) mice, and mutants of ERp57, we analyzed the function of ERp57 in laser-induced thrombosis. Fibrin deposition was decreased in Pf4-Cre/ERp57(fl/fl) mice, consistent with a role for platelet ERp57 in fibrin generation. Fibrin deposition was further decreased with infusion of mAb1 and in Tie2-Cre/ERp57(fl/fl) mice, consistent with endothelial cells also contributing to fibrin deposition. Infusion of eptibifatide inhibited platelet and fibrin deposition, confirming a role for platelets in fibrin deposition. Infusion of recombinant ERp57 corrected the defect in fibrin deposition but not platelet accumulation, suggesting a direct effect of ERp57 on coagulation. mAb1 inhibited thrombin generation in vitro, consistent with a requirement for ERp57 in coagulation. Platelet accumulation was decreased to similar extents in Pf4-Cre/ERp57(fl/fl) mice, Tie2-Cre/ERp57(fl/fl) mice and normal mice infused with mAb1. Infusion of completely inactivated ERp57 or ERp57 with a non-functional second active site inhibited fibrin deposition and platelet accumulation, indicating that the isomerase activity of the second active site is required for these processes. ERp57 regulates thrombosis via multiple targets. © 2014 International Society on Thrombosis and Haemostasis.

21 CFR 1240.65 - Psittacine birds.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Psittacine birds. 1240.65 Section 1240.65 Food and... DISEASES Specific Administrative Decisions Regarding Interstate Shipments § 1240.65 Psittacine birds. (a) The term psittacine birds shall include all birds commonly known as parrots, Amazons, Mexican double...

47 CFR 65.304 - Capital structure.

Science.gov (United States)

2010-10-01

... 47 Telecommunication 3 2010-10-01 2010-10-01 false Capital structure. 65.304 Section 65.304... OF RETURN PRESCRIPTION PROCEDURES AND METHODOLOGIES Exchange Carriers § 65.304 Capital structure. The proportion of each cost of capital component in the capital structure is equal to: Proportion in the capital...

46 CFR 46.10-65 - Construction.

Science.gov (United States)

2010-10-01

... 46 Shipping 2 2010-10-01 2010-10-01 false Construction. 46.10-65 Section 46.10-65 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) LOAD LINES SUBDIVISION LOAD LINES FOR PASSENGER VESSELS Administration § 46.10-65 Construction. (a) The watertight subdivision of every passenger vessel...

32 CFR 552.65 - Command supervision.

Science.gov (United States)

2010-07-01

... 32 National Defense 3 2010-07-01 2010-07-01 true Command supervision. 552.65 Section 552.65 National Defense Department of Defense (Continued) DEPARTMENT OF THE ARMY MILITARY RESERVATIONS AND NATIONAL CEMETERIES REGULATIONS AFFECTING MILITARY RESERVATIONS Solicitation on Military Reservations § 552.65 Command supervision. (a) All insurance...

Protective effect of bone morphogenetic protein 6 on RPE cells injury caused by H2O2

Directory of Open Access Journals (Sweden)

Li Chen

2016-01-01

Full Text Available AIM:To investigate the effect of bone morphogenetic protein 6(BMP-6on cellular morphology, proliferation and apoptosis of retinal pigment epithelial cells(ARPE-19incubated in hydrogen peroxide(H2O2. METHODS:ARPE-19 cells were cultured conventionally and divided into four groups. One group was untreated as blank group, the other three groups were incubated in 75μm/L H2O2, 150ng/mLBMP-6 or75μm/L H2O2+150ng/mL BMP-6. All the groups were incubated for 3h, 6h, 9h and 12h. We tested the cell viabilitity by MTT. We used flow cytometry to test the cell cycle and cell apoptosis.RESULTS:H2O2 significantly decreased the cell activity in time-dependent manner. The activity of cells with BMP-6+H2O2 was higher H2O2 group, and the differences between the two groups at 3h and 6h were significant(P2O2, while the cells with BMP-6 were less cell detachment and apoptosis. CONCLUSION:BMP-6 has protective effects on RPE cells from oxidative stress in certain extent.

Journal of Genetics | Indian Academy of Sciences

Indian Academy of Sciences (India)

Home; Journals; Journal of Genetics. Govindasamy Kumaramanickavel. Articles written in Journal of Genetics. Volume 81 Issue 1 April 2002 pp 19-23 Perspectives. RPE65 gene: multiplex PCR and mutation screening in patients from India with retinal degenerative diseases · Biju Joseph Anuradha Srinivasan Nagasamy ...

10 CFR 39.65 - Personnel monitoring.

Science.gov (United States)

2010-01-01

... 10 Energy 1 2010-01-01 2010-01-01 false Personnel monitoring. 39.65 Section 39.65 Energy NUCLEAR REGULATORY COMMISSION LICENSES AND RADIATION SAFETY REQUIREMENTS FOR WELL LOGGING Radiation Safety Requirements § 39.65 Personnel monitoring. (a) The licensee may not permit an individual to act as a logging...

7 CFR 953.65 - Effective time.

Science.gov (United States)

2010-01-01

... 7 Agriculture 8 2010-01-01 2010-01-01 false Effective time. 953.65 Section 953.65 Agriculture... STATES Order Regulating Handling Effective Time and Termination § 953.65 Effective time. The provisions of this subpart shall become effective at such time as the Secretary may declare above his signature...

42 CFR 483.65 - Infection control.

Science.gov (United States)

2010-10-01

... 42 Public Health 5 2010-10-01 2010-10-01 false Infection control. 483.65 Section 483.65 Public... Care Facilities § 483.65 Infection control. The facility must establish and maintain an infection control program designed to provide a safe, sanitary, and comfortable environment and to help prevent the...

9 CFR 3.65 - Terminal facilities.

Science.gov (United States)

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Terminal facilities. 3.65 Section 3.65... Transportation Standards § 3.65 Terminal facilities. No person subject to the Animal Welfare regulations shall commingle shipments of live rabbits with inanimate cargo. All animal holding areas of a terminal facility...

43 CFR 12.65 - Program income.

Science.gov (United States)

2010-10-01

... 43 Public Lands: Interior 1 2010-10-01 2010-10-01 false Program income. 12.65 Section 12.65 Public... to State and Local Governments Post-Award Requirements § 12.65 Program income. (a) General. Grantees are encouraged to earn income to defray program costs. Program income includes income from fees for...

FS65 Disposition Option Report

Energy Technology Data Exchange (ETDEWEB)

Wenz, Tracy R. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

2015-09-25

This report outlines the options for dispositioning the MOX fuel stored in FS65 containers at LANL. Additional discussion regarding the support equipment for loading and unloading the FS65 transport containers is included at the end of the report.

The unfolded protein response and the role of protein disulphide isomerase in neurodegeneration.

Directory of Open Access Journals (Sweden)

Emma ePerri

2016-01-01

Full Text Available The maintenance and regulation of proteostasis is a critical function for post-mitotic neurons and dysregulation of proteostasis is increasingly implicated in neurodegenerative diseases. Despite having different clinical manifestations, these disorders share similar pathology; an accumulation of misfolded proteins in neurons and subsequent disruption to cellular proteostasis. The endoplasmic reticulum (ER is an important component of proteostasis, and when the accumulation of misfolded proteins occurs within the ER, this disturbs ER homeostasis, giving rise to ER stress. This triggers the unfolded protein response (UPR, distinct signalling pathways that whilst initially protective, are pro-apoptotic if ER stress is prolonged. ER stress is increasingly implicated in neurodegenerative diseases, and emerging evidence highlights the complexity of the UPR in these disorders, with both protective and detrimental components being described. Protein Disulphide Isomerase (PDI is an ER chaperone induced during ER stress that is responsible for the formation of disulphide bonds in proteins. Whilst initially considered to be protective, recent studies have revealed unconventional roles for PDI in neurodegenerative diseases, distinct from its normal function in the UPR and the ER, although these mechanisms remain poorly defined. However specific aspects of PDI function may offer the potential to be exploited therapeutically in the future. This review will focus on the evidence linking ER stress and the UPR to neurodegenerative diseases, with particular emphasis on the emerging functions ascribed to PDI in these conditions.

TRIM65 negatively regulates p53 through ubiquitination

Energy Technology Data Exchange (ETDEWEB)

Li, Yang [Department of Respiration, The First Hospital of Jilin University, Changchun 130021 (China); Ma, Chengyuan [Department of Neurosurgery, The First Hospital of Jilin University, Changchun 130021 (China); Zhou, Tong [Department of Endocrinology, The First Hospital of Jilin University, Changchun 130021 (China); Liu, Ying [Department of Respiration, The First Hospital of Jilin University, Changchun 130021 (China); Sun, Luyao [Department of Infectious Diseases, The First Hospital of Jilin University, Changchun 130021 (China); Yu, Zhenxiang, E-mail: zhenxiangyu2015@gmail.com [Department of Respiration, The First Hospital of Jilin University, Changchun 130021 (China)

2016-04-22

Tripartite-motif protein family member 65 (TRIM65) is an important protein involved in white matter lesion. However, the role of TRIM65 in human cancer remains less understood. Through the Cancer Genome Atlas (TCGA) gene alteration database, we found that TRIM65 is upregulated in a significant portion of non-small cell lung carcinoma (NSCLC) patients. Our cell growth assay revealed that TRIM65 overexpression promotes cell proliferation, while knockdown of TRIM65 displays opposite effect. Mechanistically, TRIM65 binds to p53, one of the most critical tumor suppressors, and serves as an E3 ligase toward p53. Consequently, TRIM65 inactivates p53 through facilitating p53 poly-ubiquitination and proteasome-mediated degradation. Notably, chemotherapeutic reagent cisplatin induction of p53 is markedly attenuated in response to ectopic expression of TRIM65. Cell growth inhibition by TRIM65 knockdown is more significant in p53 positive H460 than p53 negative H1299 cells, and knockdown of p53 in H460 cells also shows compromised cell growth inhibition by TRIM65 knockdown, indicating that p53 is required, at least in part, for TRIM65 function. Our findings demonstrate TRIM65 as a potential oncogenic protein, highly likely through p53 inactivation, and provide insight into development of novel approaches targeting TRIM65 for NSCLC treatment, and also overcoming chemotherapy resistance. - Highlights: • TRIM65 expression is elevated in NSCLC. • TRIM65 inactivates p53 through mediating p53 ubiquitination and degradation. • TRIM65 attenuates the response of NSCLC cells to cisplatin.

The expression of Millettia pinnata chalcone isomerase in Saccharomyces cerevisiae salt-sensitive mutants enhances salt-tolerance.

Science.gov (United States)

Wang, Hui; Hu, Tangjin; Huang, Jianzi; Lu, Xiang; Huang, Baiqu; Zheng, Yizhi

2013-04-24

The present study demonstrates a new Millettia pinnata chalcone isomerase (MpCHI) whose transcription level in leaf was confirmed to be enhanced after being treated by seawater or NaCl (500 mM) via transcriptome sequencing and Real-Time Quantitative Reverse Transcription PCR (QRT-PCR) analyses. Its full length cDNA (666 bp) was obtained by 3'-end and 5'-end Rapid Amplification of cDNA Ends (RACE). The analysis via NCBI BLAST indicates that both aminoacid sequence and nucleotide sequence of the MpCHI clone share high homology with other leguminous CHIs (73%-86%). Evolutionarily, the phylogenic analysis further revealed that the MpCHI is a close relative of leguminous CHIs. The MpCHI protein consists of 221 aminoacid (23.64 KDa), whose peptide length, amino acid residues of substrate-binding site and reactive site are very similar to other leguminous CHIs reported previously. Two pYES2-MpCHI transformed salt-sensitive Saccharomyces cerevisiae mutants (Δnha1 and Δnhx1) showed improved salt-tolerance significantly compared to pYES2-vector transformed yeast mutants, suggesting the MpCHI or the flavonoid biosynthesis pathway could regulate the resistance to salt stress in M. pinnata.

28 CFR 65.51 - Recordkeeping.

Science.gov (United States)

2010-07-01

... examination, to any books, documents, and records of recipients of Federal law enforcement assistance provided... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Recordkeeping. 65.51 Section 65.51 Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) EMERGENCY FEDERAL LAW ENFORCEMENT ASSISTANCE...

40 CFR 407.65 - [Reserved

Science.gov (United States)

2010-07-01

... 40 Protection of Environment 28 2010-07-01 2010-07-01 true [Reserved] 407.65 Section 407.65 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS CANNED AND PRESERVED FRUITS AND VEGETABLES PROCESSING POINT SOURCE CATEGORY Canned and Preserved Fruits...

28 CFR 65.52 - Civil rights.

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2010-07-01

... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Civil rights. 65.52 Section 65.52... Additional Requirements § 65.52 Civil rights. The Act provides that “no person in any state shall on the... funds under the Act are also subject to the provisions of title VI of the Civil Rights Act of 1964...

40 CFR 178.65 - Judicial review.

Science.gov (United States)

2010-07-01

... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Judicial review. 178.65 Section 178.65... REQUESTS FOR HEARINGS Judicial Review § 178.65 Judicial review. An order issued under § 178.37 is final... of the order in the Federal Register. The failure to file a petition for judicial review within the...

40 CFR 65.105 - Leak repair.

Science.gov (United States)

2010-07-01

... 40 Protection of Environment 15 2010-07-01 2010-07-01 false Leak repair. 65.105 Section 65.105... FEDERAL AIR RULE Equipment Leaks § 65.105 Leak repair. (a) Leak repair schedule. The owner or operator shall repair each leak detected as soon as practical but not later than 15 calendar days after it is...

Role of Loop-Clamping Side Chains in Catalysis by Triosephosphate Isomerase.

Science.gov (United States)

Zhai, Xiang; Amyes, Tina L; Richard, John P

2015-12-09

The side chains of Y208 and S211 from loop 7 of triosephosphate isomerase (TIM) form hydrogen bonds to backbone amides and carbonyls from loop 6 to stabilize the caged enzyme-substrate complex. The effect of seven mutations [Y208T, Y208S, Y208A, Y208F, S211G, S211A, Y208T/S211G] on the kinetic parameters for TIM catalyzed reactions of the whole substrates dihydroxyacetone phosphate and d-glyceraldehyde 3-phosphate [(k(cat)/K(m))(GAP) and (k(cat)/K(m))DHAP] and of the substrate pieces glycolaldehyde and phosphite dianion (k(cat)/K(HPi)K(GA)) are reported. The linear logarithmic correlation between these kinetic parameters, with slope of 1.04 ± 0.03, shows that most mutations of TIM result in an identical change in the activation barriers for the catalyzed reactions of whole substrate and substrate pieces, so that the transition states for these reactions are stabilized by similar interactions with the protein catalyst. The second linear logarithmic correlation [slope = 0.53 ± 0.16] between k(cat) for isomerization of GAP and K(d)(⧧) for phosphite dianion binding to the transition state for wildtype and many mutant TIM-catalyzed reactions of substrate pieces shows that ca. 50% of the wildtype TIM dianion binding energy, eliminated by these mutations, is expressed at the wildtype Michaelis complex, and ca. 50% is only expressed at the wildtype transition state. Negative deviations from this correlation are observed when the mutation results in a decrease in enzyme reactivity at the catalytic site. The main effect of Y208T, Y208S, and Y208A mutations is to cause a reduction in the total intrinsic dianion binding energy, but the effect of Y208F extends to the catalytic site.

Gibberellic Acid-Induced Aleurone Layers Responding to Heat Shock or Tunicamycin Provide Insight into the N-Glycoproteome, Protein Secretion, and Endoplasmic Reticulum Stress

DEFF Research Database (Denmark)

Barba Espin, Gregorio; Dedvisitsakul, Plaipol; Hägglund, Per

2014-01-01

respond to gibberellic acid by secreting an array of proteins and provide a unique system for the analysis of plant protein secretion. Perturbation of protein secretion in gibberellic acid-induced aleurone layers by two independent mechanisms, heat shock and tunicamycin treatment, demonstrated overlapping...... and secretion, such as calreticulin, protein disulfide isomerase, proteasome subunits, and isopentenyl diphosphate isomerase. Sixteen heat shock proteins in 29 spots showed diverse responses to the treatments, with only a minority increasing in response to heat shock. The majority, all of which were small heat...... shock proteins, decreased in heat-shocked aleurone layers. Additionally, glycopeptide enrichment and N-glycosylation analysis identified 73 glycosylation sites in 65 aleurone layer proteins, with 53 of the glycoproteins found in extracellular fractions and 36 found in intracellular fractions...

Styrene Oxide Isomerase of Rhodococcus opacus 1CP, a Highly Stable and Considerably Active Enzyme

Science.gov (United States)

Gröning, Janosch A. D.; Tischler, Dirk; Kaschabek, Stefan R.; Schlömann, Michael

2012-01-01

Styrene oxide isomerase (SOI) is involved in peripheral styrene catabolism of bacteria and converts styrene oxide to phenylacetaldehyde. Here, we report on the identification, enrichment, and biochemical characterization of a novel representative from the actinobacterium Rhodococcus opacus 1CP. The enzyme, which is strongly induced during growth on styrene, was shown to be membrane integrated, and a convenient procedure was developed to highly enrich the protein in active form from the wild-type host. A specific activity of about 370 U mg−1 represents the highest activity reported for this enzyme class so far. This, in combination with a wide pH and temperature tolerance, the independence from cofactors, and the ability to convert a spectrum of substituted styrene oxides, makes a biocatalytic application imaginable. First, semipreparative conversions were performed from which up to 760 μmol of the pure phenylacetaldehyde could be obtained from 130 U of enriched SOI. Product concentrations of up to 76 mM were achieved. However, due to the high chemical reactivity of the aldehyde function, SOI was shown to be the subject of an irreversible product inhibition. A half-life of 15 min was determined at a phenylacetaldehyde concentration of about 55 mM, indicating substantial limitations of applicability and the need to modify the process. PMID:22504818

Autoimmune gastro-pancreatitis with anti-protein disulfide isomerase-associated 2 autoantibody in Aire-deficient BALB/cAnN mice.

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Hironori Kurisaki

Full Text Available Although the autoimmune regulator (Aire knockout (KO mouse model has been reported to present various organ-specific autoimmune diseases depending on genetic background, autoimmune pancreatitis in mice of BALB/c background has not yet been reported. Here, we report that Aire KO mice with BALB/cAnN background showed significant lymphoid cell infiltration in the pancreas and stomach. To examine whether the phenotype in the pancreas and stomach is due to autoimmune reaction associated with autoantibody production, indirect immunofluorescence staining followed by Western blot analysis was performed. Consequently, the autoantibody against pancreas and stomach was detected in the sera of Aire KO mice, and the target antigen of the autoantibody was identified as protein disulfide isomerase-associated 2 (Pdia2, which was reported to be expressed preferentially in the pancreas and stomach. Thus, Aire KO mice of BALB/cAnN background can serve as a useful animal model for autoimmune gastro-pancreatitis with anti-Pdia2 autoantibody production.

Autoimmune gastro-pancreatitis with anti-protein disulfide isomerase-associated 2 autoantibody in Aire-deficient BALB/cAnN mice.

Science.gov (United States)

Kurisaki, Hironori; Nagao, Yukihiro; Nagafuchi, Seiho; Mitsuyama, Masao

2013-01-01

Although the autoimmune regulator (Aire) knockout (KO) mouse model has been reported to present various organ-specific autoimmune diseases depending on genetic background, autoimmune pancreatitis in mice of BALB/c background has not yet been reported. Here, we report that Aire KO mice with BALB/cAnN background showed significant lymphoid cell infiltration in the pancreas and stomach. To examine whether the phenotype in the pancreas and stomach is due to autoimmune reaction associated with autoantibody production, indirect immunofluorescence staining followed by Western blot analysis was performed. Consequently, the autoantibody against pancreas and stomach was detected in the sera of Aire KO mice, and the target antigen of the autoantibody was identified as protein disulfide isomerase-associated 2 (Pdia2), which was reported to be expressed preferentially in the pancreas and stomach. Thus, Aire KO mice of BALB/cAnN background can serve as a useful animal model for autoimmune gastro-pancreatitis with anti-Pdia2 autoantibody production.

D-Tagatose production in the presence of borate by resting Lactococcus lactis cells harboring Bifidobacterium longum L-arabinose isomerase.

Science.gov (United States)

Salonen, Noora; Salonen, Kalle; Leisola, Matti; Nyyssölä, Antti

2013-04-01

Bifidobacterium longum NRRL B-41409 L-arabinose isomerase (L-AI) was overexpressed in Lactococcus lactis using a phosphate depletion inducible expression system. The resting L. lactis cells harboring the B. longum L-AI were used for production of D-tagatose from D-galactose in the presence of borate buffer. Multivariable analysis suggested that high pH, temperature and borate concentration favoured the conversion of D-galactose to D-tagatose. Almost quantitative conversion (92 %) was achieved at 20 g L⁻¹ substrate and at 37.5 °C after 5 days. The D-tagatose production rate of 185 g L⁻¹ day ⁻¹ was obtained at 300 g L⁻¹ galactose, at 1.15 M borate, and at 41 °C during 10 days when the production medium was changed every 24 h. There was no significant loss in productivity during ten sequential 24 h batches. The initial D-tagatose production rate was 290 g L⁻¹ day⁻¹ under these conditions.

Structural insights into conserved L-arabinose metabolic enzymes reveal the substrate binding site of a thermophilic L-arabinose isomerase.

Science.gov (United States)

Lee, Yong-Jik; Lee, Sang-Jae; Kim, Seong-Bo; Lee, Sang Jun; Lee, Sung Haeng; Lee, Dong-Woo

2014-03-18

Structural genomics demonstrates that despite low levels of structural similarity of proteins comprising a metabolic pathway, their substrate binding regions are likely to be conserved. Herein based on the 3D-structures of the α/β-fold proteins involved in the ara operon, we attempted to predict the substrate binding residues of thermophilic Geobacillus stearothermophilus L-arabinose isomerase (GSAI) with no 3D-structure available. Comparison of the structures of L-arabinose catabolic enzymes revealed a conserved feature to form the substrate-binding modules, which can be extended to predict the substrate binding site of GSAI (i.e., D195, E261 and E333). Moreover, these data implicated that proteins in the l-arabinose metabolic pathway might retain their substrate binding niches as the modular structure through conserved molecular evolution even with totally different structural scaffolds. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

TM0416, a Hyperthermophilic Promiscuous Nonphosphorylated Sugar Isomerase, Catalyzes Various C5 and C6 Epimerization Reactions.

Science.gov (United States)

Shin, Sun-Mi; Cao, Thinh-Phat; Choi, Jin Myung; Kim, Seong-Bo; Lee, Sang-Jae; Lee, Sung Haeng; Lee, Dong-Woo

2017-05-15

There is currently little information on nonphosphorylated sugar epimerases, which are of potential interest for producing rare sugars. We found a gene (the TM0416 gene) encoding a putative d-tagatose-3-epimerase-related protein from the hyperthermophilic bacterium Thermotoga maritima We overexpressed the TM0416 gene in Escherichia coli and purified the resulting recombinant protein for detailed characterization. Amino acid sequence alignment and a structural similarity search revealed that TM0416 is a putative nonphosphorylated sugar epimerase. The recombinant enzyme exhibited maximal C-3 epimerization of l-ribulose to l-xylulose at ∼80°C and pH 7 in the presence of 1 mM Mn 2+ In addition, this enzyme showed unusually high activity for the epimerization of d-tagatose to d-sorbose, with a conversion yield of 20% after 6 h at 80°C. Remarkably, the enzyme catalyzed the isomerization of d-erythrose or d-threose to d-erythrulose significantly, with conversion yields of 71% and 54.5%, respectively, after 6 h at 80°C at pH 7. To further investigate the substrate specificity of TM0416, we determined its crystal structures in complex with divalent metal ions and l-erythrulose at resolutions of 1.5 and 1.6 Å. Detailed inspection of the structural features and biochemical data clearly demonstrated that this metalloenzyme, with a freely accessible substrate-binding site and neighboring hydrophobic residues, exhibits different and promiscuous substrate preferences, compared with its mesophilic counterparts. Therefore, this study suggests that TM0416 can be functionally classified as a novel type of l-ribulose 3-epimerase (R3E) with d-erythrose isomerase activity. IMPORTANCE Rare sugars, which occur naturally in small amounts, have attracted considerable attention in the food and drug industries. However, there is little information on nonphosphorylated sugar epimerases, which might potentially be applied for the production of rare sugars. This study describes the

Involvement of the Retinoid X Receptor Ligand in the Anti-Inflammatory Effect Induced by Peroxisome Proliferator-Activated Receptor Agonist In Vivo

Directory of Open Access Journals (Sweden)

Atsuki Yamamoto

2011-01-01

Full Text Available Peroxisome proliferator-activated receptor γ (PPARγ forms a heterodimeric DNA-binding complex with retinoid X receptors (RXRs. It has been reported that the effect of the PPAR agonist is reduced in hepatocyte RXR-deficient mice. Therefore, it is suggested that the endogenous RXR ligand is involved in the PPARγ agonist-induced anti-inflammatory effect. However, the participation of the RXR ligand in the PPARγ-induced anti-inflammatory effect is unknown. Here, we investigated the influence of RXR antagonist on the anti-inflammatory effect of PPARγ agonist pioglitazone in carrageenan test. In addition, we also examined the influence of PPAR antagonist on the anti-inflammatory effect induced by RXR agonist NEt-3IP. The RXR antagonist suppressed the antiedema effect of PPARγ agonist. In addition, the anti-inflammatory effect of RXR agonist was suppressed by PPARγ antagonist. PPARγ agonist-induced anti-inflammatory effects were reversed by the RXR antagonist. Thus, we showed that the endogenous RXR ligand might contribute to the PPARγ agonist-induced anti-inflammatory effect.

Increasing human Th17 differentiation through activation of orphan nuclear receptor retinoid acid-related orphan receptor γ (RORγ) by a class of aryl amide compounds.

Science.gov (United States)

Zhang, Wei; Zhang, Jing; Fang, Leiping; Zhou, Ling; Wang, Shuai; Xiang, Zhijun; Li, Yuan; Wisely, Bruce; Zhang, Guifeng; An, Gang; Wang, Yonghui; Leung, Stewart; Zhong, Zhong

2012-10-01

In a screen for small-molecule inhibitors of retinoid acid-related orphan receptor γ (RORγ), we fortuitously discovered that a class of aryl amide compounds behaved as functional activators of the interleukin 17 (IL-17) reporter in Jurkat cells. Three of these compounds were selected for further analysis and found to activate the IL-17 reporter with potencies of ∼0.1 μM measured by EC₅₀. These compounds were shown to directly bind to RORγ by circular dichroism-based thermal stability experiments. Furthermore, they can enhance an in vitro Th17 differentiation process in human primary T cells. As RORγ remains an orphan nuclear receptor, discovery of these aryl amide compounds as functional agonists will now provide pharmacological tools for us to dissect functions of RORγ and facilitate drug discovery efforts for immune-modulating therapies.

Novel 9-cis/all-trans β-carotene isomerases from plastidic oil bodies in Dunaliella bardawil catalyze the conversion of all-trans to 9-cis β-carotene.

Science.gov (United States)

Davidi, Lital; Pick, Uri

2017-06-01

We identified and demonstrated the function of 9-cis/all-trans β-carotene isomerases in plastidic globules of Dunaliella bardawil, the species accumulating the highest levels of 9-cis β-carotene that is essential for humans. The halotolerant alga Dunaliella bardawil is unique in that it accumulates under light stress high levels of β-carotene in plastidic lipid globules. The pigment is composed of two major isomers: all-trans β-carotene, the common natural form of this pigment, and 9-cis β-carotene. The biosynthetic pathway of β-carotene is known, but it is not clear how the 9-cis isomer is formed. We identified in plastidic lipid globules that were isolated from D. bardawil two proteins with high sequence homology to the D27 protein-a 9-cis/all-trans β-carotene isomerase from rice (Alder et al. Science 335:1348-1351, 2012). The proteins are enriched in the oil globules by 6- to 17-fold compared to chloroplast proteins. The expression of the corresponding genes, 9-cis-βC-iso1 and 9-cis-βC-iso2, is enhanced under light stress. The synthetic proteins catalyze in vitro conversion of all-trans to 9-cis β-carotene. Expression of the 9-cis-βC-iso1 or of 9-cis-βC-iso2 genes in an E. coli mutant line that harbors β-carotene biosynthesis genes enhanced the conversion of all-trans into 9-cis β-carotene. These results suggest that 9-cis-βC-ISO1 and 9-cis-βC-ISO2 proteins are responsible for the formation of 9-cis β-carotene in D. bardawil under stress conditions.

Detection of GAD65 autoantibodies of type-1 diabetes using anti-GAD65-abs reagent produced from bovine brain tissue

Directory of Open Access Journals (Sweden)

Djoko W. Soeatmadji

2005-12-01

Full Text Available Clinically, type 1 diabetes may presents as type 2 diabetes which sometimes not easily differentiated. Perhaps only autoimmune markers of β-cells destruction could differentiate those two clinical conditions. Due to extremely high cost ( $ 150/test, examination of anti-glutamic acid decarboxylase-65 auto-antibodies (anti-GAD65Abs may not be routinely performed in most, if not all, clinical laboratories in Indonesia. Hence, the production of anti-GAD65 Abs reagent in Indonesia may reduce the cost and improve the quality of diabetes care in Indonesia. We produce reagent to detect anti-GAD65-Abs using bovine brain tissue as source of GAD enzyme in 3 steps. Step 1, isolation, purification of GAD65 from bovine brain tissue and used it as a primary antigen to stimulate the generation of anti-GAD65 antibodies in Wistar rat. Step 2, the purified GAD65 antibodies were than used as a secondary antibody to induce the production of anti-anti-GAD65-antibodies in Wistar rat and rabbit. Step 3. Labeling  anti-anti GAD65-antibodies with alkaline phoshpatase and peroxidase, and detecting anti-GAD65Abs previously detected using commercial kit. The anti-anti-GAD65- antibodies reagent produced in our laboratories  successfully identify anti-GAD65-Abs of type 1 diabetic patients previously detected  with commercial reagent. (Med J Indones 2005; 14: 197-203Keywords: GAD, type-1 Diabetes

The use of phosphomannose isomerase selection system for Agrobacterium-mediated transformation of tobacco and flax aimed for phytoremediation.

Science.gov (United States)

Hilgert, Jitka; Sura-De Jong, Martina; Fišer, Jiří; Tupá, Kateřina; Vrbová, Miroslava; Griga, Miroslav; Macek, Tomáš; Žiarovská, Jana

2017-05-04

A plant selection system based on the phosphomannose isomerase gene (pmi) as a selectable marker is often used to avoid selection using antibiotic resistance. Nevertheless, pmi gene is endogenous in several plant species and therefore difficult to use in such cases. Here we evaluated and compared Agrobacterium-mediated transformation of Linum usitatissimum breeding line AGT-952 (without endogenous pmi gene) and Nicotiana tabacum var. WSC-38 (with endogenous pmi gene). Transformation was evaluated for vectors bearing transgenes that have the potential to be involved in improved phytoremediation of contaminated environment. Tobacco regenerants selection resulted in 6.8% transformation efficiency when using a medium supplemented with 30 g/L mannose with stepwise decrease of the sucrose concentration. Similar transformation efficiency (5.3%) was achieved in transformation of flax. Relatively low selection efficiency was achieved (12.5% and 34.8%, respectively). The final detection of efficient pmi selection was conducted using PCR and the non-endogenous genes; pmi transgene for flax and todC2 transgene for tobacco plants.

Structural and Genetic Studies Demonstrate Neurologic Dysfunction in Triosephosphate Isomerase Deficiency Is Associated with Impaired Synaptic Vesicle Dynamics

Energy Technology Data Exchange (ETDEWEB)

Roland, Bartholomew P.; Zeccola, Alison M.; Larsen, Samantha B.; Amrich, Christopher G.; Talsma, Aaron D.; Stuchul, Kimberly A.; Heroux, Annie; Levitan, Edwin S.; VanDemark, Andrew P.; Palladino, Michael J.; Pallanck, Leo J.

2016-03-31

Triosephosphate isomerase (TPI) deficiency is a poorly understood disease characterized by hemolytic anemia, cardiomyopathy, neurologic dysfunction, and early death. TPI deficiency is one of a group of diseases known as glycolytic enzymopathies, but is unique for its severe patient neuropathology and early mortality. The disease is caused by missense mutations and dysfunction in the glycolytic enzyme, TPI. Previous studies have detailed structural and catalytic changes elicited by disease-associated TPI substitutions, and samples of patient erythrocytes have yielded insight into patient hemolytic anemia; however, the neuropathophysiology of this disease remains a mystery. This study combines structural, biochemical, and genetic approaches to demonstrate that perturbations of the TPI dimer interface are sufficient to elicit TPI deficiency neuropathogenesis. The present study demonstrates that neurologic dysfunction resulting from TPI deficiency is characterized by synaptic vesicle dysfunction, and can be attenuated with catalytically inactive TPI. Collectively, our findings are the first to identify, to our knowledge, a functional synaptic defect in TPI deficiency derived from molecular changes in the TPI dimer interface.

Techniques for intrinsically labeling wheat with 65Zn

International Nuclear Information System (INIS)

Starks, T.L.; Johnson, P.E.

1985-01-01

Several techniques of intrinsically labeling wheat with 65 Zn were compared: stem injection of 65 Zn, stem injection of 65 Zn + ZnSO 4 , foliar application of 65 Zn, and the addition of 65 Zn to a hydroponic solution. Incorporation levels of 65 Zn into the grain were 62.6% stem injection, 45.2% stem-injected 65 Zn + ZnSO 4 , 57.5% foliar application, and 2.3% hydroponic solution. Four protein fractions were extracted from fat-free whole wheat flour. Distribution of 65 Zn into the protein fractions for all treatments, was 8.5-20.3% in albumins and globulins, 47.4-60.3% in glutenins, 1-2.6% in gliadins, and 9.8-28.3% in the remaining proteins. Separation of the fractions by gel chromatography showed that protein and Zn distributions were similar among the treatments and when compared to the controls. Zinc-65 distribution was similar to the natural Zn distribution. These data illustrate that stem-injected 65 Zn is incorporated in the same manner and ratios as Zn naturally utilized by wheat

Ribose catabolism of Escherichia coli: characterization of the rpiB gene encoding ribose phosphate isomerase B and of the rpiR gene, which is involved in regulation of rpiB expression

DEFF Research Database (Denmark)

Sørensen, Kim I.; Hove-Jensen, Bjarne

1996-01-01

. The rpiB gene resided on a 4.6-kbp HindIII-EcoRV DNA fragment from phage lambda 10H5 (642) of the Kohara gene library and mapped at 92.85 min. Consistent with this map position, the cloned DNA fragment contained two divergent open reading frames of 149 and 296 codons, encoding ribose phosphate isomerase B...

Phycoerythrin-specific bilin lyase-isomerase controls blue-green chromatic acclimation in marine Synechococcus.

Science.gov (United States)

Shukla, Animesh; Biswas, Avijit; Blot, Nicolas; Partensky, Frédéric; Karty, Jonathan A; Hammad, Loubna A; Garczarek, Laurence; Gutu, Andrian; Schluchter, Wendy M; Kehoe, David M

2012-12-04

The marine cyanobacterium Synechococcus is the second most abundant phytoplanktonic organism in the world's oceans. The ubiquity of this genus is in large part due to its use of a diverse set of photosynthetic light-harvesting pigments called phycobiliproteins, which allow it to efficiently exploit a wide range of light colors. Here we uncover a pivotal molecular mechanism underpinning a widespread response among marine Synechococcus cells known as "type IV chromatic acclimation" (CA4). During this process, the pigmentation of the two main phycobiliproteins of this organism, phycoerythrins I and II, is reversibly modified to match changes in the ambient light color so as to maximize photon capture for photosynthesis. CA4 involves the replacement of three molecules of the green light-absorbing chromophore phycoerythrobilin with an equivalent number of the blue light-absorbing chromophore phycourobilin when cells are shifted from green to blue light, and the reverse after a shift from blue to green light. We have identified and characterized MpeZ, an enzyme critical for CA4 in marine Synechococcus. MpeZ attaches phycoerythrobilin to cysteine-83 of the α-subunit of phycoerythrin II and isomerizes it to phycourobilin. mpeZ RNA is six times more abundant in blue light, suggesting that its proper regulation is critical for CA4. Furthermore, mpeZ mutants fail to normally acclimate in blue light. These findings provide insights into the molecular mechanisms controlling an ecologically important photosynthetic process and identify a unique class of phycoerythrin lyase/isomerases, which will further expand the already widespread use of phycoerythrin in biotechnology and cell biology applications.

14 CFR 65.19 - Retesting after failure.

Science.gov (United States)

2010-01-01

... 14 Aeronautics and Space 2 2010-01-01 2010-01-01 false Retesting after failure. 65.19 Section 65.19 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AIRMEN CERTIFICATION: AIRMEN OTHER THAN FLIGHT CREWMEMBERS General § 65.19 Retesting after failure. An...

Production of L-allose and D-talose from L-psicose and D-tagatose by L-ribose isomerase.

Science.gov (United States)

Terami, Yuji; Uechi, Keiko; Nomura, Saki; Okamoto, Naoki; Morimoto, Kenji; Takata, Goro

2015-01-01

L-ribose isomerase (L-RI) from Cellulomonas parahominis MB426 can convert L-psicose and D-tagatose to L-allose and D-talose, respectively. Partially purified recombinant L-RI from Escherichia coli JM109 was immobilized on DIAION HPA25L resin and then utilized to produce L-allose and D-talose. Conversion reaction was performed with the reaction mixture containing 10% L-psicose or D-tagatose and immobilized L-RI at 40 °C. At equilibrium state, the yield of L-allose and D-talose was 35.0% and 13.0%, respectively. Immobilized enzyme could convert L-psicose to L-allose without remarkable decrease in the enzyme activity over 7 times use and D-tagatose to D-talose over 37 times use. After separation and concentration, the mixture solution of L-allose and D-talose was concentrated up to 70% and crystallized by keeping at 4 °C. L-Allose and d-talose crystals were collected from the syrup by filtration. The final yield was 23.0% L-allose and 7.30% D-talose that were obtained from L-psicose and D-tagatose, respectively.

The Role of S-Nitrosylation and S-Glutathionylation of Protein Disulphide Isomerase in Protein Misfolding and Neurodegeneration

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M. Halloran

2013-01-01

Full Text Available Neurodegenerative diseases involve the progressive loss of neurons, and a pathological hallmark is the presence of abnormal inclusions containing misfolded proteins. Although the precise molecular mechanisms triggering neurodegeneration remain unclear, endoplasmic reticulum (ER stress, elevated oxidative and nitrosative stress, and protein misfolding are important features in pathogenesis. Protein disulphide isomerase (PDI is the prototype of a family of molecular chaperones and foldases upregulated during ER stress that are increasingly implicated in neurodegenerative diseases. PDI catalyzes the rearrangement and formation of disulphide bonds, thus facilitating protein folding, and in neurodegeneration may act to ameliorate the burden of protein misfolding. However, an aberrant posttranslational modification of PDI, S-nitrosylation, inhibits its protective function in these conditions. S-nitrosylation is a redox-mediated modification that regulates protein function by covalent addition of nitric oxide- (NO- containing groups to cysteine residues. Here, we discuss the evidence for abnormal S-nitrosylation of PDI (SNO-PDI in neurodegeneration and how this may be linked to another aberrant modification of PDI, S-glutathionylation. Understanding the role of aberrant S-nitrosylation/S-glutathionylation of PDI in the pathogenesis of neurodegenerative diseases may provide insights into novel therapeutic interventions in the future.

The acid tolerant L-arabinose isomerase from the food grade Lactobacillus sakei 23K is an attractive D-tagatose producer.

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Rhimi, Moez; Ilhammami, Rimeh; Bajic, Goran; Boudebbouze, Samira; Maguin, Emmanuelle; Haser, Richard; Aghajari, Nushin

2010-12-01

The araA gene encoding an L-arabinose isomerase (L-AI) from the psychrotrophic and food grade Lactobacillus sakei 23K was cloned, sequenced and over-expressed in Escherichia coli. The recombinant enzyme has an apparent molecular weight of nearly 220 kDa, suggesting it is a tetramer of four 54 kDa monomers. The enzyme is distinguishable from previously reported L-AIs by its high activity and stability at temperatures from 4 to 40 degrees C, and pH from 3 to 8, and by its low metal requirement of only 0.8 mM Mn(2+) and 0.8 mM Mg(2+) for its maximal activity and thermostability. Enzyme kinetic studies showed that this enzyme displays a high catalytic efficiency allowing D-galactose bioconversion rates of 20% and 36% at 10 and 45 degrees C, respectively, which are useful for commercial production of D-tagatose. 2010 Elsevier Ltd. All rights reserved.

Peptidyl prolyl isomerase Pin1-inhibitory activity of D-glutamic and D-aspartic acid derivatives bearing a cyclic aliphatic amine moiety.

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Nakagawa, Hidehiko; Seike, Suguru; Sugimoto, Masatoshi; Ieda, Naoya; Kawaguchi, Mitsuyasu; Suzuki, Takayoshi; Miyata, Naoki

2015-12-01

Pin1 is a peptidyl prolyl isomerase that specifically catalyzes cis-trans isomerization of phosphorylated Thr/Ser-Pro peptide bonds in substrate proteins and peptides. Pin1 is involved in many important cellular processes, including cancer progression, so it is a potential target of cancer therapy. We designed and synthesized a novel series of Pin1 inhibitors based on a glutamic acid or aspartic acid scaffold bearing an aromatic moiety to provide a hydrophobic surface and a cyclic aliphatic amine moiety with affinity for the proline-binding site of Pin1. Glutamic acid derivatives bearing cycloalkylamino and phenylthiazole groups showed potent Pin1-inhibitory activity comparable with that of known inhibitor VER-1. The results indicate that steric interaction of the cyclic alkyl amine moiety with binding site residues plays a key role in enhancing Pin1-inhibitory activity. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Isolation, Characterization, and Establishment of Spontaneously Immortalized Cell Line HRPE-2S With Stem Cell Properties.

Science.gov (United States)

Shams Najafabadi, Hoda; Soheili, Zahra-Soheila; Samiei, Shahram; Ahmadieh, Hamid; Ranaei Pirmardan, Ehsan; Masoumi, Maryam

2017-10-01

The retinal pigment epithelium is a monolayer of highly specialized pigmented cells located between the neural retina and the Bruch's membrane of the choroid. RPE cells play a crucial role in the maintenance and function of the underlying photoreceptors. This study introduces a spontaneously arising human retinal pigment epithelial cell line, HRPE-2S, which was isolated from primary RPE cell culture of 2 days old male donor. We characterized morphology and functional properties of the new cell line. The immortalized cell line was maintained in culture for more than 70 passages and 240 divisions. The average doubling time of the cells was approximately 22 h and got freezed at 26th passage. The cell line expressed RPE-specific markers RPE65 and cell junction protein ZO1 as an epithelial cell marker. It also expressed CHX10, PAX6, Nestin, SOX2 as stem and retinal progenitor cell markers. Ki67 as a marker of cell proliferation was expressed in all HRPE-2S cells. It represented typical epithelial cobblestone morphology and did not phenotypically change through several passages. Stem cell-like aggregations (neurospheres) were observed in SEM microscopy. The cells represented high mitotic index. They could be viable under hypoxic conditions and serum deprivation. According to functional studies, the cell line exhibited stem cell-like behaviors with particular emphasis on its self-renewal capacity. LDH isoenzymes expression pattern confirmed the same cellular source for both of the HRPE-2S cells and primary RPE cells. Characteristics of HRPE-2S cells promise it as an in vitro model for RPE stem cell-based researches. J. Cell. Physiol. 232: 2626-2640, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

7 CFR 65.140 - Food service establishment.

Science.gov (United States)

2010-01-01

... retail establishments that provide ready-to-eat foods that are consumed either on or outside of the... 7 Agriculture 3 2010-01-01 2010-01-01 false Food service establishment. 65.140 Section 65.140..., PEANUTS, AND GINSENG General Provisions Definitions § 65.140 Food service establishment. Food service...

24 CFR 234.65 - Nature of title.

Science.gov (United States)

2010-04-01

... 24 Housing and Urban Development 2 2010-04-01 2010-04-01 false Nature of title. 234.65 Section 234.65 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued... OWNERSHIP MORTGAGE INSURANCE Eligibility Requirements-Individually Owned Units § 234.65 Nature of title. A...

The Expression of Millettia pinnata Chalcone Isomerase in Saccharomyces cerevisiae Salt-Sensitive Mutants Enhances Salt-Tolerance

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Baiqu Huang

2013-04-01

Full Text Available The present study demonstrates a new Millettia pinnata chalcone isomerase (MpCHI whose transcription level in leaf was confirmed to be enhanced after being treated by seawater or NaCl (500 mM via transcriptome sequencing and Real-Time Quantitative Reverse Transcription PCR (QRT-PCR analyses. Its full length cDNA (666 bp was obtained by 3'-end and 5'-end Rapid Amplification of cDNA Ends (RACE. The analysis via NCBI BLAST indicates that both aminoacid sequence and nucleotide sequence of the MpCHI clone share high homology with other leguminous CHIs (73%–86%. Evolutionarily, the phylogenic analysis further revealed that the MpCHI is a close relative of leguminous CHIs. The MpCHI protein consists of 221 aminoacid (23.64 KDa, whose peptide length, amino acid residues of substrate-binding site and reactive site are very similar to other leguminous CHIs reported previously. Two pYES2-MpCHI transformed salt-sensitive Saccharomyces cerevisiae mutants (Δnha1 and Δnhx1 showed improved salt-tolerance significantly compared to pYES2-vector transformed yeast mutants, suggesting the MpCHI or the flavonoid biosynthesis pathway could regulate the resistance to salt stress in M. pinnata.

Crystal structure and enzymatic properties of chalcone isomerase from the Antarctic vascular plant Deschampsia antarctica Desv.

Science.gov (United States)

Park, Sun-Ha; Lee, Chang Woo; Cho, Sung Mi; Lee, Hyoungseok; Park, Hyun; Lee, Jungeun; Lee, Jun Hyuck

2018-01-01

Chalcone isomerase (CHI) is an important enzyme for flavonoid biosynthesis that catalyzes the intramolecular cyclization of chalcones into (S)-flavanones. CHIs have been classified into two types based on their substrate specificity. Type I CHIs use naringenin chalcone as a substrate and are found in most of plants besides legumes, whereas type II CHIs in leguminous plants can also utilize isoliquiritigenin. In this study, we found that the CHI from the Antarctic plant Deschampsia antarctica (DaCHI1) is of type I based on sequence homology but can use type II CHI substrates. To clarify the enzymatic mechanism of DaCHI1 at the molecular level, the crystal structures of unliganded DaCHI1 and isoliquiritigenin-bound DaCHI1 were determined at 2.7 and 2.1 Å resolutions, respectively. The structures revealed that isoliquiritigenin binds to the active site of DaCHI1 and induces conformational changes. Additionally, the activity assay showed that while DaCHI1 exhibits substrate preference for naringenin chalcone, it can also utilize isoliquiritigenin although the catalytic activity was relatively low. Based on these results, we propose that DaCHI1 uses various substrates to produce antioxidant flavonoids as an adaptation to oxidative stresses associated with harsh environmental conditions.

Crystal structure and enzymatic properties of chalcone isomerase from the Antarctic vascular plant Deschampsia antarctica Desv.

Directory of Open Access Journals (Sweden)

Sun-Ha Park

Full Text Available Chalcone isomerase (CHI is an important enzyme for flavonoid biosynthesis that catalyzes the intramolecular cyclization of chalcones into (S-flavanones. CHIs have been classified into two types based on their substrate specificity. Type I CHIs use naringenin chalcone as a substrate and are found in most of plants besides legumes, whereas type II CHIs in leguminous plants can also utilize isoliquiritigenin. In this study, we found that the CHI from the Antarctic plant Deschampsia antarctica (DaCHI1 is of type I based on sequence homology but can use type II CHI substrates. To clarify the enzymatic mechanism of DaCHI1 at the molecular level, the crystal structures of unliganded DaCHI1 and isoliquiritigenin-bound DaCHI1 were determined at 2.7 and 2.1 Å resolutions, respectively. The structures revealed that isoliquiritigenin binds to the active site of DaCHI1 and induces conformational changes. Additionally, the activity assay showed that while DaCHI1 exhibits substrate preference for naringenin chalcone, it can also utilize isoliquiritigenin although the catalytic activity was relatively low. Based on these results, we propose that DaCHI1 uses various substrates to produce antioxidant flavonoids as an adaptation to oxidative stresses associated with harsh environmental conditions.

Peptidyl Prolyl Isomerase PIN1 Directly Binds to and Stabilizes Hypoxia-Inducible Factor-1α.

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Hyeong-Jun Han

Full Text Available Peptidyl prolyl isomerase (PIN1 regulates the functional activity of a subset of phosphoproteins through binding to phosphorylated Ser/Thr-Pro motifs and subsequently isomerization of the phosphorylated bonds. Interestingly, PIN1 is overexpressed in many types of malignancies including breast, prostate, lung and colon cancers. However, its oncogenic functions have not been fully elucidated. Here, we report that PIN1 directly interacts with hypoxia-inducible factor (HIF-1α in human colon cancer (HCT116 cells. PIN1 binding to HIF-1α occurred in a phosphorylation-dependent manner. We also found that PIN1 interacted with HIF-1α at both exogenous and endogenous levels. Notably, PIN1 binding stabilized the HIF-1α protein, given that their levels were significantly increased under hypoxic conditions. The stabilization of HIF-1α resulted in increased transcriptional activity, consequently upregulating expression of vascular endothelial growth factor, a major contributor to angiogenesis. Silencing of PIN1 or pharmacologic inhibition of its activity abrogated the angiogenesis. By utilizing a bioluminescence imaging technique, we were able to demonstrate that PIN1 inhibition dramatically reduced the tumor volume in a subcutaneous mouse xenograft model and angiogenesis as well as hypoxia-induced transcriptional activity of HIF-1α. These results suggest that PIN1 interacting with HIF-1α is a potential cancer chemopreventive and therapeutic target.

7 CFR 29.65 - Accessibility of tobacco.

Science.gov (United States)

2010-01-01

... 7 Agriculture 2 2010-01-01 2010-01-01 false Accessibility of tobacco. 29.65 Section 29.65... REGULATIONS TOBACCO INSPECTION Regulations Permissive Inspection § 29.65 Accessibility of tobacco. All tobacco... characteristics or for drawing of samples. In the case of tobacco in packages, the coverings shall be removed by...

Antagonist effect of triptolide on AKT activation by truncated retinoid X receptor-alpha.

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Na Lu

Full Text Available BACKGROUND: Retinoid X receptor-alpha (RXRα is a key member of the nuclear receptor superfamily. We recently demonstrated that proteolytic cleavage of RXRα resulted in production of a truncated product, tRXRα, which promotes cancer cell survival by activating phosphatidylinositol-3-OH kinase (PI3K/AKT pathway. However, how the tRXRα-mediated signaling pathway in cancer cells is regulated remains elusive. METHODOLOGY/PRINCIPAL FINDINGS: We screened a natural product library for tRXRα targeting leads and identified that triptolide, an active component isolated from traditional Chinese herb Trypterygium wilfordii Hook F, could modulate tRXRα-mediated cancer cell survival pathway in vitro and in animals. Our results reveal that triptolide strongly induces cancer cell apoptosis dependent on intracellular tRXRα expression levels, demonstrating that tRXRα serves as an important intracellular target of triptolide. We show that triptolide selectively induces tRXRα degradation and inhibits tRXRα-dependent AKT activity without affecting the full-length RXRα. Interestingly, such effects of triptolide are due to its activation of p38. Although triptolide also activates Erk1/2 and MAPK pathways, the effects of triptolide on tRXRα degradation and AKT activity are only reversed by p38 siRNA and p38 inhibitor. In addition, the p38 inhibitor potently inhibits tRXRα interaction with p85α leading to AKT inactivation. Our results demonstrate an interesting novel signaling interplay between p38 and AKT through tRXRα mediation. We finally show that targeting tRXRα by triptolide strongly activates TNFα death signaling and enhances the anticancer activity of other chemotherapies. CONCLUSIONS/SIGNIFICANCE: Our results identify triptolide as a new xenobiotic regulator of the tRXRα-dependent survival pathway and provide new insight into the mechanism by which triptolide acts to induce apoptosis of cancer cells. Triptolide represents one of the most

42 CFR 409.65 - Lifetime reserve days.

Science.gov (United States)

2010-10-01

... HOSPITAL INSURANCE BENEFITS Scope of Hospital Insurance Benefits § 409.65 Lifetime reserve days. (a... private insurance coverage that begins after the first 90 inpatient days in a benefit period, or if the... 42 Public Health 2 2010-10-01 2010-10-01 false Lifetime reserve days. 409.65 Section 409.65 Public...

46 CFR 98.25-65 - Filling density.

Science.gov (United States)

2010-10-01

... 46 Shipping 4 2010-10-01 2010-10-01 false Filling density. 98.25-65 Section 98.25-65 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) CARGO AND MISCELLANEOUS VESSELS SPECIAL... § 98.25-65 Filling density. (a) The filling density, or the percent ratio of the liquefied gas that may...

Obtaining a citric tristeza virus p65 protein antibody and preliminary results of p65 in vivo expression

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Yanneth Torres

2003-07-01

Full Text Available The citric tristeza virus (CTV belongs to the Closteroviridae family which indudes the only vegetal viruses possessing genes homologous to HSP70 thermal cellular shock proteins in their genome. Such is the case of the gene encoding for the CTV p65 protein which presents high homology with the HSP70 protein family. It has been shown recently that HSP70h viral proteins (such as CTV p65 are involved both in viral assembly, as a microtubule binding protein, and in cell-cell movement. Since CTV is the most deleterious citrus pathogen, understanding this protein's role in the pathogenesis process is important. Rabbits were immunised with four synthetic peptides (corresponding to CTV p65 thermal shock protein's carboxyl-terminal region to obtain polyclonal antibodies. All the peptides used were immunogenic, even though two of them showed greater response. Whilst none of the antibodies obtained reacted to non-infected plant extract, the p65 proteins was detected in extracts taken from citric plants infected with CTV Based on the antibody's reaction to two Colombian isolates having different serological characteristics, the p65 antibody's immunological behaviour appeared to be independent of the symptomatic severity of the CTV isolates. It was shown that the ORF encoded for the HSP70 homologue in CTV was expressed in vivo, even though the p65 antibody was only detected in concentrated protein extracts taken from infected plants, supporting reports from other studies that the concentration of this protein in plants infected with CTV is low. This is the first time that a polyclonal CTV antibody has been obtained in Colombia against p65 (a protein intervening in viral assembly and movement. Adapting a technique for obtaining p65 antibodies by using synthetic peptides as immunogens could be useful in the future for detecting or diagnosing p65 proteins present in different Colombian CTV isolates, especially in developing studies contributing towards greater

Identification of the 11-cis-specific retinyl-ester synthase in retinal Müller cells as multifunctional O-acyltransferase (MFAT)

Science.gov (United States)