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Sample records for 5-aminolevulinic acid dehydratase

  1. Inhibitory Effect of Gabaculine on 5-Aminolevulinate Dehydratase Activity in Radish Seedlings 1

    PubMed Central

    Tchuinmogne, Simo J.; Huault, Claude; Aoues, Abdelkader; Balangé, Alain P.

    1989-01-01

    We have compared the activity of 5-aminolevulinate dehydratase (5-ALAD) with the amount of protein detected by specific antibodies in rocket immunoelectrophoresis. Parallel kinetic evolutions of enzymic activity and amount of antigen were observed in radish (Raphanus sativus L.) cotyledons, both in complete darkness or under standard far red light involving phytochrome. However, the treatment of seedlings with gabaculine leads to an important decrease in enzymic activity, while the specific protein content is maintained. This inhibition is not overcome by the addition of glutamic acid, but by 5-aminolevulinic acid which points to a specific control of 5-ALAD activity by its substrate. As there is no discrepancy between the enzymic activity and the amount of antigen during the time course development of seedlings, this could confirm a coordinate cellular control between 5-aminolevulinic acid formation and 5-ALAD protein synthesis, both being amplified by the action of phytochrome. PMID:16666925

  2. Characterization of 5-aminolevulinate synthase from Agrobacterium radiobacter, screening new inhibitors for 5-aminolevulinate dehydratase from Escherichia coli and their potential use for high 5-aminolevulinate production.

    PubMed

    Lin, Jianping; Fu, Weiqi; Cen, Peilin

    2009-04-01

    The hemA gene encoding 5-aminolevulinate synthase (ALAS) from Agrobacterium radiobacter zju-0121 showed 92.6% homology with that from A. radiobacter ATCC4718 and contained several rare codons. To enhance the expression of this gene, Escherichia coli Rosetta(DE3), which is a rare codon optimizer strain, was used as the host to construct an efficient recombinant strain. And the encoded protein was over-expressed as fusion protein and was purified by affinity purification on Ni-NTA agarose and by gel filtration chromatography on Sephadex G-25 Medium resin. The recombinant protein was partly characterized, and D-glucose, D-fructose, D-xylose, D-mannose, L-arabinose, D-galactose, lactose, sucrose and maltose were detected to have no distinct inhibition on this recombinant ALAS. Meanwhile, 20mM D-glucose or D-xylose inhibited about 20% activity of ALA dehydratase (ALAD) from Escherichia coli Rosetta(DE3). Combining D-xylose as a new inhibitor for ALAD with D-glucose in fed-batch culture and based on the optimal culture system using Rosetta(DE3)/pET28a-hemA, the yield of ALA achieved was 7.3g/l (56 mM) under the appropriate conditions in the fermenter.

  3. Photodynamic therapy with 5-aminolevulinic acid: basic principles and applications

    NASA Astrophysics Data System (ADS)

    Pottier, Roy H.; Kennedy, James C.

    1996-01-01

    Numerous photosensitizing pigments that absorb visible light and are selectively retained in neoplastic tissue are being investigated as potential photochemotherapeutic agents. While much emphasis is being placed on the synthesis of new, far-red absorbing photosensitizers, an alternative approach has been to stimulate the human body to produce its own natural photosensitizer, namely protoporphyrin IX (PpIX). Exogenous 5-aminolevulinic acid (ALA) is rapidly bioconverted into PP by mitochondria, the process being particularly efficient in tumor cells. Since PpIX has a natural and rapid clearing mechanism (via the capture of iron in the process of being converted into heme), ALA-PDT does not suffer from lingering skin phototoxicity. ALA may be introduced orally, intravenously, or topically, and ALA-PDT has been shown to be effective in the treatment of both malignant and non-malignant lesions.

  4. Therapeutic effects of topical 5-aminolevulinic acid photodynamic therapy

    PubMed Central

    Hu, Yin-E; Dai, Shu-Fang; Wang, Bin; Qu, Wei; Gao, Jun-Ling

    2016-01-01

    Objective: To evaluate the therapeutic effects of combined 5-aminolevulinic acid (ALA) and photodynamic therapy (PDT) on genital warts and the safety. Methods: One hundred ten patients with genital warts who were treated in our hospital from June 2013 to October 2014 were selected. The warts and affected parts were disinfected with benzalkonium bromide solution, and the warts were covered with absorbent cotton that had already been added freshly prepared 20% ALA solution, packaged and fixed. Then they were wet-dressed in dark, into which ALA solution was added according to the proportion of 5:3:2 every 30 minutes for three consecutive hours. Afterwards, the warts were illuminated by using photodynamic laser apparatus. The clinical outcomes, adverse reactions and recurrence rates were observed. Results: Genital warts were relieved in 107 out of the 110 cases (cure rate: 97.3%). Male patients had significantly better treatment outcomes at the urethral orifice than those in other affected parts. In the 107 patients, the cure rate of male patients was 98.8%, and they were cured after being treated four times. In contrast, female patients, who were cured after 5 times of treatment, had the cure rate of 91.7%. Their cure rates were similar (χ2=0, P>0.05), but the males were cured after significantly fewer times of treatment than the females (t=-7.432, P<0.05). Five patients suffered from mild tingling or burning sensation upon dressing at the urethral orifice, and the others were all free from systemic adverse reactions. After illumination, a small portion of the patients had mildly red, swelling, painful affected parts, with mild edema that almost disappeared within three days. Three patients relapsed at the urethral orifice and were then cured after further treatment. Conclusion: ALA-PDT can treat genital warts safely with high cure rate and low recurrence rate, particularly working for those of males at the urethral orifice. PMID:27648048

  5. Therapeutic effects of topical 5-aminolevulinic acid photodynamic therapy

    PubMed Central

    Hu, Yin-E; Dai, Shu-Fang; Wang, Bin; Qu, Wei; Gao, Jun-Ling

    2016-01-01

    Objective: To evaluate the therapeutic effects of combined 5-aminolevulinic acid (ALA) and photodynamic therapy (PDT) on genital warts and the safety. Methods: One hundred ten patients with genital warts who were treated in our hospital from June 2013 to October 2014 were selected. The warts and affected parts were disinfected with benzalkonium bromide solution, and the warts were covered with absorbent cotton that had already been added freshly prepared 20% ALA solution, packaged and fixed. Then they were wet-dressed in dark, into which ALA solution was added according to the proportion of 5:3:2 every 30 minutes for three consecutive hours. Afterwards, the warts were illuminated by using photodynamic laser apparatus. The clinical outcomes, adverse reactions and recurrence rates were observed. Results: Genital warts were relieved in 107 out of the 110 cases (cure rate: 97.3%). Male patients had significantly better treatment outcomes at the urethral orifice than those in other affected parts. In the 107 patients, the cure rate of male patients was 98.8%, and they were cured after being treated four times. In contrast, female patients, who were cured after 5 times of treatment, had the cure rate of 91.7%. Their cure rates were similar (χ2=0, P>0.05), but the males were cured after significantly fewer times of treatment than the females (t=-7.432, P<0.05). Five patients suffered from mild tingling or burning sensation upon dressing at the urethral orifice, and the others were all free from systemic adverse reactions. After illumination, a small portion of the patients had mildly red, swelling, painful affected parts, with mild edema that almost disappeared within three days. Three patients relapsed at the urethral orifice and were then cured after further treatment. Conclusion: ALA-PDT can treat genital warts safely with high cure rate and low recurrence rate, particularly working for those of males at the urethral orifice.

  6. Binding of /sup 14/C-5-aminolevulinic acid to a stromal protein from developing pea chloroplasts

    SciTech Connect

    Thayer, S.S.; Castelfranco, P.A.; Wilkinson, J.; Benson, G.

    1987-04-01

    /sup 14/-5-Aminolevulinic acid (/sup 14/C-ALA) binds to a stromal protein with an apparent molecular weight of 42-43 KD on LDS and non-denaturing gels. The reaction is rapid. Binding is inhibited by sulfhydryl reagents, mM concentrations of levulinic, dihydroxy heptanoic acids and gabaculine, 10 ..mu..M N-methylprotoporphyrin. Dicarboxilic acids, such as deltaKG, Glu, OAA, do not inhibit. Chloramphenicol, ATP, protoporphyrin, anoxia, light, darkness have no effect. The product, once formed, is stable to treatment with 5% conc. HCl in cold acetone. It can be chased in a second incubation with unlabeled ALA, but not with levulinic acid. No activity was detected in the subplastidic membrane fractions. Western blot analysis failed to reveal any homology between the labeled protein and either cytochrome for ALA dehydratase. This ALA-binding protein was not formed in chloroplasts isolated from fully expanded pea leaves. Therefore, it is deemed likely to participate in ALA metabolism during chloroplast development.

  7. Optimization of the heme biosynthesis pathway for the production of 5-aminolevulinic acid in Escherichia coli

    PubMed Central

    Zhang, Junli; Kang, Zhen; Chen, Jian; Du, Guocheng

    2015-01-01

    5-Aminolevulinic acid (ALA), the committed intermediate of the heme biosynthesis pathway, shows significant promise for cancer treatment. Here, we identified that in addition to hemA and hemL, hemB, hemD, hemF, hemG and hemH are also the major regulatory targets of the heme biosynthesis pathway. Interestingly, up-regulation of hemD and hemF benefited ALA accumulation whereas overexpression of hemB, hemG and hemH diminished ALA accumulation. Accordingly, by combinatorial overexpression of the hemA, hemL, hemD and hemF with different copy-number plasmids, the titer of ALA was improved to 3.25 g l−1. Furthermore, in combination with transcriptional and enzymatic analysis, we demonstrated that ALA dehydratase (HemB) encoded by hemB is feedback inhibited by the downstream intermediate protoporphyrinogen IX. This work has great potential to be scaled-up for microbial production of ALA and provides new important insights into the regulatory mechanism of the heme biosynthesis pathway. PMID:25716896

  8. Photofrin and 5-aminolevulinic acid permeation through oral mucosa in vitro

    NASA Astrophysics Data System (ADS)

    Flock, Stephen T.; Alleman, Anthony; Lehman, Paul; Blevins, Steve; Stone, Angie; Fink, Louis; Dinehart, Scott; Stern, Scott J.

    1994-07-01

    Photofrin and 5-aminolevulinic acid are photosensitizers that show promise in the photodynamic treatment of cancer, port-wine stains, atherosclerosis and viral lesions. Photofrin is a mixture of porphyrins which, upon the absorption of light, become temporarily cytotoxic. One side-effect associated with the use of Photofrin is long-term cutaneous photosensitivity. It is possible that topical application of this photosensitizing dye will ameliorate such a side-effect. Another way to avoid the cutaneous photosensitivity in photodynamic therapy is to use 5- aminolevulinic acid, which is a porphyrin precursor that causes an increase in the synthesis and concentration of the photosensitizer protoporphyrin IX. 5-aminolevulinic acid is usually applied topically, and so minimizes cutaneous photosensitivity while maximizing the local protoporphyrin concentration. There are a host of disorders in oral mucosa that are potentially treatable by photodynamic therapy. However, since stratum corneum presents an impermeable barrier to many pharmaceuticals, it is not clear that topical application of the photosensitizer will result in a clinically relevant tissue concentration. We have therefore studied the permeation behavior of Photofrin and 5-aminolevulinic acid by applying them to the surface of ex vivo oral mucosa tissue positioned by a Franz diffusion cell. In order to increase the permeability of the photosensitizer across the stratum corneum, we studied the effects of four different drug carriers: phosphate buffered saline, dimethylsulfoxide, ethanol and Azone with isopropyl alcohol.

  9. Optimization of Biomass and 5-Aminolevulinic Acid Production by Rhodobacter sphaeroides ATCC17023 via Response Surface Methodology.

    PubMed

    Liu, Shuli; Zhang, Guangming; Li, Jianzheng; Li, Xiangkun; Zhang, Jie

    2016-06-01

    Microbial 5-aminolevulinic acid (ALA) produced from wastewater is considered as potential renewable energy. However, many hurdles are needed to be overcome such as the regulation of key influencing factors on ALA yield. Biomass and ALA production by Rhodobacter sphaeroides was optimized using response surface methodology. The culturing medium was artificial volatile fatty acids wastewater. Three additives were optimized, namely succinate and glycine that are precursors of ALA biosynthesis, and D-glucose that is an inhibitor of ALA dehydratase. The optimal conditions were achieved by analyzing the response surface plots. Statistical analysis showed that succinate at 8.56 mmol/L, glycine at 5.06 mmol/L, and D-glucose at 7.82 mmol/L were the best conditions. Under these optimal conditions, the highest biomass production and ALA yield of 3.55 g/L and 5.49 mg/g-biomass were achieved. Subsequent verification experiments at optimal values had the maximum biomass production of 3.41 ± 0.002 g/L and ALA yield of 5.78 ± 0.08 mg/g-biomass. PMID:26875086

  10. A New Strategy for Production of 5-Aminolevulinic Acid in Recombinant Corynebacterium glutamicum with High Yield

    PubMed Central

    Yang, Peng; Liu, Wenjing; Cheng, Xuelian; Wang, Jing; Qi, Qingsheng

    2016-01-01

    ABSTRACT 5-Aminolevulinic acid (ALA), a nonprotein amino acid involved in tetrapyrrole synthesis, has been widely applied in agriculture, medicine, and food production. Many engineered metabolic pathways have been constructed; however, the production yields are still low. In this study, several 5-aminolevulinic acid synthases (ALASs) from different sources were evaluated and compared with respect to their ALA production capacities in an engineered Corynebacterium glutamicum CgS1 strain that can accumulate succinyl-coenzyme A (CoA). A codon-optimized ALAS from Rhodobacter capsulatus SB1003 displayed the best potential. Recombinant strain CgS1/pEC-SB produced 7.6 g/liter ALA using a mineral salt medium in a fed-batch fermentation mode. Employing two-stage fermentation, 12.46 g/liter ALA was produced within 17 h, with a productivity of 0.73 g/liter/h, in recombinant C. glutamicum. Through overexpression of the heterologous nonspecific ALA exporter RhtA from Escherichia coli, the titer was further increased to 14.7 g/liter. This indicated that strain CgS1/pEC-SB-rhtA holds attractive industrial application potential for the future. IMPORTANCE In this study, a two-stage fermentation strategy was used for production of the value-added nonprotein amino acid 5-aminolevulinic acid from glucose and glycine in a generally recognized as safe (GRAS) host, Corynebacterium glutamicum. The ALA titer represented the highest in the literature, to our knowledge. This high production capacity, combined with the potential easy downstream processes, made the recombinant strain an attractive candidate for industrial use in the future. PMID:26921424

  11. Evidence that isolated developing chloroplasts are capable of synthesizing chlorophyll b from 5-aminolevulinic acid

    SciTech Connect

    Huang, Laiqiang; Hoffman, N.E. )

    1990-09-01

    Developing chloroplasts isolated from cucumber (Cucumis sativus L. var Beit Alpha) cotyledons are capable of incorporating ({sup 14}C)5-aminolevulinic acid into chlorophyll (Chl) b and Chl a when incubated under photosynthetic illumination. Thin layer chromatography and high pressure liquid chromatography were employed to analyze the pigments. The specific radioactivity in Chl a was over three times higher than that found in Chl b. Both Chl a and b synthesizing activities in organello decayed rapidly at approximately the same rate. We conclude that concomitant synthesis of Chl a/b-binding apoprotein is not required for Chl b synthesis.

  12. Enhanced 5-aminolevulinic acid-gold nanoparticle conjugate-based photodynamic therapy using pulse laser

    NASA Astrophysics Data System (ADS)

    Xu, Hao; Yao, Cuiping; Wang, Jing; Chang, Zhennan; Zhang, Zhenxi

    2016-02-01

    The low bioavailability is a crucial limitation for the application of 5-aminolevulinic acid (ALA) in theranostics. In this research, 5-aminolevulinic acid and gold nanoparticle conjugates (ALA-GNPs) were synthesized to improve the bioavailability of ALA and to investigate the impact of ALA photodynamic therapy (ALA-PDT) in Hela cells. A 532 nm pulse laser and light-emitting diode (central wavelengths 502 nm) were jointly used as light sources in PDT research. The results show a 532 nm pulse laser can control ALA release from ALA-GNPs by adjusting the pulse laser dose. This laser control release may be attributed to the heat generation from GNPs under pulse laser irradiation, which indicates accurately adjusting the pulse laser dose to control the drug release in the cell interior can be considered as a new cellular surgery modality. Furthermore, the PDT results in Hela cells indicate the enhancement of ALA release by pulse laser before PDT can promote the efficacy of cell eradication in the light-emitting diode PDT (LED-PDT). This laser mediated drug release system can provide a new online therapy approach in PDT and it can be utilized in the optical monitor technologies based individual theranostics.

  13. Production of 5-aminolevulinic acid by cell free multi-enzyme catalysis.

    PubMed

    Meng, Qinglong; Zhang, Yanfei; Ju, Xiaozhi; Ma, Chunling; Ma, Hongwu; Chen, Jiuzhou; Zheng, Ping; Sun, Jibin; Zhu, Jun; Ma, Yanhe; Zhao, Xueming; Chen, Tao

    2016-05-20

    5-Aminolevulinic acid (ALA) is the precursor for the biosynthesis of tetrapyrroles and has broad agricultural and medical applications. Currently ALA is mainly produced by chemical synthesis and microbial fermentation. Cell free multi-enzyme catalysis is a promising method for producing high value chemicals. Here we reported our work on developing a cell free process for ALA production using thermostable enzymes. Cheap substrates (succinate and glycine) were used for ALA synthesis by two enzymes: 5-aminolevulinic acid synthase (ALAS) from Laceyella sacchari (LS-ALAS) and succinyl-CoA synthase (Suc) from Escherichia coli. ATP was regenerated by polyphosphate kinase (Ppk) using polyphosphate as the substrate. Succinate was added into the reaction system in a fed-batch mode to avoid its inhibition effect on Suc. After reaction for 160min, ALA concentration was increased to 5.4mM. This is the first reported work on developing the cell free process for ALA production. Through further process and enzyme optimization the cell free process could be an effective and economic way for ALA production.

  14. Utilization of 5-aminolevulinic acid in the photodynamic therapy of tumors: biochemical and photobiological aspects

    NASA Astrophysics Data System (ADS)

    Pottier, Roy H.; Kennedy, James C.

    1994-03-01

    Inherent in both plants and animals is the natural porphyrin, Protoporphyrin IX (Pp). Although Pp does not appear to have any intrinsic biological activity, it is a potent natural photosensitizer. When activated with ultraviolet or visible light, this photosensitizer can induce significant photodynamic effects on tissues, cells, subcellular elements, and macromolecules via the production of singlet oxygen. The biosynthesis of endogenous Pp is under strict enzymatic control. It is possible to bypass a rate controlling step and induce large, transient concentrations of Pp by the addition of exogenous 5-aminolevulinic acid (ALA). ALA may be administered systemically or topically. Much larger amounts of Pp are produced in certain types of tumor tissue than in adjacent normal tissue. Topically applied ALA can be used to treat a variety of skin lesions, including actinic keratosis, basal cell carcinomas and psoriasis.

  15. Clinical utility of 5-aminolevulinic acid HCl to better visualize and more completely remove gliomas

    PubMed Central

    Halani, Sameer H; Adamson, D Cory

    2016-01-01

    Surgical resection is typically the first line of treatment for gliomas. However, the neurosurgeon faces a major challenge in achieving maximal resection in high-grade gliomas as these infiltrative tumors make it difficult to discern tumor margins from normal brain with conventional white-light microscopy alone. To aid in resection of these infiltrative tumors, fluorescence-guided surgery has gained much popularity in intraoperative visualization of malignant gliomas, with 5-aminolevulinic acid (5-ALA) leading the way. First introduced in an article in Neurosurgery, 5-ALA has since become a safe, effective, and inexpensive method to visualize and improve resection of gliomas. This has undoubtedly led to improvements in the clinical course of patients as demonstrated by the increased overall and progression-free survival in patients with such devastating disease. This literature review aims to discuss the major studies and trials demonstrating the clinical utility of 5-ALA and its ability to aid in complete resection of malignant gliomas.

  16. 5-aminolevulinic acid guidance during awake craniotomy to maximise extent of safe resection of glioblastoma multiforme.

    PubMed

    Corns, Robert; Mukherjee, Soumya; Johansen, Anja; Sivakumar, Gnanamurthy

    2015-01-01

    Overall survival for patients with glioblastoma multiforme (GBM) has been consistently shown to improve when the surgeon achieves a gross total resection of the tumour. It has also been demonstrated that surgical adjuncts such as 5-aminolevulinic acid (5-ALA) fluorescence--which delineates malignant tumour tissue--normal brain tissue margin seen using violet-blue excitation under an operating microscope--helps achieve this. We describe the case of a patient with recurrent left frontal GBM encroaching on Broca's area (eloquent brain). Gross total resection of the tumour was achieved by combining two techniques, awake resection to prevent damage to eloquent brain and 5-ALA fluorescence guidance to maximise the extent of tumour resection.This technique led to gross total resection of all T1-enhancing tumour with the avoidance of neurological deficit. The authors recommend this technique in patients when awake surgery can be tolerated and gross total resection is the aim of surgery. PMID:26177997

  17. Fluorescence-guided resections and photodynamic therapy for malignant gliomas using 5-aminolevulinic acid

    NASA Astrophysics Data System (ADS)

    Stepp, Herbert G.; Beck, Tobias; Beyer, Wolfgang; Pongratz, Thomas; Sroka, Ronald; Baumgartner, Reinhold; Stummer, Walter; Olzowy, Bernhard; Mehrkens, Jan H.; Tonn, Joerg C.; Reulen, Hans J.

    2005-04-01

    Oral application of 20 mg/kg bw of 5-aminolevulinic acid results in a highly specific accumulation of fluorescent and phototoxic Protoporphyrin IX in malignant glioma tissue. Surgical removal with fluorescence guidance is studied in a phase III clinical trial, adjuvant Photodynamic Therapy (PDT) to the surgical cavity is in phase II and for interstitial PDT of recurrent gliomas, a phase I/II study has started. Fluorescence guided resections have been shown to be safe and effective in augmenting neurosurgical removal of malignant gliomas in 52 consecutive patients. Intra-operative fluorescence spectroscopy showed statistically significant higher sensitizer accumulation in vital brain tumor versus the infiltration zone and in the infiltration zone versus adjacent normal brain, which contained very little PPIX. This is promisingly exploited for PDT - both to the surgical cavity by surface irradiation and for stereotactically guided interstitial irradiation.

  18. Review of dermatology use of 5-aminolevulinic acid photodynamic therapy in China from 1997 to 2013

    NASA Astrophysics Data System (ADS)

    Wang, Peiru; Zhang, Guolong; Wang, Xiuli

    2015-07-01

    The prodrug 5-aminolevulinic acid (ALA) and its ester derivatives have been used in photodynamic therapy (PDT) in dermatology worldwide. In China, ALA-PDT was first used to treat urethral condylomata acuminata and non-melanoma skin cancers in 1997. A powder formulation of ALA hydrochloride was approved by the Chinese Food and Drug Administration for the treatment of condylomata acuminata in 2007. Large successful experience of treating condylomatas was accumulated compared with Western countries. Meanwhile, numerous clinical studies as well as off-label use of ALAPDT have been carried out in China. To reflect the progress of ALA-PDT in China, several major Chinese and English databases were searched and published data were reviewed in this article.

  19. Physiological and Metabolic Effects of 5-Aminolevulinic Acid for Mitigating Salinity Stress in Creeping Bentgrass

    PubMed Central

    Yang, Zhimin; Chang, Zuoliang; Sun, Lihong; Yu, Jingjin; Huang, Bingru

    2014-01-01

    The objectives of this study were to determine whether foliar application of a chlorophyll precursor, 5-aminolevulinic acid (ALA), could mitigate salinity stress damages in perennial grass species by regulating photosynthetic activities, ion content, antioxidant metabolism, or metabolite accumulation. A salinity-sensitive perennial grass species, creeping bentgrass (Agrostis stolonifera), was irrigated daily with 200 mM NaCl for 28 d, which were foliar sprayed with water or ALA (0.5 mg L−1) weekly during the experiment in growth chamber. Foliar application of ALA was effective in mitigating physiological damage resulting from salinity stress, as manifested by increased turf quality, shoot growth rate, leaf relative water content, chlorophyll content, net photosynthetic rate, stomatal conductance and transpiration rate. Foliar application of ALA also alleviated membrane damages, as shown by lower membrane electrolyte leakage and lipid peroxidation, which was associated with increases in the activities of antioxidant enzymes. Leaf content of Na+ was reduced and the ratio of K+/Na+ was increased with ALA application under salinity stress. The positive effects of ALA for salinity tolerance were also associated with the accumulation of organic acids (α-ketoglutaric acid, succinic acid, and malic acid), amino acids (alanine, 5-oxoproline, aspartic acid, and γ -aminobutyric acid), and sugars (glucose, fructose, galactose, lyxose, allose, xylose, sucrose, and maltose). ALA-mitigation of physiological damages by salinity could be due to suppression of Na+ accumulation and enhanced physiological and metabolic activities related to photosynthesis, respiration, osmotic regulation, and antioxidant defense. PMID:25551443

  20. Physiological and metabolic effects of 5-aminolevulinic acid for mitigating salinity stress in creeping bentgrass.

    PubMed

    Yang, Zhimin; Chang, Zuoliang; Sun, Lihong; Yu, Jingjin; Huang, Bingru

    2014-01-01

    The objectives of this study were to determine whether foliar application of a chlorophyll precursor, 5-aminolevulinic acid (ALA), could mitigate salinity stress damages in perennial grass species by regulating photosynthetic activities, ion content, antioxidant metabolism, or metabolite accumulation. A salinity-sensitive perennial grass species, creeping bentgrass (Agrostis stolonifera), was irrigated daily with 200 mM NaCl for 28 d, which were foliar sprayed with water or ALA (0.5 mg L-1) weekly during the experiment in growth chamber. Foliar application of ALA was effective in mitigating physiological damage resulting from salinity stress, as manifested by increased turf quality, shoot growth rate, leaf relative water content, chlorophyll content, net photosynthetic rate, stomatal conductance and transpiration rate. Foliar application of ALA also alleviated membrane damages, as shown by lower membrane electrolyte leakage and lipid peroxidation, which was associated with increases in the activities of antioxidant enzymes. Leaf content of Na+ was reduced and the ratio of K+/Na+ was increased with ALA application under salinity stress. The positive effects of ALA for salinity tolerance were also associated with the accumulation of organic acids (α-ketoglutaric acid, succinic acid, and malic acid), amino acids (alanine, 5-oxoproline, aspartic acid, and γ -aminobutyric acid), and sugars (glucose, fructose, galactose, lyxose, allose, xylose, sucrose, and maltose). ALA-mitigation of physiological damages by salinity could be due to suppression of Na+ accumulation and enhanced physiological and metabolic activities related to photosynthesis, respiration, osmotic regulation, and antioxidant defense.

  1. Regulation of the hemA gene during 5-aminolevulinic acid formation in Pseudomonas aeruginosa.

    PubMed Central

    Hungerer, C; Troup, B; Römling, U; Jahn, D

    1995-01-01

    The general tetrapyrrole precursor 5-aminolevulinic acid is formed in bacteria via two different biosynthetic pathways. Members of the alpha group of the proteobacteria use 5-aminolevulinic acid synthase for the condensation of succinyl-coenzyme A and glycine, while other bacteria utilize a two-step pathway from aminoacylated tRNA(Glu). The tRNA-dependent pathway, involving the enzymes glutamyl-tRNA reductase (encoded by hemA) and glutamate-1-semialdehyde-2,1-aminomutase (encoded by hemL), was demonstrated to be used by Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas stutzeri, Comamonas testosteroni, Azotobacter vinelandii, and Acinetobacter calcoaceticus. To study the regulation of the pathway, the glutamyl-tRNA reductase gene (hemA) from P. aeruginosa was cloned by complementation of an Escherichia coli hemA mutant. The hemA gene was mapped to the SpeI A fragment and the DpnIL fragment of the P. aeruginosa chromosome corresponding to min 24.1 to 26.8. The cloned hemA gene, coding for a protein of 423 amino acids with a calculated molecular mass of 46,234 Da, forms an operon with the gene for protein release factor 1 (prf1). This translational factor mediates the termination of the protein chain at the ribosome at amber and ochre codons. Since the cloned hemA gene did not possess one of the appropriate stop codons, an autoregulatory mechanism such as that postulated for the enterobacterial system was ruled out. Three open reading frames of unknown function transcribed in the opposite direction to the hemA gene were found. hemM/orf1 and orf2 were found to be homologous to open reading frames located in the 5' region of enterobacterial hemA genes. Utilization of both transcription start sites was changed in a P. aeruginosa mutant missing the oxygen regulator Anr (Fnr analog), indicating the involvement of the transcription factor in hemA expression. DNA sequences homologous to one half of an Anr binding site were detected at one of the determined

  2. Mechanism for enhanced 5-aminolevulinic acid fluorescence in isocitrate dehydrogenase 1 mutant malignant gliomas

    PubMed Central

    Kim, Ji Young; Kim, Sung Kwon; Kim, Seung-Ki; Park, Sung-Hye; Kim, Hyeonjin; Lee, Se-Hoon; Choi, Seung Hong; Park, Sunghyouk; Park, Chul-Kee

    2015-01-01

    Fluorescence-guided surgery using 5-aminolevulinic acid (5-ALA) has become the main treatment modality in malignant gliomas. However unlike glioblastomas, there are inconsistent result about fluorescence status in WHO grade III gliomas. Here, we show that mutational status of IDH1 is linked to 5-ALA fluorescence. Using genetically engineered malignant glioma cells harboring wild type (U87MG-IDH1WT) or mutant (U87MG-IDH1R132H) IDH1, we demonstrated a lag in 5-ALA metabolism and accumulation of protoporphyrin IX (PpIX) in U87MG-IDH1R132H cells. Next, we used liquid chromatography–mass spectrometry (LC-MS) to screen for tricarboxylic acid (TCA) cycle-related metabolite changes caused by 5-ALA exposure. We observed low baseline levels of NADPH, an essential cofactor for the rate-limiting step of heme degradation, in U87MG-IDH1R132H cells. High levels of NADPH are required to metabolize excessive 5-ALA, giving a plausible reason for the temporarily enhanced 5-ALA fluorescence in mutant IDH1 cells. This hypothesis was supported by the results of metabolic screening in human malignant glioma samples. In conclusion, we have discovered a relationship between enhanced 5-ALA fluorescence and IDH1 mutations in WHO grade III gliomas. Low levels of NADPH in tumors with mutated IDH1 is responsible for the enhanced fluorescence. PMID:26008980

  3. Transcriptomic analysis for elucidating the physiological effects of 5-aminolevulinic acid accumulation on Corynebacterium glutamicum.

    PubMed

    Yu, Xiaoli; Jin, Haiying; Cheng, Xuelian; Wang, Qian; Qi, Qingsheng

    2016-11-01

    5-Aminolevulinic acid (ALA), the committed intermediate of the heme biosynthetic pathway, attracts close attention among researchers because of its potential applications to cancer treatment and agriculture. Overexpression of heterologous hemA and hemL, which encode glutamyl-tRNA reductase and glutamate-1-semialdehyde aminotransferase, respectively, in Corynebacterium glutamicum produces ALA, although whether ALA accumulation causes unintended effects on the host is unknown. Here we used an integrated systems approach to compare global transcriptional changes induced by the expression of hemA and hemL. Metabolic pathway such as glycolysis was inhibited, but tricarboxylic acid cycle, pentose phosphate pathway, and respiratory metabolism were stimulated. Moreover, the transcriptional levels of certain genes involved in heme biosynthesis were up-regulated, and the data implicate the two-component system (TCS) HrrSA was involved in the regulation of heme synthesis. With these understandings, it is proposed that ALA accumulation stimulates heme synthesis pathway and respiratory metabolism. Our study illuminates the physiological effects of overexpressing hemA and hemL on the phenotype of C. glutamicum and contributes important insights into the regulatory mechanisms of the heme biosynthetic pathways. PMID:27664748

  4. Alternative Routes for the Synthesis of 5-Aminolevulinic Acid in Maize Leaves 1

    PubMed Central

    Harel, Eitan; Ne'Eman, Emma

    1983-01-01

    Intact plastids from greening maize (Zea mays L.) leaves converted [14C]glutamate and [14C]2-ketoglutarate (KG) to [14C]5-aminolevulinic acid (ALA). Glutamate appeared to be the immediate precursor of ALA, while KG was first converted to glutamate, as shown by the effect of various inhibitors of amino acid metabolism. Plastids from greening leaves contained markedly higher activity as compared with etioplasts or chloroplasts. The synthesis of ALA by intact plastids was light dependent. The enzyme system resides in the stroma of plastids or may be lightly bound to membranes. The solubilized system showed maximal activity around pH 7.9 and required Mg2+, ATP, and NADPH although dependence on the latter was not clear-cut. A relatively high level of activity could be extracted from etioplasts. Maximal activity was obtained from plastids of leaves which had been illuminated for 90 minutes, after which activity declined sharply. The enzyme system solubilized from plastids also catalyzed the conversion of putative glutamate 1-semialdehyde to ALA in a reaction which was not dependent on the addition of an amino donor. The system in maize greatly resembled the one which had been reported from barley. It is suggested that this system is the one responsible for the biosynthesis of ALA destined for chlorophyll formation. PMID:16663121

  5. Role of 5-aminolevulinic acid-conjugated gold nanoparticles for photodynamic therapy of cancer

    NASA Astrophysics Data System (ADS)

    Zhang, Zhenxi; Wang, Sijia; Xu, Hao; Wang, Bo; Yao, Cuiping

    2015-05-01

    There are three possible mechanisms for 5-aminolevulinic acid (5-ALA) conjugated gold nanoparticles (GNPs) through electrostatic bonding for photodynamic therapy (PDT) of cancer: GNPs delivery function, singlet oxygen generation (SOG) by GNPs irradiated by light, and surface resonance enhancement (SRE) of SOG. Figuring out the exact mechanism is important for further clinical treatment. 5-ALA-GNPs and human chronic myeloid leukemia K562 cells were used to study delivery function and SOG by GNPs. The SRE of SOG enabled by GNPs was explored by protoporphyrin IX (PpIX)-GNPs conjugate through electrostatic bonding. Cell experiments show that the GNPs can improve the efficiency of PDT, which is due to the vehicle effect of GNPs. PpIX-GNPs conjugate experiments demonstrated that SOG can be improved about 2.5 times over PpIX alone. The experiments and theoretical results show that the local field enhancement (LFE) via localized surface plasmon resonance (LSPR) of GNPs is the major role; the LFE was dependent on the irradiation wavelength and the GNP's size. The LFE increased with an increase of the GNP size (2R ≤50 nm). However, the LSPR function of the GNPs was not found in cell experiments. Our study shows that in 5-ALA-conjugated GNPs PDT, the delivery function of GNPs is the major role.

  6. Rational design of 5-aminolevulinic acid derivatives aimed at improving photodynamic therapy.

    PubMed

    Casas, Adriana; Batlle, Alcira

    2002-07-01

    5-aminolevulinic acid (ALA) is the first intermediate in heme biosynthesis and is therefore a precursor of protoporphyrin IX (PpIX). PpIX is used as an endogenous photosensitizer in photodynamic therapy (PDT). Several chemical modifications have been made, both on the amino and carboxyl groups of ALA to induce higher PpIX production and photosensitisation. Esterification of ALA with aliphatic lineal and cyclic alcohols was found to reduce the amount of ALA required for photosensitization. Esterification by aliphatic alcohols with carbohydrate chains equal or lower than C4 leads to porphyrin accumulation lower than ALA, whereas equal or longer than C6 chains leads to greater synthesis of porphyrin. A branch point in the alcohol located next to the site of ester cleavage limits access of the esters to the esterase active site, resulting in lower PpIX production. ALA esters of the polyethylenglycol family can induce high levels of PpIX, with some selectivity for endothelial cells toward tumor cells. On the basis of the differential expression of some aminopeptidases in tumor vasculature when compared to normal vasculature, some ALA-pseudopeptides were synthesized. In a rational design of ALA derivatives, the transport mechanism of these aminoacids into the cell is central. Due to the similar characteristics between ALA and GABA transport, a novel approach for designing new ALA derivatives which could penetrate more easily into tumoral cells, would be to take into account the structures of the inhibitors of GABA transport. PMID:12678731

  7. Clinical utility of 5-aminolevulinic acid HCl to better visualize and more completely remove gliomas.

    PubMed

    Halani, Sameer H; Adamson, D Cory

    2016-01-01

    Surgical resection is typically the first line of treatment for gliomas. However, the neurosurgeon faces a major challenge in achieving maximal resection in high-grade gliomas as these infiltrative tumors make it difficult to discern tumor margins from normal brain with conventional white-light microscopy alone. To aid in resection of these infiltrative tumors, fluorescence-guided surgery has gained much popularity in intraoperative visualization of malignant gliomas, with 5-aminolevulinic acid (5-ALA) leading the way. First introduced in an article in Neurosurgery, 5-ALA has since become a safe, effective, and inexpensive method to visualize and improve resection of gliomas. This has undoubtedly led to improvements in the clinical course of patients as demonstrated by the increased overall and progression-free survival in patients with such devastating disease. This literature review aims to discuss the major studies and trials demonstrating the clinical utility of 5-ALA and its ability to aid in complete resection of malignant gliomas. PMID:27672334

  8. Clinical utility of 5-aminolevulinic acid HCl to better visualize and more completely remove gliomas

    PubMed Central

    Halani, Sameer H; Adamson, D Cory

    2016-01-01

    Surgical resection is typically the first line of treatment for gliomas. However, the neurosurgeon faces a major challenge in achieving maximal resection in high-grade gliomas as these infiltrative tumors make it difficult to discern tumor margins from normal brain with conventional white-light microscopy alone. To aid in resection of these infiltrative tumors, fluorescence-guided surgery has gained much popularity in intraoperative visualization of malignant gliomas, with 5-aminolevulinic acid (5-ALA) leading the way. First introduced in an article in Neurosurgery, 5-ALA has since become a safe, effective, and inexpensive method to visualize and improve resection of gliomas. This has undoubtedly led to improvements in the clinical course of patients as demonstrated by the increased overall and progression-free survival in patients with such devastating disease. This literature review aims to discuss the major studies and trials demonstrating the clinical utility of 5-ALA and its ability to aid in complete resection of malignant gliomas. PMID:27672334

  9. Role of 5-aminolevulinic acid-conjugated gold nanoparticles for photodynamic therapy of cancer.

    PubMed

    Zhang, Zhenxi; Wang, Sijia; Xu, Hao; Wang, Bo; Yao, Cuiping

    2015-05-01

    There are three possible mechanisms for 5-aminolevulinic acid (5-ALA) conjugated gold nanoparticles (GNPs) through electrostatic bonding for photodynamic therapy (PDT) of cancer: GNPs delivery function,singlet oxygen generation (SOG) by GNPs irradiated by light, and surface resonance enhancement (SRE) of SOG. Figuring out the exact mechanism is important for further clinical treatment. 5-ALA-GNPs and human chronic myeloid leukemia K562 cells were used to study delivery function and SOG by GNPs. The SRE of SOG enabled by GNPs was explored by protoporphyrin IX (PpIX)-GNPs conjugate through electrostatic bonding.Cell experiments show that the GNPs can improve the efficiency of PDT, which is due to the vehicle effect of GNPs. PpIX–GNPs conjugate experiments demonstrated that SOG can be improved about 2.5 times over PpIX alone. The experiments and theoretical results show that the local field enhancement (LFE) via localized surface plasmon resonance (LSPR) of GNPs is the major role; the LFE was dependent on the irradiation wavelength and the GNP's size. The LFE increased with an increase of the GNP size (2R ≤ 50 nm). However, the LSPR function of the GNPs was not found in cell experiments. Our study shows that in 5-ALA-conjugated GNPs PDT, the delivery function of GNPs is the major role.

  10. Comparison of 5-aminolevulinic acid-encapsulated liposome versus ethosome for skin delivery for photodynamic therapy.

    PubMed

    Fang, Yi-Ping; Tsai, Yi-Hung; Wu, Pao-Chu; Huang, Yaw-Bin

    2008-05-22

    Topical photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA) is an alternative therapy for many non-melanoma skin cancers. The major limitation of this therapy, however, is the low permeability of ALA through the stratum corneum (SC) of the skin. The objective of the present work was to characterize ethosomes containing ALA and to enhance the skin production of protoporphyrin IX (PpIX), compared to traditional liposomes. Results showed that the average particle sizes of the ethosomes were less than those of liposomes. Moreover, the entrapment efficiency of ALA in the ethosome formulations was 8-66% depending on the surfactant added. The particle size of the ethosomes was still approximately <200 nm after 32 days of storage. An in vivo animal study observed the presence of PpIX in the skin by confocal laser scanning microscopy (CLSM). The results indicated that the penetration ability of ethosomes was greater than that of liposomes. The enhancements of all the formulations were ranging from 11- to 15-fold in contrast to that of control (ALA in an aqueous solution) in terms of PpIX intensity. In addition, colorimetry detected no erythema in the irradiated skin. The results demonstrated that the enhancement ratio of ethosome formulations did not significantly differ between the non-irradiated and irradiated groups except for PE/CH/SS, which may have been due to a photobleaching effect of the PDT-irradiation process. PMID:18325699

  11. Physicochemical characterisation of a novel thermogelling formulation for percutaneous penetration of 5-aminolevulinic acid.

    PubMed

    Grüning, Nadja; Müller-Goymann, Christel Charlotte

    2008-06-01

    The present contribution was dedicated to the development and characterisation of a semisolid formulation of 5-aminolevulinic acid (5-ALA), appropriate for the diagnosis and treatment of actinic keratosis in photodynamic therapy. To achieve sufficiently high concentrations of the polar substance within the living epithelium after topical application, the semisolid base was enriched with penetration enhancers. A semisolid liquid crystalline system for drug delivering was the formulation of choice. It was composed of isopropyl alcohol, dimethyl isosorbide, medium chain triglycerides, water, and Pluronic F 127 as a polyoxyethylene-polyoxypropylene surface-active block copolymer. Rheometrical investigations were performed in the oscillatory mode and showed a thermo reversible gelification behaviour of the formulation, which therefore was denoted Thermogel. Permeation studies through human stratum corneum revealed higher permeation coefficients for 5-ALA from the Thermogel than from different German Pharmacopoeia creams. For example a 7.5-fold increase in comparison with Basiscreme DAC, and a 19.5-fold increase compared to water containing hydrophilic ointment. With respect to Dolgit(R) Mikrogel, the permeation coefficient from the Thermogel was 6.4-fold higher. These results were in accordance with those of differential scanning calorimetry measurements. Thermogel disclosed the strongest interactions with stratum corneum lipids.

  12. Effects of 5-aminolevulinic acid on a murine model of diet-induced obesity

    PubMed Central

    Koganei, Megumi; Saitou, Yuri; Tsuchiya, Kyoko; Abe, Fuminori; Tanaka, Toru; Horinouchi, Izumi; Izumi, Yoshiya; Yamaji, Taketo; Takahashi, Takeshi

    2015-01-01

    The effects of 5-aminolevulinic acid (5-ALA) on obesity were investigated using a murine model (diet-induced obese mice). Diet-induced obese mice were divided into 4 groups: a control group (C group), which was fed a high-fat diet; a low-5-ALA dose (10 mg/kg/day) group (10A group); a moderate-5-ALA dose (30 mg/kg/day) group (30A group); and a high-5-ALA dose (100 mg/kg/day) group (100A group). 5-ALA was administered by mixing the high fat diet for 8 weeks. Body weight increases in the 30A and 100A groups were significantly smaller compared with those of the C group. Body fat measurements by X-ray computed tomography indicated that the 100A group showed a tendency toward low visceral fat quantities during the final week of the study. Visceral fat weights in the 30A and 100A groups were slightly low. The levels of serum alanine aminotransferase (ALT) and total cholesterol (TC) in the 10A group was slightly low, whereas the 30A and 100A groups showed significantly lower ALT and TC values. Liver lipid concentration showed a dose-dependent decrease with ALA. Thus, in this diet-induced obese murine model, administration of 5-ALA had a significantly beneficial impact on the visceral fat, serum ALT and TC, and liver lipid concentration. PMID:26388673

  13. Sonodynamic therapy using 5-aminolevulinic acid enhances the efficacy of bleomycin.

    PubMed

    Osaki, Tomohiro; Ono, Misato; Uto, Yoshihiro; Ishizuka, Masahiro; Tanaka, Tohru; Yamanaka, Nobuyasu; Kurahashi, Tsukasa; Azuma, Kazuo; Murahata, Yusuke; Tsuka, Takeshi; Ito, Norihiko; Imagawa, Tomohiro; Okamoto, Yoshiharu

    2016-04-01

    Sonodynamic therapy (SDT) kills tumor cells through the synergistic effects of ultrasound and a sonosensitizer agent. We examined whether 5-aminolevulinic acid (5-ALA)-based SDT at 1 or 3 MHz could enhance the cytotoxicity of bleomycin (BLM) toward mouse mammary tumor cells both in vitro and in vivo. At 1 MHz, cell viability in the 5-ALA-based SDT group at 1, 2, and 3 W/cm(2) was 34.30%, 50.90%, and 60.16%, respectively. Cell viability in the 5-ALA-based SDT+BLM group at 1, 2, and 3 W/cm(2) was 0.09%, 0.32%, and 0.17%, respectively. In contrast, at 3 MHz, 5-ALA-based SDT+BLM did not show pronounced cytotoxicity. In the in vivo study, 5-ALA-based SDT+BLM was significantly more cytotoxic than 5-ALA-based SDT at 1 MHz and 3 MHz. These findings suggest that the mechanism of tumor shrinkage induced by 5-ALA-based SDT+BLM might involve not only direct cell killing, but also vascular shutdown. Thus, we show here that 5-ALA-based SDT enhances the efficacy of BLM both in vitro and in vivo. PMID:26799128

  14. 5-aminolevulinic acid and neuronavigation in high-grade glioma surgery: results of a combined approach.

    PubMed

    Panciani, Pier Paolo; Fontanella, Marco; Garbossa, Diego; Agnoletti, Alessandro; Ducati, Alessandro; Lanotte, Michele

    2012-02-01

    In high-grade glioma surgery, several techniques are used to achieve the maximum cytoreductive treatment preserving neurological functions. However, the effectiveness of all the methods used alone is reduced by specific limitations of each. We assessed the reliability of a multimodal strategy based on 5-aminolevulinic acid (5-ALA) and neuronavigation. We prospectively studied 18 patients with suspected, non eloquent-area malignant gliomas amenable for complete resection. Conventional illumination was used until the excision appeared complete. The cavity was then systematically inspected in violet-blue light to identify any residual tumour. Multiple biopsies of both fluorescent and non-fluorescent tissue were performed in all cases. Each specimen was labelled according to the sampling location (inside or outside the boundary set by the neuronavigator). The samples were analysed by a neuropathologist blinded to the intraoperative classification. We reviewed the results of both methods, either singly or in combination. Individual analysis showed higher 5-ALA reliability compared to neuronavigation. However, several false-negative fluorescent specimens were detected. With the combined use of fluorescence and neuroimaging, only 1 sample (negative for both 5-ALA and navigation) was tumoral tissue. In our experience, the combined approach showed the best sensitivity and it is recommended in cases of lesions involving non-eloquent areas.

  15. Effectiveness of 5-aminolevulinic acid photodynamic therapy in the treatment of hidradenitis suppurativa: a report of 5 cases.

    PubMed

    Andino Navarrete, R; Hasson Nisis, A; Parra Cares, J

    2014-01-01

    Hidradenitis suppurativa has been described as a chronic, recurrent, and disabling inflammatory disease involving the entire hair follicle. Several treatments, including photodynamic therapy, have been used, but the results have been inconsistent and recurrence is high. In this prospective study, we evaluated disease severity, quality of life, and treatment tolerance in 5 patients with moderate to severe hidradenitis suppurativa treated with photodynamic therapy using 5-aminolevulinic acid and a 635-nm light source. Treatment effectiveness was evaluated using the Sartorius severity score, the Dermatology Life Quality Index, and a visual analog scale for pain and disease activity. Significant improvements were observed with all 3 instruments and the effects remained visible at 8 weeks. Our results suggest that photodynamic therapy with 5-aminolevulinic acid and a light wavelength of 635 nm could reduce disease severity and improve quality of life in patients with difficult-to-treat hidradenitis suppurativa.

  16. Hydrokolloid occlusive dressings for photodynamic therapy (PDT) of cutaneous lesions with endogenous porphyrins induced by 5-aminolevulinic acid

    NASA Astrophysics Data System (ADS)

    Gahlen, Johannes; Stern, Josef; Herfarth, Christian

    1995-03-01

    Protoporphyrin (Pp IX) is the final intermediate product before haem and can be stimulated to a phototoxic reaction with light. The presence of 5-aminolevulinic acid can increase the intracellular biosynthesis of Pp IX in certain types of tumor cells. The photosensitizing concentrations of Pp IX make laser light induced fluorescence diagnostics (LIFD) and photodynamic therapy possible. A topical application of a 5-aminolevulinic acid solution requires a waterproof occlusive dressing for several hours. We developed a simple technique for a practical preparation for PDT using a hydrocolloid dressing. The normal surrounding skin can be spared. We present our first therapeutic experience with a case of cutaneous breast cancer in a 65-year-old female patient. Six hours after topical application of 10% isotonic 5- aminolevulinic acid under the hydrocolloid dressing PDT was performed (Ar-Dye Laser, 630 nm wavelength). Twenty four hours after PDT a superficial tumor necrosis could be observed with a maximum depth of tumor necrosis of 2 - 3 mm. The surrounding normal skin was without any inflammation.

  17. Alleviation of salt-induced oxidative damage by 5-aminolevulinic acid in wheat seedlings

    NASA Astrophysics Data System (ADS)

    Genişel, Mucip; Erdal, Serkan

    2016-04-01

    The aim of this study was to elucidate how 5-aminolevulinic acid (ALA), the precursor of chlorophyll compounds, affects the defence mechanisms of wheat seedlings induced by salt stress. To determine the possible stimulative effects of ALA against salinity, 11-day old wheat seedlings were sprayed with ALA at two different concentrations (10 and 20 mg.l-1) and then stressed by exposure to salt (150 mM NaCl). The salt stress led to significant changes in the antioxidant activity. While guaiacol peroxidase activity decreased, the activities of superoxide dismutase, catalase, and ascorbate peroxidase markedly increased under salt stress. Compared to the salt stress alone, the application of ALA beforehand further increased the activity of these enzymes. This study is the first time the effects of ALA have been monitored with regard to protein content and the isoenzyme profiles of the antioxidant enzymes. Although the salt stress reduced both the soluble protein content and protein band intensities, pre-treating with ALA significantly mitigated these stress-induced reductions. The data for the isoenzyme profiles of the antioxidant enzymes paralleled that of the ALA-induced increases in antioxidant activity. As a consequence of the high antioxidant activity in the seedlings pre-treated with ALA, the stress-induced elevations in the reactive oxygen species, superoxide anion, and hydrogen peroxide contents and lipid peroxidation levels were markedly diminished. Taken together, this data demonstrated that pre-treating with ALA confers resistance to salt stress by modulating the protein synthesis and antioxidant activity in wheat seedlings.

  18. Photodynamic therapy of urethral condylomata acuminata using topically 5-aminolevulinic acid

    NASA Astrophysics Data System (ADS)

    Wang, Xiuli; Wang, Hongwei; Wang, Haishan; Xu, Shizheng; Liao, Kanghuang; Hillemanns, Peter

    2005-07-01

    Background Electrocoagulation and laser evaporation for urethral condylomata acuminata have high recurrence rates and can be associated with urethral malformations. Objective To investigate the effect of photodynamic therapy (PDT) with topical 5-aminolevulinic acid (ALA) on urethral condylomata acuminata and to examine the histological changes in lesions of condylomata acuminata after ALA-PDT. Methods One hundred and sixty-four urethral condylomata patients were given topical ALA followed by intraurethral PDT through a cylindrical fiber. Among the cases, 16 penile and vulval condylomatous lesions in 11 patients were treated with topical ALA-PDT at same time. After the treatment, biopsy specimens were collected from the 16 penile and vulval lesions. The histological changes were then evaluated by light microscope and electron microscope. Results The complete response rate for urethral condylomata by topical ALA-PDT was 95.12% and the recurrence rate was 5.13% after 6 to 24 months follow-up. Keratinocytes in middle and upper layers of the epidermis with marked vacuolation and some necrocytosis were detected one and three hours after PDT. Necrosis in all layers of the epidermis was noted five hours after PDT by microscopy. In electron microscopy of kerationcytes, distinct ultrastructural abnormalities of mitochondrion, endoplasmic reticulum and membrane damage were observed. Apoptotic bodies were detected three hours after PDT and a large number of the keratinocytes exhibited necrosis five hours after PDT by electron microscope. Conclusions Results suggests that topical ALA-PDT is a simple, effective, relatively safe, less recurrent and comparatively well tolerated treatment for urethral condylomata acuminata. The mechanisms might be that ALA-PDT could trigger apoptotic process and necrosis in the HPV infected keratinocytes. Key words:

  19. [Effect of 5-aminolevulinic acid on photosynthetic characteristics of tomato seedlings under NaCl stress].

    PubMed

    Zhao, Yan-Yan; Yan, Fei; Hu, Li-Pan; Zhou, Xiao-Ting; Zou, Zhi-Rong

    2014-10-01

    In this research, the possibility of exogenous application of 5-aminolevulinic acid (ALA) on photosynthetic characteristics of tomato seedlings under NaCl stress was investigated. Five leaves seedlings of tomato (Solanum lycopersicum cv. Jinpeng No. 1) were used as starting materials, applied with 50 mg · L(-1) ALA by foliage spray or 10 mg · L(-1) ALA by root soaking to study the changes in their photosynthesis and fluorescence parameters under 100 mmol · L(-1) NaCl. The result showed that, photosynthetic gas exchange parameters (net photosynthetic rate P,, stomata conductance g(s), intercellular CO2 concentration Ci, transpiration Tr) and chlorophyll fluorescence parameters (Fv'/Fm', Fm', ΦPS II, ETR, qP, Pc) were severely reduced under NaCl treatment and ALA application by foliage spray or root soaking with proper concentrations exerted positive influences on tomato seedlings under salt stress, while there were some differences between foliage spray and root soaking in the influence on chlorophyll content, photosynthesis and chlorophyll fluorescence. Both foliage spray with 50 mg · L(-1) ALA and root soaking with 10 mg L(-1) ALA significantly increased Pn, Ci, g(s) and Tr of tomato seedlings under NaCl stress, alleviated photosynthetic inhibition. Root application of ALA had a better effect on the chlorophyll content than foliage application. However, the photosynthetic parameters showed that foliage application of ALA had a better effect than root application, and both treatments had no difference in the influence on chlorophyll fluorescence parameters of tomato seedlings. It could be deduced that the regulating effect of ALA on enhancing salt tolerance of tomato seedlings is attributed to its effect on improving chlorophyll biosynthesis and metabolism, increasing stomatal conductance and reducing stomatal limitation, thus, enhancing the photosynthetic capacity and PS II photochemical efficiency of tomato leaves under NaCl stress. PMID:25796901

  20. [Effect of 5-aminolevulinic acid on photosynthetic characteristics of tomato seedlings under NaCl stress].

    PubMed

    Zhao, Yan-Yan; Yan, Fei; Hu, Li-Pan; Zhou, Xiao-Ting; Zou, Zhi-Rong

    2014-10-01

    In this research, the possibility of exogenous application of 5-aminolevulinic acid (ALA) on photosynthetic characteristics of tomato seedlings under NaCl stress was investigated. Five leaves seedlings of tomato (Solanum lycopersicum cv. Jinpeng No. 1) were used as starting materials, applied with 50 mg · L(-1) ALA by foliage spray or 10 mg · L(-1) ALA by root soaking to study the changes in their photosynthesis and fluorescence parameters under 100 mmol · L(-1) NaCl. The result showed that, photosynthetic gas exchange parameters (net photosynthetic rate P,, stomata conductance g(s), intercellular CO2 concentration Ci, transpiration Tr) and chlorophyll fluorescence parameters (Fv'/Fm', Fm', ΦPS II, ETR, qP, Pc) were severely reduced under NaCl treatment and ALA application by foliage spray or root soaking with proper concentrations exerted positive influences on tomato seedlings under salt stress, while there were some differences between foliage spray and root soaking in the influence on chlorophyll content, photosynthesis and chlorophyll fluorescence. Both foliage spray with 50 mg · L(-1) ALA and root soaking with 10 mg L(-1) ALA significantly increased Pn, Ci, g(s) and Tr of tomato seedlings under NaCl stress, alleviated photosynthetic inhibition. Root application of ALA had a better effect on the chlorophyll content than foliage application. However, the photosynthetic parameters showed that foliage application of ALA had a better effect than root application, and both treatments had no difference in the influence on chlorophyll fluorescence parameters of tomato seedlings. It could be deduced that the regulating effect of ALA on enhancing salt tolerance of tomato seedlings is attributed to its effect on improving chlorophyll biosynthesis and metabolism, increasing stomatal conductance and reducing stomatal limitation, thus, enhancing the photosynthetic capacity and PS II photochemical efficiency of tomato leaves under NaCl stress.

  1. Blood interference in fiber-optical based fluorescence guided resection of glioma using 5-aminolevulinic acid

    NASA Astrophysics Data System (ADS)

    Haj-Hosseini, Neda; Lowndes, Shannely; Salerud, Göran; Wårdell, Karin

    2011-03-01

    Fluorescence guidance in brain tumor resection is performed intra-operatively where bleeding is included. When using fiber-optical probes, the transmission of light to and from the tissue is totally or partially blocked if a small amount of blood appears in front of the probe. Sometimes even after rinsing with saline, the remnant blood cells on the optical probe head, disturb the measurements. In such a case, the corresponding spectrum cannot be reliably quantified and is therefore discarded. The optimal case would be to calculate and take out the blood effect systematically from the collected signals. However, the first step is to study the pattern of blood interference in the fluorescence spectrum. In this study, a fiber-optical based fluorescence spectroscopy system with a laser excitation light of 405 nm (1.4 J/cm2) was used during fluorescence guided brain tumor resection using 5-aminolevulinic acid (5-ALA). The blood interference pattern in the fluorescence spectrum collected from the brain was studied in two patients. The operation situation was modeled in the laboratory by placing blood drops from the finger tip on the skin of forearm and the data was compared to the brain in vivo measurements. Additionally, a theoretical model was developed to simulate the blood interference pattern on the skin autofluorescence. The blood affects the collected fluorescence intensity and leaves traces of oxy and deoxy-hemoglobin absorption peaks. According to the developed theoretical model, the autofluorescence signal is considered to be totally blocked by an approximately 500 μm thick blood layer.

  2. Quantification of topically delivered 5-aminolevulinic acid by lontophoresis across ex vivo human stratum corneum.

    PubMed

    Boddé, Harry E; Roemelé, Peter E H; Star, Willem M

    2002-04-01

    Iontophoretic transport of the prodrug 5-aminolevulinic acid (ALA), which is used for photodynamic therapy (PDT), across human stratum corneum (SC) was studied quantitatively in vitro. The experiments were carried out in a three-compartment iontophoresis cell consisting of two electrode chambers equipped with Ag-AgCl electrodes, each separated from a central acceptor chamber by a sheet of SC, supported by a dialysis membrane, to mimic the side-by-side configuration normally used in vivo. Acceptor fluid samples were collected every hour for a period of 30 h in a fraction collector and analyzed by high-performance liquid chromatography-fluorometry after derivatization of the ALA. The iontophoretic ALA flux was studied as a function of the applied current density and the ALA concentration in the donor solution (1, 2.5 or 10% ALA). Depending on the ALA concentration in the donor cell, iontophoresis enhances the flux from close to the detection limit of 0.23 nmol cm(-2) h(-1) at zero current density (passive diffusion) to several hundred or thousand nanomoles per square centimeter per hour at current densities ranging from 100 to 1000 microA cm(-2). For example, interpolating our data we find that with an ALA concentration of 2% in the donor chamber, a current density of 0.255 mA cm(-2) transports 0.065 micromol cm(-2) ALA across the SC in 10 min (conditions of Rhodes et al. (1997), J. Invest. Dermatol. 108, 87-91). For passive diffusion we find that a 5 h topical application of 20% ALA results in the transport of 0.05 micromol cm(-2). Thus, the amount of ALA that passively diffuses through the SC in several hours, leading to therapeutic levels of protoporphyrin IX (PpIX) in the epidermis, can be delivered by iontophoresis in 10 min or less. However, because the formation of sufficient PpIX also requires several hours and also because the SC overlying skin lesions such as basal cell carcinoma (BCC) is not intact, the clinical benefit of topical ALA delivery by

  3. Risk factors for developing oral 5-aminolevulinic acid-induced side effects in patients undergoing fluorescence guided resection.

    PubMed

    Chung, Ivan Wong Hin; Eljamel, Sam

    2013-12-01

    Oral 5 aminolevulinic acid (5-ALA) is used to assist surgical resection of malignant tumours in the brain and other locations. Hypotension and alteration of liver functions have been reported as potential adverse effects. This study was designed to assess the incidence and contributing factors that cause 5-ALA induced side effects in a cohort of 90 patients. Hypotension occurred in 11% of patients irrespective of 5-ALA dose. The only contributing factor was the presence of cardiovascular disease and antihypertensive drug therapy with an odd ratio of 17.7. Liver function were disturbed in 2% in patients who received 20mg or less/kg body weight compared to 4% in those who received a dose of >20mg/kg 5-ALA. The liver dysfunction was minor and was not clinically significant. We concluded that 5-ALA induced side effects were minimal and hypotension more likely to occur in patients receiving antihypertensive drug therapy.

  4. Optimization of Influencing Factors on Biomass Accumulation and 5-Aminolevulinic Acid (ALA) Yield in Rhodobacter sphaeroides Wastewater Treatment.

    PubMed

    Liu, Shuli; Li, Xiangkun; Zhang, Guangming; Zhang, Jie

    2015-11-01

    This study aimed to optimize four factors affecting biomass accumulation and 5-aminolevulinic acid (ALA) yield together with pollutants removal in Rhodobacter sphaeroides wastewater treatment. Results showed that it was feasible to produce biomass and ALA in R. sphaeroides wastewater treatment. Microaerobic, 1,000-3,000 lux, and pH 7.0 were optimal conditions for the highest ALA yield of 4.5 ± 0.5 mg/g-biomass. Under these conditions, COD removal and biomass production rate were 93.3 ± 0.9% and 31.8 ± 0.5 mg/l/h, respectively. In addition, trace elements Fe(2+), Mg(2+), Ni(2+), and Zn(2+) further improved the ALA yield, COD removal, and biomass production rate. Specifically, the highest ALA yield (12.5 ± 0.6 mg/g-biomass) was achieved with Fe(2+) addition.

  5. The heme precursor 5-aminolevulinic acid disrupts the Warburg effect in tumor cells and induces caspase-dependent apoptosis.

    PubMed

    Sugiyama, Yuta; Hagiya, Yuichiro; Nakajima, Motowo; Ishizuka, Masahiro; Tanaka, Tohru; Ogura, Shun-Ichiro

    2014-03-01

    Our previous study demonstrated that 5-aminolevulinic acid (ALA) administered to mice stimulates oxidative phosphorylation by upregulation of the mitochondrial respiratory chain complex IV enzyme cytochrome c oxidase (COX). The present study investigated whether ALA disrupts the Warburg effect, which represents a shift in ATP generation from oxidative phosphorylation to glycolysis, protecting tumor cells against oxidative stress-mediated apoptosis. The human lung carcinoma cell line A549 exposed to ALA exhibited enhanced oxidative phosphorylation, which was indicated by an increase in COX protein expression and oxygen consumption. Furthermore, ALA suppressed glycolysis-mediated acidosis. This normalization of the ATP metabolic pathways significantly increased the generation of superoxide anion radical (O2•-) and the functional expression of active caspase-3, leading to caspase-dependent apoptosis. These data demonstrate that ALA inhibits the Warburg effect and induces cancer cell death. Use of this endogenous compound might constitute a novel approach to cancer therapy. PMID:24366173

  6. Photodynamic therapy using systemic administration of 5-aminolevulinic acid and a 410-nm wavelength light-emitting diode for methicillin-resistant Staphylococcus aureus-infected ulcers in mice.

    PubMed

    Morimoto, Kuniyuki; Ozawa, Toshiyuki; Awazu, Kunio; Ito, Nobuhisa; Honda, Norihiro; Matsumoto, Sohkichi; Tsuruta, Daisuke

    2014-01-01

    Bacterial resistance to antibiotics has become a worldwide problem. One potential alternative for bacterial control is photodynamic therapy. 5-aminolevulinic acid is a natural precursor of the photosensitizer protoporphyrin IX. Relatively little is known about the antibacterial efficacy of photodynamic therapy using the systemic administration of 5-aminolevulinic acid; a few reports have shown that 5-aminolevulinic acid exerts photodynamic effects on methicillin-resistant Staphylococcus aureus (MRSA) in vitro. In this study, we evaluated the effectiveness of photodynamic therapy using 5-aminolevulinic acid and a 410-nm wavelength light-emitting diode in vitro and in vivo for the treatment of MRSA. We found that 5-aminolevulinic acid photodynamic therapy with the light-emitting diode had an in-vitro bactericidal effect on MRSA. In vivo, protoporphyrin IX successfully accumulated in MRSA on ulcer surfaces after intraperitoneal administration of 5-aminolevulinic acid to mice. Furthermore, 5-aminolevulinic acid photodynamic therapy accelerated wound healing and decreased bacterial counts on ulcer surfaces; in contrast, vancomycin treatment did not accelerate wound healing. Our findings indicate that 5-aminolevulinic acid photodynamic therapy may be a new treatment option for MRSA-infected wounds.

  7. Photodynamic Therapy Using Systemic Administration of 5-Aminolevulinic Acid and a 410-nm Wavelength Light-Emitting Diode for Methicillin-Resistant Staphylococcus aureus-Infected Ulcers in Mice

    PubMed Central

    Morimoto, Kuniyuki; Ozawa, Toshiyuki; Awazu, Kunio; Ito, Nobuhisa; Honda, Norihiro; Matsumoto, Sohkichi; Tsuruta, Daisuke

    2014-01-01

    Bacterial resistance to antibiotics has become a worldwide problem. One potential alternative for bacterial control is photodynamic therapy. 5-aminolevulinic acid is a natural precursor of the photosensitizer protoporphyrin IX. Relatively little is known about the antibacterial efficacy of photodynamic therapy using the systemic administration of 5-aminolevulinic acid; a few reports have shown that 5-aminolevulinic acid exerts photodynamic effects on methicillin-resistant Staphylococcus aureus (MRSA) in vitro. In this study, we evaluated the effectiveness of photodynamic therapy using 5-aminolevulinic acid and a 410-nm wavelength light-emitting diode in vitro and in vivo for the treatment of MRSA. We found that 5-aminolevulinic acid photodynamic therapy with the light-emitting diode had an in-vitro bactericidal effect on MRSA. In vivo, protoporphyrin IX successfully accumulated in MRSA on ulcer surfaces after intraperitoneal administration of 5-aminolevulinic acid to mice. Furthermore, 5-aminolevulinic acid photodynamic therapy accelerated wound healing and decreased bacterial counts on ulcer surfaces; in contrast, vancomycin treatment did not accelerate wound healing. Our findings indicate that 5-aminolevulinic acid photodynamic therapy may be a new treatment option for MRSA-infected wounds. PMID:25140800

  8. Effect of 5-aminolevulinic acid on erythropoiesis: A preclinical in vitro characterization for the treatment of congenital sideroblastic anemia

    SciTech Connect

    Fujiwara, Tohru; Takahashi, Kiwamu; Okitsu, Yoko; Fukuhara, Noriko; Onishi, Yasushi; Ishizawa, Kenichi; Ichinohasama, Ryo; Nakamura, Yukio; Nakajima, Motowo; Tanaka, Tohru; Harigae, Hideo

    2014-11-07

    Highlights: • Treatment with ALA induces erythroid differentiation of K562 cells. • Transportation of ALA into erythroid cells occurs predominantly via SLC36A1. • ALA restores defects in ALAS2 in human iPS cell-derived erythroblasts. • ALA may represent a novel therapeutic option for CSA caused by ALAS2 mutations. - Abstract: Congenital sideroblastic anemia (CSA) is a hereditary disorder characterized by microcytic anemia and bone marrow sideroblasts. The most common form of CSA is attributed to mutations in the X-linked gene 5-aminolevulinic acid synthase 2 (ALAS2). ALAS2 is a mitochondrial enzyme, which utilizes glycine and succinyl-CoA to form 5-aminolevulinic acid (ALA), a crucial precursor in heme synthesis. Therefore, ALA supplementation could be an effective therapeutic strategy to restore heme synthesis in CSA caused by ALAS2 defects. In a preclinical study, we examined the effects of ALA in human erythroid cells, including K562 cells and human induced pluripotent stem cell-derived erythroid progenitor (HiDEP) cells. ALA treatment resulted in significant dose-dependent accumulation of heme in the K562 cell line. Concomitantly, the treatment substantially induced erythroid differentiation as assessed using benzidine staining. Quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis confirmed significant upregulation of heme-regulated genes, such as the globin genes [hemoglobin alpha (HBA) and hemoglobin gamma (HBG)] and the heme oxygenase 1 (HMOX1) gene, in K562 cells. Next, to investigate the mechanism by which ALA is transported into erythroid cells, quantitative RT-PCR analysis was performed on previously identified ALA transporters, including solute carrier family 15 (oligopeptide transporter), member (SLC15A) 1, SLC15A2, solute carrier family 36 (proton/amino acid symporter), member (SLC36A1), and solute carrier family 6 (neurotransmitter transporter), member 13 (SLC6A13). Our analysis revealed that SLC36A1 was abundantly

  9. Formation of protoporphyrin IX in mouse skin after topical application of 5-aminolevulinic acid and its methyl esther

    NASA Astrophysics Data System (ADS)

    Sorensen, Roar; Juzenas, Petras; Iani, Vladimir; Moan, Johan

    1999-02-01

    Normal skin of nude mice (Balb/c) was treated topically with 5-aminolevulinic acid (ALA) and its methyl ester (ALA-Me) for 24 hours. Approximately 0.1 gram of freshly prepared cream was applied to a spot of 1 cm2 on the flank of the mice, which was then covered with a transparent dressing. The ALA induced protoporphyrin IX (PpIX) was studied by means of a noninvasive fiber-optic fluorescence probe connected to a luminescence spectrometer. The excitation wavelength was 407 nm, and the emission wavelength was 637 nm. For the first hour a slight lag in PpIX production was observed for the mice treated with ALA-Me compared to the mice treated with ALA. After approximately 12 hours the ALA and the ALA-Me treated mice showed the same PpIX fluorescence intensity. From 12 hours until 24 hours the PpIX fluorescence intensity decreased for both treatment modalities, even though ALA and ALA-Me were continuously present. At 24 hours ALA-Me-treated mice had less than half the amount of PpIX in their skin compared with ALA- treated mice.

  10. Clearance of protoporphyrin IX from mouse skin after topical application of 5-aminolevulinic acid and its methyl ester

    NASA Astrophysics Data System (ADS)

    Juzenas, Petras; Sorensen, Roar; Iani, Vladimir; Moan, Johan

    1999-02-01

    The clearance of protoporphyrin IX (PpIX) from the skin of hairless BALB/c mice after topical application of 5- aminolevulinic acid (ALA) and its methyl ester (ALA-Me) was investigated. Creams containing 2 or 20% of ALA or ALA-Me were topically applied on spots of approximately 1 cm2 for 12 hours. The PpIX fluorescence was detected by the means of a Perkin Elmer LS50B luminescence spectrometer equipped with a fiber-optic probe. The emission spectrum was identical with that of cell-bound PpIX. After 12 hours application of ALA and ALA-Me similar amounts of PpIX were found. After creme removal the ALA-induced PpIX fluorescence decayed with a half-life of about 20 hours (20% ALA cream). The ALA-Me-induced PpIX was faster cleared from the skin than ALA-induced PpIX, and had a half-life of about 7 hours (20% ALA-Me cream).

  11. Photosensitizer fluorescence and singlet oxygen luminescence as dosimetric predictors of topical 5-aminolevulinic acid photodynamic therapy induced clinical erythema

    PubMed Central

    Mallidi, Srivalleesha; Anbil, Sriram; Lee, Seonkyung; Manstein, Dieter; Elrington, Stefan; Kositratna, Garuna; Schoenfeld, David; Pogue, Brian; Davis, Steven J.; Hasan, Tayyaba

    2014-01-01

    Abstract. The need for patient-specific photodynamic therapy (PDT) in dermatologic and oncologic applications has triggered several studies that explore the utility of surrogate parameters as predictive reporters of treatment outcome. Although photosensitizer (PS) fluorescence, a widely used parameter, can be viewed as emission from several fluorescent states of the PS (e.g., minimally aggregated and monomeric), we suggest that singlet oxygen luminescence (SOL) indicates only the active PS component responsible for the PDT. Here, the ability of discrete PS fluorescence-based metrics (absolute and percent PS photobleaching and PS re-accumulation post-PDT) to predict the clinical phototoxic response (erythema) resulting from 5-aminolevulinic acid PDT was compared with discrete SOL (DSOL)-based metrics (DSOL counts pre-PDT and change in DSOL counts pre/post-PDT) in healthy human skin. Receiver operating characteristic curve (ROC) analyses demonstrated that absolute fluorescence photobleaching metric (AFPM) exhibited the highest area under the curve (AUC) of all tested parameters, including DSOL based metrics. The combination of dose-metrics did not yield better AUC than AFPM alone. Although sophisticated real-time SOL measurements may improve the clinical utility of SOL-based dosimetry, discrete PS fluorescence-based metrics are easy to implement, and our results suggest that AFPM may sufficiently predict the PDT outcomes and identify treatment nonresponders with high specificity in clinical contexts. PMID:24503639

  12. Improvement of systemic 5-aminolevulinic acid-based photodynamic therapy in vivo using light fractionation with a 75-minute interval.

    PubMed

    de Bruijn, H S; van der Veen, N; Robinson, D J; Star, W M

    1999-02-15

    We have studied different single and fractionated illumination schemes after systemic administration of 5-aminolevulinic acid (ALA) to Improve the response of nodular tumors to ALA-mediated photodynamic therapy. Tumors transplanted on the thigh of female WAG/Rij rats were transdermally illuminated with red light (633 nm) after systemic ALA administration (200 mg/kg). The effectiveness of each treatment scheme was determined from the tumor volume doubling time. A single illumination (100 J/cm2 at 100 mW/cm2, 2.5 h after ALA administration) yielded a doubling time of 6.6+/-1.2 days. This was significantly different from the untreated control (doubling time, 1.7+/-0.1 days). The only treatment scheme that yielded a significant improvement compared to all other schemes studied was illumination at both 1 and 2.5 h after ALA administration (both 100 J/cm2 at 100 mW/cm2) and resulted in a tumor volume doubling time of 18.9+/-2.9 days. A possible mechanism to explain this phenomenon is that the protoporphyrin IX formed after administration of ALA is photodegraded by the first illumination. In the 75-min interval, new porphyrin is formed enhancing the effect of the second illumination. PMID:10029082

  13. Prospective study of topical 5-aminolevulinic acid photodynamic therapy for the treatment of severe adolescent acne in Chinese patients.

    PubMed

    Ma, Ying; Liu, Ye; Wang, Qianqian; Ren, Jie; Xiang, Leihong

    2015-05-01

    Acne vulgaris is one of the most common skin diseases in adolescents. In the present study, we aimed to evaluate the effectiveness and safety of topical 5-aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) for the treatment of severe acne in Chinese adolescent patients. Twenty-one Chinese adolescent patients aged 12-18 years with Pillsbury III-IV severe facial acne were treated with three courses of ALA-PDT. A 5% ALA lotion was applied topically for 60 min followed by irradiation with light-emitting diode light at 633 nm with a light intensity of 75-80 mW/cm(2) and a light dose of 90-96 J/cm(2) . Clinical assessment was conducted before and after each treatment, and at each follow-up session. The total effective rates were 85.71%, 90.48%, and 95.23% after the three PDT sessions, and at the 4- and 8-week follow ups, respectively. ALA-PDT is an effective treatment for severe adolescent acne vulgaris, and is associated with mild and reversible side-effects.

  14. Topical photodynamic therapy with 5-aminolevulinic acid in the treatment of actinic keratoses: a first clinical study

    NASA Astrophysics Data System (ADS)

    Karrer, Sigrid; Szeimies, Rolf-Markus; Sauerwald, Angela; Landthaler, Michael

    1996-01-01

    In this first clinical study performed according to GCP- (good clinical practice) guidelines, efficacy, and tolerability of topical photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA) were tested in the treatment of actinic keratoses. Ten patients (6 f, 4 m) with 36 lesions (19 located on hands and arms, 17 on the head) received ALA-PDT once. Five to six hours after occlusive application of ALA (water-in-oil-emulsion containing 10% ALA) irradiation was performed with an incoherent light source. Up to 3 months after treatment patients were monitored. A score evaluating infiltration and keratosis of treated actinic keratoses allowed us to estimate therapeutic efficacy. Compared to the initial score (100%) significantly lower score-sums were observed at the 28 day follow-up at both localizations (head: 15%; hand: 67%). Complete remission (score sum 0) resulted in 71% of actinic keratoses localized on the head. Except for slight pain and burning sensations during and after irradiation there were no notable side effects. This study proved good efficacy and tolerability of topical PDT in the treatment of actinic keratoses. Whether PDT is able to compete with established treatment modalities remains to be shown in further studies.

  15. 5-Aminolevulinic Acid-Mediated Sonodynamic Therapy Inhibits RIPK1/RIPK3-Dependent Necroptosis in THP-1-Derived Foam Cells

    PubMed Central

    Tian, Fang; Yao, Jianting; Yan, Meng; Sun, Xin; Wang, Wei; Gao, Weiwei; Tian, Zhen; Guo, Shuyuan; Dong, Zengxiang; Li, Bicheng; Gao, Tielei; Shan, Peng; Liu, Bing; Wang, Haiyang; Cheng, Jiali; Gao, Qianping; Zhang, Zhiguo; Cao, Wenwu; Tian, Ye

    2016-01-01

    Necroptosis, or programmed necrosis, contributes to the formation of necrotic cores in atherosclerotic plaque in animal models. However, whether inhibition of necroptosis ameliorates atherosclerosis is largely unknown. In this study, we demonstrated that necroptosis occurred in clinical atherosclerotic samples, suggesting that it may also play an important role in human atherosclerosis. We established an in vitro necroptotic model in which necroptosis was induced in THP-1-derived foam cells by serum deprivation. With this model, we demonstrated that 5-aminolevulinic acid-mediated sonodynamic therapy (ALA-SDT) inhibited necroptosis while promoting apoptosis. ALA-SDT activated the caspase-3 and caspase-8 pathways in foam cells, which is responsible for the switch from necroptosis to apoptosis. The inhibition of either caspase-8 or caspase-3 abolished the anti-necroptotic effect of ALA-SDT. In addition, we found that caspase-3 activation peaked 4 hours after ALA-SDT treatment, 2 hours earlier than maximal caspase-8activation. Taken together, our data indicate that ALA-SDT mediates the switch from necroptosis to apoptosis by activating the caspase-3 and caspase-8 pathways and may improve the prognosis of atherosclerosis. PMID:26911899

  16. 5-Aminolevulinic Acid-Mediated Sonodynamic Therapy Inhibits RIPK1/RIPK3-Dependent Necroptosis in THP-1-Derived Foam Cells.

    PubMed

    Tian, Fang; Yao, Jianting; Yan, Meng; Sun, Xin; Wang, Wei; Gao, Weiwei; Tian, Zhen; Guo, Shuyuan; Dong, Zengxiang; Li, Bicheng; Gao, Tielei; Shan, Peng; Liu, Bing; Wang, Haiyang; Cheng, Jiali; Gao, Qianping; Zhang, Zhiguo; Cao, Wenwu; Tian, Ye

    2016-01-01

    Necroptosis, or programmed necrosis, contributes to the formation of necrotic cores in atherosclerotic plaque in animal models. However, whether inhibition of necroptosis ameliorates atherosclerosis is largely unknown. In this study, we demonstrated that necroptosis occurred in clinical atherosclerotic samples, suggesting that it may also play an important role in human atherosclerosis. We established an in vitro necroptotic model in which necroptosis was induced in THP-1-derived foam cells by serum deprivation. With this model, we demonstrated that 5-aminolevulinic acid-mediated sonodynamic therapy (ALA-SDT) inhibited necroptosis while promoting apoptosis. ALA-SDT activated the caspase-3 and caspase-8 pathways in foam cells, which is responsible for the switch from necroptosis to apoptosis. The inhibition of either caspase-8 or caspase-3 abolished the anti-necroptotic effect of ALA-SDT. In addition, we found that caspase-3 activation peaked 4 hours after ALA-SDT treatment, 2 hours earlier than maximal caspase-8activation. Taken together, our data indicate that ALA-SDT mediates the switch from necroptosis to apoptosis by activating the caspase-3 and caspase-8 pathways and may improve the prognosis of atherosclerosis. PMID:26911899

  17. Needle-free injection of 5-aminolevulinic acid in photodynamic therapy for the treatment of condylomata acuminata

    PubMed Central

    LI, XIULI; WANG, XIUXIU; GU, JUNYING; MA, YUE’E; LIU, ZHIYU; SHI, YULING

    2013-01-01

    The external application of 5-aminolevulinic acid (ALA) in photodynamic therapy (PDT) results in a shallow penetration depth in thick or extensive condylomata acuminata (CA) lesions, thus demonstrating a poor therapeutic effect for those patients. To compare the efficacy of needle-free injection with external application of ALA in PDT for the treatment of CA, 160 CA patients with thick or extensive warts received ALA-PDT by means of external application or needle-free injection of ALA, respectively. The complete response (CR) rate and recurrence rate in the two groups were analyzed. The CR rate after the first treatment in the needle-free injection group (68.8%) was significantly higher compared with that in the external application group (52.5%; P=0.035). The recurrence rates in the needle-free injection group and external application group were 4.1 and 15.4%, respectively (P=0.022). The needle-free injection of ALA increases the therapeutic effect of PDT for CA patients with thick or extensive lesions. It shortens the treatment time and reduces the recurrence rate, and has great potential in the treatment of CA. PMID:23935753

  18. 5-aminolevulinic acid for quantitative seek-and-treat of high-grade dysplasia in Barrett's esophagus cellular models

    NASA Astrophysics Data System (ADS)

    Yeh, Shu-Chi Allison; Ling, Celine S. N.; Andrews, David W.; Patterson, Michael S.; Diamond, Kevin R.; Hayward, Joseph E.; Armstrong, David; Fang, Qiyin

    2015-02-01

    High-grade dysplasia (HGD) in Barrett's esophagus (BE) poses increased risk for developing esophageal adenocarcinoma. To date, early detection and treatment of HGD regions are still challenging due to the sampling error from tissue biopsy and relocation error during the treatment after histopathological analysis. In this study, CP-A (metaplasia) and CP-B (HGD) cell lines were used to investigate the "seek-and-treat" potential using 5-aminolevulinic acid-induced protoporphyrin IX (PpIX). The photodynamic therapy photosensitizer then provides both a phototoxic effect and additional image contrast for automatic detection and real-time laser treatment. Complementary to our studies on automatic classification, this work focused on characterizing subcellular irradiation and the potential phototoxicity on both metaplasia and HGD. The treatment results showed that the HGD cells are less viable than metaplastic cells due to more PpIX production at earlier times. Also, due to mitochondrial localization of PpIX, a better killing effect was achieved by involving mitochondria or whole cells compared with just nucleus irradiation in the detected region. With the additional toxicity given by PpIX and potential morphological/textural differences for pattern recognition, this cellular platform serves as a platform to further investigate real-time "seek-and-treat" strategies in three-dimensional models for improving early detection and treatment of BE.

  19. Cystic acne improved by photodynamic therapy with short-contact 5-aminolevulinic acid and sequential combination of intense pulsed light and blue light activation.

    PubMed

    Melnick, Stuart

    2005-01-01

    Photodynamic therapy with short-contact 5-aminolevulinic acid (Levulan Kerastick, Dusa Pharmaceuticals, Inc.) and activation by intense pulsed light in an initial treatment and blue light in 3 subsequent treatments has resulted in significant improvement in severity of acne, reduction in the number of lesions, improvement in skin texture, and smoothing of scar edges in an Asian patient with severe (class 4) facial cystic acne and scarring. PMID:16302560

  20. In Vivo Curative and Protective Potential of Orally Administered 5-Aminolevulinic Acid plus Ferrous Ion against Malaria

    PubMed Central

    Suzuki, Shigeo; Hikosaka, Kenji; Balogun, Emmanuel O.; Komatsuya, Keisuke; Niikura, Mamoru; Kobayashi, Fumie; Takahashi, Kiwamu; Tanaka, Tohru; Nakajima, Motowo

    2015-01-01

    5-Aminolevulinic acid (ALA) is a naturally occurring amino acid present in diverse organisms and a precursor of heme biosynthesis. ALA is commercially available as a component of cosmetics, dietary supplements, and pharmaceuticals for cancer diagnosis and therapy. Recent reports demonstrated that the combination of ALA and ferrous ion (Fe2+) inhibits the in vitro growth of the human malaria parasite Plasmodium falciparum. To further explore the potential application of ALA and ferrous ion as a combined antimalarial drug for treatment of human malaria, we conducted an in vivo efficacy evaluation. Female C57BL/6J mice were infected with the lethal strain of rodent malaria parasite Plasmodium yoelii 17XL and orally administered ALA plus sodium ferrous citrate (ALA/SFC) as a once-daily treatment. Parasitemia was monitored in the infected mice, and elimination of the parasites was confirmed using diagnostic PCR. Treatment of P. yoelii 17XL-infected mice with ALA/SFC provided curative efficacy in 60% of the mice treated with ALA/SFC at 600/300 mg/kg of body weight; no mice survived when treated with vehicle alone. Interestingly, the cured mice were protected from homologous rechallenge, even when reinfection was attempted more than 230 days after the initial recovery, indicating long-lasting resistance to reinfection with the same parasite. Moreover, parasite-specific antibodies against reported vaccine candidate antigens were found and persisted in the sera of the cured mice. These findings provide clear evidence that ALA/SFC is effective in an experimental animal model of malaria and may facilitate the development of a new class of antimalarial drug. PMID:26324278

  1. In vitro skin permeation and retention of 5-aminolevulinic acid ester derivatives for photodynamic therapy.

    PubMed

    De Rosa, Fernanda Scarmato; Tedesco, Antônio Cláudio; Lopez, Renata Fonseca Vianna; Pierre, Maria Bernadete Riemma; Lange, Norbert; Marchetti, Juliana Maldonado; Rotta, Jeane Cristina Gomes; Bentley, Maria Vitória Lopes Badra

    2003-04-29

    In photodynamic therapy (PDT), 5-aminiolevulinic acid (5-ALA) applied topically is converted, via the heme cycle, into protoporphyrin IX (PpIX), a photosensitizing agent, which upon excitation with light can induce tumor destruction. Due to its hydrophilic and zwitterionic characteristics, 5-ALA has limited penetration into the skin. More lipophilic 5-ALA ester derivatives are expected to cross stratum corneum more easily than 5-ALA. According to the determination of the partition coefficients of 5-ALA methyl, n-butyl, n-hexyl and n-octyl esters, these compounds showed an increased affinity to the SC, with 5-ALA hexyl ester and 5-ALA-octyl ester having the highest partition coefficients. Our in vitro skin permeation studies demonstrated an increased permeated amount for hexyl-ALA after 6 h of incubation, compared to other esters and 5-ALA. After 6 h, more 5-ALA-hexyl ester and -octyl ester were retained at viable epidermis and dermis than 5-ALA. According to these results, and considering that the conversion of 5-ALA into PpIX occurs preferentially in epidermis, it can be supposed that topical use of ester derivatives with longer chains (C(6) or C(8)) is an interesting proposal to optimize topical 5-ALA-PDT

  2. Nitric Oxide Mediates 5-Aminolevulinic Acid-Induced Antioxidant Defense in Leaves of Elymus nutans Griseb. Exposed to Chilling Stress

    PubMed Central

    Fu, Juanjuan; Chu, Xitong; Sun, Yongfang; Miao, Yanjun; Xu, Yuefei; Hu, Tianming

    2015-01-01

    Nitric oxide (NO) and 5-aminolevulinic acid (ALA) are both extremely important signalling molecules employed by plants to control many aspects of physiology. In the present study, the role of NO in ALA-induced antioxidant defense in leaves of two sources of Elymus nutans Griseb. (Damxung, DX and Zhengdao, ZD) was investigated. Chilling stress enhanced electrolyte leakage, accumulation of malondialdehyde (MDA), hydrogen peroxide (H2O2) and superoxide radical in two E. nutans, which were substantially alleviated by exogenous ALA and NO application. Pretreatment with NO scavenger PTIO or NOS inhibitor L-NNA alone and in combination with ALA induced enhancements in electrolyte leakage and the accumulation of MDA, H2O2 and superoxide radical in leaves of DX and ZD exposed to chilling stress, indicating that the inhibition of NO biosynthesis reduced the chilling resistance of E. nutans and the ALA-enhanced chilling resistance. Further analyses showed that ALA and NO enhanced antioxidant defense and activated plasma membrane (PM) H+-ATPase and decreased the accumulation of ROS induced by chilling stress. A pronounced increase in nitric oxide synthase (NOS) activity and NO release by exogenous ALA treatment was found in chilling-resistant DX plants exposed to chilling stress, while only a little increase was observed in chilling-sensitive ZD. Furthermore, inhibition of NO accumulation by PTIO or L-NNA blocked the protective effect of exogenous ALA, while both exogenous NO treatment and inhibition of endogenous NO accumulation did not induce ALA production. These results suggested that NO might be a downstream signal mediating ALA-induced chilling resistance in E. nutans. PMID:26151364

  3. Regulation of Cadmium-Induced Proteomic and Metabolic Changes by 5-Aminolevulinic Acid in Leaves of Brassica napus L.

    PubMed

    Ali, Basharat; Gill, Rafaqat A; Yang, Su; Gill, Muhammad B; Farooq, Muhammad A; Liu, Dan; Daud, Muhammad K; Ali, Shafaqat; Zhou, Weijun

    2015-01-01

    It is evident from previous reports that 5-aminolevulinic acid (ALA), like other known plant growth regulators, is effective in countering the injurious effects of heavy metal-stress in oilseed rape (Brassica napus L.). The present study was carried out to explore the capability of ALA to improve cadmium (Cd2+) tolerance in B. napus through physiological, molecular, and proteomic analytical approaches. Results showed that application of ALA helped the plants to adjust Cd2+-induced metabolic and photosynthetic fluorescence changes in the leaves of B. napus under Cd2+ stress. The data revealed that ALA treatment enhanced the gene expressions of antioxidant enzyme activities substantially and could increase the expression to a certain degree under Cd2+ stress conditions. In the present study, 34 protein spots were identified that differentially regulated due to Cd2+ and/or ALA treatments. Among them, 18 proteins were significantly regulated by ALA, including the proteins associated with stress related, carbohydrate metabolism, catalysis, dehydration of damaged protein, CO2 assimilation/photosynthesis and protein synthesis/regulation. From these 18 ALA-regulated proteins, 12 proteins were significantly down-regulated and 6 proteins were up-regulated. Interestingly, it was observed that ALA-induced the up-regulation of dihydrolipoyl dehydrogenase, light harvesting complex photo-system II subunit 6 and 30S ribosomal proteins in the presence of Cd2+ stress. In addition, it was also observed that ALA-induced the down-regulation in thioredoxin-like protein, 2, 3-bisphosphoglycerate, proteasome and thiamine thiazole synthase proteins under Cd2+ stress. Taken together, the present study sheds light on molecular mechanisms involved in ALA-induced Cd2+ tolerance in B. napus leaves and suggests a more active involvement of ALA in plant physiological processes than previously proposed.

  4. Regulation of Cadmium-Induced Proteomic and Metabolic Changes by 5-Aminolevulinic Acid in Leaves of Brassica napus L.

    PubMed Central

    Ali, Basharat; Gill, Rafaqat A.; Yang, Su; Gill, Muhammad B.; Farooq, Muhammad A.; Liu, Dan; Daud, Muhammad K.; Ali, Shafaqat; Zhou, Weijun

    2015-01-01

    It is evident from previous reports that 5-aminolevulinic acid (ALA), like other known plant growth regulators, is effective in countering the injurious effects of heavy metal-stress in oilseed rape (Brassica napus L.). The present study was carried out to explore the capability of ALA to improve cadmium (Cd2+) tolerance in B. napus through physiological, molecular, and proteomic analytical approaches. Results showed that application of ALA helped the plants to adjust Cd2+-induced metabolic and photosynthetic fluorescence changes in the leaves of B. napus under Cd2+ stress. The data revealed that ALA treatment enhanced the gene expressions of antioxidant enzyme activities substantially and could increase the expression to a certain degree under Cd2+ stress conditions. In the present study, 34 protein spots were identified that differentially regulated due to Cd2+ and/or ALA treatments. Among them, 18 proteins were significantly regulated by ALA, including the proteins associated with stress related, carbohydrate metabolism, catalysis, dehydration of damaged protein, CO2 assimilation/photosynthesis and protein synthesis/regulation. From these 18 ALA-regulated proteins, 12 proteins were significantly down-regulated and 6 proteins were up-regulated. Interestingly, it was observed that ALA-induced the up-regulation of dihydrolipoyl dehydrogenase, light harvesting complex photo-system II subunit 6 and 30S ribosomal proteins in the presence of Cd2+ stress. In addition, it was also observed that ALA-induced the down-regulation in thioredoxin-like protein, 2, 3-bisphosphoglycerate, proteasome and thiamine thiazole synthase proteins under Cd2+ stress. Taken together, the present study sheds light on molecular mechanisms involved in ALA-induced Cd2+ tolerance in B. napus leaves and suggests a more active involvement of ALA in plant physiological processes than previously proposed. PMID:25909456

  5. Comparative split-face study of 5-aminolevulinic acid photodynamic therapy with intense pulsed light for photorejuvenation of Asian skin.

    PubMed

    Kosaka, Sachiko; Yasumoto, Minako; Akilov, Oleg E; Hasan, Tayyaba; Kawana, Seiji

    2010-12-01

    Photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA) (ALA-PDT) using intense pulsed light (IPL) as a light source (IPL-ALA-PDT) has been used for photorejuvenation, but it is unclear if this protocol can be applied to darker skin types. We performed this study to assess our IPL-ALA-PDT protocol for photorejuvenation in Asian skin. To determine an appropriate dose, ALA ointment (0-20%) was applied to the upper arm of five healthy volunteers and the fluorescence intensity (FI) was measured using a spectrofluorometer. Non-linear regression analysis of FI 2 h after ALA application with global fitting gave a typical sigmoid dose-response curve with R² = 0.9705 and saturation after 5% ALA. The entire faces of 16 Japanese women with photodamage were then treated with IPL (500-670 and 870-1400 nm, 23-30 J/cm²) 2 h after application of 5% ALA to one side of the face. Three treatments were delivered at 4-week intervals with follow-up visits. Comparative analysis of photorejuvenation showed noticeable improvements on both sides of the face, although the reduction in the photoaging score from baseline did not differ significantly between the two sides in all subjects. Despite this finding, 75% of the patients felt that the IPL-ALA-PDT-treated side of the face showed greater improvement than the IPL-treated side. However, all IPL-ALA-PDT-treated sides showed adverse effects such as erythema and pain. Therefore, we conclude that the IPL-ALA-PDT protocol requires optimization for photorejuvenation in Asians.

  6. Efficacy of a Complex of 5-Aminolevulinic Acid and Glycyl-Histidyl-Lysine Peptide on Hair Growth

    PubMed Central

    Sim, Hyun Bo; Jang, Yong Hyun; Lee, Seok-Jong; Kim, Do Won; Yim, Soon-Ho

    2016-01-01

    Background Pattern hair loss is a very common problem. Although effective therapeutics for the treatment of pattern hair loss have been used, novel therapeutic modalities are still required to enhance hair growth. Objective We investigated the efficacy and safety of a complex (ALAVAX) of 5-aminolevulinic acid (5-ALA) and glycyl-histidyl-lysine (GHK) peptide for the treatment of pattern hair loss. Methods Forty-five patients with male pattern hair loss were treated with ALAVAX 100 mg/ml (group A), ALAVAX 50 mg/ml (group B) or placebo (group C) once a day for 6 months. Total hair count, hair length, hair thickness, patient's assessment and adverse events were evaluated at month 1, 3, and 6. Results An increase in hair count for 6 months was 52.6 (p<0.05) in group A, 71.5 (p<0.05) in group B, and 9.6 in group C. The ratio of changes in hair count between group B (2.38) and group C (1.21) at 6 months showed a statistically significant difference (p<0.05). The proportion above good satisfaction was higher in group A (26.7%) than in the other groups (group B: 14.3%, group C: 7.1%). There was no statistically significant difference in hair length and hair thickness among 3 groups at 6 months. There was no adverse event in 3 groups. Conclusion Our study showed that a complex of 5-ALA and GHK peptide may be considered as one of the complementary agents for the treatment of male pattern hair loss. PMID:27489425

  7. In Vitro Comparison of Hypericin and 5-Aminolevulinic Acid-Derived Protoporphyrin IX for Photodynamic Inactivation of Medulloblastoma Cells

    PubMed Central

    Ritz, Rainer; Scheidle, Christian; Noell, Susan; Roser, Florian; Schenk, Martin; Dietz, Klaus; Strauss, Wolfgang S. L.

    2012-01-01

    Background Hypericin (HYP) is a naturally occurring photosensitizer. Cellular uptake and photodynamic inactivation after incubation with this photosensitizer have neither been examined in medulloblastoma cells in vitro, nor compared with 5-aminolevulinic acid-derived protoporphyrin IX (5-ALA-derived PpIX). Methods In 3 medulloblastoma cell lines (D283 Med, Daoy, and D341 Med) the time- and concentration-dependent intracellular accumulation of HYP and 5-ALA-derived PpIX was analyzed by fluorescence microscopy (FM) and FACS. Photocytotoxicity was measured after illumination at 595 nm (HYP) and 635 nm (5-ALA-derived PpIX) in D283 Med cells and compared to U373 MG glioma cells. Results All medulloblastoma cell lines exhibited concentration- and time-dependent uptake of HYP. Incubation with HYP up to 10 µM resulted in a rapid increase in fluorescence intensity, which peaked between 2 and 4 hours. 5-ALA-derived PpIX accumulation increased in D283 Med cells by 22% over baseline after 5-ALA incubation up to 1.2 mM. Photocytotoxicity of 5-ALA-derived PpIX was higher in D283 Med medulloblastoma compared to U373MG glioma. The [lethal dose (light dose that is required to reduce cell survival to 50% of control)] of 5-ALA-derived PpIX was 3.8 J/cm2 in D283 Med cells versus 5.7 J/cm2 in U373MG glioma cells. Photocytotoxicity of HYP in D283 Med cells was determined at 2.5 µM after an incubation time of 2 h and an illumination wavelength of 595 nm. The value was 0.47 J/cm2. Conclusion By its 5-fold increase in fluorescence over autofluorescence levels HYP has excellent properties for tumor visualization in medulloblastomas. The high photocytotoxicity of HYP, compared to 5-ALA-derived PpIX, is convincingly demonstrated by its 8- to 13-fold lower . Therefore HYP might be a promising molecule for intraoperative visualization and photodynamic treatment of medulloblastomas. PMID:23251668

  8. 5-Aminolevulinic acid ameliorates cadmium-induced morphological, biochemical, and ultrastructural changes in seedlings of oilseed rape.

    PubMed

    Ali, Basharat; Huang, C R; Qi, Z Y; Ali, Shafaqat; Daud, M K; Geng, X X; Liu, H B; Zhou, W J

    2013-10-01

    Due to its prolific growth, oilseed rape (Brassica napus L.) can be grown successfully for phytoremediation of cadmium (Cd)-contaminated soils. Nowadays, use of plant growth regulators against heavy metals stress is one of the major objectives of researchers. The present study evaluates the ameliorate effects of 5-aminolevulinic acid (ALA, 0, 0.4, 2, and 10 mg/l) on the growth of oilseed rape (B. napus L. cv. ZS 758) seedlings under Cd stress (0, 100, and 500 μM). Results have shown that Cd stress hampered the seedling growth by decreasing the radical and hypocotyls length, shoot and root biomass, chlorophyll content, and antioxidants enzymes. On the other hand, Cd stress increased the level of malondialdehyde (MDA) and production of H2O2 and accumulation of Cd in the shoots. The microscopic study of leaf mesophyll cells showed that toxicity of Cd totally destroyed the whole cell structure, and accumulation of Cd also appeared in micrographs. Application of ALA at lower dosage (2 mg/l) enhanced the seedling growth and biomass. The results showed that 2 mg/l ALA significantly improved chlorophyll content under Cd stress and decreased the level of Cd contents in shoots. Application of ALA reduced the MDA and H2O2 levels in the cotyledons. The antioxidants enzymes (ascorbate peroxidase, peroxidase, catalase, glutathione reductase, and superoxide dismutase) enhanced their activities significantly with the application of 2 mg/l ALA under Cd stress. This study also indicated that higher dosage of ALA (10 mg/l) imposed the negative effect on the growth of oilseed rape. Microscopic study showed that application of ALA alleviated the toxic effects of Cd in the mesophyll cell and improved the cell structure. Use of 2 mg/l ALA under 500 μM Cd was found to be more effective, and under this dosage, cell structure was clear, with obvious cell wall and cell membrane as well as a big nucleus, which was found with well-developed two or more nucleoli. Chloroplast was almost round

  9. Treating cutaneous squamous cell carcinoma using 5-aminolevulinic acid polylactic-co-glycolic acid nanoparticle-mediated photodynamic therapy in a mouse model

    PubMed Central

    Wang, Xiaojie; Shi, Lei; Tu, Qingfeng; Wang, Hongwei; Zhang, Haiyan; Wang, Peiru; Zhang, Linglin; Huang, Zheng; Zhao, Feng; Luan, Hansen; Wang, Xiuli

    2015-01-01

    Background Squamous cell carcinoma (SCC) is a common skin cancer, and its treatment is still difficult. The aim of this study was to evaluate the effectiveness of nanoparticle (NP)-assisted 5-aminolevulinic acid (ALA) delivery for topical photodynamic therapy (PDT) of cutaneous SCC. Materials and methods Ultraviolet-induced cutaneous SCCs were established in hairless mice. ALA-loaded polylactic-co-glycolic acid (PLGA) NPs were prepared and characterized. The kinetics of ALA PLGA NP-induced protoporphyrin IX fluorescence in SCCs, therapeutic efficacy of ALA NP-mediated PDT, and immune responses were examined. Results PLGA NPs enhanced protoporphyrin IX production in SCC. ALA PLGA NP-mediated topical PDT was more effective than free ALA of the same concentration in treating cutaneous SCC. Conclusion PLGA NPs provide a promising strategy for delivering ALA in topical PDT of cutaneous SCC. PMID:25609949

  10. Generation of reactive oxygen species from 5-aminolevulinic acid and Glutamate in cooperation with excited CdSe/ZnS QDs

    NASA Astrophysics Data System (ADS)

    Duong, Hong Dinh; Lee, Jee Won; Rhee, Jong Il

    2014-08-01

    CdSe/ZnS quantum dots (QDs) can be joined in the reductive pathway involving the electron transfer to an acceptor or in the oxidative pathway involving the hole transfer to a donor. They were exploited in the oxidation reactions of 5-aminolevulinic acid (ALA) and glutamate (GLU) for the generation of reactive oxygen species (ROS) such as hydroxyl radical (HO●) and superoxide anion (O2 ● -). Fast and highly efficient oxidation reactions of ALA to produce HO● and of GLU to produce O2 ●- were observed in the cooperation of mercaptopropionic acid (MPA)-capped CdSe/ZnS QDs under LED irradiation. Fluorescence spectroscopy and electron spin resonance (ESR) spectroscopy were used to evaluate the generation of different forms of ROS. Confocal fluorescent microscopic images of the size and morphology of HeLa cells confirmed the ROS generation from ALA or GLU in cooperation with CdSe/ZnS QDs under LED irradiation.

  11. Homology modeling of human γ-butyric acid transporters and the binding of pro-drugs 5-aminolevulinic acid and methyl aminolevulinic acid used in photodynamic therapy.

    PubMed

    Baglo, Yan; Gabrielsen, Mari; Sylte, Ingebrigt; Gederaas, Odrun A

    2013-01-01

    Photodynamic therapy (PDT) is a safe and effective method currently used in the treatment of skin cancer. In ALA-based PDT, 5-aminolevulinic acid (ALA), or ALA esters, are used as pro-drugs to induce the formation of the potent photosensitizer protoporphyrin IX (PpIX). Activation of PpIX by light causes the formation of reactive oxygen species (ROS) and toxic responses. Studies have indicated that ALA and its methyl ester (MAL) are taken up into the cells via γ-butyric acid (GABA) transporters (GATs). Uptake via GATs into peripheral sensory nerve endings may also account for one of the few adverse side effects of ALA-based PDT, namely pain. In the present study, homology models of the four human GAT subtypes were constructed using three x-ray crystal structures of the homologous leucine transporter (LeuT) as templates. Binding of the native substrate GABA and the possible substrates ALA and MAL was investigated by molecular docking of the ligands into the central putative substrate binding sites in the outward-occluded GAT models. Electrostatic potentials (ESPs) of the putative substrate translocation pathway of each subtype were calculated using the outward-open and inward-open homology models. Our results suggested that ALA is a substrate of all four GATs and that MAL is a substrate of GAT-2, GAT-3 and BGT-1. The ESP calculations indicated that differences likely exist in the entry pathway of the transporters (i.e. in outward-open conformations). Such differences may be exploited for development of inhibitors that selectively target specific GAT subtypes and the homology models may hence provide tools for design of therapeutic inhibitors that can be used to reduce ALA-induced pain. PMID:23762315

  12. Photodynamic therapy of superficial basal cell carcinoma with 5-aminolevulinic acid with dimethylsulfoxide and ethylendiaminetetraacetic acid: a comparison of two light sources.

    PubMed

    Soler, A M; Angell-Petersen, E; Warloe, T; Tausjø, J; Steen, H B; Moan, J; Giercksky, K E

    2000-06-01

    The aim of this prospective randomized study was to compare the clinical and cosmetic outcome of superficial basal cell carcinomas (BCC), using either laser or broadband halogen light, in photodynamic therapy with topical 5-aminolevulinic acid (ALA). A total of 83 patients with 245 superficial BCC were included in the study. Standard treatment involved 15 min of local pretreatment with 99% dimethylsulfoxide (DMSO) before topical application of 20% ALA with DMSO (2%) and ethylendiaminetetraacetic acid (2%) as cofactors for 3 h before light exposure with either laser or a broadband lamp (BL). A complete response was achieved in 95 lesions (86%) in the laser group and 110 lesions (82%) in the BL group 6 months after treatment. Of these, 80 lesions (84%) in the laser group and 101 lesions (92%) in the lamp group were independently evaluated to have an excellent or good cosmetic post-treatment score. No serious adverse events were reported. This study shows that there is no statistical significant difference in cure the rate (P = 0.49) and the cosmetic outcome (P = 0.075) with topical application of a modified ALA-cream between light exposure from a simple BL with continuous spectrum (570-740 nm) or from a red-light laser (monochromatic 630 nm). Cost and safety are further elements in favor of the BL in this setting.

  13. Treatment of moderate to severe inflammatory acne vulgaris: photodynamic therapy with 5-aminolevulinic acid and a novel advanced fluorescence technology pulsed light source.

    PubMed

    Gold, Michael H; Biron, Julie A; Boring, Molly; Bridges, Tancy M; Bradshaw, Virginia L

    2007-03-01

    The use of photodynamic therapy (PDT) with 20% 5-aminolevulinic acid (ALA) for the treatment of acne vulgaris has been explored. This study evaluates the safety and efficacy of a new Advanced Fluorescence Technology (AFT) pulsed light source (420-950 nm) for photoactivation in ALA PDT for the treatment of moderate to severe inflammatory facial acne vulgaris. Nineteen subjects received 4 ALA PDT treatments with the AFT pulsed light source. Treatments were spaced 2 weeks apart. ALA was incubated for 15 to 30 minutes. At the end of the fourth treatment, the total reductions in inflammatory and noninflammatory lesion counts were 54.5% and 37.5%, respectively. Median Global Severity Scores suggest a trend toward reduction after several treatments. Investigator and subject assessments show moderate to marked improvement in most patients. The new AFT pulsed light source with ALA PDT appears to be a safe and effective modality for the treatment of moderate to severe inflammatory acne vulgaris.

  14. Complete resolution of a squamous cell carcinoma of the skin using intralesional 5-aminolevulinic acid photodynamic therapy intralesional PDT for SCC.

    PubMed

    Sotiriou, Eleni; Apalla, Zoi; Ioannides, Demetris

    2010-10-01

    We present an 82-year-old female patient with a 2-year history of an infiltrative squamous cell carcinoma (SCC) on her right cheek. The patient was treated with one intralesional photodynamic therapy (PDT) session using 10% 5-aminolevulinic acid solution. We used red light by a non-coherent light source at a light dose of 100 J/cm² and a fluency rate of 100 mW/cm². Complete clinical and histological response was achieved 3 months after the treatment procedure. Cosmetic outcome was evaluated as fair. The patient remains disease free with the absence of any clinical sign of recurrence 16 months after PDT. Long-term follow-up is needed for assessment of recurrences. Optimization of the therapeutic protocol, as well as justification of our results in larger studies are needed in order to elicit safe conclusions.

  15. A Pilot Cost-Effectiveness Analysis of Treatments in Newly Diagnosed High-Grade Gliomas: The Example of 5-Aminolevulinic Acid Compared With White-Light Surgery

    PubMed Central

    Alves, Marta; Castel-Branco, Marta; Stummer, Walter

    2015-01-01

    BACKGROUND: High-grade gliomas are aggressive, incurable tumors characterized by extensive diffuse invasion of the normal brain parenchyma. Novel therapies at best prolong survival; their costs are formidable and benefit is marginal. Economic restrictions thus require knowledge of the cost-effectiveness of treatments. Here, we show the cost-effectiveness of enhanced resections in malignant glioma surgery using a well-characterized tool for intraoperative tumor visualization, 5-aminolevulinic acid (5-ALA). OBJECTIVE: To evaluate the cost-effectiveness of 5-ALA fluorescence-guided neurosurgery compared with white-light surgery in adult patients with newly diagnosed high-grade glioma, adopting the perspective of the Portuguese National Health Service. METHODS: We used a Markov model (cohort simulation). Transition probabilities were estimated with the use of data from 1 randomized clinical trial and 1 noninterventional prospective study. Utility values and resource use were obtained from published literature and expert opinion. Unit costs were taken from official Portuguese reimbursement lists (2012 values). The health outcomes considered were quality-adjusted life-years, life-years, and progression-free life-years. Extensive 1-way and probabilistic sensitivity analyses were performed. RESULTS: The incremental cost-effectiveness ratios are below €10 000 in all evaluated outcomes, being around €9100 per quality-adjusted life-year gained, €6700 per life-year gained, and €8800 per progression-free life-year gained. The probability of 5-ALA fluorescence-guided surgery cost-effectiveness at a threshold of €20000 is 96.0% for quality-adjusted life-year, 99.6% for life-year, and 98.8% for progression-free life-year. CONCLUSION: 5-ALA fluorescence-guided surgery appears to be cost-effective in newly diagnosed high-grade gliomas compared with white-light surgery. This example demonstrates cost-effectiveness analyses for malignant glioma surgery to be feasible on

  16. eEF1A1 binds and enriches protoporphyrin IX in cancer cells in 5-aminolevulinic acid based photodynamic therapy.

    PubMed

    Fan, Zhichao; Cui, Xiaojun; Wei, Dan; Liu, Wei; Li, Buhong; He, Hao; Ye, Huamao; Zhu, Naishuo; Wei, Xunbin

    2016-01-01

    Photodynamic therapy (PDT) with protoporphyrin IX (PpIX), which is endogenously derived from 5-aminolevulinic acid (5-ALA) or its derivatives, is a promising modality for the treatment of both pre-malignant and malignant lesions. However, the mechanisms of how ALA-induced PpIX selectively accumulated in the tumors are not fully elucidated. Here we discovered that eukaryotic translation elongation factor 1 alpha 1 (eEF1A1) interacted with PpIX (with an affinity constant of 2.96 × 10(6) M(-1)). Microscopy imaging showed that ALA-induced PpIX was co-localized with eEF1A1 in cancer cells. eEF1A1 was found to enrich ALA-induced PpIX in cells by competitively blocking the downstream bioavailability of PpIX. Taken together, our study discovered eEF1A1 as a novel photosensitizer binding protein, which may play an essential role in the enrichment of ALA-induced PpIX in cancer cells during PDT. These suggested eEF1A1 as a molecular marker to predict the selectivity and efficiency of 5-ALA based PDT in cancer therapy. PMID:27150264

  17. Alleviation of lead toxicity by 5-aminolevulinic acid is related to elevated growth, photosynthesis, and suppressed ultrastructural damages in oilseed rape.

    PubMed

    Tian, Tian; Ali, Basharat; Qin, Yebo; Malik, Zaffar; Gill, Rafaqat A; Ali, Shafaqat; Zhou, Weijun

    2014-01-01

    Lead (Pb) is a widely spread pollutant and leads to diverse morphological and structural changes in the plants. In this study, alleviating role of 5-aminolevulinic acid (ALA) in oilseed rape (Brassica napus L.) was investigated with or without foliar application of ALA (25 mg L(-1)) in hydroponic environment under different Pb levels (0, 100, and 400 µM). Outcomes stated that plant morphology and photosynthetic attributes were reduced under the application of Pb alone. However, ALA application significantly increased the plant growth and photosynthetic parameters under Pb toxicity. Moreover, ALA also lowered the Pb concentration in shoots and roots under Pb toxicity. The microscopic studies depicted that exogenously applied ALA ameliorated the Pb stress and significantly improved the cell ultrastructures. After application of ALA under Pb stress, mesophyll cell had well-developed nucleus and chloroplast having a number of starch granules. Moreover, micrographs illustrated that root tip cell contained well-developed nucleus, a number of mitochondria, and golgi bodies. These results proposed that under 15-day Pb-induced stress, ALA improved the plant growth, chlorophyll content, photosynthetic parameters, and ultrastructural modifications in leaf mesophyll and root tip cells of the B. napus plants.

  18. Topical glycerol monooleate/propylene glycol formulations enhance 5-aminolevulinic acid in vitro skin delivery and in vivo protophorphyrin IX accumulation in hairless mouse skin.

    PubMed

    Steluti, Regilene; De Rosa, Fernanda Scarmato; Collett, John; Tedesco, Antônio Cláudio; Bentley, Maria Vitória Lopes Badra

    2005-08-01

    Photodynamic therapy (PDT), a potential therapy for cancer treatment, utilizes exogenously applied or endogenously formed photosensitizers, further activated by light in an appropriate wavelength and dose to induce cell death through free radical formation. 5-Aminolevulinic acid (5-ALA) is a pro-drug which can be converted to the effective photosensitizer, protoporphyrin IX (PpIX). However, the use of 5-ALA in PDT is limited by the low penetration capacity of this highly hydrophilic molecule into appropriate skin layers. In the present study, we propose to increase 5-ALA penetration by using formulations containing glycerol monooleate (GMO), an interesting and useful component of pharmaceutical formulations. Propylene glycol solutions containing different concentrations of GMO significantly increased the in vitro skin permeation/retention of 5-ALA in comparison to control solutions. In vivo studies also showed increased PpIX accumulation in mouse hairless skin, after the use of topical 5-ALA formulations containing GMO in a concentration-dependent manner. The results show that skin 5-ALA penetration and PpIX accumulation, important factors for the success of topical 5-ALA-PDT in skin cancer, are optimized by GMO/propylene glycol formulations.

  19. Inhibition of MAPK signaling pathways enhances cell death induced by 5-Aminolevulinic acid-photodynamic therapy in skin squamous carcinoma cells.

    PubMed

    Ge, Xinhong; Liu, Jianping; Shi, Zhiyun; Jing, Li; Yu, Nan; Zhang, Xiujuan; Jiao, Yaning; Wang, Yili; Li, P Andy

    2016-04-01

    Combination of a photosensitizer, 5-aminolevulinic acid (ALA), with photodynamic therapy (PDT) has been widely used to treat skin squamous cell carcinoma (SCC). However, a portion of SCC patients do not respond well to PDT. The molecular reason for this resistance is not clear. We hypothesize that mitogen-activated phosphorylation kinase (MAPK) plays a key role in mediating SCC resistance to PDT. To determine whether inhibition of MAPK signaling enhances the anti-tumor effect of ALA-PDT in SCC. The human squamous carcinoma cell line, SCL-1, was either untreated or treated with various combinations of ALA, PDT light source and inhibitors of MAPK signaling components. ALA-PDT treatment significantly decreased cell viability, increased the percentage of annexin-V positive cells and resulted in formation of apoptotic bodies. ALA-PDT treated cells showed increased levels of p-MEK, p-ERK1/2, p-p38, p-Elk-1, p-JNK and p-c-Jun. Addition of inhibitors for ERK1/2 (PD98059), p38 (SB203580) and JNK (SP60125) reversed the changes and led to a more dramatic decrease in SCL-1 cell viability than seen with ALA-PDT alone. Inhibition of the MAPK pathway enhances the cytotoxic effect of ALA-PDT on SCL-1.

  20. eEF1A1 binds and enriches protoporphyrin IX in cancer cells in 5-aminolevulinic acid based photodynamic therapy

    NASA Astrophysics Data System (ADS)

    Fan, Zhichao; Cui, Xiaojun; Wei, Dan; Liu, Wei; Li, Buhong; He, Hao; Ye, Huamao; Zhu, Naishuo; Wei, Xunbin

    2016-05-01

    Photodynamic therapy (PDT) with protoporphyrin IX (PpIX), which is endogenously derived from 5-aminolevulinic acid (5-ALA) or its derivatives, is a promising modality for the treatment of both pre-malignant and malignant lesions. However, the mechanisms of how ALA-induced PpIX selectively accumulated in the tumors are not fully elucidated. Here we discovered that eukaryotic translation elongation factor 1 alpha 1 (eEF1A1) interacted with PpIX (with an affinity constant of 2.96 × 106 M‑1). Microscopy imaging showed that ALA-induced PpIX was co-localized with eEF1A1 in cancer cells. eEF1A1 was found to enrich ALA-induced PpIX in cells by competitively blocking the downstream bioavailability of PpIX. Taken together, our study discovered eEF1A1 as a novel photosensitizer binding protein, which may play an essential role in the enrichment of ALA-induced PpIX in cancer cells during PDT. These suggested eEF1A1 as a molecular marker to predict the selectivity and efficiency of 5-ALA based PDT in cancer therapy.

  1. Wavelength-dependent in-vitro and in-vivo photodynamic effects after sensitization with 5-aminolevulinic acid induced protoporphyrin IX

    NASA Astrophysics Data System (ADS)

    Szeimies, Rolf-Markus; Abels, Christoph; Fritsch, Clemens; Steinbach, Pia; Baeumler, Wolfgang; Messmann, Helmut; Goetz, Alwin E.; Goerz, Guenter; Landthaler, Michael

    1996-01-01

    Photodynamic therapy (PDT) with topically applied 5-aminolevulinic acid (ALA) is of growing interest, in particular in dermatology. Due to the fact that PDT with intravenously administered Photofrin is the only clinically approved sensitizer so far and is performed at a wavelength of 630 nm, this wavelength is also used in most experimental and clinical trials with ALA. In this study influence of irradiation with coherent light from a tunable dye laser at different wavelengths ranging from 625 to 649 nm was investigated. In in vitro experiments HaCaT immortalized human keratinocytes were sensitized with 30 (mu) g/ml ALA for 24 hrs. By determination of cell viability with the MTT test, best cell-killing effects were observed following irradiation at 635 nm. In an in vivo setting using an amelanotic melanoma (A-Mel-3) grown subcutaneously in Syrian Golden hamsters, these results were confirmed: tumor growth determined by measuring tumor volume increase after 28 days was less pronounced in animals treated with 100 mg/kg ALA i.v. and irradiated 2.5 hrs. later at 635 nm, as compared to animals receiving an equal dose and irradiated at 630 nm. This observation in vitro is probably due to large amounts of photosensitizing protoporphyrin IX (PP) localized in cell membranes which is visualized by confocal laser scanning microscopy (CLSM) and determined by HPLC analysis. These results suggest that in ALA-PDT when a coherent light source is used probably better results are achieved irradiating at 635 nm.

  2. Differential antioxidant defense and detoxification mechanisms in photodynamically stressed rice plants treated with the deregulators of porphyrin biosynthesis, 5-aminolevulinic acid and oxyfluorfen.

    PubMed

    Phung, Thu-Ha; Jung, Sunyo

    2015-04-01

    This study focuses on differential molecular mechanisms of antioxidant and detoxification systems in rice plants under two different types of photodynamic stress imposed by porphyrin deregulators, 5-aminolevulinic acid (ALA) and oxyfluorfen (OF). The ALA-treated plants with white necrosis exhibited a greater decrease in photochemical quantum efficiency, Fv/Fm, as well as a greater increase in activity of superoxide dismutase, compared to the OF-treated plants. By contrast, the brown necrosis in OF-treated plants resulted in not only more widely dispersed H2O2 production and greater increases in H2O2-decomposing enzymes, catalase and peroxidase, but also lower ascorbate redox state. In addition, ALA- and OF-treated plants markedly up-regulated transcript levels of genes involved in detoxification processes including transport and movement, cellular homeostasis, and xenobiotic conjugation, with prominent up-regulation of serine/threonine kinase and chaperone only in ALA-treated plants. Our results demonstrate that different photodynamic stress imposed by ALA and OF developed differential actions of antioxidant enzymes and detoxification. Particularly, detoxification system may play potential roles in plant protection against photodynamic stress imposed by porphyrin deregulators, thereby contributing to alleviation of photodynamic damage.

  3. Mechanism of rat osteosarcoma cell apoptosis induced by a combination of low-intensity ultrasound and 5-aminolevulinic acid in vitro.

    PubMed

    Li, Y N; Zhou, Q; Yang, B; Hu, Z; Wang, J H; Li, Q S; Cao, W W

    2015-01-01

    We investigated the killing effect of low-intensity ultrasound combined with 5-aminolevulinic acid (5-ALA) on the rat osteosarcoma cell line UMR-106. Logarithmic-phase UMR-106 cells were divided into a control group, ultrasound group and 5-ALA group. The cell apoptotic rate, production of reactive oxygen species, and the change in mitochondrial membrane potential were analyzed by flow cytometry; ultrastructural changes were observed by transmission electron microscopy. Using low-intensity ultrasound at 1.0 MHz and 2.0 W/cm(2) plus 5-ALA at a concentration of 2 mM, the apoptotic rate of the sonodynamic therapy group was 27.2 ± 3.4% which was significantly higher than that of the control group, ultrasound group, and 5-ALA group (P < 0.05). The production of reactive oxygen species was 32.6 ± 2.2% and the decrease in mitochondrial membrane potential was 39.5 ± 2.5%. The 33342 staining showed nuclear condensation and fragmentation in the ultrasound group and 5-ALA group. Structural changes in the cell membrane, mitochondria, Golgi apparatus, and other organelles observed by transmission electron microscopy included formation of apoptotic bodies. The killing effect of low-intensity ultrasound combined with 5-ALA on UMR-106 cells was significant. Cell apoptosis played a vital role in the killing effect, and the mitochondria pathway contributed to the apoptosis of UMR-106 cells. PMID:26345893

  4. The Safety and Tolerability of 5-Aminolevulinic Acid Phosphate with Sodium Ferrous Citrate in Patients with Type 2 Diabetes Mellitus in Bahrain

    PubMed Central

    Al-Saber, Feryal; Aldosari, Waleed; Alselaiti, Mariam; Khalfan, Hesham; Kaladari, Ahmed; Khan, Ghulam; Harb, George; Rehani, Riyadh; Kudo, Sizuka; Koda, Aya; Tanaka, Tohru

    2016-01-01

    Type 2 diabetes mellitus is prevalent especially in Gulf countries and poses serious long-term risks to patients. A multifaceted treatment approach can include nutritional supplements with antioxidant properties such as 5-aminolevulinic acid (5-ALA) with sodium ferrous citrate (SFC). This prospective, randomized, single-blind, placebo-controlled, dose escalating pilot clinical trial assessed the safety of 5-ALA with SFC at doses up to 200 mg 5-ALA/229.42 mg SFC per day in patients living in Bahrain with type 2 diabetes mellitus that was uncontrolled despite the use of one or more antidiabetic drugs. Fifty-three patients (n = 53) from 3 sites at one center were enrolled by Dr. Feryal (Site #01), Dr. Hesham (Site #02), and Dr. Waleed (Site #03) (n = 35, 5-ALA-SFC; n = 18, placebo). There was no significant difference in incidence of adverse events reported, and the most frequent events reported were gastrointestinal in nature, consistent with the known safety profile of 5-ALA in patients with diabetes. No significant changes in laboratory values and no difference in hypoglycemia between patients receiving 5-ALA and placebo were noted. Overall, the current results support that use of 5-ALA-SFC up to 200 mg per day taken as 2 divided doses is safe in patients taking concomitant oral antidiabetic medications and may offer benefits in the diabetic population. This trial is registered with ClinicalTrials.gov NCT02481141. PMID:27738640

  5. Synergism of herbicide toxicity by 5-aminolevulinic acid is related to physiological and ultra-structural disorders in crickweed (Malachium aquaticum L.).

    PubMed

    Xu, Ling; Zhang, Wenfang; Ali, Basharat; Islam, Faisal; Zhu, Jinwen; Zhou, Weijun

    2015-11-01

    Selection of effective herbicides to control weeds has been one of the major objectives of scientists. This study determines the differential tolerance or susceptibility of crickweed (Malachium aquaticum L.) to various concentration combinations of 5-aminolevulinic acid (ALA) (1, 10 and 100mg/L) and propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy)benzylamino)benzoate (ZJ0273) (100, 200, and 500mg/L). ALA was applied as pre- and post-treatment alone or in combination with ZJ0273. Results showed that ZJ0273 stress alone imposed negative effects on M. aquaticum seedling's growth, net photosynthetic rates and SPAD values, and the rate of decline was consistently increased with the increase in ZJ0273 concentration. The ZJ0273 treatment showed a gradual decrease in the activities of antioxidant enzymes peroxidase (POD), superoxide dismutase (SOD), and ascorbate peroxidase (APX), and increase in the accumulation of malondialdehyde (MDA). Changes in chloroplast swelling, increased number of plastoglobuli, disruption of thylakoid, disintegrated mitochondria and turbid nucleoplasm were noticed. Moreover, SDS-PAGE analysis of total proteins revealed that herbicide stress in the leaves was associated with the decrease or disappearance of some protein bands. Further, two-dimensional gel electrophoresis (2-DE) results showed that proteins in different spots were classified into three types for M. aquaticum. These results indicate that the combined treatment of ALA and ZJ0273 synergizes the herbicide toxicity which is different from its independent effects on M. aquaticum and thus, could improve weed control efficacy. PMID:26615151

  6. Inhibition of MAPK signaling pathways enhances cell death induced by 5-Aminolevulinic acid-photodynamic therapy in skin squamous carcinoma cells.

    PubMed

    Ge, Xinhong; Liu, Jianping; Shi, Zhiyun; Jing, Li; Yu, Nan; Zhang, Xiujuan; Jiao, Yaning; Wang, Yili; Li, P Andy

    2016-04-01

    Combination of a photosensitizer, 5-aminolevulinic acid (ALA), with photodynamic therapy (PDT) has been widely used to treat skin squamous cell carcinoma (SCC). However, a portion of SCC patients do not respond well to PDT. The molecular reason for this resistance is not clear. We hypothesize that mitogen-activated phosphorylation kinase (MAPK) plays a key role in mediating SCC resistance to PDT. To determine whether inhibition of MAPK signaling enhances the anti-tumor effect of ALA-PDT in SCC. The human squamous carcinoma cell line, SCL-1, was either untreated or treated with various combinations of ALA, PDT light source and inhibitors of MAPK signaling components. ALA-PDT treatment significantly decreased cell viability, increased the percentage of annexin-V positive cells and resulted in formation of apoptotic bodies. ALA-PDT treated cells showed increased levels of p-MEK, p-ERK1/2, p-p38, p-Elk-1, p-JNK and p-c-Jun. Addition of inhibitors for ERK1/2 (PD98059), p38 (SB203580) and JNK (SP60125) reversed the changes and led to a more dramatic decrease in SCL-1 cell viability than seen with ALA-PDT alone. Inhibition of the MAPK pathway enhances the cytotoxic effect of ALA-PDT on SCL-1. PMID:27032574

  7. Alleviation of Lead Toxicity by 5-Aminolevulinic Acid Is Related to Elevated Growth, Photosynthesis, and Suppressed Ultrastructural Damages in Oilseed Rape

    PubMed Central

    Tian, Tian; Qin, Yebo; Gill, Rafaqat A.; Ali, Shafaqat

    2014-01-01

    Lead (Pb) is a widely spread pollutant and leads to diverse morphological and structural changes in the plants. In this study, alleviating role of 5-aminolevulinic acid (ALA) in oilseed rape (Brassica napus L.) was investigated with or without foliar application of ALA (25 mg L−1) in hydroponic environment under different Pb levels (0, 100, and 400 µM). Outcomes stated that plant morphology and photosynthetic attributes were reduced under the application of Pb alone. However, ALA application significantly increased the plant growth and photosynthetic parameters under Pb toxicity. Moreover, ALA also lowered the Pb concentration in shoots and roots under Pb toxicity. The microscopic studies depicted that exogenously applied ALA ameliorated the Pb stress and significantly improved the cell ultrastructures. After application of ALA under Pb stress, mesophyll cell had well-developed nucleus and chloroplast having a number of starch granules. Moreover, micrographs illustrated that root tip cell contained well-developed nucleus, a number of mitochondria, and golgi bodies. These results proposed that under 15-day Pb-induced stress, ALA improved the plant growth, chlorophyll content, photosynthetic parameters, and ultrastructural modifications in leaf mesophyll and root tip cells of the B. napus plants. PMID:24683549

  8. eEF1A1 binds and enriches protoporphyrin IX in cancer cells in 5-aminolevulinic acid based photodynamic therapy

    PubMed Central

    Fan, Zhichao; Cui, Xiaojun; Wei, Dan; Liu, Wei; Li, Buhong; He, Hao; Ye, Huamao; Zhu, Naishuo; Wei, Xunbin

    2016-01-01

    Photodynamic therapy (PDT) with protoporphyrin IX (PpIX), which is endogenously derived from 5-aminolevulinic acid (5-ALA) or its derivatives, is a promising modality for the treatment of both pre-malignant and malignant lesions. However, the mechanisms of how ALA-induced PpIX selectively accumulated in the tumors are not fully elucidated. Here we discovered that eukaryotic translation elongation factor 1 alpha 1 (eEF1A1) interacted with PpIX (with an affinity constant of 2.96 × 106 M−1). Microscopy imaging showed that ALA-induced PpIX was co-localized with eEF1A1 in cancer cells. eEF1A1 was found to enrich ALA-induced PpIX in cells by competitively blocking the downstream bioavailability of PpIX. Taken together, our study discovered eEF1A1 as a novel photosensitizer binding protein, which may play an essential role in the enrichment of ALA-induced PpIX in cancer cells during PDT. These suggested eEF1A1 as a molecular marker to predict the selectivity and efficiency of 5-ALA based PDT in cancer therapy. PMID:27150264

  9. 5-Aminolevulinic acid combined with ferrous iron induces carbon monoxide generation in mouse kidneys and protects from renal ischemia-reperfusion injury.

    PubMed

    Hou, Jiangang; Cai, Songjie; Kitajima, Yuya; Fujino, Masayuki; Ito, Hidenori; Takahashi, Kiwamu; Abe, Fuminori; Tanaka, Tohru; Ding, Qiang; Li, Xiao-Kang

    2013-10-15

    Renal ischemia reperfusion injury (IRI) is a major factor responsible for acute renal failure. An intermediate in heme synthesis, 5-aminolevulinic acid (5-ALA) is fundamental in aerobic energy metabolism. Heme oxygenase (HO)-1 cleaves heme to form biliverdin, carbon monoxide (CO), and iron (Fe(2+)), which is used with 5-ALA. In the present study, we investigated the role of 5-ALA in the attenuation of acute renal IRI using a mouse model. Male Balb/c mice received 30 mg/kg 5-ALA with Fe(2+) 48, 24, and 2 h before IRI and were subsequently subjected to bilateral renal pedicle occlusion for 45 min. The endogenous CO concentration of the kidneys from the mice administered 5-ALA/Fe(2+) increased significantly, and the peak concentrations of serum creatinine and blood urea nitrogen decreased. 5-ALA/Fe(2+) treatments significantly decreased the tubular damage and number of apoptotic cells. IRI-induced renal thiobarbituric acid-reactive substance levels were also significantly decreased in the 5-ALA/Fe(2+) group. Furthermore, mRNA expression of HO-1, TNF-α, and interferon-γ was significantly increased after IRI. Levels of HO-1 were increased and levels of TNF-α and interferon-γ were decreased in the 5-ALA/Fe(2+)-pretreated renal parenchyma after IRI. F4/80 staining showed reduced macrophage infiltration, and TUNEL staining revealed that there were fewer interstitial apoptotic cells. These findings suggest that 5-ALA/Fe(2+) can protect the kidneys against IRI by reducing macrophage infiltration and decreasing renal cell apoptosis via the generation of CO.

  10. 5-Aminolevulinic Acid Thins Pear Fruits by Inhibiting Pollen Tube Growth via Ca(2+)-ATPase-Mediated Ca(2+) Efflux.

    PubMed

    An, Yuyan; Li, Jie; Duan, Chunhui; Liu, Longbo; Sun, Yongping; Cao, Rongxiang; Wang, Liangju

    2016-01-01

    Chemical fruit thinning has become a popular practice in modern fruit orchards for achieving high quality fruits, reducing costs of hand thinning and promoting return bloom. However, most of the suggested chemical thinners are often concerned for their detrimental effects and environmental problems. 5-Aminolevulic acid (ALA) is a natural, nontoxic, biodegradable, and environment-friendly plant growth regulator. One of its outstanding roles is improving plant photosynthesis and fruit quality. Here, results showed that applying 100-200 mg/L ALA at full bloom stage significantly reduced pear fruit set. Both in vivo and in vitro studies showed that ALA significantly inhibited pollen germination and tube growth. ALA decreased not only cytosolic Ca(2+) concentration ([Ca(2+)]cyt) but also "tip-focused" [Ca(2+)]cyt gradient, indicating that ALA inhibited pollen tube growth by down-regulating calcium signaling. ALA drastically enhanced pollen Ca(2+)-ATPase activity, suggesting that ALA-induced decrease of calcium signaling probably resulted from activating calcium pump. The significant negative correlations between Ca(2+)-ATPase activity and pollen germination or pollen tube length further demonstrated the critical role of calcium pump in ALA's negative effect on pollen germination. Taken together, our results suggest that ALA at low concentrations is a potential biochemical thinner, and it inhibits pollen germination and tube growth via Ca(2+) efflux by activating Ca(2+)-ATPase, thereby thinning fruits by preventing fertilization.

  11. 5-Aminolevulinic Acid Thins Pear Fruits by Inhibiting Pollen Tube Growth via Ca2+-ATPase-Mediated Ca2+ Efflux

    PubMed Central

    An, Yuyan; Li, Jie; Duan, Chunhui; Liu, Longbo; Sun, Yongping; Cao, Rongxiang; Wang, Liangju

    2016-01-01

    Chemical fruit thinning has become a popular practice in modern fruit orchards for achieving high quality fruits, reducing costs of hand thinning and promoting return bloom. However, most of the suggested chemical thinners are often concerned for their detrimental effects and environmental problems. 5-Aminolevulic acid (ALA) is a natural, nontoxic, biodegradable, and environment-friendly plant growth regulator. One of its outstanding roles is improving plant photosynthesis and fruit quality. Here, results showed that applying 100–200 mg/L ALA at full bloom stage significantly reduced pear fruit set. Both in vivo and in vitro studies showed that ALA significantly inhibited pollen germination and tube growth. ALA decreased not only cytosolic Ca2+ concentration ([Ca2+]cyt) but also “tip-focused” [Ca2+]cyt gradient, indicating that ALA inhibited pollen tube growth by down-regulating calcium signaling. ALA drastically enhanced pollen Ca2+-ATPase activity, suggesting that ALA-induced decrease of calcium signaling probably resulted from activating calcium pump. The significant negative correlations between Ca2+-ATPase activity and pollen germination or pollen tube length further demonstrated the critical role of calcium pump in ALA's negative effect on pollen germination. Taken together, our results suggest that ALA at low concentrations is a potential biochemical thinner, and it inhibits pollen germination and tube growth via Ca2+ efflux by activating Ca2+-ATPase, thereby thinning fruits by preventing fertilization. PMID:26904082

  12. Differential antioxidant defense and detoxification mechanisms in photodynamically stressed rice plants treated with the deregulators of porphyrin biosynthesis, 5-aminolevulinic acid and oxyfluorfen

    SciTech Connect

    Phung, Thu-Ha; Jung, Sunyo

    2015-04-03

    This study focuses on differential molecular mechanisms of antioxidant and detoxification systems in rice plants under two different types of photodynamic stress imposed by porphyrin deregulators, 5-aminolevulinic acid (ALA) and oxyfluorfen (OF). The ALA-treated plants with white necrosis exhibited a greater decrease in photochemical quantum efficiency, F{sub v}/F{sub m}, as well as a greater increase in activity of superoxide dismutase, compared to the OF-treated plants. By contrast, the brown necrosis in OF-treated plants resulted in not only more widely dispersed H{sub 2}O{sub 2} production and greater increases in H{sub 2}O{sub 2}-decomposing enzymes, catalase and peroxidase, but also lower ascorbate redox state. In addition, ALA- and OF-treated plants markedly up-regulated transcript levels of genes involved in detoxification processes including transport and movement, cellular homeostasis, and xenobiotic conjugation, with prominent up-regulation of serine/threonine kinase and chaperone only in ALA-treated plants. Our results demonstrate that different photodynamic stress imposed by ALA and OF developed differential actions of antioxidant enzymes and detoxification. Particularly, detoxification system may play potential roles in plant protection against photodynamic stress imposed by porphyrin deregulators, thereby contributing to alleviation of photodynamic damage. - Highlights: • We employ two different types of photodynamic stress, white and brown necrosis. • We examine molecular mechanisms of antioxidative and detoxification systems. • ALA and OF develop differential actions of antioxidant and detoxification systems. • Coordinated mechanism of antioxidants and detoxification works against toxic ROS. • Detoxification system plays critical roles in protection against photodynamic stress.

  13. Comparative Study of Photodynamic Therapy with Topical Methyl Aminolevulinate versus 5-Aminolevulinic Acid for Facial Actinic Keratosis with Long-Term Follow-Up

    PubMed Central

    Ko, Dong-Yeob; Kim, Ki-Ho

    2014-01-01

    Background Few studies have compared the efficacy, cosmetic outcomes, and adverse events between 5-aminolevulinic acid photodynamic therapy (ALA-PDT) and methyl aminolevulinate-PDT (MAL-PDT) for actinic keratoses (AKs) in Asian ethnic populations with dark-skin. Objective We retrospectively compared the long-term efficacy, recurrence rates, cosmetic outcomes, and safety of ALA-PDT versus MAL-PDT for facial AKs in Koreans. Methods A total of 222 facial AKs in 58 patients were included in this study. A total of 153 lesions (29 patients) were treated with 5-ALA, and 69 lesions (29 patients) with MAL. ALA and MAL creams were applied for 6 hours and 3 hours, respectively; the lesions were then illuminated with a halogen lamp at 150 J/cm2 for ALA-PDT and a diode lamp at 37 J/cm2 for MAL-PDT. Results The complete response rates of ALA-PDT and MAL-PDT were 56.9% and 50.7%, respectively, with no significant difference at 12 months after treatment. No significant difference in recurrence rates was observed between the 2 PDT modalities at either 6 or 12 months after treatment. There was no significant difference in the cosmetic outcomes between the 2 treatment modalities at 12 months after PDT. However, ALA-PDT caused significantly more painful than MAL-PDT (p=0.005). The adverse events were mild to moderate, transient, and self-limiting for both modalities. Conclusion MAL-PDT was similar to ALA-PDT in terms of long-term efficacy, recurrence rates, cosmetic outcomes, and adverse events; however, it was a significantly less painful procedure than ALA-PDT in our study. PMID:24966631

  14. Effects of exogenous 5-aminolevulinic acid on photosynthesis, stomatal conductance, transpiration rate, and PIP gene expression of tomato seedlings subject to salinity stress.

    PubMed

    Zhao, Y Y; Yan, F; Hu, L P; Zhou, X T; Zou, Z R; Cui, L R

    2015-01-01

    The effects of exogenous 5-aminolevulinic acid (ALA) on photosynthesis, plant growth, and the expression of two aquaporin genes in tomato seedlings under control and salinity conditions were investigated. Exogenous ALA application significantly improved net photosynthetic rate (Pn), total chlorophyll content, and plant biomass accumulation of tomato seedlings under salinity stress. As revealed by real-time PCR analyses, after treatment with ALA alone, expression of both LePIP1 and LePIP2 in the two tomato cultivars was up-regulated at 2 h and subsequently decreased to normal levels. Under salinity stress, transcript levels of LePIP1 in both leaves and roots of salt-sensitive cultivars (cv. Zhongza No.9) increased significantly and were considerably higher than in cultivars exposed to ALA alone. In contrast, the expression levels of LePIP1 and LePIP2 in cvs. Jinpeng No.1 cultivars were slightly lower under salinity stress than under ALA treatment. In addition, transcript levels of both LePIP1 and LePIP2 in the roots of Jinpeng No. 1 cultivars were considerably lower than those in the roots of Zhongza No. 9 cultivars under salinity stress, regardless of ALA supplementation, implying that Jinpeng No. 1 cultivars had a better capacity to maintain membrane intrinsic protein stability. Further, ALA application distinctly counteracted the up- or down-regulation of LePIP1 and LePIP2 in both cultivars under salinity stress, in accordance with the improvements instomatal conductance, transpiration rate, and Pn of tomato leaves. The results presented here indicate that ALA controls aquaporin expression, thus, presumably ALA regulates water homeostasis and enhances salt tolerance of tomato seedlings. PMID:26125845

  15. Impact of 5-aminolevulinic acid with iron supplementation on exercise efficiency and home-based walking training achievement in older women.

    PubMed

    Masuki, Shizue; Morita, Atsumi; Kamijo, Yoshi-ichiro; Ikegawa, Shigeki; Kataoka, Yufuko; Ogawa, Yu; Sumiyoshi, Eri; Takahashi, Kiwamu; Tanaka, Tohru; Nakajima, Motowo; Nose, Hiroshi

    2016-01-01

    A reduction in exercise efficiency with aging limits daily living activities. We examined whether 5-aminolevulinic acid (ALA) with sodium ferrous citrate (SFC) increased exercise efficiency and voluntary achievement of interval walking training (IWT) in older women. Ten women [65 ± 3(SD) yr] who had performed IWT for >12 mo and were currently performing IWT participated in this study. The study was conducted in a placebo-controlled, double-blind crossover design. All subjects underwent two trials for 7 days each in which they performed IWT with ALA+SFC (100 and 115 mg/day, respectively) or placebo supplement intake (CNT), intermittently with a 2-wk washout period. Before and after each trial, subjects underwent a graded cycling test at 27.0 °C atmospheric temperature and 50% relative humidity, and oxygen consumption rate, carbon dioxide production rate, and lactate concentration in plasma were measured. Furthermore, for the first 6 days of each trial, exercise intensity for IWT was measured by accelerometry. We found that, in the ALA+SFC trial, oxygen consumption rate and carbon dioxide production rate during graded cycling decreased by 12% (P < 0.001) and 11% (P = 0.001) at every workload, respectively, accompanied by a 16% reduction in lactate concentration in plasma (P < 0.001), although all remained unchanged in the CNT trial (P > 0.2). All of the reductions were significantly greater in the ALA+SFC than the CNT trial (P < 0.05). Furthermore, the training days, impulse, and time at fast walking were 42% (P = 0.028), 102% (P = 0.027), and 69% (P = 0.039) higher during the ALA+SFC than the CNT intake period, respectively. Thus ALA+SFC supplementation augmented exercise efficiency and thereby improved IWT achievement in older women.

  16. Topical delivery of 5-aminolevulinic acid-encapsulated ethosomes in a hyperproliferative skin animal model using the CLSM technique to evaluate the penetration behavior.

    PubMed

    Fang, Yi-Ping; Huang, Yaw-Bin; Wu, Pao-Chu; Tsai, Yi-Hung

    2009-11-01

    Psoriasis, an inflammatory skin disease, exhibits recurring itching, soreness, and cracked and bleeding skin. Currently, the topical delivery of 5-aminolevulinic acid-photodynamic therapy (ALA-PDT) is an optional treatment for psoriasis which provides long-term therapeutic effects, is non-toxic and enjoys better compliance with patients. However, the precursor of ALA is hydrophilic, and thus its ability to penetrate the skin is limited. Also, little research has provided a platform to investigate the penetration behavior in disordered skin. We employed a highly potent ethosomal carrier (phosphatidylethanolamine; PE) to investigate the penetration behavior of ALA and the recovery of skin in a hyperproliferative murine model. We found that the application of ethosomes produced a significant increase in cumulative amounts of 5-26-fold in normal and hyperproliferative murine skin samples when compared to an ALA aqueous solution; and the ALA aqueous solution appeared less precise in terms of the penetration mode in hyperproliferative murine skin. After the ethosomes had been applied, the protoporphyrin IX (PpIX) intensity increased about 3.64-fold compared with that of the ALA aqueous solution, and the penetration depth reached 30-80 microm. The results demonstrated that the ethosomal carrier significantly improved the delivery of ALA and the formation of PpIX in both normal and hyperproliferative murine skin samples, and the expression level of tumor necrosis factor (TNF)-alpha was reduced after the ALA-ethosomes were applied to treat hyperproliferative murine skin. Furthermore, the results of present study encourage more investigations on the mechanism of the interaction with ethosomes and hyperproliferative murine skin.

  17. Impact of 5-aminolevulinic acid with iron supplementation on exercise efficiency and home-based walking training achievement in older women

    PubMed Central

    Masuki, Shizue; Morita, Atsumi; Kamijo, Yoshi-ichiro; Ikegawa, Shigeki; Kataoka, Yufuko; Ogawa, Yu; Sumiyoshi, Eri; Takahashi, Kiwamu; Tanaka, Tohru; Nakajima, Motowo

    2015-01-01

    A reduction in exercise efficiency with aging limits daily living activities. We examined whether 5-aminolevulinic acid (ALA) with sodium ferrous citrate (SFC) increased exercise efficiency and voluntary achievement of interval walking training (IWT) in older women. Ten women [65 ± 3(SD) yr] who had performed IWT for >12 mo and were currently performing IWT participated in this study. The study was conducted in a placebo-controlled, double-blind crossover design. All subjects underwent two trials for 7 days each in which they performed IWT with ALA+SFC (100 and 115 mg/day, respectively) or placebo supplement intake (CNT), intermittently with a 2-wk washout period. Before and after each trial, subjects underwent a graded cycling test at 27.0°C atmospheric temperature and 50% relative humidity, and oxygen consumption rate, carbon dioxide production rate, and lactate concentration in plasma were measured. Furthermore, for the first 6 days of each trial, exercise intensity for IWT was measured by accelerometry. We found that, in the ALA+SFC trial, oxygen consumption rate and carbon dioxide production rate during graded cycling decreased by 12% (P < 0.001) and 11% (P = 0.001) at every workload, respectively, accompanied by a 16% reduction in lactate concentration in plasma (P < 0.001), although all remained unchanged in the CNT trial (P > 0.2). All of the reductions were significantly greater in the ALA+SFC than the CNT trial (P < 0.05). Furthermore, the training days, impulse, and time at fast walking were 42% (P = 0.028), 102% (P = 0.027), and 69% (P = 0.039) higher during the ALA+SFC than the CNT intake period, respectively. Thus ALA+SFC supplementation augmented exercise efficiency and thereby improved IWT achievement in older women. PMID:26514619

  18. Topical delivery of 5-aminolevulinic acid-encapsulated ethosomes in a hyperproliferative skin animal model using the CLSM technique to evaluate the penetration behavior.

    PubMed

    Fang, Yi-Ping; Huang, Yaw-Bin; Wu, Pao-Chu; Tsai, Yi-Hung

    2009-11-01

    Psoriasis, an inflammatory skin disease, exhibits recurring itching, soreness, and cracked and bleeding skin. Currently, the topical delivery of 5-aminolevulinic acid-photodynamic therapy (ALA-PDT) is an optional treatment for psoriasis which provides long-term therapeutic effects, is non-toxic and enjoys better compliance with patients. However, the precursor of ALA is hydrophilic, and thus its ability to penetrate the skin is limited. Also, little research has provided a platform to investigate the penetration behavior in disordered skin. We employed a highly potent ethosomal carrier (phosphatidylethanolamine; PE) to investigate the penetration behavior of ALA and the recovery of skin in a hyperproliferative murine model. We found that the application of ethosomes produced a significant increase in cumulative amounts of 5-26-fold in normal and hyperproliferative murine skin samples when compared to an ALA aqueous solution; and the ALA aqueous solution appeared less precise in terms of the penetration mode in hyperproliferative murine skin. After the ethosomes had been applied, the protoporphyrin IX (PpIX) intensity increased about 3.64-fold compared with that of the ALA aqueous solution, and the penetration depth reached 30-80 microm. The results demonstrated that the ethosomal carrier significantly improved the delivery of ALA and the formation of PpIX in both normal and hyperproliferative murine skin samples, and the expression level of tumor necrosis factor (TNF)-alpha was reduced after the ALA-ethosomes were applied to treat hyperproliferative murine skin. Furthermore, the results of present study encourage more investigations on the mechanism of the interaction with ethosomes and hyperproliferative murine skin. PMID:19660544

  19. 5-Aminolevulinic Acid Protects against Cisplatin-Induced Nephrotoxicity without Compromising the Anticancer Efficiency of Cisplatin in Rats In Vitro and In Vivo

    PubMed Central

    Matsumoto, Tatsuki; Ishihara, Masayuki; Hamada, Kazu; Shimamura, Yoshiko; Ogata, Koji; Inoue, Kosuke; Taniguchi, Yoshinori; Horino, Taro; Karashima, Takashi; Tamura, Kenji; Fukuhara, Hideo; Fujimoto, Shimpei; Tsuda, Masayuki; Shuin, Taro

    2013-01-01

    Background/Aims Nephrotoxicity is a frequent and major limitation in cisplatin (CDDP)-based chemotherapy. 5-Aminolevulinic acid (ALA) is widely distributed in animal cells, and it is a precursor of tetrapyrole compounds such as heme that is fundamentally important in aerobic energy metabolism. The aim of this study is to evaluate the protective role of ALA in CDDP-induced acute kidney injury (AKI). Method We used CDDP-induced AKI rat model and cultured renal tubular cells (NRK-52E). We divided four groups of rats: control, CDDP only, CDDP + ALA(post);(ALA 10 mg/kg + Fe in drinking water) after CDDP, CDDP + ALA(pre & post). Result CDDP increased Cr up to 6.5 mg/dl, BUN up to 230 mg/dl, and ALA significantly reduced these changes. ALA ameliorates CDDP-induced morphological renal damages, and reduced tubular apoptosis evaluated by TUNEL staining and cleaved caspase 3. Protein and mRNA levels of ATP5α, complex(COX) IV, UCP2, PGC-1α in renal tissue were significantly decreased by CDDP, and ALA ameliorates reduction of these enzymes. In contrast, Heme Oxigenase (HO)-1 level is induced by CDDP treatment, and ALA treatment further up-regulates HO-1 levels. In NRK-52E cells, the CDDP-induced reduction of protein and mRNA levels of mitochondrial enzymes was significantly recovered by ALA + Fe. CDDP-induced apoptosis were ameliorated by ALA + Fe treatment. Furthermore, we evaluated the size of transplantated bladder carcinoma to the rat skin, and ALA did not change the anti cancer effects of CDDP. Conclusion These data suggested that the protective role of ALA in cisplatin-induced AKI is via protection of mitochondrial viability and prevents tubular apoptosis. Also there are no significant effects of ALA on anticancer efficiency of CDDP in rats. Thus, ALA has the potential to prevent CDDP nephrotoxicity without compromising its anticancer efficacy. PMID:24324635

  20. Effects of exogenous 5-aminolevulinic acid on photosynthesis, stomatal conductance, transpiration rate, and PIP gene expression of tomato seedlings subject to salinity stress.

    PubMed

    Zhao, Y Y; Yan, F; Hu, L P; Zhou, X T; Zou, Z R; Cui, L R

    2015-06-11

    The effects of exogenous 5-aminolevulinic acid (ALA) on photosynthesis, plant growth, and the expression of two aquaporin genes in tomato seedlings under control and salinity conditions were investigated. Exogenous ALA application significantly improved net photosynthetic rate (Pn), total chlorophyll content, and plant biomass accumulation of tomato seedlings under salinity stress. As revealed by real-time PCR analyses, after treatment with ALA alone, expression of both LePIP1 and LePIP2 in the two tomato cultivars was up-regulated at 2 h and subsequently decreased to normal levels. Under salinity stress, transcript levels of LePIP1 in both leaves and roots of salt-sensitive cultivars (cv. Zhongza No.9) increased significantly and were considerably higher than in cultivars exposed to ALA alone. In contrast, the expression levels of LePIP1 and LePIP2 in cvs. Jinpeng No.1 cultivars were slightly lower under salinity stress than under ALA treatment. In addition, transcript levels of both LePIP1 and LePIP2 in the roots of Jinpeng No. 1 cultivars were considerably lower than those in the roots of Zhongza No. 9 cultivars under salinity stress, regardless of ALA supplementation, implying that Jinpeng No. 1 cultivars had a better capacity to maintain membrane intrinsic protein stability. Further, ALA application distinctly counteracted the up- or down-regulation of LePIP1 and LePIP2 in both cultivars under salinity stress, in accordance with the improvements instomatal conductance, transpiration rate, and Pn of tomato leaves. The results presented here indicate that ALA controls aquaporin expression, thus, presumably ALA regulates water homeostasis and enhances salt tolerance of tomato seedlings.

  1. Interstitial photodynamic therapy of canine prostate with meso-tetra-(m-hydroxyphenyl) chlorin and 5-aminolevulinic acid: a preliminary study

    NASA Astrophysics Data System (ADS)

    Chang, Shi-Chung; Buonaccorsi, Giovanni A.; MacRobert, Alexander J.; Bown, Stephen G.

    1996-01-01

    Photodynamic therapy (PDT) is proved to have potential for managing various malignancies. We investigated tissue biodistribution and photodynamic effects on a canine model in vivo using second generation photosensitizers, meso-tetra(m-hydroxyphenyl)chlorin (mTHPC) and 5-aminolaevulinic acid (ALA) to evaluate the feasibility and possible future application of PDT on the prostate. Using fluorescence microscopy, the optimal sensitization time of the prostate was between 24 - 72 hours with mTHPC and, 3 hours with ALA. After optimum time of sensitization, prostates of mature beagle were treated with laser at various sites by placing fiber interstitially under the guidance of transrectal ultrasound. The light dose for each treatment site was 100 J (100 mW for 1,000 seconds at the wavelength of 650 and 630 nm, respectively). With mTHPC, single laser fiber was able to induce organ confined PDT lesion as large as 20 by 18 by 18 mm in size. However, the PDT lesion with ALA was negligible 3 days after treatment. Physical distress manifested as urinary retention, poor appetite and body weigh loss, was more prominent with increasing number of treatment sites as a result of extensive prostatic swelling and urethral damages. However, these problems usually alleviated spontaneously 7 to 10 days after PDT. The characteristic histological changes were hemorrhagic necrosis and glandular destruction with preservation of interlobular collagen fibers. Urethral damage seen at the early stage healed by regeneration of urothelium in 4 weeks. We conclude that interstitial PDT with mTHPC is technically possible to produce extensive glandular necrosis in the normal prostate which heals safely and does not change the prostatic architecture. ALA, although it seems promising for bladder tumors, is much less effective than mTHPC on the prostate. With mTHPC, it might have the potential for treating prostate cancers localized in the periphery of the gland.

  2. Evaluating the efficacy and safety of a novel endoscopic fluorescence imaging modality using oral 5-aminolevulinic acid for colorectal tumors

    PubMed Central

    Tsuruki, Eriko So; Saito, Yutaka; Abe, Seiichiro; Takamaru, Hiroyuki; Yamada, Masayoshi; Sakamoto, Taku; Nakajima, Takeshi; Matsuda, Takahisa; Sekine, Shigeki; Taniguchi, Hirokazu

    2016-01-01

    Background and study aims: Five-aminolevulinic acid (5-ALA) is being increasingly used for photodynamic diagnosis and therapy of various types of tumors including brain, urologic, and other neoplasias. The use of 5-ALA to treat Barrett’s carcinomas has been documented, but its clinical effectiveness for diagnosis of gastrointestinal tumors, particularly early cancers, remains unknown. Patients and methods: The aim of our feasibility study was to evaluate the visibility of colorectal tumors using endoscopic fluorescence imaging (EFI) after oral administration of 5-ALA. The lesions identified by direct visualization and by the spectrums produced using EFI modality with 5-ALA were compared to the clinicopathologic features of resected specimens. Results: Twenty-three patients with a total of 27 known colorectal lesions were enrolled in the study. The median tumor size was 30 mm (range 10 – 75). Eleven of the lesions were flat or depressed lesions and 16 were sessile. Red fluorescence was observed in 22 out of 27 lesions. Red fluorescence was negative in 4 out of 11 flat or depressed lesions. In comparison with histopathologic findings, the rates of red fluorescence visibility were 62.5 % in low-grade intraepithelial neoplasia, 77.8 % in high-grade neoplasia, and 100 % in submucosal carcinoma. Red fluorescence visibility increased with the degree of dysplasia. There were no significant adverse events identified in this study. Conclusions: This feasibility study using EFI with 5-ALA demonstrated high visibility of superficial colorectal neoplasia. EFI with 5-ALA appears to be a novel, safe technique for improving real-time colorectal tumor imaging. PMID:26793782

  3. Low-Dose Topical 5-Aminolevulinic Acid Photodynamic Therapy in the Treatment of Different Severity of Acne Vulgaris.

    PubMed

    Tao, Shi-Qin; Li, Fei; Cao, Lei; Xia, Ru-Shan; Fan, Hua; Fan, Ying; Sun, Hui; Jing, Cheng; Yang, Li-Jia

    2015-12-01

    The objective of this article is to investigate the effectiveness and safety of photodynamic therapy (PDT) with 3.6 % topical aminolevulinic acid (ALA) and a short incubation time with red light in moderate to severe acne. One hundred and thirty-six patients with moderate to severe acne were treated with 3.6 % topical ALA-PDT for three sessions with an interval of 2 weeks. Patients were evaluated for efficacy and safety on week 2, 4, 6, 8, and 12 after the initial treatment. Most patients showed apparent clearance of acne lesions at the treated site after three sessions. The effective treatment rates were increased after the multiple therapies. The clinical outcomes are the best at 4 weeks after the final treatment. The total effectiveness rate and cure rate of the low-dose ALA-PDT procedure is 92.65 and 47.06 %, respectively. Thirty-one patients and nineteen patients showed apparent exacerbation of acne lesions before the 2nd and 3rd treatment, respectively, but all of them showed good or excellent improvement after a three-course treatment. A few patients showed mild relapse including papules and comedos at 8 weeks after the final treatment. No significant differences are found in the effects of different acne severity and different genders. Adverse reactions are mild and transient. A 3.6 % topical ALA-PDT with a short time incubation with red light is a simple and an effective treatment option for moderate to severe acne with mild side effects in Chinese people.

  4. In vitro study of cell death with 5-aminolevulinic acid based photodynamic therapy to improve the efficiency of cancer treatment

    NASA Astrophysics Data System (ADS)

    Firdous, S.; Nawaz, M.; Ikram, M.; Ahmed, M.

    2012-03-01

    Photodynamic therapy (PDT) is a kind of photochemo therapeutic treatment that exerts its effect mainly through the induction of cell death. Distinct types of cell death may be elicited by different PDT regimes. In this study, efforts are underway to optimize PDT protocols for improved efficacy and combination of all three PDT mechanisms involved in the different human carcinomas cell narcosis. Our in vitro cell culture experiments with 5-aminolevulanic acid (ALA) a clinically approved photiosensitizer (PS) and 635 nm laser light have yielded promising results, as follow: (1) (human cervical cancer (HeLa) cell line incubated, for 18 h, with 30 μg/ml of 5-ALA, treated with laser light dose of 50 J/cm2 can produce 85% of cell killing (2) human larynx carcinoma (Hep2c) cell line incubated, for 7 h, with 55 μg/ml of 5-ALA, treated with laser light dose of 85 J/cm2 can produce 75% of cell killing (3) human liver cancer (HepG2) cell line incubated, for 22-48 h, with 262 μg/ml of 5-ALA, treated with laser light dose of 120 J/cm2 can produce 95% of cell killing (4) human muscle cancer (RD) cell line incubated, for 47 h, with 250 μg/ml of 5-ALA, treated with laser light dose of 80 J/cm2 can produce 76% of cell killing (5) Human embryonic kidney (HEK293T) cell line incu-bated, for 18 h, with 400 μg/ml of 5-ALA, treated with laser light dose of 40 J/cm2 can produce 82% of cell killing confirming the efficacy of photodynamic therapy.

  5. Clinical efficacy of 5-aminolevulinic acid photodynamic therapy in the treatment of moderate to severe facial acne vulgaris

    PubMed Central

    CHEN, XIANGQI; SONG, HONGTAO; CHEN, SHENGPING; ZHANG, JING; NIU, GAOXIANG; LIU, XIANGNONG

    2015-01-01

    Acne vulgaris is considered as a therapeutic challenge in terms of managing ongoing symptoms and preventing scar formation. Although there are many available treatments for alleviating acne, therapies for resistant or moderate-to-severe forms have been limited to systemic agents that are accompanied by potentially severe side-effects. While, aminolevulinic acid (ALA) photodynamic therapy (PDT) has increasingly been used as a simple and safe therapeutic option of acne vulgaris, the clinical efficacy requires confirmation in further studies. The aim of this study was to investigate the efficacy and safety of 5-ALA-PDT in the treatment of moderate-to-severe facial acne vulgaris. A total of 50 patients with moderate-to-severe facial acne were enrolled in the study and randomly divided equally into a therapy group and a control group. In the therapy group, the patients were treated with 5% 5-ALA for 1.5 h, followed by three 20-min doses of infrared radiation once a week; in the control group, the patients were treated with three 20 min doses of infrared radiation without 5-ALA once a week. Both treatments lasted for 3 weeks. The clinical efficacy was determined by evaluating acne lesion counts at weeks 0, 2, 4 and 6. Total efficacy rate (TER) was the primary endpoint of the study, and was defined as the proportion of the patients whose treatment effectiveness evaluation was cured (≥90% of skin lesions improved) and excellent (60–89% improvement). Adverse effects were recorded throughout the study. The study was completed by 24 patients in the therapy group and 23 patients in the control group. The numbers of acne lesions significantly decreased. The TER of the therapy group was significantly higher than that of the control group at weeks 4 and 6. Adverse effects were observed in 12 patients of the therapy group and 2 patients of the control group. In the therapy group the most common adverse effect was a burning sensation (n=7), followed by transient

  6. Effect of cell cycle phase on the sensitivity of SAS cells to sonodynamic therapy using low-intensity ultrasound combined with 5-aminolevulinic acid in vitro.

    PubMed

    Li, Nan; Sun, Miao; Wang, Yao; Lv, Yanhong; Hu, Zheng; Cao, Wenwu; Zheng, Jinhua; Jiao, Xiaohui

    2015-08-01

    Sonodynamic therapy (SDT) with 5-aminolevulinic acid (5-ALA) can effectively inhibit various types of tumor in vitro and in vivo. However, the association between the efficacy of SDT and the phase of the cell cycle remains to be elucidated. 5-ALA may generate different quantities of sonosensitizer, protoporphyrin IX (PpIX), in different phases of the cell cycle, which may result in differences in sensitivity to 5-ALA-induced SDT. The present study aimed to investigate the effect of the cell cycle on the susceptibility of SAS cells to SDT following synchronization to different cell cycle phases. These results indicates that the rates of cell death and apoptosis of the SAS cells in the S and G2/M phases were significantly higher following SDT, compared with those in the G1-phase cells and unsynchronized cells, with a corresponding increase in PpIX in the S and G2/M cells. In addition, the expression of caspase-3 increased, while that of B-cell lymphoma (Bcl)-2 decreased markedly in theS and G2/M cells following SDT. Cyclin A was also expressed at higher levels in the S and G2/M cells, compared with the G1-phase cells. SDT also caused a significant upregulation of cyclin A in all phases of the cell cycle, however this was most marked in the S and G2/M cells. It was hypothesized that high expression levels of cyclin A in the S and G2/M cells may promote the induction of caspase-3 and reduce the induction of Bcl-2 by SDT and, therefore, enhance apoptosis. Taken together, these data demonstrated that cells in The S and G2/M phases generate more intracellular PpIX, have higher levels of cyclin A and are, therefore, more sensitive to SDT-induced cytotoxicity. These findings indicate the potential novel approach to preventing the onset of cancer by combining cell-cycle regulators with SDT. This sequential combination therapy may be a simple and cost-effective way of enhancing the effects of SDT in clinical settings.

  7. Photodynamic therapy of human skin tumors using topical application of 5-aminolevulinic acid, dimethylsulfoxide (DMSO), and edetic acid disodium salt (EDTA)

    NASA Astrophysics Data System (ADS)

    Orenstein, Arie; Kostenich, Gennady; Tsur, H.; Roitman, Leonid; Ehrenberg, Benjamin; Malik, Zvi

    1995-01-01

    The results of photodynamic therapy (PDT) in 48 patients bearing basal cell carcinoma (BCC) and 7 patients with squamous cell carcinoma (SCC) of the skin are described. Five- aminolevulinic acid (5-ALA) was applied topically in two formulations. The first formulation contained 20% of 5-ALA in a base cream, and the second formulation (5-ALA composite cream), contained an additional 2% of dimethylsulfoxide (DMSO) and 2% of edetic acid disodium salt (EDTA). The creams were left on the skin for 2 - 5 hours. Production of protoporphyrin (PP) was measured in situ by a laser-induced fluorescence (LIF) method. The results of fluorescence measurement clearly indicate that PP accumulation in tumors induced by the 5-ALA composite cream was markedly higher than that induced by the 5-ALA cream. The tumors were light-irradiated (600 - 720 nm) after 4 - 5 hours of cream applications, using the light delivery system Versa-Light by a light dose of 100 J/cm2. The clinically superficial BCC tumors were highly responsive to PDT; the overall result in BCC patients was an 85.4% complete response. Histological examination showed an initial edematous reaction, followed by necrosis and complete disappearance of the tumor. The superficial SCC tumors showed a 100% complete response after PDT. The ulcerated nodular SCC showed partial responses.

  8. The Value of 5-Aminolevulinic Acid in Low-grade Gliomas and High-grade Gliomas Lacking Glioblastoma Imaging Features: An Analysis Based on Fluorescence, Magnetic Resonance Imaging, 18F-Fluoroethyl Tyrosine Positron Emission Tomography, and Tumor Molecular Factors

    PubMed Central

    Jaber, Mohammed; Wölfer, Johannes; Ewelt, Christian; Holling, Markus; Hasselblatt, Martin; Niederstadt, Thomas; Zoubi, Tarek; Weckesser, Matthias

    2015-01-01

    BACKGROUND: Approximately 20% of grade II and most grade III gliomas fluoresce after 5-aminolevulinic acid (5-ALA) application. Conversely, approximately 30% of nonenhancing gliomas are actually high grade. OBJECTIVE: The aim of this study was to identify preoperative factors (ie, age, enhancement, 18F-fluoroethyl tyrosine positron emission tomography [18F-FET PET] uptake ratios) for predicting fluorescence in gliomas without typical glioblastomas imaging features and to determine whether fluorescence will allow prediction of tumor grade or molecular characteristics. METHODS: Patients harboring gliomas without typical glioblastoma imaging features were given 5-ALA. Fluorescence was recorded intraoperatively, and biopsy specimens collected from fluorescing tissue. World Health Organization (WHO) grade, Ki-67/MIB-1 index, IDH1 (R132H) mutation status, O6-methylguanine DNA methyltransferase (MGMT) promoter methylation status, and 1p/19q co-deletion status were assessed. Predictive factors for fluorescence were derived from preoperative magnetic resonance imaging and 18F-FET PET. Classification and regression tree analysis and receiver-operating-characteristic curves were generated for defining predictors. RESULTS: Of 166 tumors, 82 were diagnosed as WHO grade II, 76 as grade III, and 8 as glioblastomas grade IV. Contrast enhancement, tumor volume, and 18F-FET PET uptake ratio >1.85 predicted fluorescence. Fluorescence correlated with WHO grade (P < .001) and Ki-67/MIB-1 index (P < .001), but not with MGMT promoter methylation status, IDH1 mutation status, or 1p19q co-deletion status. The Ki-67/MIB-1 index in fluorescing grade III gliomas was higher than in nonfluorescing tumors, whereas in fluorescing and nonfluorescing grade II tumors, no differences were noted. CONCLUSION: Age, tumor volume, and 18F-FET PET uptake are factors predicting 5-ALA-induced fluorescence in gliomas without typical glioblastoma imaging features. Fluorescence was associated with an increased

  9. 5-Aminolevulinic Acid-Mediated Sonodynamic Therapy Promotes Phenotypic Switching from Dedifferentiated to Differentiated Phenotype via Reactive Oxygen Species and p38 Mitogen-Activated Protein Kinase in Vascular Smooth Muscle Cells.

    PubMed

    Dan, Juhua; Sun, Xin; Li, Wanlu; Zhang, Yun; Li, Xuesong; Xu, Haobo; Li, Zhitao; Tian, Zhen; Guo, Shuyuan; Yao, Jianting; Gao, Weidong; Tian, Ye

    2015-06-01

    Sonodynamic therapy (SDT) has been found to inhibit in-stent restenosis in animal models. However, the mechanism is not fully elucidated. Here, we investigated the effects of 5-aminolevulinic acid (ALA)-mediated SDT (ALA-SDT) on vascular smooth muscle cells (VSMCs), a cause of restenosis, with a focus on SDT-induced phenotypic switching. Serum-induced dedifferentiated VSMCs were cultured with ALA (1 mm, 24 h) and exposed to ultrasound (0.8 W/cm(2)) for 5 min. Results indicated that ALA-SDT inhibited the migration and proliferation of VSMCs and enhanced the expression of differentiated phenotypic markers in VSMCs. Additionally, ALA-SDT increased intracellular reactive oxygen species accumulation and phosphorylated p38 mitogen-activated protein kinase in VSMCs. Inhibition of reactive oxygen species elevation or p38 mitogen-activated protein kinase activity abolished the expression of smooth muscle 22α (SM22α) in VSMCs induced by ALA-SDT. Taken together, these results suggest that ALA-SDT promotes transformation of the VSMC phenotype from the dedifferentiated to differentiated status via reactive oxygen species and activated p38 mitogen-activated protein kinase.

  10. The application of 5-aminolevulinic acid in the treatment of precancerous lesions, skin cancer, and a new approach to the control of therapy

    NASA Astrophysics Data System (ADS)

    Kulas, Zbigniew; Bereś-Pawlik, Elżbieta; Bieniek, Andrzej; Matusiak, Łukasz

    2009-02-01

    The aim of our work was to determine a therapeutic effect of photodynamic therapy (PDT). Twenty five patients with the Bowen's disease, actinic keratosis and basal cell carcinoma (superficial, nodular) were examined. They were treated with photosensitizer - aminolevulinic acid (metabolized in protoporphyrin IX), and the new red light source built of high-power diodes. A new method, based on numerical analysis of fluorescence imaging of tissues, was proposed as a way for controlling therapy.

  11. {Delta}-Aminolevulinic acid dehydratase: A sensitive indicator of lead exposure in broiler chicks: (Gallus domesticus)

    SciTech Connect

    Bakalli, R.I.; Pesti, G.M.; Konjufca, V.

    1995-12-01

    Delta-aminolevulinic acid dehydratase, EC 4.2.1.24 (ALAD) is one of the enzymes participating in heme synthesis. The study reported in this paper was designed to determine the activity of erythrocyte ALAD anbd the relationship between this enzyme and tissue lead levels in chickens, during Pb intake and after withdrawing Bv from the feed. 20 refs., 3 tabs.

  12. In vitro study of 5-aminolevulinic acid-based photodynamic therapy for apoptosis in human cervical HeLa cell line

    NASA Astrophysics Data System (ADS)

    Atif, M.; Firdous, S.; Khurshid, A.; Noreen, L.; Zaidi, S. S. Z.; Ikram, M.

    2009-12-01

    5-aminolevulanic acid (ALA), belonging among the promising second generation of sensitizers, was evaluated as an inducer of photodamage on HeLa (human cervical adenocarcinoma) cell line. A diode laser (635 nm) was used as a source for initiation of the photodynamic effect. We studied the influence of different incubation times, various concentrations of sensitizer, different irradiation doses and various combinations of sensitizer and light doses on the photodamage of HeLa cells. Viability of cells was determined by means of neutral red assay. The quantitative cellular uptake of ALA sensitizer was done by spectrophotometric measurements. No prominent cytotoxic or phototoxic effects on HeLa were observed due to sensitizer or light doses when studied independently of each other. However phototoxicity evoked by laser irradiated sensitizer was detected in HeLa cell line.

  13. Photodynamic Therapy (PDT) using intratumoral injection of the 5- aminolevulinic acid (5-ALA) for the treatment of eye cancer in cattle

    NASA Astrophysics Data System (ADS)

    Hage, Raduan; Mancilha, Geraldo; Zângaro, Renato A.; Munin, Egberto; Plapler, Hélio

    2007-02-01

    A six-year old Holstein cow with an eye cancer (ocular squamous cell carcinoma) involving the third eyelid and conjunctiva was submitted to photodynamic therapy using intratumoral 20% aminolevulinic acid (5-ALA - Aldrich Chemical Company, Milwaukee, USA) and a light emitting diode (LED - VET LED - MMOptics (R)) with wavelength between 600 and 700 nm, 2 cm diameter circular light beam, power of 150 mW, light dose of 50 J/cm2 as a source of irradiation. Fifteen days after the experimental procedure we observed about 50% tumor reduction and complete remission after 3 months. Relapse was not observed up to 12 months after the treatment. Although the study only includes one animal not allowing definite conclusions, it indicates that PDT represents a safe and technically feasible approach in the treatment of eye cancer in cattle.

  14. Dihydroxy acid dehydratase from spinach contains a [2Fe-2S] cluster.

    PubMed

    Flint, D H; Emptage, M H

    1988-03-15

    Dihydroxy acid dehydratase, the third enzyme in the branched-chain amino acid biosynthetic pathway, has been purified to homogeneity (5000-fold) from spinach leaves. The molecular weights of dihydroxy acid dehydratase as determined by sodium dodecyl sulfate and native gel electrophoresis are 63,000 and 110,000, respectively, suggesting the native enzyme is a dimer. 2 moles of iron were found per mol of protein monomer. Chemical analyses of iron and labile sulfide gave an Fe/S2- ratio of 0.95. The EPR spectrum of dithionite-reduced enzyme (gavg = 1.91) is similar to spectra characteristic of Rieske Fe-S proteins and has a spin concentration of 1 spin/1.9 irons. These results strongly suggest that dihydroxy acid dehydratase contains a [2Fe-2S] cluster, a novel finding for enzymes of the hydrolyase class. In contrast to the Rieske Fe-S proteins, the redox potential of the Fe-S cluster is quite low (-470 mV). Upon addition of substrate, the EPR signal of the reduced enzyme changes to one typical of 2Fe ferredoxins (gavg = 1.95), and the visible absorption spectrum of the native enzyme shows substantial changes between 400 and 600 nm. Reduction of the Fe-S cluster decreases the enzyme activity by 6-fold under Vmax conditions. These results suggest the direct involvement of the [2Fe-2S] cluster of dihydroxy acid dehydratase in catalysis. Similar conclusions have been reached for the catalytic involvement of the [4Fe-4S] cluster of the hydrolyase aconitase (Emptage, M. H., Kent, T. A., Kennedy, M. C., Beinert, H., and Münck, E. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 4674-4678).

  15. Catalytic residues are shared between two pseudosubunits of the dehydratase domain of the animal fatty acid synthase.

    PubMed

    Pasta, Saloni; Witkowski, Andrzej; Joshi, Anil K; Smith, Stuart

    2007-12-01

    Expression, characterization, and mutagenesis of a series of N-terminal fragments of an animal fatty acid synthase, containing the beta-ketoacyl synthase, acyl transferase, and dehydratase domains, demonstrate that the dehydratase domain consists of two pseudosubunits, derived from contiguous regions of the same polypeptide, in which a single active site is formed by the cooperation of the catalytic histidine 878 residue of the first pseudosubunit with aspartate 1032 of the second pseudosubunit. Mutagenesis and modeling studies revealed an essential role for glutamine 1036 in anchoring the position of the catalytic aspartate. These findings establish that sequence elements previously assigned to a central structural core region of the type I fatty acid synthases and some modular polyketide synthase counterparts play an essential catalytic role as part of the dehydratase domain.

  16. The role and properties of the iron-sulfur cluster in Escherichia coli dihydroxy-acid dehydratase.

    PubMed

    Flint, D H; Emptage, M H; Finnegan, M G; Fu, W; Johnson, M K

    1993-07-15

    Dihydroxy-acid dehydratase has been purified from Escherichia coli and characterized as a homodimer with a subunit molecular weight of 66,000. The combination of UV visible absorption, EPR, magnetic circular dichroism, and resonance Raman spectroscopies indicates that the native enzyme contains a [4Fe-4S]2+,+ cluster, in contrast to spinach dihydroxy-acid dehydratase which contains a [2Fe-2S]2+,+ cluster (Flint, D. H., and Emptage, M. H. (1988) J. Biol. Chem. 263, 3558-3564). In frozen solution, the reduced [4Fe-4S]+ cluster has a S = 3/2 ground state with minor contributions from forms with S = 1/2 and possibly S = 5/2 ground states. Resonance Raman studies of the [4Fe-4S]2+ cluster in E. coli dihydroxy-acid dehydratase indicate non-cysteinyl coordination of a specific iron, which suggests that it is likely to be directly involved in catalysis as is the case with aconitase (Emptage, M. H., Kent, T. A., Kennedy, M. C., Beinert, H., and Münck, E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4674-4678). Dihydroxy-acid dehydratase from E. coli is inactivated by O2 in vitro and in vivo as a result of oxidative degradation of the [4Fe-4S]cluster. Compared to aconitase, the oxidized cluster of E. coli dihydroxy-acid dehydratase appears to be less stable as either a cubic or linear [3Fe-4S] cluster or a [2Fe-2S] cluster. Oxidative degradation appears to lead to a complete breakdown of the Fe-S cluster, and the resulting protein cannot be reactivated with Fe2+ and thiol reducing agents.

  17. Delta-aminolevulinic acid dehydratase: Inhibition in ducks dosed with lead shot

    USGS Publications Warehouse

    Finley, M.T.; Dieter, M.P.; Locke, L.N.

    1976-01-01

    Lead concentration in blood and erythrocyte delta-aminolevulinic acid dehydratase (ALAD) activity was measured in mallard ducks dosed with one all-lead shot or one lead-iron combination shot. For 2 weeks after dosage, lead in blood of ducks given an all-lead shot was fourfold higher than in those dosed with lead-iron shot. At 3 and 4 weeks, the differences in lead residues were directly proportional to lead content of the shots. ALAD activities measured at these intervals were inversely correlated (P < 0.01) with the concentration of lead in the blood, suggesting that biochemical responses to the two types of shot were dependent upon the quantity of lead present.

  18. Effects of zinc, copper, and lead toxicity on. cap alpha. -aminolevulinic acid dehydratase activity. [Rats

    SciTech Connect

    Shafiq-ur-Rehman

    1984-07-01

    The distribution of lead, zinc and copper in the human environment has been recognized as a major toxicological factor. Lead ions have been shown to inhibit the activity of delta-aminolevulinic acid dehydratase (delta-ALAD), which is involved in the biosynthesis of heme. Copper also has its inhibitory effect on delta-ALAD activity. A study has shown that the delta-ALAD was activated by zinc ions at physiological concentrations. In view of these reports, it was considered worthwhile to study the poisoning effects of lead, zinc and copper on delta-ALAD activity along with the concentrations of these metal ions in the blood. A possible role of Zn/sup + +/, Cu/sup + +/, and Pb/sup + +/ interaction and their influence on delta-ALAD has been explored in the present paper.

  19. Hereditary Tyrosinemia and the Heme Biosynthetic Pathway. PROFOUND INHIBITION OF δ-AMINOLEVULINIC ACID DEHYDRATASE ACTIVITY BY SUCCINYLACETONE

    PubMed Central

    Sassa, Shigeru; Kappas, Attallah

    1983-01-01

    Succinylacetone (4,6-dioxoheptanoic acid) is an abnormal metabolite produced in patients with hereditary tyrosinemia as a consequence of an inherited deficiency of fumarylacetoacetate hydrolase. It is known that patients with this hereditary disease excrete excessive amounts of δ-aminolevulinic acid (ALA) in urine and that certain patients have an accompanying clinical syndrome resembling that of acute intermittent porphyria (AIP). In order to elucidate the relation of succinylacetone to the heme biosynthetic pathway, we have examined the effects of this metabolite on the cellular heme content of cultured avian hepatocytes and on the activity of purified ALA dehydratase from normal human erythrocytes and from mouse and bovine liver. Our data indicate that succinylacetone is an extremely potent competitive inhibitor of ALA dehydratase in human as well as in animal tissues. By using purified preparations of the enzyme from human erythrocytes and mouse and bovine liver, an inhibitor constant ranging from 2 × 10-7 M to 3 × 10-7 M was obtained. In cultured hepatocytes, succinylacetone also inhibited ALA dehydratase activity, decreased the cellular content of heme and cytochrome P-450, and greatly potentiated the induction response of ALA synthase to drugs such as phenobarbital, chemicals such as allylisopropylacetamide and 3,5-dicarbethoxy-1,4-dihydrocollidine, and natural steroids such as etiocholanolone. Four patients with hereditary tyrosinemia have been studied and all were found to have greatly depressed levels of erythrocyte ALA dehydratase activity and elevated concentrations of this inhibitor in urine. These findings indicate that tyrosinemia is a disorder of special pharmacogenetic interest because succinylacetone, an abnormal product of the tyrosine metabolic pathway, resulting from the primary gene defect of the disease, profoundly inhibits heme biosynthesis in normal cells through a blockade at the ALA dehydratase level, leading to clinical and metabolic

  20. Association between delta-aminolevulinic acid dehydratase polymorphism and placental lead levels.

    PubMed

    Kayaaltı, Zeliha; Sert, Selda; Kaya-Akyüzlü, Dilek; Söylemez, Esma; Söylemezoğlu, Tülin

    2016-01-01

    Lead inhibits the delta-aminolevulinic acid dehydratase (ALAD) activity and results in neurotoxic aminolevulinic acid accumulation in the blood. During pregnancy, lead in the maternal blood can easily cross the placenta. The aim of this study was to determine whether the maternal ALAD G177C polymorphism (rs1800435) was related to the placental lead levels. The study population comprised 97 blood samples taken from mothers to investigate ALAD G177C polymorphism and their placentas to measure lead levels. ALAD G177C polymorphism was detected by standard polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) technique and atomic absorption spectrometry (AAS) equipped with a graphite furnace and Zeeman background correction system was used for lead determination. The median placental lead levels for ALAD1-1, ALAD1-2 and ALAD2-2 genotypes were 7.54 μg/kg, 11.78 μg/kg and 18.53 μg/kg, respectively. Statistically significant association was found between the maternal ALAD G177C polymorphism and placental lead levels (p<0.05). This study suggested that maternal ALAD G177C polymorphism was associated with placental lead levels.

  1. Structure and functional characterization of a bile aciddehydratase BaiE in secondary bile acid synthesis.

    PubMed

    Bhowmik, Shiva; Chiu, Hsien-Po; Jones, David H; Chiu, Hsiu-Ju; Miller, Mitchell D; Xu, Qingping; Farr, Carol L; Ridlon, Jason M; Wells, James E; Elsliger, Marc-André; Wilson, Ian A; Hylemon, Phillip B; Lesley, Scott A

    2016-03-01

    Conversion of the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) to the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA) is performed by a few species of intestinal bacteria in the genus Clostridium through a multistep biochemical pathway that removes a 7α-hydroxyl group. The rate-determining enzyme in this pathway is bile acid 7α-dehydratase (baiE). In this study, crystal structures of apo-BaiE and its putative product-bound [3-oxo-Δ(4,6) -lithocholyl-Coenzyme A (CoA)] complex are reported. BaiE is a trimer with a twisted α + β barrel fold with similarity to the Nuclear Transport Factor 2 (NTF2) superfamily. Tyr30, Asp35, and His83 form a catalytic triad that is conserved across this family. Site-directed mutagenesis of BaiE from Clostridium scindens VPI 12708 confirm that these residues are essential for catalysis and also the importance of other conserved residues, Tyr54 and Arg146, which are involved in substrate binding and affect catalytic turnover. Steady-state kinetic studies reveal that the BaiE homologs are able to turn over 3-oxo-Δ(4) -bile acid and CoA-conjugated 3-oxo-Δ(4) -bile acid substrates with comparable efficiency questioning the role of CoA-conjugation in the bile acid metabolism pathway. PMID:26650892

  2. Expression of dehydratase domains from a polyunsaturated fatty acid synthase increases the production of fatty acids in Escherichia coli

    PubMed Central

    Oyola-Robles, Delise; Rullán-Lind, Carlos; Carballeira, Néstor M.; Baerga-Ortiz, Abel

    2014-01-01

    Increasing the production of fatty acids by microbial fermentation remains an important step towards the generation of biodiesel and other portable liquid fuels. In this work, we report an Escherichia coli strain engineered to overexpress a fragment consisting of four dehydratase domains from the polyunsaturated fatty acid (PUFA) synthase enzyme complex from the deep-sea bacterium, Photobacterium profundum. The DH1-DH2-UMA enzyme fragment was excised from its natural context within a multi-enzyme PKS and expressed as a stand-alone protein. Fatty acids were extracted from the cell pellet, esterified with methanol and quantified by GC-MS analysis. Results show that the E. coli strain expressing the DH tetradomain fragment was capable of producing up to a 5-fold increase (80.31 mg total FA/L culture) in total fatty acids over the negative control strain lacking the recombinant enzyme. The enhancement in production was observed across the board for all the fatty acids that are typically made by E. coli. The overexpression of the DH tetradomain did not affect E. coli cell growth, thus showing that the observed enhancement in fatty acid production was not a result of effects associated with cell density. The observed enhancement was more pronounced at lower temperatures (3.8-fold at 16 °C, 3.5-fold at 22 °C and 1.5-fold at 30 °C) and supplementation of the media with 0.4% glycerol did not result in an increase in fatty acid production. All these results taken together suggest that either the dehydration of fatty acid intermediates are a limiting step in the E. coli fatty acid biosynthesis machinery, or that the recombinant dehydratase domains used in this study are also capable of catalyzing thioester hydrolysis of the final products. The enzyme in this report is a new tool which could be incorporated into other existing strategies aimed at improving fatty acid production in bacterial fermentations towards accessible biodiesel precursors. PMID:24411456

  3. δ-Aminolevulinic acid dehydratase genotype predicts toxic effects of lead on workers' peripheral nervous system.

    PubMed

    Zheng, Guang; Tian, Liting; Liang, Yihuai; Broberg, Karin; Lei, Lijian; Guo, Weijun; Nilsson, Johan; Bergdahl, Ingvar A; Skerfving, Staffan; Jin, Taiyi

    2011-08-01

    There is a wide variation in sensitivity to lead (Pb) exposure, which may be due to genetic susceptibility towards Pb. We investigated whether a polymorphism (rs1800435) in the δ-aminolevulinic acid dehydratase (ALAD) gene affected the toxicokinetics and toxicodynamics of Pb. Among 461 Chinese Pb-exposed storage battery and 175 unexposed workers, allele frequencies for the ALAD1 and ALAD2 alleles were 0.968 and 0.032, respectively. The Pb-exposed workers had a higher fraction of the ALAD1-2/2-2 genotype than unexposed workers (7.8% vs. 2.3%, p=0.01). The Pb levels in blood (B-Pb) and urine (U-Pb) were higher in Pb-exposed workers carrying the ALAD2 allele compared to homozygotes for ALAD1 (median B-Pb: 606 vs. 499 μg/L; U-Pb: 233 vs. 164 μg/g creatinine), while there was no statistically significant difference in the unexposed controls (median: 24 vs. 37 μg/L, and 3.9 vs. 6.4μg/g creatinine, respectively). High B-Pb and U-Pb were associated with statistically significantly lower sensory and motor conduction velocities in the median, ulnar and peroneal nerves. At the same B-Pb and U-Pb, ALAD1 homozygotes had lower conduction velocities than the ALAD2 carriers. There were similar trends for toxic effects on haem synthesis (zinc protoporphyrin and haemoglobin in blood) and renal function (albumin and N-acetyl-d-β-acetylglucosaminidase in urine), but without statistical significance. There was no difference in Pb toxicokinetics and toxicodynamics associated with VDR BsmI polymorphism. Our results show that the ALAD genotype modifies the relationship between Pb and its toxic effects on the peripheral nervous system. This must be considered in the assessment of risks at Pb exposure.

  4. Aminolevulinic acid dehydratase activity in American dippers (Cinclus mexicanus) from a metal-impacted stream.

    PubMed

    Strom, Sean M; Ramsdell, Howard S; Archuleta, Andrew S

    2002-01-01

    Blood samples collected from adult and nestling American dippers (Cinclus mexicanus) along the Arkansas River (CO, USA), a stream impacted by discharges from historical mining operations, and a reference stream were analyzed for lead concentration and delta-aminolevulinic acid dehydratase (ALAD) activity. Median ALAD activities of adult and nestling dippers from the Arkansas River were found to be significantly different from median ALAD activities of reference adults and nestlings (p = 0.002 and p = 0.028). Median ALAD activity for adult dippers from the Arkansas River was more than 50% lower relative to reference adults and activity approached a level close to 50% lower in nestlings from the same site. Median blood lead concentrations from adult (range 15.4-386.0 ppb) and nestling (range 12.1-323.0 ppb) dippers from the Arkansas River were found to be significantly different from median blood lead concentrations of reference adult (range 4.2-29.6 ppb) and nestling (range 4.2-8.2 ppb) dippers (p < 0.001 and p = 0.011). The median hematocrit level in adult dippers did not vary between sites (p = 0.73), whereas the median hematocrit level of nestling dippers from the reference site was significantly lower compared to Arkansas River nestlings (p = 0.042). Blood lead concentration in both adult and nestling dippers was found to be significantly correlated with invertebrate lead concentration (r = 0.81, p < 0.001 and r = 0.62, p = 0.01, respectively). Highly significant negative correlations were observed between blood lead concentration and ALAD activity in both adult and nestling dippers (r = -0.86, p < 0.001 and r = -0.84, p < 0.001, respectively). This study suggests that dippers (both adults and nestlings) from the Arkansas River have significantly lower ALAD activity and significantly higher blood lead concentrations compared to reference values. The measurement of ALAD activity may be a sensitive and accurate biomarker for environmental lead exposure in dippers

  5. Neurotransmitter transporter family including SLC6A6 and SLC6A13 contributes to the 5-aminolevulinic acid (ALA)-induced accumulation of protoporphyrin IX and photodamage, through uptake of ALA by cancerous cells.

    PubMed

    Tran, Tai Tien; Mu, Anfeng; Adachi, Yuka; Adachi, Yasushi; Taketani, Shigeru

    2014-01-01

    δ-Aminolevulinic acid (ALA)-induced protoporphyrin accumulation is widely used in the treatment of cancer, as photodynamic therapy (PDT). To clarify the mechanisms of ALA uptake by tumor cells, we have examined the ALA-induced accumulation of protoporphyrin by the treatment of colon cancer DLD-1 and epithelial cancer HeLa cells with γ-aminobutyric acid (GABA)-related compounds. When the cells were treated with GABA, taurine and β-alanine, the level of protoporphyrin was decreased, suggesting that plasma membrane transporters involved in the transport of neurotransmitters contribute to the uptake of ALA. By transfection with neurotransmitter transporters SLC6A6, SLC6A8 and SLC6A13 cDNA, the ALA- and ALA methylester-dependent accumulation of protoporphyrin markedly increased in HEK293T cells, dependent on an increase in the uptake of ALA. When ALA-treated cells were exposed to white light, the extent of photodamage increased in SLC6A6- and SLC6A13-expressing cells. Conversely, knockdown of SLC6A6 or SLC6A13 with siRNAs in DLD-1 and HeLa cells decreased the ALA-induced accumulation. The expression of SLC6A6 and SLC6A13 was found in some cancer cell lines. Immunohistochemical studies revealed that the presence of these transporters was elevated in colon cancerous cells. These results indicated that neurotransmitter transporters including SLC6A6 and SLC6A13 mediate the uptake of ALA and can play roles in the enhancement of ALA-induced accumulation of protoporphyrin in cancerous cells.

  6. Delta-aminolevulinic acid dehydratase enzyme activity in blood, brain, and liver of lead-dosed ducks

    USGS Publications Warehouse

    Dieter, M.P.; Finley, M.T.

    1979-01-01

    Mallard ducks were dosed with a single shotgun pellet (ca. 200 mg lead). After 1 month there was about 1 ppm lead in blood, 2.5 in liver, and 0.5 in brain. Lead-induced inhibition of delta-aminolevulinic acid dehydratase enzyme in blood and cerebellum was much greater than in cerebral hemisphere or liver and was strongly correlated with the lead concentration in these tissues. The cerebellar portion of the brain was more sensitive to delta-aminolevulinic acid dehydratase enzyme inhibition by lead than were the other tissues examined. There was also a greater increase in the glial cell marker enzyme, butyrylcholinesterase, in cerebellum than in cerebral hemisphere, suggesting that nonregenerating neuronal cells were destroyed by lead and replaced by glial cells in that portion of the brain. Even partial loss of cerebellar tissue is severely debilitating in waterfowl, because functions critical to survival such as visual, auditory, motor, and reflex responses are integrated at this brain center.

  7. Campylobacter jejuni fatty acid synthase II: Structural and functional analysis of [beta]-hydroxyacyl-ACP dehydratase (FabZ)

    SciTech Connect

    Kirkpatrick, Andrew S.; Yokoyama, Takeshi; Choi, Kyoung-Jae; Yeo, Hye-Jeong

    2009-08-14

    Fatty acid biosynthesis is crucial for all living cells. In contrast to higher organisms, bacteria use a type II fatty acid synthase (FAS II) composed of a series of individual proteins, making FAS II enzymes excellent targets for antibiotics discovery. The {beta}-hydroxyacyl-ACP dehydratase (FabZ) catalyzes an essential step in the FAS II pathway. Here, we report the structure of Campylobacter jejuni FabZ (CjFabZ), showing a hexamer both in crystals and solution, with each protomer adopting the characteristic hot dog fold. Together with biochemical analysis of CjFabZ, we define the first functional FAS II enzyme from this pathogen, and provide a framework for investigation on roles of FAS II in C. jejuni virulence

  8. Expression of a hemA gene from Agrobacterium radiobacter in a rare codon optimizing Escherichia coli for improving 5-aminolevulinate production.

    PubMed

    Fu, Weiqi; Lin, Jianping; Cen, Peilin

    2010-01-01

    The 5-aminolevulinate (ALA) synthase gene (hemA) from Agrobacterium radiobacter zju-0121, which was cloned previously in our laboratory, contains several rare codons. To enhance the expression of this gene, Escherichia coli Rosetta(DE3), which is a rare codon optimizer strain, was picked out as the host to construct an efficient recombinant strain. Cell extracts of the recombinant E. coli were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under the appropriate conditions. The results indicated that the activity of ALA synthase expressed in Rosetta(DE3)/pET-28a(+)-hemA was about 20% higher than that in E. coli BL21(DE3). Then the effects of precursors (glycine and succinate) and glucose, which is an inhibitor for ALA dehydratase as well as the carbon sources for cell growth, on the production of 5-aminolevulinate were investigated. Based on an optimal fed-batch culture system described in our previous work, up to 6.5 g/l (50 mM) ALA was produced in a 15-l fermenter.

  9. Delta-aminolevulinic acid dehydratase (ALAD) polymorphism in lead exposed Bangladeshi children and its effect on urinary aminolevulinic acid (ALA)

    SciTech Connect

    Tasmin, Saira; Furusawa, Hana; Ahmad, Sk. Akhtar; Watanabe, Chiho

    2015-01-15

    Background and objective: Lead has long been recognized as a harmful environmental pollutant. People in developing countries like Bangladesh still have a higher risk of lead exposure. Previous research has suggested that the delta-aminolevulinic acid dehydratase (ALAD) genotype can modify lead toxicity and individual susceptibility. As children are more susceptible to lead-induced toxicity, this study investigated whether the ALAD genotype influenced urinary excretion of delta-aminolevulinic acid (U-ALA) among children exposed to environmental lead in Bangladesh. Methods: Subjects were elementary schoolchildren from a semi-urban industrialized area in Bangladesh. A total of 222 children were studied. Blood and urine were collected to determine ALAD genotypes, blood lead levels and urinary aminolevulinic acid (U-ALA). Results: The mean BPb level was 9.7 µg/dl for the study children. BPb was significantly positively correlated with hemoglobin (p<0.01). In total, allele frequency for ALAD 1 and 2 was 0.83 and 0.17 respectively. The mean U-ALA concentration was lower in ALAD1-2/2-2 carriers than ALAD1-1 carriers for boys (p=0.001). But for girls, U-ALA did not differ significantly by genotype (p=0.26). When U-ALA was compared by genotype at the same exposure level in a multiple linear regression analysis, boys who were ALAD1-2/2-2 carriers still had a lower level of U-ALA compared to ALAD1-1carriers. Conclusion: This study provides information about the influence of ALAD polymorphism and its association with U-ALA in Bangladeshi children. Our results indicate that the ALAD1-2/2-2 genotype may have a protective effect in terms of U-ALA for environmentally lead exposed boys. - Highlights: • High blood lead level for the environmentally exposed schoolchildren. • BPb was significantly correlated with U-ALA and Hb. • Effect of ALAD genotype on U-ALA is differed by sex. • Lower U-ALA in ALAD2 than ALAD1 carriers only for boys at same exposure.

  10. Levels of delta-aminolevulinate dehydratase, uroporphyrinogen-I synthase, and protoporphyrin IX in erythrocytes from anemic mutant mice.

    PubMed Central

    Sassa, S; Bernstein, S E

    1977-01-01

    Levels of erythrocyte delta-aminolevulinate dehydratase [ALA-dehydratase; porphobilinogen synthase; 5-aminolevulinate hydro-lyase (adding 5-aminolevulinate and cyclizing), EC 4.2.1.24], UROPORPHYRINOGEN-I synthase [Uro-synthase; porphobilinogen ammonia-lyase (polymerizing), EC 4.3;1.8], AND PROTOPORPHYRIN IX (Proto) were measured by sensitive semimicroassays using 2-5 mul of whole blood obtained from normal and anemic mutant mice. The levels of erythrocyte ALA-dehydratase and Uro-synthase showed marked developmental changes and ALA-dehydratase was influenced by the Lv gene. Mice with overt hemolytic diseases (ja/ja, sph/sph, nb/nb, ha/ha) had 10- to 20-fold increases in ALA-dehydratase, Uro-synthase, and Proto compared with their normal controls. Mice with an iron deficiency (mk/mk) and mice with hypoplastic anemias (W/Wv, Sl/Sld, an/an) had mild to moderate increases in these parameters. Elevated enzyme activities and Proto correlated well with the number of reticulocytes. Because all mice with anemias possessed elevated levels of ALA-dehydratase, Uro-synthase, and Proto independent of differences in their genotypes, the increase in these parameters is not likely to be the result of a specific gene defect. The increased enzyme activities and Proto concentration probably reflect increased frequency of young red cells that are still active in heme biosynthesis. PMID:265562

  11. Membrane topology and essential amino acid residues of Phs1, a 3-hydroxyacyl-CoA dehydratase involved in very long-chain fatty acid elongation.

    PubMed

    Kihara, Akio; Sakuraba, Hiroko; Ikeda, Mika; Denpoh, Aki; Igarashi, Yasuyuki

    2008-04-25

    Yeast Phs1 is the 3-hydroxyacyl-CoA dehydratase that catalyzes the third reaction of the four-step cycle in the elongation of very long-chain fatty acids (VLCFAs). In yeast, the hydrophobic backbone of sphingolipids, ceramide, consists of a long-chain base and an amide-linked C26 VLCFA. Therefore, defects in VLCFA synthesis would be expected to greatly affect sphingolipid synthesis. In fact, in this study we found that reduced Phs1 levels result in significant impairment of the conversion of ceramide to inositol phosphorylceramide. Phs1 proteins are conserved among eukaryotes, constituting a novel protein family. Phs1 family members exhibit no sequence similarity to other dehydratase families, so their active site sequence and catalytic mechanism have been completely unknown. Here, by mutating 22 residues conserved among Phs1 family members, we identified six amino acid residues important in Phs1 function, two of which (Tyr-149 and Glu-156) are indispensable. We also examined the membrane topology of Phs1 using an N-glycosylation reporter assay. Our results suggest that Phs1 is a membrane-spanning protein that traverses the membrane six times and has an N terminus and C terminus facing the cytosol. The important amino acids are concentrated in or near two of the six proposed transmembrane regions. Thus, we also propose a catalytic mechanism for Phs1 that is not unlike mechanisms used by other hydratases active in lipid synthesis.

  12. Effects of lead on delta-aminolevulinic acid dehydratase activity, growth, hemoglobin content, and reproduction in Daphnia magna

    SciTech Connect

    Berglind, R.; Dave, G.; Sjoebeck, M.L.

    1985-04-01

    The effects of continuous exposure to lead for various periods and recovery in clean water on delta-aminolevulinic acid dehydratase (ALA-D) activity, hemoglobin content, growth, and reproduction were studied in Daphnia magna. Steady-state inhibition of ALA-D activity was reached within 2 days in 16, 64, and 256 micrograms Pb liter-1, but restoration in clean water was prolonged in relation to previous exposure. In spite of the inhibition of ALA-D activity hemoglobin content increased after 2 days in 16 and 24 micrograms Pb liter-1. Furthermore, hemoglobin content in previously exposed animals increased during recovery in clean water. Maximum hemoglobin content (2.9 times control value) was found after 2 days recovery of animals exposed to 64 micrograms Pb liter-1. These findings suggest that some enzyme(s) other than ALA-D in the biosynthetic pathway of hemoglobin formation is (are) more sensitive to lead. Growth, in contrast to reproduction, was stimulated by low concentrations of lead (less than 64 micrograms Pb liter-1), although in 256 micrograms Pb liter-1 growth was also significantly impaired. After 19 days the 16 and 50% reproductive impairment concentrations were less than or equal to 1 and 10 micrograms Pb liter-1, respectively.

  13. Relationships between blood lead concentration and aminolevulinic acid dehydratase in alcoholics and workers industrially exposed to lead

    SciTech Connect

    Bortoli, A.; Fazzin, G.; Marin, V.; Trabuio, G.; Zotti, S.

    1986-07-01

    Blood lead concentration (Pb-B), aminolevulinic acid dehydratase (ALAD), and gamma-GT were measured in 265 workers industrially exposed to lead and in 184 patients with liver disease resulting from alcohol consumption. The first group was divided according to alcohol use, i.e., nondrinkers, moderate drinkers, and heavy drinkers. The second group was divided according to the following criteria: hepatopatic without cirrhosis, hepatopatic with compensated cirrhosis, and hepatopatic with decompensated cirrhosis. Heavy drinkers who were industrially exposed had the highest Pb-B (40.4 +/- 14.6 micrograms/dl) and the lowest ALAD (22.2 +/- 9.1 U/L). The correlations between Pb-B and ALAD show no significant change with the increase of Pb-B. In the alcoholic group, 76 patients with alcoholic liver disease without cirrhosis had the highest Pb-B (40.3-9.1 micrograms/dl) and ALAD the lowest (18.6 +/- 7.7 U/L). The negative correlation between Pb-B and log ALAD disappeared completely in individuals with Pb-B that exceeded 50 micrograms/dl, independent from the seriousness of illness.

  14. Effects of Delta-Aminolevulinic Acid Dehydratase Polymorphisms on Susceptibility to Lead in Han Subjects from Southwestern China

    PubMed Central

    Yang, Yuelin; Wu, Jin; Sun, Pin

    2012-01-01

    This study is to determine the distribution of the delta-aminolevulinic acid dehydratase (ALAD) polymorphism among Han subjects of the Chinese population and to study whether the polymorphism in the ALAD gene modifies the toxicity of lead in lead-exposed workers. For this purpose we conducted a cross-sectional study on 156 Chinese workers who were exposed to lead in lead-acid battery and electric-flex manufacturing plants. The authors found that the allele frequencies of ALAD1 and ALAD2 were 0.9679 and 0.0321, respectively. Workers with the ALAD 1-1 genotype were associated with higher blood lead levels than those with the ALAD 1-2 genotype. Blood and urine lead levels were much higher in storage battery workers than in cable workers. The self-conscious symptom survey showed that the incidences of debilitation, amnesia and dreaminess were much higher in those had more than five years of tenure or contact with lead on the job within the ALAD 1-1 genotype subgroup. Laboratory examinations showed that serum iron and zinc levels in workers’ with the ALAD 1-2 genotype were higher than those with the ALAD 1-1 genotype, especially in storage-battery workers. Correlation analysis indicated that the blood lead level negatively correlated with serum calcium, iron and zinc level. The data of this study suggest that the ALAD gene polymorphism and serum ion levels may modify the kinetics of lead in blood. Therefore, the authors recommend that an adequate intake of dietary calcium, iron, and zinc or the calcium, iron, and zinc supplementation should be prescribed to Chinese lead exposed workers. PMID:22851944

  15. Effects of delta-aminolevulinic acid dehydratase polymorphisms on susceptibility to lead in Han subjects from southwestern China.

    PubMed

    Yang, Yuelin; Wu, Jin; Sun, Pin

    2012-07-01

    This study is to determine the distribution of the delta-aminolevulinic acid dehydratase (ALAD) polymorphism among Han subjects of the Chinese population and to study whether the polymorphism in the ALAD gene modifies the toxicity of lead in lead-exposed workers. For this purpose we conducted a cross-sectional study on 156 Chinese workers who were exposed to lead in lead-acid battery and electric-flex manufacturing plants. The authors found that the allele frequencies of ALAD1 and ALAD2 were 0.9679 and 0.0321, respectively. Workers with the ALAD 1-1 genotype were associated with higher blood lead levels than those with the ALAD 1-2 genotype. Blood and urine lead levels were much higher in storage battery workers than in cable workers. The self-conscious symptom survey showed that the incidences of debilitation, amnesia and dreaminess were much higher in those had more than five years of tenure or contact with lead on the job within the ALAD 1-1 genotype subgroup. Laboratory examinations showed that serum iron and zinc levels in workers' with the ALAD 1-2 genotype were higher than those with the ALAD 1-1 genotype, especially in storage-battery workers. Correlation analysis indicated that the blood lead level negatively correlated with serum calcium, iron and zinc level. The data of this study suggest that the ALAD gene polymorphism and serum ion levels may modify the kinetics of lead in blood. Therefore, the authors recommend that an adequate intake of dietary calcium, iron, and zinc or the calcium, iron, and zinc supplementation should be prescribed to Chinese lead exposed workers.

  16. A Formulation Study of 5-Aminolevulinic Encapsulated in DPPC Liposomes in Melanoma Treatment

    PubMed Central

    Lin, Ming-Wei; Huang, Yaw-Bin; Chen, Chun-Lin; Wu, Pao-Chu; Chou, Chien-Ying; Wu, Ping-Ching; Hung, Shih-Ya

    2016-01-01

    Photodynamic therapy (PDT) is a widely used technique for epithelial skin cancer treatment. 5-aminolevulinic acid (5-ALA) is a drug currently used for PDT and is a hydrophilic molecule at its physiological pH, and this limits its capacity to cross the stratum corneum of skin. Since skin penetration is a key factor in the efficacy of topical 5-ALA-mediated PDT, numerous strategies have been proposed to improve skin penetration. Yet this problem is still ongoing. The results of a previous study showed a low rate of 5-ALA encapsulated in liposomes (5.7%) that were 400 nm in size. In the present study, we used 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes as vehicles and tested their delivery efficacy of 5-ALA-medicated PDT both in vitro and in vivo. Our data shows that 5-ALA encapsulated in 0.1 or 0.5% DPPC liposomes (5-ALA/DPPC) had a better encapsulated rate (15~16%) and were smaller in size (84~89 nm). We found the 5-ALA/DPPC formulation reduced cell viability, mitochondria membrane potential, and enhanced intracellular ROS accumulation as compared to 5-ALA alone in melanoma cells. Furthermore, the 5-ALA/DPPC formulation also had better skin penetration ability as compared to the 5-ALA in our ex vivo data by assaying 5-ALA converted into protoporphyrin IX (PpIX) in the skin of the mice that were experimented on. In melanoma xenograft models, 5-ALA/DPPC enhanced PpIX accumulation only in tumor tissue but not normal skin. In conclusion, we found DPPC liposomes to be good carriers for 5-ALA delivery and believe that they may prove useful in 5-ALA-mediated PDT in the future. PMID:27429584

  17. Inhibition of erythrocytes δ-aminolevulinic acid dehydratase (ALAD) activity in fish from waters affected by lead smelters

    USGS Publications Warehouse

    Schmitt, Christopher J.; Caldwell, Colleen A.; Olsen, Bill; Serdar, Dave; Coffey, Mike

    2002-01-01

    We assessed the effects on fish of lead (Pb) released to streamsby smelters located in Trail, BC (Canada), E. Helena, MT, Herculaneum, MO, and Glover, MO. Fish were collected by electrofishing from sites located downstream of smelters and from reference sites. Blood from each fish was analyzed for δ-aminolevulinic acid dehydratase (ALAD) activity and hemoglobin (Hb), and samples of blood, liver, or carcass were analyzed for Pb, zinc (Zn), or both. Fish collected downstreamof all four smelters sites had elevated Pb concentrations, decreased ALAD activity, or both relative to their respectivereference sites. At E. Helena, fish from the downstream site also had lower Hb concentrations than fish from upstream. Differences among taxa were also apparent. Consistent with previous studies, ALAD activity in catostomids (Pisces: Catostomidae-northern hog sucker,Hypentelium nigricans;river carpsucker, Carpiodes carpio; largescale sucker, Catostomus macrocheilus; and mountain sucker, C. platyrhynchus) seemed more sensitive to Pb-induced ALADinhibition than the salmonids (Pisces: Salmonidae-rainbow trout,Oncorhynchus mykiss; brook trout,Salvelinus fontinalis) or common carp (Cyprinus carpio). Some of these differences may have resulted from differential accumulation of Zn, which was not measured at all sites. We detected noALAD activity in channel catfish (Ictaluruspunctatus) from either site on the Mississippi River at Herculaneum, MO. Our findings confirmed that Pb is releasedto aquatic ecosystems by smelters and accumulated by fish, andwe documented potentially adverse effects of Pb in fish. We recommend that Zn be measured along with Pb when ALAD activityis used as a biomarker and the collection of at least 10 fish ofa species at each site to facilitate statistical analysis.

  18. In Silico Structure Prediction of Human Fatty Acid Synthase–Dehydratase: A Plausible Model for Understanding Active Site Interactions

    PubMed Central

    John, Arun; Umashankar, Vetrivel; Samdani, A.; Sangeetha, Manoharan; Krishnakumar, Subramanian; Deepa, Perinkulam Ravi

    2016-01-01

    Fatty acid synthase (FASN, UniProt ID: P49327) is a multienzyme dimer complex that plays a critical role in lipogenesis. Consequently, this lipogenic enzyme has gained tremendous biomedical importance. The role of FASN and its inhibition is being extensively researched in several clinical conditions, such as cancers, obesity, and diabetes. X-ray crystallographic structures of some of its domains, such as β-ketoacyl synthase, acetyl transacylase, malonyl transacylase, enoyl reductase, β-ketoacyl reductase, and thioesterase, (TE) are already reported. Here, we have attempted an in silico elucidation of the uncrystallized dehydratase (DH) catalytic domain of human FASN. This theoretical model for DH domain was predicted using comparative modeling methods. Different stand-alone tools and servers were used to validate and check the reliability of the predicted models, which suggested it to be a highly plausible model. The stereochemical analysis showed 92.0% residues in favorable region of Ramachandran plot. The initial physiological substrate β-hydroxybutyryl group was docked into active site of DH domain using Glide. The molecular dynamics simulations carried out for 20 ns in apo and holo states indicated the stability and accuracy of the predicted structure in solvated condition. The predicted model provided useful biochemical insights into the substrate–active site binding mechanisms. This model was then used for identifying potential FASN inhibitors using high-throughput virtual screening of the National Cancer Institute database of chemical ligands. The inhibitory efficacy of the top hit ligands was validated by performing molecular dynamics simulation for 20 ns, where in the ligand NSC71039 exhibited good enzyme inhibition characteristics and exhibited dose-dependent anticancer cytotoxicity in retinoblastoma cancer cells in vitro. PMID:27559295

  19. In Silico Structure Prediction of Human Fatty Acid Synthase-Dehydratase: A Plausible Model for Understanding Active Site Interactions.

    PubMed

    John, Arun; Umashankar, Vetrivel; Samdani, A; Sangeetha, Manoharan; Krishnakumar, Subramanian; Deepa, Perinkulam Ravi

    2016-01-01

    Fatty acid synthase (FASN, UniProt ID: P49327) is a multienzyme dimer complex that plays a critical role in lipogenesis. Consequently, this lipogenic enzyme has gained tremendous biomedical importance. The role of FASN and its inhibition is being extensively researched in several clinical conditions, such as cancers, obesity, and diabetes. X-ray crystallographic structures of some of its domains, such as β-ketoacyl synthase, acetyl transacylase, malonyl transacylase, enoyl reductase, β-ketoacyl reductase, and thioesterase, (TE) are already reported. Here, we have attempted an in silico elucidation of the uncrystallized dehydratase (DH) catalytic domain of human FASN. This theoretical model for DH domain was predicted using comparative modeling methods. Different stand-alone tools and servers were used to validate and check the reliability of the predicted models, which suggested it to be a highly plausible model. The stereochemical analysis showed 92.0% residues in favorable region of Ramachandran plot. The initial physiological substrate β-hydroxybutyryl group was docked into active site of DH domain using Glide. The molecular dynamics simulations carried out for 20 ns in apo and holo states indicated the stability and accuracy of the predicted structure in solvated condition. The predicted model provided useful biochemical insights into the substrate-active site binding mechanisms. This model was then used for identifying potential FASN inhibitors using high-throughput virtual screening of the National Cancer Institute database of chemical ligands. The inhibitory efficacy of the top hit ligands was validated by performing molecular dynamics simulation for 20 ns, where in the ligand NSC71039 exhibited good enzyme inhibition characteristics and exhibited dose-dependent anticancer cytotoxicity in retinoblastoma cancer cells in vitro. PMID:27559295

  20. Associations of Uric Acid with Polymorphisms in the δ-Aminolevulinic Acid Dehydratase, Vitamin D Receptor, and Nitric Oxide Synthase Genes in Korean Lead Workers

    PubMed Central

    Weaver, Virginia M.; Schwartz, Brian S.; Jaar, Bernard G.; Ahn, Kyu-Dong; Todd, Andrew C.; Lee, Sung-Soo; Kelsey, Karl T.; Silbergeld, Ellen K.; Lustberg, Mark E.; Parsons, Patrick J.; Wen, Jiayu; Lee, Byung-Kook

    2005-01-01

    Recent research suggests that uric acid may be nephrotoxic at lower levels than previously recognized and that it may be one mechanism for lead-related nephrotoxicity. Therefore, in understanding mechanisms for lead-related nephrotoxicity, it would be of value to determine whether genetic polymorphisms that are associated with renal outcomes in lead workers and/or modify associations between lead dose and renal function are also associated with uric acid and/or modify associations between lead dose and uric acid. We analyzed data on three such genetic polymorphisms: δ-aminolevulinic acid dehydratase (ALAD), endothelial nitric oxide synthase (eNOS), and the vitamin D receptor (VDR). Mean (± SD) tibia, blood, and dimercaptosuccinic acid–chelatable lead levels were 37.2 ± 40.4 μg/g bone mineral, 32.0± 15.0 g/dL, and 0.77± 0.86 μg/mg creatinine, respectively, in 798 current and former lead workers. Participants with the eNOS Asp allele had lower mean serum uric acid compared with those with the Glu/Glu genotype. Among older workers (age ≥ median of 40.6 years), ALAD genotype modified associations between lead dose and uric acid levels. Higher lead dose was significantly associated with higher uric acid in workers with the ALAD1-1 genotype; associations were in the opposite direction in participants with the variant ALAD1-2 genotype. In contrast, higher tibia lead was associated with higher uric acid in those with the variant VDR B allele; however, modification was dependent on participants with the bb genotype and high tibia lead levels. We conclude that genetic polymorphisms may modify uric acid mediation of lead-related adverse renal effects. PMID:16263504

  1. Comparison of the beluga whale (Delphinapterus leucas) expressed genes for 5-aminolevulinate synthase with those in other vertebrates.

    PubMed

    Kreiling, J A; Duncan, R; Faggart, M A; Cornell, N W

    1999-06-01

    The cDNA and inferred amino acid sequences were determined for beluga whale (Delphinapterus leucas) erythroid (E) and housekeeping (H) forms of 5-aminolevulinate synthase (ALS), and they were compared with known sequences for five other vertebrates with particular attention to regulatory features. The cDNAs for whale ALS-E and -H encode, respectively, proteins of 582 and 640 amino acids. Sequence alignments suggest that the whale ALS-H, like those for rat and chicken, has an N-terminal mitochondrial targeting sequence of 56 amino acids. There is a high degree of amino acid conservation between the beluga whale proteins and those of other vertebrates, including regulatory elements and functional residues that have been defined in other ALSs. Both whale proteins contain three heme regulatory motifs suggesting that mitochondrial uptake may be regulated by heme. The ALS-E mRNA contains an iron responsive element in its 5'-untranslated region indicating that its expression may be post-transcriptionally regulated by cellular iron. This extensive structural similarity and the presence of the same regulatory elements found in other ALSs indicate that regulation of ALS in beluga whale is similar to that in other vertebrates.

  2. The Burkholderia cenocepacia BDSF Quorum Sensing Fatty Acid is Synthesized by a Bifunctional Crotonase Homologue Having Both Dehydratase and Thioesterase Activities

    PubMed Central

    Bi, Hongkai; Christensen, Quin H.; Feng, Youjun; Wang, Haihong; Cronan, John E.

    2012-01-01

    Summary Signal molecules of the Diffusible Signal Factor (DSF) family have been shown recently to be involved in regulation of pathogenesis and biofilm formation in diverse Gram-negative bacteria. DSF signals are reported to be active not only on their cognate bacteria, but also on unrelated bacteria and the pathogenic yeast, Candida albicans. DSFs are monounsaturated fatty acids of medium chain length containing an unusual cis-2 double bond. Although genetic analyses had identified genes involved in DSF synthesis, the pathway of DSF synthesis was unknown. The DSF of the important human pathogen Burkholderia cenocepacia (called BDSF) is cis-2-dodecenoic acid. We report that BDSF is synthesized from a fatty acid synthetic intermediate, the acyl carrier protein (ACP) thioester of 3-hydroxydodecanoic acid. This intermediate is intercepted by protein Bcam0581 and converted to cis-2-dodecenoyl-ACP. Bcam0581 is annotated as a homologue of crotonase, the first enzyme of the fatty acid degradation pathway. We demonstrated Bcam0581to be a bifunctional protein that not only catalyzed dehydration of 3-hydroxydodecanoyl-ACP to cis-2-dodecenoyl-ACP, but also cleaved the thioester bond to give the free acid. Both activities required the same set of active site residues. Although dehydratase and thioesterase activities are known activities of the crotonase superfamily, Bcam0581 is the first protein shown to have both activities. PMID:22221091

  3. Responses to hexyl 5-aminolevulinate-induced photodynamic treatment in rat bladder cancer model

    NASA Astrophysics Data System (ADS)

    Arum, Carl-Jørgen; Gederas, Odrun; Larsen, Eivind; Randeberg, Lise; Zhao, Chun-Mei

    2010-02-01

    OBJECTIVES: In this study, we evaluated histologically the effects of hexyl 5-aminolevulinateinduced photodynamic treatment in the AY-27 tumor cell induced rat bladder cancer model. MATERIAL & METHODS: The animals (fischer-344 female rats) were divided into 2 groups, half of which were orthotopically implanted with 400,000 syngeniec AY-27 urothelia1 rat bladder cancer cells and half sham implanted. 14 days post implantation 6 rats from each group were treated with hexyl 5-aminolevulinate-induced photodynamic treatment (8mM HAL and light fluence of 20 J/cm2). Additional groups of animals were only given HAL instillation, only light treatment, or no treatment. All animals were sacrificed 7 days after the PDT/only HAL/only light or no treatment. Each bladder was removed, embedded in paraffin and stained with hematoxylin, eosin, and saferin for histological evaluation at high magnification for features of tissue damage by a pathologist blinded to the sample source. RESULTS: In all animals that were AY-27 implanted and not given complete PDT treatment, viable tumors were found in the bladder mucosa and wall. In the animals treated with complete HAL-PDT only 3 of 6 animals had viable tumor. In the 3 animals with viable tumor it was significantly reduced in volume compared to the untreated animals. It was also noted that in the PDT treated animals there was a significantly increased inflammatory response (lymphocytic and mononuclear cell infiltration) in the peri-tumor area compared to implanted animals without complete HAL-PDT. CONCLUSION: Our results suggest that hexyl 5-aminolevulinate-induced photodynamic treatment in a rat bladder cancer model involves both direct effects on cell death (necrosis and apoptosis) and indirect effects to evoke the host immune-response, together contributing to tumor eradication.

  4. Catalytically active alkaline molten globular enzyme: Effect of pH and temperature on the structural integrity of 5-aminolevulinate synthase.

    PubMed

    Stojanovski, Bosko M; Breydo, Leonid; Hunter, Gregory A; Uversky, Vladimir N; Ferreira, Gloria C

    2014-12-01

    5-Aminolevulinate synthase (ALAS), a pyridoxal-5'phosphate (PLP)-dependent enzyme, catalyzes the first step of heme biosynthesis in mammals. Circular dichroism (CD) and fluorescence spectroscopies were used to examine the effects of pH (1.0-3.0 and 7.5-10.5) and temperature (20 and 37°C) on the structural integrity of ALAS. The secondary structure, as deduced from far-UV CD, is mostly resilient to pH and temperature changes. Partial unfolding was observed at pH2.0, but further decreasing pH resulted in acid-induced refolding of the secondary structure to nearly native levels. The tertiary structure rigidity, monitored by near-UV CD, is lost under acidic and specific alkaline conditions (pH10.5 and pH9.5/37°C), where ALAS populates a molten globule state. As the enzyme becomes less structured with increased alkalinity, the chiral environment of the internal aldimine is also modified, with a shift from a 420nm to 330nm dichroic band. Under acidic conditions, the PLP cofactor dissociates from ALAS. Reaction with 8-anilino-1-naphthalenesulfonic acid corroborates increased exposure of hydrophobic clusters in the alkaline and acidic molten globules, although the reaction is more pronounced with the latter. Furthermore, quenching the intrinsic fluorescence of ALAS with acrylamide at pH1.0 and 9.5 yielded subtly different dynamic quenching constants. The alkaline molten globule state of ALAS is catalytically active (pH9.5/37°C), although the kcat value is significantly decreased. Finally, the binding of 5-aminolevulinate restricts conformational fluctuations in the alkaline molten globule. Overall, our findings prove how the structural plasticity of ALAS contributes to reaching a functional enzyme.

  5. Catalytically active alkaline molten globular enzyme: Effect of pH and temperature on the structural integrity of 5-aminolevulinate synthase.

    PubMed

    Stojanovski, Bosko M; Breydo, Leonid; Hunter, Gregory A; Uversky, Vladimir N; Ferreira, Gloria C

    2014-12-01

    5-Aminolevulinate synthase (ALAS), a pyridoxal-5'phosphate (PLP)-dependent enzyme, catalyzes the first step of heme biosynthesis in mammals. Circular dichroism (CD) and fluorescence spectroscopies were used to examine the effects of pH (1.0-3.0 and 7.5-10.5) and temperature (20 and 37°C) on the structural integrity of ALAS. The secondary structure, as deduced from far-UV CD, is mostly resilient to pH and temperature changes. Partial unfolding was observed at pH2.0, but further decreasing pH resulted in acid-induced refolding of the secondary structure to nearly native levels. The tertiary structure rigidity, monitored by near-UV CD, is lost under acidic and specific alkaline conditions (pH10.5 and pH9.5/37°C), where ALAS populates a molten globule state. As the enzyme becomes less structured with increased alkalinity, the chiral environment of the internal aldimine is also modified, with a shift from a 420nm to 330nm dichroic band. Under acidic conditions, the PLP cofactor dissociates from ALAS. Reaction with 8-anilino-1-naphthalenesulfonic acid corroborates increased exposure of hydrophobic clusters in the alkaline and acidic molten globules, although the reaction is more pronounced with the latter. Furthermore, quenching the intrinsic fluorescence of ALAS with acrylamide at pH1.0 and 9.5 yielded subtly different dynamic quenching constants. The alkaline molten globule state of ALAS is catalytically active (pH9.5/37°C), although the kcat value is significantly decreased. Finally, the binding of 5-aminolevulinate restricts conformational fluctuations in the alkaline molten globule. Overall, our findings prove how the structural plasticity of ALAS contributes to reaching a functional enzyme. PMID:25240868

  6. Associations of blood lead, dimercaptosuccinic acid-chelatable lead, and tibia lead with polymorphisms in the vitamin D receptor and [delta]-aminolevulinic acid dehydratase genes.

    PubMed Central

    Schwartz, B S; Lee, B K; Lee, G S; Stewart, W F; Simon, D; Kelsey, K; Todd, A C

    2000-01-01

    A cross-sectional study was performed to evaluate the influence of polymorphisms in the [delta]-aminolevulinic acid dehydratase (ALAD) and vitamin D receptor (VDR) genes on blood lead, tibia lead, and dimercaptosuccinic acid (DMSA)-chelatable lead levels in 798 lead workers and 135 controls without occupational lead exposure in the Republic of Korea. Tibia lead was assessed with a 30-min measurement by (109)Cd-induced K-shell X-ray fluorescence, and DMSA-chelatable lead was estimated as 4-hr urinary lead excretion after oral administration of 10 mg/kg DMSA. The primary goals of the analysis were to examine blood lead, tibia lead, and DMSA-chelatable lead levels by ALAD and VDR genotypes, controlling for covariates; and to evaluate whether ALAD and VDR genotype modified relations among the different lead biomarkers. There was a wide range of blood lead (4-86 microg/dL), tibia lead (-7-338 microg Pb/g bone mineral), and DMSA-chelatable lead (4.8-2,103 microg) levels among lead workers. Among lead workers, 9.9% (n = 79) were heterozygous for the ALAD(2) allele and there were no homozygotes. For VDR, 10.7% (n = 85) had the Bb genotype, and 0.5% (n = 4) had the BB genotype. Although the ALAD and VDR genes are located on different chromosomes, lead workers homozygous for the ALAD(1) allele were much less likely to have the VDR bb genotype (crude odds ratio = 0.29, 95% exact confidence interval = 0.06-0.91). In adjusted analyses, subjects with the ALAD(2) allele had higher blood lead levels (on average, 2.9 microg/dL, p = 0.07) but no difference in tibia lead levels compared with subjects without the allele. In adjusted analyses, lead workers with the VDR B allele had significantly (p < 0.05) higher blood lead levels (on average, 4.2 microg/dL), chelatable lead levels (on average, 37.3 microg), and tibia lead levels (on average, 6.4 microg/g) than did workers with the VDR bb genotype. The current data confirm past observations that the ALAD gene modifies the

  7. [delta-Aminolevulinate dehydratase deficiency].

    PubMed

    Fujita, H; Ishida, N; Akagi, R

    1995-06-01

    delta-Aminolevulinate dehydratase (ALAD: E. C. 4.2.1.24), the second enzyme in the heme biosynthetic pathway, condenses two moles of delta-aminolevulinic acid to form porphobilinogen. ALAD deficiency is well known to develop signs and symptoms of typical hepatic porphyria, and classified into three categories as follows: (i) ALAD porphyria, a genetic defect of the enzyme, (ii) tyrosinemia type I, a genetic defect of fumarylacetoacetase in the tyrosine catabolic pathway, producing succinylacetone (a potent inhibitor of ALAD), and (iii) ALAD inhibition by environmental hazards, such as lead, trichloroethylene, and styrene. In the present article, we will describe molecular and biochemical mechanisms to cause the enzyme defect to discuss the significance of ALAD defect on human health.

  8. Biomonitoring of lead-contaminated Missouri streams with an assay for erythrocyte δ-aminolevulinic acid dehydratase activity in fish blood

    USGS Publications Warehouse

    Schmitt, C. J.; Wildhaber, M.L.; Hunn, J.B.; Nash, T.; Tieger, M. N.; Steadman, B. L.

    1993-01-01

    The activity of the enzyme δ-aminolevulinic acid dehydratase (ALA-D) in erythrocytes has long been used as a biomarker of lead exposure in humans and waterfowl and, more recently, in fishes. The assay was tested for ALA-D activity in fishes from streams affected by lead in combination with other metals from lead-zinc mining and related activities. Fishes (mostly catostomids) were collected from sites affected by historic and current mining activities, and from sites considered to be unaffected by mining (reference sites). A group of potentially toxic elements was measured in blood and carcass samples of individual fish, as were ALA-D activity, total protein (TP), and hemoglobin (Hb) in blood. Concentrations of mining-related metals (lead, zinc, and cadmium) were significantly greater (P<0.05) in fish blood and carcass at sites affected by historic mining activities than at reference and active mining sites. When analyzed by multiple regression, ALA-D activity, Hb, and TP accounted for 66% of blood-lead and 69% of carcass-lead variability. Differences among species were small. ALA-D activity as a biomarker adequately distinguished sites affected by bioavailable environmental lead. Zinc was the only other metal that affected ALA-D activity; it appeared to ameliorate the inactivation of ALA-D by lead.

  9. Delta-aminolevulinic acid dehydratase activity (ALA-D) in red mullet (Mullus barbatus) from Mediterranean waters as biomarker of lead exposure.

    PubMed

    Fernández, B; Martínez-Gómez, C; Benedicto, J

    2015-05-01

    The enzyme delta-aminolevulinic acid dehydratase (ALA-D) has been investigated as biomarker of lead (Pb) exposure in red mullet (Mullus barbatus) from the Spanish continental shelf. Concentrations of Pb and Zn in muscle and organosomatic indices were also measured to explore causality. Blood ALA-D assay conditions were optimized; the optimum pH for this species has been set to 6.5. Results showed that ALA-D activity ranged from 3.2 to 16.9 nmol PBGmin(-1)mg(-1). No significant differences on ALA-D levels between genders have been detected. ALA-D Baseline level and Background Assessment Criteria (BAC) for this species have been set to 9.1 and 6.6 nmol PBGmin(-1)mg(-1), respectively. There have been detected significant differences on ALA-D activity levels among areas, though the markedly low levels of Pb measured in fish muscle seemed not to be able to produce a relevant suppression on ALA-D. In spite of this, a weak inverse relationship detected between ALA-D and Pb concentrations pointed out the potential of this biomarker in red mullet to reflect Pb bioavailability in marine environment. Nevertheless, subsequent research on ALA-D in marine fish species is recommended to be limited to areas where environmental Pb is effectively accumulated by fish.

  10. Purification and characterization of 5-aminolaevulinic acid dehydratase from Escherichia coli and a study of the reactive thiols at the metal-binding domain.

    PubMed Central

    Spencer, P; Jordan, P M

    1993-01-01

    5-Aminolaevulinic acid dehydratase (ALAD) from a recombinant strain of Escherichia coli was purified to homogeneity. The enzyme is a homo-octamer of subunit M(r) 36554 +/- 17. Enzyme activity was dependent on the presence of Zn2+ ions and an exogenous thiol. Two molar equivalents of Zn2+ are bound/mol of subunit under reducing conditions. On exposure to the metal chelator EDTA, the two Zn2+ ions are removed, giving an inactive metal-depleted apo-ALAD. On oxidation of holo-ALAD, two disulphide bonds are formed with the loss of 1 mol of Zn2+/mol of subunit. The formation of the first disulphide led to the loss of catalytic activity. Replacement of the two bound Zn2+ ions with Co2+ resulted in the formation of a green protein with a spectrum indicative of the presence of charge-transfer bands from one or more cysteine-Co2+ ligands. While Mg2+ could not activate apo-ALAD alone, it was able to substitute for the second molar equivalent of bound Zn2+, leading to a further 4-fold stimulation in activity. The four cysteine residues involved in the formation of the two disulphide bonds were identified by protein-chemistry studies and were all located in a region of the protein extending from amino acid residues 120-134. Protein sequence data obtained in the present study has permitted the resolution of several differences between the published gene-derived protein sequences for ALAD from E. coli. PMID:8439296

  11. 5-Aminolevulinate production by Escherichia coli containing the Rhodobacter sphaeroides hemA gene

    SciTech Connect

    Van Der Werf, M.J.; Zeikus, J.G. |

    1996-10-01

    The Rhodobacter sphaeroides hemA gene codes for 5-aminolevulinate (ALA) synthase. This enzyme catalyzes the pyridoxal phosphate-dependent condensation of succinyl coenzyme A and glycine-forming ALA. The R. sphaeroides hemA gene in the pUC18/19 vector system was transformed into Escherichia coli. The effects of both genetic and physiological factors on the expression of ALA synthase and the production of ALA were studied. ALA synthase activity levels were maximal when hemA had the same transcription direction as the lac promoter. The distance between the lac promoter and hemA affected the expression of ALA synthase on different growth substrates. The E. coli host strain used had an enormous effect on the ALA synthase activity level and on the production of ALA, with E. coli DH1 being best suited. The ALA synthase activity level was also dependent on the carbon source. Succinate, L-malate, fumarate, and L-aspartate gave the highest levels of ALA synthase activity, while the use of lactose as a carbon source resulted in a repression of ALA synthase. After growth on succinate, ALA synthase represented {approx}5% of total cellular protein. The ALA synthase activity level was also dependent on the pH of the medium, with maximal activity occurring at pH 6.5. ALA production by whole cells was limited by the availability of glycine, and the addition of 2 g of glycine per liter to the growth medium increased the production of ALA fivefold, to 2.25 mM. In recombinant E. coli extracts, up to 22 mM ALA was produced from succinate, glycine, and ATP. 58 refs., 4 figs., 7 tabs.

  12. The protective effect of delta-aminolevulinic acid dehydratase 1-2 and 2-2 isozymes against blood lead with higher hematologic parameters.

    PubMed Central

    Kim, Hee-Seon; Lee, Sung-Soo; Lee, Gap-Soo; Hwangbo, Young; Ahn, Kyu-Dong; Lee, Byung-Kook

    2004-01-01

    Previous studies have suggested that delta-aminolevulinic acid dehydratase (ALAD) types 1-2 or 2-2 are protective against the toxicity of blood lead (PbB) when zinc protoporphyrin (ZPP) levels are low because of differential binding of lead in erythrocytes. The hypothesis is that subjects with the ALAD 1-1 genotype are more susceptible to lead exposure with impaired hematologic synthesis and therefore that iron nutrition is more important in those with the ALAD 1-1 genotype. The purpose of this study was to prove the protective effect of ALAD 1-2/2-2 against PbB with higher hematologic parameters. Data on 1,219 male workers from eight lead-using factories in the Republic of Korea were examined in this cross-sectional study. Blood samples were evaluated for PbB, ZPP, hemoglobin (Hb), and serum iron (SFe) concentrations and ALAD genotypes. The overall prevalence of the ALAD 1-2/2-2 genotype was 9.3%, which was associated with lower log ZPP (p < 0.001) and higher Hb (p = 0.014) levels. For the subjects with normal iron status (SFe levels > 60 micro g/dL), those with the ALAD 1-1 genotype were more likely to be anemic (adjusted odds ratio of 5.2; 95% confidence interval, 1.2-22.6) than those with ALAD 1-2/2-2. The study confirms the protective effects of ALAD 1-2/2-2 polymorphisms against PbB on hematologic pathways. In order to promote health and to minimize the toxicity of lead exposure more effectively, the nutritional management of iron in Korean workers should take both their ALAD genotypes and occupational lead exposures into account. PMID:15064157

  13. Effect of the delta-aminolevulinic acid dehydratase gene polymorphism on renal and neurobehavioral function in workers exposed to lead in China.

    PubMed

    Gao, Ai; Lu, Xiao-Ting; Li, Qiu-Ying; Tian, Lin

    2010-09-01

    Effects of delta-aminolevulinic acid dehydratase (ALAD) polymorphisms on the renal and neurobehavioral functions were investigated in Chinese workers from a storage battery plant exposed to inorganic lead. Blood and urine were collected from each worker to determine the ALAD genotypes, blood lead levels (PbB), urinary beta2-MG and urinary NAG activity. The World Health Organization Neurobehavioral Core Test Battery (WHO-NCTB) was used. Of the 135 lead workers tested for ALAD genotype, 126 were ALAD1-1, 9 were ALAD1-2 but none were ALAD2-2. The gene frequencies of ALADl-1 and ALADl-2 were 93.33% and 6.67%, respectively. The workers with ALAD1-2 genotype had significantly higher concentrations of PbB (62.52microg/dl vs. 41.02microg/dl), urinary NAG (22.01U/gCr vs. 13.49U/gCr), urinary beta2-MG (194.98microg/gCr vs. 112.88microg/gCr), and digit span backward (DSB) score (6.67 vs. 5.33) than those of ALAD1-1 genotype. Urinary NAG of ALAD1-2 genotype carriers was significantly higher than that of ALAD1-1 genotype under the same blood lead level (b(i) 0.75 vs. b(i) 0.29). Interaction between PbB and ALAD genotypes has a significant influence on NAG (P=0.02) and beta(2)-MG (P=0.01). It is postulated that the workers with the ALAD2 allele appear to be more susceptible to the effects of lead on renal injury, whereas neurobehavioral functions in ALAD1 homozygote tend to be more vulnerable.

  14. [Inhibition rate of gamma-aminolevulinic acid dehydratase activity in erythrocytes as a reliable index for individual workers of low lead exposure].

    PubMed

    Hirano, H; Omichi, M; Ohishi, H; Ishikawa, K; Hirashima, N

    1983-09-01

    As the delta-aminolevulinic acid dehydratase (ALAD) activity in erythrocytes is decreased by lead exposure, we considered that a net reduction of ALAD activity by lead in blood should be the difference between the activity fully activated with zinc (Zn2+) and dithiothreitol (DTT) and that without activation. The optimal condition of activation of ALAD was found by addition of 0.25 mM of Zn2+ and 10 mM of DTT in the reaction mixture. Judging from our previous results that the amount of inhibition of ALAD activity can be represented as the rate of inhibition and is closely correlated with the dose of lead administered to rabbits, the inhibition rate of ALAD activity and lead content in blood (Pb-B) of lead workers were measured. The scatter diagram obtained from the inhibition rate and lead content in blood has two groups being divided at 50 micrograms/ml of Pb-B. In one group less than 50 micrograms/100 ml of Pb-B, the inhibition rate has been closely related to Pb-B., the regression equation being Y = 1.82 X + 11.7, and the correlation coefficient + 0.926. In another group more than 50 micrograms/100 ml of Pb-B the inhibition rate remained constant at the 90% level. Measurement of the inhibition rate suggests to have practical validity for monitoring lead exposure in workers, and by means of a nomograph lead content in blood can be estimated from the inhibition rate.

  15. Effects of cyanobacterial extracellular products and gibberellic acid on salinity tolerance in Oryza sativa L

    PubMed Central

    Rodríguez, AA; Stella, AM; Storni, MM; Zulpa, G; Zaccaro, MC

    2006-01-01

    Salt stress is one of the most serious factors limiting the productivity of rice, the staple diet in many countries. Gibberellic acid has been reported to reduce NaCl-induced growth inhibition in some plants including rice. Most paddy soils have a natural population of Cyanobacteria, prokaryotic photosynthethic microorganisms, which synthesize and liberate plant growth regulators such as gibberellins that could exert a natural beneficial effect on salt stressed rice plants. The aim of this work was to evaluate the effect of the cyanobacterium Scytonema hofmanni extracellular products on the growth of rice seedlings inhibited by NaCl and to compare it with the effect of the gibberellic acid in the same stress condition. Growth (length and weight of the seedlings) and biochemical parameters (5-aminolevulinate dehydratase activity, total free porphyrin and pigments content) were evaluated. Salt exposure negatively affected all parameters measured, with the exception of chlorophyll. Chlrorophyll concentrations nearly doubled upon exposure to high salt. Gibberellic acid counteracted the effect of salt on the length and dry weight of the shoot, and on carotenoid and chlorophyll b contents. Extracellular products nullified the salt effect on shoot dry weight and carotenoid content; partially counteracted the effect on shoot length (from 54% to 38% decrease), root dry weight (from 59% to 41% decrease) and total free porphyrin (from 31 to 13% decrease); reduced by 35% the salt increase of chlorophyll a; had no effect on root length and chlorophyll b. Gibberellic acid and extracellular products increased 5-aminolevulinate dehydratase activity over the control without salt. When coincident with high salinity, exposure to either EP or GA3, resulted in a reversal of shoot-related responses to salt stress. We propose that Scytonema hofmanni extracellular products may counteract altered hormone homeostasis of rice seedlings under salt stress by producing gibberellin-like plant

  16. Fractionated PDT with 5-aminolevulinic acid: effective, cost effective, and patient friendly

    NASA Astrophysics Data System (ADS)

    de Vijlder, Hannah C.; Middelburg, Tom A.; de Bruijn, Henriette S.; Robinson, Dominic J.; Neumann, H. A. Martino; de Haas, Ellen R. M.

    2009-06-01

    PDT with ALA and MAL is established as a relatively effective treatment for non-melanoma skin cancer and premalignancies. PDT is often repeated, because a single treatment gives poor long term results. Preclinical studies showed that ALA-PDT applying a fractionated illumination scheme with a small first light fraction and a second larger light fraction separated by a dark interval of two hours resulted in a significant increase in efficacy. Whereas the efficacy was not enhanced by fractionating MAL-PDT, indicating that ALA-PDT mechanism is not the same as MAL-PDT mechanism. The increase in efficacy using fractionated PDT was confirmed clinically. A randomized comparative clinical study comparing fractionated ALA-PDT versus non-fractionated ALA-PDT in the treatment of superficial basal cell carcinoma showed a significant higher response rate in the lesions treated with fractionated ALA-PDT after a follow-up of one year ( p<0.002, log-rank test). The five year follow-up is studied at moment. So far the complete response in the group treated with fractionated ALA-PDT seems to be only a few percentages lower compared to the one year follow-up. Besides the gain in response rate, fractionated ALA PDT is cost effective. ALA gel is less expensive than the commercially available MAL (Metvix) and moreover fractionated ALA-PDT takes one treatment day, instead of two treatment days using the Metvix treatment protocol (two MAL-PDT treatments separated by one week), both reducing direct and indirect costs and the burden to the patient.

  17. Associations of blood pressure and hypertension with lead dose measures and polymorphisms in the vitamin D receptor and delta-aminolevulinic acid dehydratase genes.

    PubMed Central

    Lee, B K; Lee, G S; Stewart, W F; Ahn, K D; Simon, D; Kelsey, K T; Todd, A C; Schwartz, B S

    2001-01-01

    Evidence suggests that lead and selected genes known to modify the toxicokinetics of lead--namely, those for the vitamin D receptor (VDR) and delta-aminolevulinic acid dehydratase (ALAD)--may independently influence blood pressure and hypertension risk. We report the relations among ALAD and VDR genotypes, three lead dose measures, and blood pressure and hypertension status in 798 Korean lead workers and 135 controls without occupational exposure to lead. Lead dose was assessed by blood lead, tibia lead measured by X-ray fluorescence, and dimercaptosuccinic acid (DMSA)-chelatable lead. Among lead workers, 9.9% (n = 79) were heterozygous for the ALAD(2) allele, and there were no ALAD(2) homozygotes; 11.2% (n = 89) had at least one copy of the VDR B allele, and 0.5% (n = 4) had the BB genotype. In linear regression models to control for covariates, VDR genotype (BB and Bb vs. bb), blood lead, tibia lead, and DMSA-chelatable lead were all positive predictors of systolic blood pressure. On average, lead workers with the VDR B allele, mainly heterozygotes, had systolic blood pressures that were 2.7-3.7 mm Hg higher than did workers with the bb genotype. VDR genotype was also associated with diastolic blood pressure; on average, lead workers with the VDR B allele had diastolic blood pressures that were 1.9-2.5 mm Hg higher than did lead workers with the VDR bb genotype (p = 0.04). VDR genotype modified the relation of age with systolic blood pressure; compared to lead workers with the VDR bb genotype, workers with the VDR B allele had larger elevations in blood pressure with increasing age. Lead workers with the VDR B allele also had a higher prevalence of hypertension compared to lead workers with the bb genotype [adjusted odds ratio (95% confidence interval) = 2.1 (1.0, 4.4), p = 0.05]. None of the lead biomarkers was associated with diastolic blood pressure, and tibia lead was the only lead dose measure that was a significant predictor of hypertension status. In

  18. Lead accumulation and depression of delta-aminolevulinic acid dehydratase (ALAD) in young birds fed automotive waste oil

    USGS Publications Warehouse

    Eastin, W.C.; Hoffman, D.J.; O'Leary, C.T.

    1983-01-01

    The effects of a 3-week dietary exposure to automotive waste crankcase oil (WCO) were examined in 1-week-old mallard (Anas platyrhynchos) ducklings and pheasant (Phasianus colchicus) chicks. Treatment groups consisted of birds exposed to 0.5, 1.5, or 4.5% WCO, to 4.5% clean crankcase oil (CCO), or untreated controls. In both species, red blood cell ALAD activity was significantly inhibited after one week by 50 to 60% in the 0.5% WCO group and by 85 to 90% in the 4.5% WCO group due to the presence of lead. Growth, hematocrit, and hemoglobin were not significantly affected at the end of three weeks. Plasma aspartate aminotransferase (AST) activity was higher in mallards after three weeks of ingesting either 4.5% WCO or 4.5% CCO, suggesting an oil-related effect due to components other than lead. Treatment had no effect on plasma concentration of uric acid, glucose, triglycerides, total protein, or cholesterol. Lead analysis showed the WCO to contain 4,200 ppm Pb and the CCO to contain 2 ppm. Tissues of mallards were examined for accumulation of lead and the order of accumulation at the end of three weeks was kidney > liver > blood ~ brain.

  19. Characterization of recombinantly expressed dihydroxy-acid dehydratase from Sulfobus solfataricus-A key enzyme for the conversion of carbohydrates into chemicals.

    PubMed

    Carsten, Jörg M; Schmidt, Anja; Sieber, Volker

    2015-10-10

    Dihydroxyacid dehydratases (DHADs) are excellent biocatalysts for the defunctionalization of biomass. Here, we report on the recombinant production of DHAD from Sulfolobus solfataricus (SsDHAD) in E. coli and its characterization with special focus on activity toward non-natural substrates, thermo-stability, thermo-inactivation kinetics and activation capabilities and its application within multi-step cascades for chemicals production. Using a simple heat treatment of cell lysate as major purification step we achieved a specific activity of 4.4 units per gram cell mass toward the substrate d-gluconate. The optimal temperature and pH value for this reaction are 77°C and pH 6.2. The inhibitory concentration (IC50, 50% residual activity) of different alcohols was determined to be 15% (v/v) for ethanol, 4.5% (v/v) for butanol and 4% (v/v) for isobutanol. Besides d-gluconate and the natural substrate 2,3-dihydroxyisovalerate (DHIV) SsDHAD is able to convert the C3-sugar-acid d-glycerate to pyruvate, a reaction, which does not occur in natural metabolic pathways, with a specific activity of 10.7±0.4mU/mg. The specific activity of the enzyme can be increased 3-fold by incubation with 2-mercaptoethanol. The activation has no impact on temperature dependence, but modulates the thermo-inactivation tolerance at 50°C. The total turnover numbers for all of the three reactions was found to be 35.5×10(3)±1.0×10(3) for the conversion of d-gluconate to 2-keto-3-deoxygluconate (KDG), 28.2×10(3)±0.8×10(3) for DHIV to 2-ketovalerate (KIV) and 943±0.28×10(2) for d-glycerate to pyruvate. With activated SsDHAD these values could be further increased 5- and 4-fold for the d-gluconate and d-glycerate conversion, respectively.

  20. Characterization of recombinantly expressed dihydroxy-acid dehydratase from Sulfobus solfataricus-A key enzyme for the conversion of carbohydrates into chemicals.

    PubMed

    Carsten, Jörg M; Schmidt, Anja; Sieber, Volker

    2015-10-10

    Dihydroxyacid dehydratases (DHADs) are excellent biocatalysts for the defunctionalization of biomass. Here, we report on the recombinant production of DHAD from Sulfolobus solfataricus (SsDHAD) in E. coli and its characterization with special focus on activity toward non-natural substrates, thermo-stability, thermo-inactivation kinetics and activation capabilities and its application within multi-step cascades for chemicals production. Using a simple heat treatment of cell lysate as major purification step we achieved a specific activity of 4.4 units per gram cell mass toward the substrate d-gluconate. The optimal temperature and pH value for this reaction are 77°C and pH 6.2. The inhibitory concentration (IC50, 50% residual activity) of different alcohols was determined to be 15% (v/v) for ethanol, 4.5% (v/v) for butanol and 4% (v/v) for isobutanol. Besides d-gluconate and the natural substrate 2,3-dihydroxyisovalerate (DHIV) SsDHAD is able to convert the C3-sugar-acid d-glycerate to pyruvate, a reaction, which does not occur in natural metabolic pathways, with a specific activity of 10.7±0.4mU/mg. The specific activity of the enzyme can be increased 3-fold by incubation with 2-mercaptoethanol. The activation has no impact on temperature dependence, but modulates the thermo-inactivation tolerance at 50°C. The total turnover numbers for all of the three reactions was found to be 35.5×10(3)±1.0×10(3) for the conversion of d-gluconate to 2-keto-3-deoxygluconate (KDG), 28.2×10(3)±0.8×10(3) for DHIV to 2-ketovalerate (KIV) and 943±0.28×10(2) for d-glycerate to pyruvate. With activated SsDHAD these values could be further increased 5- and 4-fold for the d-gluconate and d-glycerate conversion, respectively. PMID:26102631

  1. Noninvasive fluorescence monitoring of protoporphyrin IX production and clinical outcomes in actinic keratoses following short-contact application of 5-aminolevulinate

    NASA Astrophysics Data System (ADS)

    Warren, Christine B.; Lohser, Sara; Wene, Lauren C.; Pogue, Brian W.; Bailin, Philip L.; Maytin, Edward V.

    2010-09-01

    Topical 5-aminolevulinic acid (ALA) is widely used in photodynamic therapy (PDT) of actinic keratoses (AK), a type of premalignant skin lesion. However, the optimal time between ALA application and exposure to light has not been carefully investigated. Our objective is to study the kinetics of protoporphyrin IX (PpIX) accumulation in AK after short contact ALA and relate this to erythemal responses. Using a noninvasive dosimeter, PpIX fluorescence measurements (5 replicates) were taken at 20-min intervals for 2 h following ALA application, in 63 AK in 20 patients. Data were analyzed for maximal fluorescent signal obtained, kinetic slope, and changes in erythema. Our results show that PpIX accumulation was linear over time, becoming statistically higher than background in 48% of all lesions by 20 min, 92% of lesions by 1 h, and 100% of lesions by 2 h. PpIX accumulation was roughly correlated with changes in lesional erythema post-PDT. We conclude that significant amounts of PpIX are produced in all AK lesions by 2 h. The linear kinetics of accumulation suggest that shorter ALA application times may be efficacious in many patients. Noninvasive fluorescence monitoring of PpIX may be useful to delineate areas of high PpIX accumulation within precancerous areas of the skin.

  2. Source and impact of lead contamination on δ-aminolevulinic acid dehydratase activity in several marine bivalve species along the Gulf of Cadiz.

    PubMed

    Company, R; Serafim, A; Lopes, B; Cravo, A; Kalman, J; Riba, I; DelValls, T A; Blasco, J; Delgado, J; Sarmiento, A M; Nieto, J M; Shepherd, T J; Nowell, G; Bebianno, M J

    2011-01-17

    Coastal areas and estuaries are particularly sensitive to metal contamination from anthropogenic sources and in the last few decades the study of space-time distribution and variation of metals has been extensively researched. The Gulf of Cadiz is no exception, with several rivers draining one of the largest concentrations of sulphide deposits in the world, the Iberian Pyrite Belt (IPB). Of these rivers, the Guadiana, one of the most important in the Iberian Peninsula, together with smaller rivers like the Tinto and Odiel, delivers a very high metal load to the adjacent coastal areas. The purpose of this work was to study the source and impact of lead (Pb) drained from historical or active mining areas in the IPB on the activity of a Pb inhibited enzyme (δ-aminolevulinic acid dehydratase, ALAD) in several bivalve species along the Gulf of Cadiz. Seven marine species (Chamelea gallina, Mactra corallina, Donax trunculus, Cerastoderma edule, Mytilus galloprovincialis, Scrobicularia plana and Crassostrea angulata) were collected at 12 sites from Mazagón, near the mouth of the rivers Tinto and Odiel (Spain), to Cacela Velha (Ria Formosa lagoon system, Portugal). Lead concentrations, ALAD activity and lead isotope ratios ((206)Pb/(204)Pb, (207)Pb/(204)Pb and (208)Pb/(204)Pb) were determined in the whole soft tissues. The highest Pb concentrations were determined in S. plana (3.50±1.09 μg g(-1) Pb d.w.) and D. trunculus (1.95±0.10 μg g(-1) Pb d.w.), while M. galloprovincialis and C. angulata showed the lowest Pb levels (<0.38 μg g(-1) Pb d.w.). In general, ALAD activity is negatively correlated with total Pb concentration. However this relationship is species dependent (e.g. linear for C. gallina ALAD=-0.36[Pb]+0.79; r=0.837; or exponential for M. galloprovincialis ALAD=2.48e(-8.3[Pb]); r=0.911). This indicates that ALAD activity has considerable potential as a biomarker of Pb and moreover, in marine bivalve species with different feeding habits. Lead isotope data

  3. Source and impact of lead contamination on δ-aminolevulinic acid dehydratase activity in several marine bivalve species along the Gulf of Cadiz.

    PubMed

    Company, R; Serafim, A; Lopes, B; Cravo, A; Kalman, J; Riba, I; DelValls, T A; Blasco, J; Delgado, J; Sarmiento, A M; Nieto, J M; Shepherd, T J; Nowell, G; Bebianno, M J

    2011-01-17

    Coastal areas and estuaries are particularly sensitive to metal contamination from anthropogenic sources and in the last few decades the study of space-time distribution and variation of metals has been extensively researched. The Gulf of Cadiz is no exception, with several rivers draining one of the largest concentrations of sulphide deposits in the world, the Iberian Pyrite Belt (IPB). Of these rivers, the Guadiana, one of the most important in the Iberian Peninsula, together with smaller rivers like the Tinto and Odiel, delivers a very high metal load to the adjacent coastal areas. The purpose of this work was to study the source and impact of lead (Pb) drained from historical or active mining areas in the IPB on the activity of a Pb inhibited enzyme (δ-aminolevulinic acid dehydratase, ALAD) in several bivalve species along the Gulf of Cadiz. Seven marine species (Chamelea gallina, Mactra corallina, Donax trunculus, Cerastoderma edule, Mytilus galloprovincialis, Scrobicularia plana and Crassostrea angulata) were collected at 12 sites from Mazagón, near the mouth of the rivers Tinto and Odiel (Spain), to Cacela Velha (Ria Formosa lagoon system, Portugal). Lead concentrations, ALAD activity and lead isotope ratios ((206)Pb/(204)Pb, (207)Pb/(204)Pb and (208)Pb/(204)Pb) were determined in the whole soft tissues. The highest Pb concentrations were determined in S. plana (3.50±1.09 μg g(-1) Pb d.w.) and D. trunculus (1.95±0.10 μg g(-1) Pb d.w.), while M. galloprovincialis and C. angulata showed the lowest Pb levels (<0.38 μg g(-1) Pb d.w.). In general, ALAD activity is negatively correlated with total Pb concentration. However this relationship is species dependent (e.g. linear for C. gallina ALAD=-0.36[Pb]+0.79; r=0.837; or exponential for M. galloprovincialis ALAD=2.48e(-8.3[Pb]); r=0.911). This indicates that ALAD activity has considerable potential as a biomarker of Pb and moreover, in marine bivalve species with different feeding habits. Lead isotope data

  4. An extreme-halophile archaebacterium possesses the interlock type of prephenate dehydratase characteristic of the Gram-positive eubacteria.

    PubMed

    Jensen, R A; d'Amato, T A; Hochstein, L I

    1988-01-01

    The focal point of phenylalanine biosynthesis is a dehydratase reaction which in different organisms may be prephenate dehydratase, arogenate dehydratase, or cyclohexadienyl dehydratase. Gram-positive, Gram-negative, and cyanobacterial divisions of the eubacterial kingdom exhibit different dehydratase patterns. A new extreme-halophile isolate, which grows on defined medium and is tentatively designated as Halobacterium vallismortis CH-1, possesses the interlock type of prephenate dehydratase present in Gram-positive bacteria. In addition to the conventional sensitivity to feedback inhibition by L-phenylalanine, the phenomenon of metabolic interlock was exemplified by the sensitivity of prephenate dehydratase to allosteric effects produced by extra-pathway (remote) effectors. Thus, L-tryptophan inhibited activity while L-tyrosine, L-methionine, L-leucine and L-isoleucine activated the enzyme. L-Isoleucine and L-phenylalanine were effective at micromolar levels; other effectors operated at mM levels. A regulatory mutant selected for resistance to growth inhibition caused by beta-2-thienylalanine possessed an altered prephenate dehydratase in which a phenomenon of disproportionately low activity at low enzyme concentration was abolished. Inhibition by L-tryptophan was also lost, and activation by allosteric activators was diminished. Not only was sensitivity to feedback inhibition by L-phenylalanine lost, but the mutant enzyme was now activated by this amino acid (a mutation type previously observed in Bacillus subtilis). It remains to be seen whether this type of prephenate dehydratase will prove to be characteristic of all archaebacteria or of some archaebacterial subgroup cluster.

  5. An extreme-halophile archaebacterium possesses the interlock type of prephenate dehydratase characteristic of the Gram-positive eubacteria

    NASA Technical Reports Server (NTRS)

    Jensen, R. A.; d'Amato, T. A.; Hochstein, L. I.

    1988-01-01

    The focal point of phenylalanine biosynthesis is a dehydratase reaction which in different organisms may be prephenate dehydratase, arogenate dehydratase, or cyclohexadienyl dehydratase. Gram-positive, Gram-negative, and cyanobacterial divisions of the eubacterial kingdom exhibit different dehydratase patterns. A new extreme-halophile isolate, which grows on defined medium and is tentatively designated as Halobacterium vallismortis CH-1, possesses the interlock type of prephenate dehydratase present in Gram-positive bacteria. In addition to the conventional sensitivity to feedback inhibition by L-phenylalanine, the phenomenon of metabolic interlock was exemplified by the sensitivity of prephenate dehydratase to allosteric effects produced by extra-pathway (remote) effectors. Thus, L-tryptophan inhibited activity while L-tyrosine, L-methionine, L-leucine and L-isoleucine activated the enzyme. L-Isoleucine and L-phenylalanine were effective at micromolar levels; other effectors operated at mM levels. A regulatory mutant selected for resistance to growth inhibition caused by beta-2-thienylalanine possessed an altered prephenate dehydratase in which a phenomenon of disproportionately low activity at low enzyme concentration was abolished. Inhibition by L-tryptophan was also lost, and activation by allosteric activators was diminished. Not only was sensitivity to feedback inhibition by L-phenylalanine lost, but the mutant enzyme was now activated by this amino acid (a mutation type previously observed in Bacillus subtilis). It remains to be seen whether this type of prephenate dehydratase will prove to be characteristic of all archaebacteria or of some archaebacterial subgroup cluster.

  6. Human Erythroid 5-Aminolevulinate Synthase Mutations Associated with X-Linked Protoporphyria Disrupt Conformational Equilibrium and Enhance Product Release†

    PubMed Central

    Fratz, Erica J.; Clayton, Jerome; Hunter, Gregory A.; Ducamp, Sarah; Breydo, Leonid; Uversky, Vladimir N.; Deybach, Jean-Charles; Gouya, Laurent; Puy, Hervé; Ferreira, Gloria C.

    2015-01-01

    Regulation of 5-aminolevulinate synthase (ALAS) is at the origin of balanced heme production in mammals. Mutations in the C-terminal region of human erythroid-specific ALAS (hALAS2) are associated with X-linked protoporphyria (XLPP), a disease characterized by extreme photosensitivity, with elevated blood concentrations of free protoporphyrin IX and zinc protoporphyrin. To investigate the molecular basis for this disease, recombinant hALAS2 and variants of the enzyme harboring the gain-of-function XLPP mutations were constructed, purified, and analyzed kinetically, spectroscopically and thermodynamically. Enhanced activities of the XLPP variants resulted from accelerations in the rate at which the product 5-aminolevulinate (ALA) was released from the enzyme. Circular dichroism spectroscopy revealed that the XLPP mutations altered the microenvironment of the pyridoxal 5’-phosphate cofactor, which underwent further and specific alterations upon succinyl-CoA binding. Transient kinetic analyses of the variant-catalyzed reactions and protein fluorescence quenching upon ALA binding to the XLPP variants demonstrated that the protein conformational transition step associated with product release was predominantly affected. Of relevance, XLPP could also be modeled in cell culture. We propose that 1) the XLPP mutations destabilize the succinyl-CoA-induced hALAS2 closed conformation and thus accelerate ALA release, 2) the extended C-terminus of wild-type mammalian ALAS2 provides a regulatory role that allows for allosteric modulation of activity, thereby controlling the rate of erythroid heme biosynthesis, and 3) this control is disrupted in XLPP, resulting in porphyrin accumulation. PMID:26300302

  7. Asn-150 of Murine Erythroid 5-Aminolevulinate Synthase Modulates the Catalytic Balance between the Rates of the Reversible Reaction.

    PubMed

    Stojanovski, Bosko M; Ferreira, Gloria C

    2015-12-25

    5-Aminolevulinate synthase (ALAS) catalyzes the first step in mammalian heme biosynthesis, the pyridoxal 5'-phosphate (PLP)-dependent and reversible reaction between glycine and succinyl-CoA to generate CoA, CO2, and 5-aminolevulinate (ALA). Apart from coordinating the positioning of succinyl-CoA, Rhodobacter capsulatus ALAS Asn-85 has a proposed role in regulating the opening of an active site channel. Here, we constructed a library of murine erythroid ALAS variants with substitutions at the position occupied by the analogous bacterial asparagine, screened for ALAS function, and characterized the catalytic properties of the N150H and N150F variants. Quinonoid intermediate formation occurred with a significantly reduced rate for either the N150H- or N150F-catalyzed condensation of glycine with succinyl-CoA during a single turnover. The introduced mutations caused modifications in the ALAS active site such that the resulting variants tipped the balance between the forward- and reverse-catalyzed reactions. Although wild-type ALAS catalyzes the conversion of ALA into the quinonoid intermediate at a rate 6.3-fold slower than the formation of the same quinonoid intermediate from glycine and succinyl-CoA, the N150F variant catalyzes the forward reaction at a mere 1.2-fold faster rate than that of the reverse reaction, and the N150H variant reverses the rate values with a 1.7-fold faster rate for the reverse reaction than that for the forward reaction. We conclude that the evolutionary selection of Asn-150 was significant for optimizing the forward enzymatic reaction at the expense of the reverse, thus ensuring that ALA is predominantly available for heme biosynthesis.

  8. Propanediol utilization genes (pdu) of Salmonella typhimurium: three genes for the propanediol dehydratase.

    PubMed Central

    Bobik, T A; Xu, Y; Jeter, R M; Otto, K E; Roth, J R

    1997-01-01

    The propanediol utilization (pdu) operon of Salmonella typhimurium encodes proteins required for the catabolism of propanediol, including a coenzyme B12-dependent propanediol dehydratase. A clone that expresses propanediol dehydratase activity was isolated from a Salmonella genomic library. DNA sequence analysis showed that the clone included part of the pduF gene, the pduABCDE genes, and a long partial open reading frame (ORF1). The clone included 3.9 kbp of pdu DNA which had not been previously sequenced. Complementation and expression studies with subclones constructed via PCR showed that three genes (pduCDE) are necessary and sufficient for propanediol dehydratase activity. The function of ORF1 was not determined. Analyses showed that the S. typhimurium propanediol dehydratase was related to coenzyme B12-dependent glycerol dehydratases from Citrobacter freundii and Klebsiella pneumoniae. Unexpectedly, the S. typhimurium propanediol dehydratase was found to be 98% identical in amino acid sequence to the Klebsiella oxytoca propanediol dehydratase; this is a much higher identity than expected, given the relationship between these organisms. DNA sequence analyses also supported previous studies indicating that the pdu operon was inherited along with the adjacent cobalamin biosynthesis operon by a single horizontal gene transfer. PMID:9352910

  9. The concentration of some metals (Pb, Cd, Zn and Cu) and delta aminolevulinic acid dehydratase activity of fruit fly (Drosophila melanogaster) living near lead and zinc smelter ``Trepça'' in Kosova

    NASA Astrophysics Data System (ADS)

    Elezaj, I. R.; Letaj, K. Rr.; Selimi, Q. I.; Zhushi-Etemi, F.

    2003-05-01

    The concentration of Pb, Cd, Zn and Cu, δ-aminolevulinic acid dehydratase activity (ALA-D: EC.4.2.1.24) hemoglobin and protein amount have been determined in three different populations of fruit fly (Drosophila melanogaster) caught at two urban sites (Mitrovica town, which is situated close to smelter of “Trepça” don close and Prishtina the capital of Kosova) and in Luki village as uncontaminated area. The results show that in the fruit fly of Mitrovica the concentration of Pb, Cd and Zn was significantly higher (P<0.00l) in comparison with that on the f-Liit fly of Prishtina and Luki. The concentration of Pb of fruit fly from Mitrovica was 3.1 times higher in comparison with that on fruit fly of Prishtina and 4.9 times higher in comparison with uncontaminated group of fruit fly. The ALA-D activity was significantly inhibited in the homogenate of fruit fly from Mitrovica in comparison with Prishtina and Luki localities (P<0.00l). ALA-D activity was also inhibited in the homogenate of Prishtina fruit fly in comparison with Luki group (P<0.00l). The amount of proteins was significantly lower in Mitrovica fruit fly in comparison with that in control and Prishtina group. The hemoglobin value was relatively unchanged.

  10. Quantitative fluorescence using 5-aminolevulinic acid–induced protoporphyrin IX biomarker as a surgical adjunct in low-grade glioma surgery

    PubMed Central

    Valdés, Pablo A.; Jacobs, Valerie; Harris, Brent T.; Wilson, Brian C.; Leblond, Frederic; Paulsen, Keith D.; Roberts, David W.

    2015-01-01

    OBJECT Previous studies in high-grade gliomas (HGGs) have indicated that protoporphyrin IX (PpIX) accumulates in higher concentrations in tumor tissue, and, when used to guide surgery, it has enabled improved resection leading to increased progression-free survival. Despite the benefits of complete resection and the advances in fluorescence-guided surgery, few studies have investigated the use of PpIX in low-grade gliomas (LGGs). Here, the authors describe their initial experience with 5-aminolevulinic acid (ALA)–induced PpIX fluorescence in a series of patients with LGG. METHODS Twelve patients with presumed LGGs underwent resection of their tumors after receiving 20 μg/kg of ALA approximately 3 hours prior to surgery under an institutional review board–approved protocol. Intraoperative assessments of the resulting PpIX emissions using both qualitative, visible fluorescence and quantitative measurements of PpIX concentration were obtained from tissue locations that were subsequently biopsied and evaluated histopathologically. Mixed models for random effects and receiver operating characteristic curve analysis for diagnostic performance were performed on the fluorescence data relative to the gold-standard histopathology. RESULTS Five of the 12 LGGs (1 ganglioglioma, 1 oligoastrocytoma, 1 pleomorphic xanthoastrocytoma, 1 oligodendroglioma, and 1 ependymoma) demonstrated at least 1 instance of visible fluorescence during surgery. Visible fluorescence evaluated on a specimen-by-specimen basis yielded a diagnostic accuracy of 38.0% (cutoff threshold: visible fluorescence score ≥ 1, area under the curve = 0.514). Quantitative fluorescence yielded a diagnostic accuracy of 67% (for a cutoff threshold of the concentration of PpIX [CPpIX] > 0.0056 μg/ml, area under the curve = 0.66). The authors found that 45% (9/20) of nonvisibly fluorescent tumor specimens, which would have otherwise gone undetected, accumulated diagnostically significant levels of CPpIX that were

  11. Characterization and mutagenesis of two novel iron-sulphur cluster pentonate dehydratases.

    PubMed

    Andberg, Martina; Aro-Kärkkäinen, Niina; Carlson, Paul; Oja, Merja; Bozonnet, Sophie; Toivari, Mervi; Hakulinen, Nina; O'Donohue, Michael; Penttilä, Merja; Koivula, Anu

    2016-09-01

    We describe here the identification and characterization of two novel enzymes belonging to the IlvD/EDD protein family, the D-xylonate dehydratase from Caulobacter crescentus, Cc XyDHT, (EC 4.2.1.82), and the L-arabonate dehydratase from Rhizobium leguminosarum bv. trifolii, Rl ArDHT (EC 4.2.1.25), that produce the corresponding 2-keto-3-deoxy-sugar acids. There is only a very limited amount of characterization data available on pentonate dehydratases, even though the enzymes from these oxidative pathways have potential applications with plant biomass pentose sugars. The two bacterial enzymes share 41 % amino acid sequence identity and were expressed and purified from Escherichia coli as homotetrameric proteins. Both dehydratases were shown to accept pentonate and hexonate sugar acids as their substrates and require Mg(2+) for their activity. Cc XyDHT displayed the highest activity on D-xylonate and D-gluconate, while Rl ArDHT functioned best on D-fuconate, L-arabonate and D-galactonate. The configuration of the OH groups at C2 and C3 position of the sugar acid were shown to be critical, and the C4 configuration also contributed substantially to the substrate recognition. The two enzymes were also shown to contain an iron-sulphur [Fe-S] cluster. Our phylogenetic analysis and mutagenesis studies demonstrated that the three conserved cysteine residues in the aldonic acid dehydratase group of IlvD/EDD family members, those of C60, C128 and C201 in Cc XyDHT, and of C59, C127 and C200 in Rl ArDHT, are needed for coordination of the [Fe-S] cluster. The iron-sulphur cluster was shown to be crucial for the catalytic activity (kcat) but not for the substrate binding (Km) of the two pentonate dehydratases. PMID:27102126

  12. Photodynamic therapy (PDT) and photodiagnosis (PD) using endogenous photosensitization induced by 5-aminolevulinic acid (ALA): current clinical and development status

    NASA Astrophysics Data System (ADS)

    Marcus, Stuart L.; Sobel, Russel S.; Golub, Allyn L.; Carroll, Ronald L.; Lundahl, Scott L.; Shulman, D. Geoffrey

    1996-04-01

    Exogenous provision of ALA to many tissues results in the accumulation of sufficient quantities of the endogenous photosensitizer protoporphyrin IX, (PpIX), to produce a photodynamic effect. Therefore, ALA may be considered the only current PDT agent in clinical development which is a biochemical precursor of a photosensitizer. Topical ALA application, followed by exposure to activating light (ALA PDT), has been reported effective for the treatment of a variety of dermatologic diseases including cutaneous T-cell lymphoma, superficial basal cell carcinoma, Bowen's disease, and actinic (solar) keratoses, and is also being examined for treatment of acne and hirsutism. PpIX induced by ALA application also may serve as a fluorescence detection marker for photodiagnosis (PD) of malignant and pre- malignant conditions of the urinary bladder and other organs. Local internal application of ALA has also been used for selective endometrial ablation in animal model systems and is beginning to be examined in human clinical studies. Systemic, oral administration of ALA has been used for ALA PDT of superficial head and neck cancer, various gastrointestinal cancers, and the condition known as Barrett's esophagus. This brief paper reviews the current clinical and development status of ALA PDT.

  13. Red versus blue light illumination in hexyl 5-aminolevulinate photodynamic therapy: the influence of light color and irradiance on the treatment outcome in vitro.

    PubMed

    Helander, Linda; Krokan, Hans E; Johnsson, Anders; Gederaas, Odrun A; Plaetzer, Kristjan

    2014-08-01

    Hexyl 5-aminolevulinate (HAL) is a lipophilic derivative of 5-aminolevulinate, a key intermediate in biosynthesis of the photosensitizer protoporphyrin IX (PpIX). The photodynamic efficacy and cell death mode after red versus blue light illumination of HAL-induced PpIX have been examined and compared using five different cancer cell lines. LED arrays emitting at 410 and 624 nm served as homogenous and adjustable light sources. Our results show that the response after HAL-PDT is cell line specific, both regarding the shape of the dose-survival curve, the overall dose required for efficient cell killing, and the relative amount of apoptosis. The ratio between 410 and 624 nm in absorption coefficient correlates well with the difference in cell killing at the same wavelengths. In general, the PDT efficacy was several folds higher for blue light as compared with red light, as expected. However, HAL-PDT₆₂₄ induced more apoptosis than HAL-PDT₄₁₀ and illumination with low irradiance resulted in more apoptosis than high irradiance at the same lethal dose. This indicates differences in death modes after low and high irradiance after similar total light doses. From a treatment perspective, these differences may be important.

  14. Red versus blue light illumination in hexyl 5-aminolevulinate photodynamic therapy: the influence of light color and irradiance on the treatment outcome in vitro

    NASA Astrophysics Data System (ADS)

    Helander, Linda; Krokan, Hans E.; Johnsson, Anders; Gederaas, Odrun A.; Plaetzer, Kristjan

    2014-08-01

    Hexyl 5-aminolevulinate (HAL) is a lipophilic derivative of 5-aminolevulinate, a key intermediate in biosynthesis of the photosensitizer protoporphyrin IX (PpIX). The photodynamic efficacy and cell death mode after red versus blue light illumination of HAL-induced PpIX have been examined and compared using five different cancer cell lines. LED arrays emitting at 410 and 624 nm served as homogenous and adjustable light sources. Our results show that the response after HAL-PDT is cell line specific, both regarding the shape of the dose-survival curve, the overall dose required for efficient cell killing, and the relative amount of apoptosis. The ratio between 410 and 624 nm in absorption coefficient correlates well with the difference in cell killing at the same wavelengths. In general, the PDT efficacy was several folds higher for blue light as compared with red light, as expected. However, HAL-PDT624 induced more apoptosis than HAL-PDT410 and illumination with low irradiance resulted in more apoptosis than high irradiance at the same lethal dose. This indicates differences in death modes after low and high irradiance after similar total light doses. From a treatment perspective, these differences may be important.

  15. Mn(2+) in D-Glucosaminate Dehydratase from Pseudomonas fluorescens.

    PubMed

    Iwamoto, R; Nakura, S

    1993-01-01

    D-Glucosaminate (D-GlcNA) dehydratase from Pseudomonas fluorescens was inhibited stoichiometrically by metal-chelating agents (EDTA, 8-hydroxyquinoline-5-sulfonic acid, α,α'-dipyridyl and o-phenan-throline). The activity of EDTA-treated enzyme was restored by incubation with Mn(2+) (0.4mM) or Ca(2+) (2mM) in the presence of pyridoxal 5'-phosphate (PLP, 0.2mM) in veronal buffer (VB, 40 mM, pH 8) at 37°C for 30 min. The atomic absorption spectrum of the native enzyme showed that the enzyme contained 1 mol of Mn(2+) per mole of enzyme. Although the EDTA-treated enzyme was unstable at 4°C, addition of Mn(2+) and PLP to the solution of the EDTA-treated enzyme prevented the inactivation. The Km of the restored enzyme for D-GlcNA was somewhat lower than that of the original enzyme. However, the Km for PLP increased 14-fold. These results suggest that D-GlcNA dehydratase contains Mn(2+) near the PLP-binding site, and the metal ion appears to stabilize the structure of the active site.

  16. Pen and Pal are nucleotide-sugar dehydratases that convert UDP-GlcNAc to UDP-6-deoxy-D-GlcNAc-5,6-ene and then to UDP-4-keto-6-deoxy-L-AltNAc for CMP-pseudaminic acid synthesis in Bacillus thuringiensis.

    PubMed

    Li, Zi; Hwang, Soyoun; Ericson, Jaime; Bowler, Kyle; Bar-Peled, Maor

    2015-01-01

    CMP-pseudaminic acid is a precursor required for the O-glycosylation of flagellin in some pathogenic Gram-negative bacteria, a process known to be critical in bacterial motility and infection. However, little is known about flagellin glycosylation in Gram-positive bacteria. Here, we identified and functionally characterized an operon, named Bti_pse, in Bacillus thuringiensis israelensis ATCC 35646, which encodes seven different enzymes that together convert UDP-GlcNAc to CMP-pseudaminic acid. In contrast, Gram-negative bacteria complete this reaction with six enzymes. The first enzyme, which we named Pen, converts UDP-d-GlcNAc to an uncommon UDP-sugar, UDP-6-deoxy-D-GlcNAc-5,6-ene. Pen contains strongly bound NADP(+) and has distinct UDP-GlcNAc 4-oxidase, 5,6-dehydratase, and 4-reductase activities. The second enzyme, which we named Pal, converts UDP-6-deoxy-D-GlcNAc-5,6-ene to UDP-4-keto-6-deoxy-L-AltNAc. Pal is NAD(+)-dependent and has distinct UDP-6-deoxy-d-GlcNAc-5,6-ene 4-oxidase, 5,6-reductase, and 5-epimerase activities. We also show here using NMR spectroscopy and mass spectrometry that in B. thuringiensis, the enzymatic product of Pen and Pal, UDP-4-keto-6-deoxy-L-AltNAc, is converted to CMP-pseudaminic acid by the sequential activities of a C4″-transaminase (Pam), a 4-N-acetyltransferase (Pdi), a UDP-hydrolase (Phy), an enzyme (Ppa) that adds phosphoenolpyruvate to form pseudaminic acid, and finally a cytidylyltransferase that condenses CTP to generate CMP-pseudaminic acid. Knowledge of the distinct dehydratase-like enzymes Pen and Pal and their role in CMP-pseudaminic acid biosynthesis in Gram-positive bacteria provides a foundation to investigate the role of pseudaminic acid and flagellin glycosylation in Bacillus and their involvement in bacterial motility and pathogenicity.

  17. Diol Dehydratase-Reactivase Is Essential for Recycling of Coenzyme B12 in Diol Dehydratase.

    PubMed

    Toraya, Tetsuo; Tanokuchi, Aya; Yamasaki, Ai; Nakamura, Takehiro; Ogura, Kenichi; Tobimatsu, Takamasa

    2016-01-12

    Holoenzymes of adenosylcobalamin-dependent diol and glycerol dehydratases undergo mechanism-based inactivation by glycerol and O2 inactivation in the absence of substrate, which accompanies irreversible cleavage of the coenzyme Co-C bond. The inactivated holodiol dehydratase and the inactive enzyme·cyanocobalamin complex were (re)activated by incubation with NADH, ATP, and Mg(2+) (or Mn(2+)) in crude extracts of Klebsiella oxytoca, suggesting the presence of a reactivating system in the extract. The reducing system with NADH could be replaced by FMNH2. When inactivated holoenzyme or the enzyme·cyanocobalamin complex, a model of inactivated holoenzyme, was incubated with purified recombinant diol dehydratase-reactivase (DD-R) and an ATP:cob(I)alamin adenosyltransferase in the presence of FMNH2, ATP, and Mg(2+), diol dehydratase activity was restored. Among the three adenosyltransferases (PduO, EutT, and CobA) of this bacterium, PduO and CobA were much more efficient for the reactivation than EutT, although PduO showed the lowest adenosyltransfease activity toward free cob(I)alamin. These results suggest that (1) diol dehydratase activity is maintained through coenzyme recycling by a reactivating system for diol dehydratase composed of DD-R, PduO adenosyltransferase, and a reducing system, (2) the releasing factor DD-R is essential for the recycling of adenosycobalamin, a tightly bound, prosthetic group-type coenzyme, and (3) PduO is a specific adenosylating enzyme for the DD reactivation, whereas CobA and EutT exert their effects through free synthesized coenzyme. Although FMNH2 was mainly used as a reductant in this study, a natural reducing system might consist of PduS cobalamin reductase and NADH.

  18. Tissue responses to hexyl 5-aminolevulinate-induced photodynamic treatment in syngeneic orthotopic rat bladder cancer model: possible pathways of action

    NASA Astrophysics Data System (ADS)

    Arum, Carl-Jørgen; Gederaas, Odrun A.; Larsen, Eivind L. P.; Randeberg, Lise L.; Hjelde, Astrid; Krokan, Hans E.; Svaasand, Lars O.; Chen, Duan; Zhao, Chun-Mei

    2011-02-01

    Orthotopic bladder cancer model in rats mimics human bladder cancer with respect to urothelial tumorigenesis and progression. Utilizing this model at pT1 (superficial stage), we analyze the tissue responses to hexyl 5-aminolevulinate-induced photodynamic therapy (HAL-PDT). In comparison to untreated rats, HAL-PDT causes little change in tumor-free rat bladder but induces inflammatory changes with increased lymphocytes and mononuclear cell infiltration in rat bladders with tumor. Immunohistochemistry reveals that HAL-PDT is without effect on proliferating cell nuclear antigen expression within the tumor and increases caspase-3 expression in both normal urothelium and the tumor. Transmission electron microscopy reveals severe mitochondrial damage, formations of apoptotic bodies, vacuoles, and lipofuscin bodies, but no microvillus-formed niches in HAL-PDT-treated bladder cancer rats. Bioinformatics analysis of the gene expression profile indicates an activation of T-cell receptor signaling pathway in bladder cancer rats without PDT. HAL-PDT increases the expression of CD3 and CD45RA in the tumor (determined by immunohistochemistry). We suggest that pathways of action of HAL-PDT may include, at least, activations of mitochondrial apoptosis and autophagy, breakdown of cancer stem cell niches, and importantly, enhancement of T-cell activation.

  19. Pen and Pal Are Nucleotide-Sugar Dehydratases That Convert UDP-GlcNAc to UDP-6-Deoxy-d-GlcNAc-5,6-ene and Then to UDP-4-Keto-6-deoxy-l-AltNAc for CMP-Pseudaminic Acid Synthesis in Bacillus thuringiensis*♦

    PubMed Central

    Li, Zi; Hwang, Soyoun; Ericson, Jaime; Bowler, Kyle; Bar-Peled, Maor

    2015-01-01

    CMP-pseudaminic acid is a precursor required for the O-glycosylation of flagellin in some pathogenic Gram-negative bacteria, a process known to be critical in bacterial motility and infection. However, little is known about flagellin glycosylation in Gram-positive bacteria. Here, we identified and functionally characterized an operon, named Bti_pse, in Bacillus thuringiensis israelensis ATCC 35646, which encodes seven different enzymes that together convert UDP-GlcNAc to CMP-pseudaminic acid. In contrast, Gram-negative bacteria complete this reaction with six enzymes. The first enzyme, which we named Pen, converts UDP-d-GlcNAc to an uncommon UDP-sugar, UDP-6-deoxy-d-GlcNAc-5,6-ene. Pen contains strongly bound NADP+ and has distinct UDP-GlcNAc 4-oxidase, 5,6-dehydratase, and 4-reductase activities. The second enzyme, which we named Pal, converts UDP-6-deoxy-d-GlcNAc-5,6-ene to UDP-4-keto-6-deoxy-l-AltNAc. Pal is NAD+-dependent and has distinct UDP-6-deoxy-d-GlcNAc-5,6-ene 4-oxidase, 5,6-reductase, and 5-epimerase activities. We also show here using NMR spectroscopy and mass spectrometry that in B. thuringiensis, the enzymatic product of Pen and Pal, UDP-4-keto-6-deoxy-l-AltNAc, is converted to CMP-pseudaminic acid by the sequential activities of a C4″-transaminase (Pam), a 4-N-acetyltransferase (Pdi), a UDP-hydrolase (Phy), an enzyme (Ppa) that adds phosphoenolpyruvate to form pseudaminic acid, and finally a cytidylyltransferase that condenses CTP to generate CMP-pseudaminic acid. Knowledge of the distinct dehydratase-like enzymes Pen and Pal and their role in CMP-pseudaminic acid biosynthesis in Gram-positive bacteria provides a foundation to investigate the role of pseudaminic acid and flagellin glycosylation in Bacillus and their involvement in bacterial motility and pathogenicity. PMID:25414257

  20. Short Communication: Molecular cloning and expression pattern of the porcine 5-aminolevulinate synthase 1 (ALAS1) gene and its association with reproductive traits.

    PubMed

    Liu, L Q; Li, F E; Deng, C Y

    2016-01-01

    5-Aminolevulinate synthase 1 (ALAS1) is the first enzyme in the heme biosynthetic pathway and is upregulated in follicular tissue during the early stages of ovulation. In this study, we isolated the complete coding sequence of the porcine ALAS1 gene and its 2-9 intron sequence, identified a single nucleotide polymorphism (SNP; T/C) in intron 9, and developed a PCR-MspI-restriction fragment length polymorphism genotyping assay. Association of the SNP with litter size was assessed in two populations [purebred Large White and the experimental synthetic (DIV) line]. Statistical analysis demonstrated that for total number of piglets born (TNB) in all parities, pigs with the CC genotype had an additional 0.68 and 0.74 piglets compared to the TC and TT animals (P < 0.05) in the DIV line, respectively. Purebred Large White sows inheriting the CC and TC genotypes gave birth to an additional 0.96 and 0.70 piglets compared to the TT animals (P < 0.05) in all parities, respectively. In addition, for TNB in all parities, a significant additive effect of 0.48 ± 0.23 and 0.37 ± 0.17 piglets/ litter was detected in sows of both populations (P < 0.05), respectively. The highest levels of ALAS1 gene expression were observed in isolated ovarian granulosa cells 2 and 12 h after stimulation with pregnant mare serum gonadotropin human chorionic gonadotropin, which represents the time of follicular development and ovulation, respectively. Therefore, the ALAS1 gene was significantly associated with litter size in two populations and could be a useful molecular marker for the selection of increasing litter size in pigs. PMID:26910002

  1. Structure and mechanism of the tRNA-dependent lantibiotic dehydratase NisB.

    PubMed

    Ortega, Manuel A; Hao, Yue; Zhang, Qi; Walker, Mark C; van der Donk, Wilfred A; Nair, Satish K

    2015-01-22

    Lantibiotics are a class of peptide antibiotics that contain one or more thioether bonds. The lantibiotic nisin is an antimicrobial peptide that is widely used as a food preservative to combat food-borne pathogens. Nisin contains dehydroalanine and dehydrobutyrine residues that are formed by the dehydration of Ser/Thr by the lantibiotic dehydratase NisB (ref. 2). Recent biochemical studies revealed that NisB glutamylates Ser/Thr side chains as part of the dehydration process. However, the molecular mechanism by which NisB uses glutamate to catalyse dehydration remains unresolved. Here we show that this process involves glutamyl-tRNA(Glu) to activate Ser/Thr residues. In addition, the 2.9-Å crystal structure of NisB in complex with its substrate peptide NisA reveals the presence of two separate domains that catalyse the Ser/Thr glutamylation and glutamate elimination steps. The co-crystal structure also provides insights into substrate recognition by lantibiotic dehydratases. Our findings demonstrate an unexpected role for aminoacyl-tRNA in the formation of dehydroamino acids in lantibiotics, and serve as a basis for the functional characterization of the many lantibiotic-like dehydratases involved in the biosynthesis of other classes of natural products. PMID:25363770

  2. Structure and Mechanism of the tRNA-Dependent Lantibiotic Dehydratase NisB

    PubMed Central

    Ortega, Manuel A.; Hao, Yue; Zhang, Qi; Walker, Mark C.; van der Donk, Wilfred A.; Nair, Satish K.

    2015-01-01

    The lantibiotic nisin is an antimicrobial peptide that is widely used as a food preservative to combat food-borne pathogens1. Nisin contains dehydroalanine and dehydrobutyrine residues that are formed via dehydration of Ser/Thr by the lantibiotic dehydratase NisB2. Recent biochemical studies revealed that NisB glutamylates Ser/Thr side chains as part of the dehydration process3. However, the molecular mechanism by which NisB utilizes glutamate to catalyze dehydration remains unresolved. Here we show that this process involves glutamyl-tRNAGlu to activate Ser/Thr residues. In addition, the 2.9 Å crystal structure of NisB in complex with its substrate peptide NisA reveals the presence of two separate domains that catalyze the Ser/Thr glutamylation and glutamate elimination steps. The co-crystal structure also provides the first insights into substrate recognition by lantibiotic dehydratases. Our findings demonstrate a non-anticipated role for aminoacyl-tRNA in the formation of dehydroamino acids in lantibiotics, and serve as a basis for the functional characterization of the many lantibiotic-like dehydratases involved in the biosynthesis of other classes of natural products. PMID:25363770

  3. Acid-, base-, and lewis-acid-catalyzed heterolysis of methoxide from an alpha-hydroxy-beta-methoxy radical: models for reactions catalyzed by coenzyme B12-dependent diol dehydratase.

    PubMed

    Xu, Libin; Newcomb, Martin

    2005-11-11

    [Reaction: see text].A model for glycol radicals was employed in laser flash photolysis kinetic studies of catalysis of the fragmentation of a methoxy group adjacent to an alpha-hydroxy radical center. Photolysis of a phenylselenylmethylcyclopropane precursor gave a cyclopropylcarbinyl radical that rapidly ring opened to the target alpha-hydroxy-beta-methoxy radical (3). Heterolysis of the methoxy group in 3 gave an enolyl radical (4a) or an enol ether radical cation (4b), depending upon pH. Radicals 4 contain a 2,2-diphenylcyclopropane reporter group, and they rapidly opened to give UV-observable diphenylalkyl radicals as the final products. No heterolysis was observed for radical 3 under neutral conditions. In basic aqueous acetonitrile solutions, specific base catalysis of the heterolysis was observed; the pK(a) of radical 3 was determined to be 12.5 from kinetic titration plots, and the ketyl radical formed by deprotonation of 3 eliminated methoxide with a rate constant of 5 x 10(7) s(-1). In the presence of carboxylic acids in acetonitrile solutions, radical 3 eliminated methanol in a general acid-catalyzed reaction, and rate constants for protonation of the methoxy group in 3 by several acids were measured. Radical 3 also reacted by fragmentation of methoxide in Lewis-acid-catalyzed heterolysis reactions; ZnBr2, Sc(OTf)3, and BF3 were found to be efficient catalysts. Catalytic rate constants for the heterolysis reactions were in the range of 3 x 10(4) to 2 x 10(6) s(-1). The Lewis-acid-catalyzed heterolysis reactions are fast enough for kinetic competence in coenzyme B12 dependent enzyme-catalyzed reactions of glycols, and Lewis-acid-catalyzed cleavages of beta-ethers in radicals might be applied in synthetic reactions.

  4. 5-aminolevulinic acid induced protoporphyrin IX as a fluorescence marker for quantitative image analysis of high-grade dysplasia in Barrett's esophagus cellular models

    NASA Astrophysics Data System (ADS)

    Yeh, Shu-Chi Allison; Sahli, Samir; Andrews, David W.; Patterson, Michael S.; Armstrong, David; Provias, John; Fang, Qiyin

    2015-03-01

    Early detection and treatment of high-grade dysplasia (HGD) in Barrett's esophagus may reduce the risk of developing esophageal adenocarcinoma. Confocal endomicroscopy (CLE) has shown advantages over routine white-light endoscopic surveillance with biopsy for histological examination; however, CLE is compromised by insufficient contrast and by intra- and interobserver variation. An FDA-approved PDT photosensitizer was used here to reveal morphological and textural features similar to those found in histological analysis. Support vector machines were trained using the aforementioned features to obtain an automatic and robust detection of HGD. Our results showed 95% sensitivity and 87% specificity using the optimal feature combination and demonstrated the potential for extension to a three-dimensional cell model.

  5. In vitro activity of the nisin dehydratase NisB.

    PubMed

    Garg, Neha; Salazar-Ocampo, Luis M A; van der Donk, Wilfred A

    2013-04-30

    The biosynthesis of several classes of ribosomally synthesized and posttranslationally modified peptides involves dehydration of serine and threonine residues. For class I lantibiotics, thiopeptides, and goadsporin, this dehydration is catalyzed by lanthionine biosynthetic enzyme B (LanB) or LanB-like proteins. Although LanB proteins have been studied since 1992, in vitro reconstitution of their dehydration activity has been elusive. We show here the in vitro activity of the dehydratase involved in the biosynthesis of the food preservative nisin (NisB). In vitro, NisB dehydrated its substrate peptide NisA eight times in the presence of glutamate, ATP, Mg(2+), and the ribosomal/membrane fraction of bacterial cell extract. Mutation of 23 highly conserved residues of NisB identified a number of amino acids that are essential for dehydration activity. In addition, these mutagenesis studies identified three mutants, R786A, R826A, and H961A, that result in multiple glutamylations of the NisA substrate. Glutamylation was observed during both Escherichia coli coexpression of NisA with these mutants and in vitro assays. Treatment of the glutamylated substrate with WT NisB results in dehydrated NisA, suggesting that the glutamylated peptide is an intermediate in dehydration. Collectively, these studies suggest that dehydration involves glutamylation of the side chains of Ser and Thr followed by elimination. The latter step has precedent in the virginiamycin resistance protein virginiamycin B lyase. These studies will facilitate investigation of other LanB proteins involved in the biosynthesis of lantibiotics, thiopeptides, and goadsporin. PMID:23589847

  6. Ameliorative effects of ferulic Acid against lead acetate-induced oxidative stress, mitochondrial dysfunctions and toxicity in prepubertal rat brain.

    PubMed

    Lalith Kumar, Venkareddy; Muralidhara

    2014-12-01

    Epidemiological evidence has shown higher susceptibility of Children to the adverse effects of lead (Pb) exposure. However, experimental studies on Pb-induced neurotoxicity in prepubertal (PP) rats are limited. The present study aimed to examine the propensity of ferulic acid (FA), a commonly occurring phenolic acid in staple foods (fruits, vegetables, cereals, coffee etc.) to abrogate Pb-induced toxicity. Initially, we characterized Pb-induced adverse effects among PP rats exposed to Pb acetate (1,000-3,000 ppm in drinking water) for 5 weeks in terms of locomotor phenotype, activity of 5-aminolevulinic acid dehydratase (ALAD) in the blood, blood Pb levels and oxidative stress in brain regions. Further, the ameliorative effects of oral supplements of FA (25 mg/kg bw/day) were investigated in PP rats exposed to Pb (3,000 ppm). Pb intoxication increased the locomotor activity and FA supplements partially reversed the phenotype, while the reduced ALAD activity was also restored. FA significantly abrogated the enhanced oxidative stress in cerebellum (Cb) and hippocampus (Hc) as evidenced in terms of ROS generation, lipid peroxidation and protein carbonyls. Further, Pb-mediated perturbations in the glutathione levels and activity of enzymic antioxidants were also markedly restored. Furthermore, the protective effect of FA was discernible in striatum in terms of reduced oxidative stress, restored cholinergic activity and dopamine levels. Interestingly, reduced activity levels of mitochondrial complex I in Cb and enhanced levels in Hc among Pb-intoxicated rats were ameliorated by FA supplements. FA also decreased the number of damaged cells in cornu ammonis area CA1 and dentate gyrus as reflected by the histoarchitecture of Hc among Pb intoxicated rats. Collectively, our findings in the PP model allow us to hypothesize that ingestion of common phenolics such as FA may significantly alleviate the neurotoxic effects of Pb which may be largely attributed to its ability

  7. Redesign of MST enzymes to target lyase activity instead promotes mutase and dehydratase activities

    PubMed Central

    Meneely, Kathleen M.; Luo, Qianyi; Lamb, Audrey L.

    2013-01-01

    The isochorismate and salicylate synthases are members of the MST family of enzymes. The isochorismate synthases establish an equilibrium for the conversion chorismate to isochorismate and the reverse reaction. The salicylate synthases convert chorismate to salicylate with an isochorismate intermediate; therefore, the salicylate synthases perform isochorismate synthase and isochorismate-pyruvate lyase activities sequentially. While the active site residues are highly conserved, there are two sites that show trends for lyase-activity and lyase-deficiency. Using steady state kinetics and HPLC progress curves, we tested the “interchange” hypothesis that interconversion of the amino acids at these sites would promote lyase activity in the isochorismate synthases and remove lyase activity from the salicylate synthases. An alternative, “permute” hypothesis, that chorismate-utilizing enzymes are designed to permute the substrate into a variety of products and tampering with the active site may lead to identification of adventitious activities, is tested by more sensitive NMR time course experiments. The latter hypothesis held true. The variant enzymes predominantly catalyzed chorismate mutase-prephenate dehydratase activities, sequentially generating prephenate and phenylpyruvate, augmenting previously debated (mutase) or undocumented (dehydratase) adventitious activities. PMID:24055536

  8. Redesign of MST enzymes to target lyase activity instead promotes mutase and dehydratase activities.

    PubMed

    Meneely, Kathleen M; Luo, Qianyi; Lamb, Audrey L

    2013-11-01

    The isochorismate and salicylate synthases are members of the MST family of enzymes. The isochorismate synthases establish an equilibrium for the conversion chorismate to isochorismate and the reverse reaction. The salicylate synthases convert chorismate to salicylate with an isochorismate intermediate; therefore, the salicylate synthases perform isochorismate synthase and isochorismate-pyruvate lyase activities sequentially. While the active site residues are highly conserved, there are two sites that show trends for lyase-activity and lyase-deficiency. Using steady state kinetics and HPLC progress curves, we tested the "interchange" hypothesis that interconversion of the amino acids at these sites would promote lyase activity in the isochorismate synthases and remove lyase activity from the salicylate synthases. An alternative, "permute" hypothesis, that chorismate-utilizing enzymes are designed to permute the substrate into a variety of products and tampering with the active site may lead to identification of adventitious activities, is tested by more sensitive NMR time course experiments. The latter hypothesis held true. The variant enzymes predominantly catalyzed chorismate mutase-prephenate dehydratase activities, sequentially generating prephenate and phenylpyruvate, augmenting previously debated (mutase) or undocumented (dehydratase) adventitious activities.

  9. The iron-sulfur clusters of dehydratases are primary intracellular targets of copper toxicity.

    PubMed

    Macomber, Lee; Imlay, James A

    2009-05-19

    Excess copper is poisonous to all forms of life, and copper overloading is responsible for several human pathologic processes. The primary mechanisms of toxicity are unknown. In this study, mutants of Escherichia coli that lack copper homeostatic systems (copA cueO cus) were used to identify intracellular targets and to test the hypothesis that toxicity involves the action of reactive oxygen species. Low micromolar levels of copper were sufficient to inhibit the growth of both WT and mutant strains. The addition of branched-chain amino acids restored growth, indicating that copper blocks their biosynthesis. Indeed, copper treatment rapidly inactivated isopropylmalate dehydratase, an iron-sulfur cluster enzyme in this pathway. Other enzymes in this iron-sulfur dehydratase family were similarly affected. Inactivation did not require oxygen, in vivo or with purified enzyme. Damage occurred concomitant with the displacement of iron atoms from the solvent-exposed cluster, suggesting that Cu(I) damages these proteins by liganding to the coordinating sulfur atoms. Copper efflux by dedicated export systems, chelation by glutathione, and cluster repair by assembly systems all enhance the resistance of cells to this metal. PMID:19416816

  10. Mutation in the 4a-carbinolamine dehydratase gene leads to mild hyperphenylalaninemia with defective cofactor metabolism

    SciTech Connect

    Citron, B.A.; Kaufman, S.; Milstien, S.; Naylor, E.W. ); Greene, C.L. )

    1993-09-01

    Hyperphenylalaninemias represent a major class of inherited metabolic disorders. They are most often caused by mutations in the phenylalanine hydroxylase gene and, less frequently but with usually more serious consequences, in genes necessary for the synthesis and regeneration of the cofactor, tetrahydrobiopterin. This cofactor is absolutely required for all aromatic amino acid hydroxylations, and, recently, nitric oxide production from L-arginine has also been found to be dependent on tetrahydrobiopterin. Phenylalanine hydroxylase catalyzes a coupled reaction in which phenylalanine is converted to tyrosine and in which tetrahydrobiopterin is converted to the unstable carbinolamine, 4a-hydroxytetrahydrobiopterin. The enzyme, carbinolamine dehydratase, catalyzes the dehydration of the carbinolamine to quinonoid dihydropterin. A decreased rate of dehydration of this compound has been hypothesized to be responsible for the production of 7-biopterin found in certain mildly hyperphenylalaninemic individuals. The authors have now identified nonsense and missense mutations in the 4a-carbinolamine dehydratase gene in a hyperphenylalaninemic child who excretes large amounts of 7-biopterin. This finding is consistent with the role of the carbinolamine dehydratase in the phenylalanine hydroxylation reaction. Together with previously identified inherited disorders in phenylalanine hydroxylase and dihydropteridine reductase, there are now identified mutations in the three enzymes involved in the phenylalanine hydroxylation system. In addition, the genetics of this system may have broader implications, since the product of the dehydratase gene has previously been shown to play an additional role (as dimerization cofactor for hepatocyte nuclear factor-1[alpha]) in the regulation of transcription, through interaction with hepatocyte nuclear factor-1[alpha].

  11. Discovery of Function in the Enolase Superfamily: d-Mannonate and d-Gluconate Dehydratases in the d-Mannonate Dehydratase Subgroup

    PubMed Central

    2015-01-01

    The continued increase in the size of the protein sequence databases as a result of advances in genome sequencing technology is overwhelming the ability to perform experimental characterization of function. Consequently, functions are assigned to the vast majority of proteins via automated, homology-based methods, with the result that as many as 50% are incorrectly annotated or unannotated (Schnoes et al. PLoS Comput. Biol.2009, 5 (12), e100060520011109). This manuscript describes a study of the d-mannonate dehydratase (ManD) subgroup of the enolase superfamily (ENS) to investigate how function diverges as sequence diverges. Previously, one member of the subgroup had been experimentally characterized as ManD [dehydration of d-mannonate to 2-keto-3-deoxy-d-mannonate (equivalently, 2-keto-3-deoxy-d-gluconate)]. In this study, 42 additional members were characterized to sample sequence–function space in the ManD subgroup. These were found to differ in both catalytic efficiency and substrate specificity: (1) high efficiency (kcat/KM = 103 to 104 M–1 s–1) for dehydration of d-mannonate, (2) low efficiency (kcat/KM = 101 to 102 M–1 s–1) for dehydration of d-mannonate and/or D-gluconate, and 3) no-activity with either d-mannonate or d-gluconate (or any other acid sugar tested). Thus, the ManD subgroup is not isofunctional and includes d-gluconate dehydratases (GlcDs) that are divergent from the GlcDs that have been characterized in the mandelate racemase subgroup of the ENS (Lamble et al. FEBS Lett.2004, 576, 133–13615474024) (Ahmed et al. Biochem. J.2005, 390, 529–54015869466). These observations signal caution for functional assignment based on sequence homology and lay the foundation for the studies of the physiological functions of the GlcDs and the promiscuous ManDs/GlcDs. PMID:24697546

  12. X-ray structure of linalool dehydratase/isomerase from Castellaniella defragrans reveals enzymatic alkene synthesis.

    PubMed

    Weidenweber, Sina; Marmulla, Robert; Ermler, Ulrich; Harder, Jens

    2016-05-01

    Linalool dehydratase/isomerase (Ldi), an enzyme of terpene degradation in Castellaniella defragrans, isomerizes the primary monoterpene alcohol geraniol into the tertiary alcohol (S)-linalool and dehydrates (S)-linalool to the alkene β-myrcene. Here we report on the crystal structures of Ldi with and without terpene substrates, revealing a cofactor-free homopentameric enzyme. The substrates were embedded inside a hydrophobic channel between two monomers of the (α,α)6 barrel fold class and flanked by three clusters of polar residues involved in acid-base catalysis. The detailed view into the active site will guide future biotechnological applications of Ldi, in particular, for industrial butadiene and isoprene production from renewable sources.

  13. Crystal structure of substrate free form of glycerol dehydratase

    SciTech Connect

    Liao, Der-Ing; Dotson, Garry; Turner, Jr., Ivan; Reiss, Lisa; Emptage, Mark

    2010-03-08

    Glycerol dehydratase (GDH) and diol dehydratase (DDH) are highly homologous isofunctional enzymes that catalyze the elimination of water from glycerol and 1,2-propanediol (1,2-PD) to the corresponding aldehyde via a coenzyme B{sub 12}-dependent radical mechanism. The crystal structure of substrate free form of GDH in complex with cobalamin and K{sup +} has been determined at 2.5 {angstrom} resolution. Its overall fold and the subunit assembly closely resemble those of DDH. Comparison of this structure and the DDH structure, available only in substrate bound form, shows the expected change of the coordination of the essential K{sup +} from hexacoordinate to heptacoordinate with the displacement of a single coordinated water by the substrate diol. In addition, there appears to be an increase in the rigidity of the K{sup +} coordination (as measured by lower B values) upon the binding of the substrate. Structural analysis of the locations of conserved residues among various GDH and DDH sequences has aided in identification of residues potentially important for substrate preference or specificity of protein-protein interactions.

  14. Crystallization and X-ray diffraction analysis of an L-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii and a D-xylonate dehydratase from Caulobacter crescentus.

    PubMed

    Rahman, Mohammad Mubinur; Andberg, Martina; Koivula, Anu; Rouvinen, Juha; Hakulinen, Nina

    2016-08-01

    L-Arabinonate dehydratase (EC 4.2.1.25) and D-xylonate dehydratase (EC 4.2.1.82) are two enzymes that are involved in a nonphosphorylative oxidation pathway of pentose sugars. L-Arabinonate dehydratase converts L-arabinonate into 2-dehydro-3-deoxy-L-arabinonate, and D-xylonate dehydratase catalyzes the dehydration of D-xylonate to 2-dehydro-3-deoxy-D-xylonate. L-Arabinonate and D-xylonate dehydratases belong to the IlvD/EDD family, together with 6-phosphogluconate dehydratases and dihydroxyacid dehydratases. No crystal structure of any L-arabinonate or D-xylonate dehydratase is available in the PDB. In this study, recombinant L-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii (RlArDHT) and D-xylonate dehydratase from Caulobacter crescentus (CcXyDHT) were heterologously expressed in Escherichia coli and purified by the use of affinity chromatography followed by gel-filtration chromatography. The purified proteins were crystallized using the hanging-drop vapour-diffusion method at 293 K. Crystals of RlArDHT that diffracted to 2.40 Å resolution were obtained using sodium formate as a precipitating agent. They belonged to space group P21, with unit-cell parameters a = 106.07, b = 208.61, c = 147.09 Å, β = 90.43°. Eight RlArDHT molecules (two tetramers) in the asymmetric unit give a VM value of 3.2 Å(3) Da(-1) and a solvent content of 62%. Crystals of CcXyDHT that diffracted to 2.66 Å resolution were obtained using sodium formate and polyethylene glycol 3350. They belonged to space group C2, with unit-cell parameters a = 270.42, b = 236.13, c = 65.17 Å, β = 97.38°. Four CcXyDHT molecules (a tetramer) in the asymmetric unit give a VM value of 4.0 Å(3) Da(-1) and a solvent content of 69%. PMID:27487924

  15. Crystallization and X-ray diffraction analysis of an l-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii and a d-xylonate dehydratase from Caulobacter crescentus

    PubMed Central

    Rahman, Mohammad Mubinur; Andberg, Martina; Koivula, Anu; Rouvinen, Juha; Hakulinen, Nina

    2016-01-01

    l-Arabinonate dehydratase (EC 4.2.1.25) and d-xylonate dehydratase (EC 4.2.1.82) are two enzymes that are involved in a nonphosphorylative oxidation pathway of pentose sugars. l-Arabinonate dehydratase converts l-arabinonate into 2-dehydro-3-deoxy-l-arabinonate, and d-xylonate dehydratase catalyzes the dehydration of d-xylonate to 2-dehydro-3-deoxy-d-xylonate. l-Arabinonate and d-xylonate dehydratases belong to the IlvD/EDD family, together with 6-phosphogluconate dehydratases and dihydroxyacid dehydratases. No crystal structure of any l-arabinonate or d-xylonate dehydratase is available in the PDB. In this study, recombinant l-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii (RlArDHT) and d-xylonate dehydratase from Caulobacter crescentus (CcXyDHT) were heterologously expressed in Escherichia coli and purified by the use of affinity chromatography followed by gel-filtration chromatography. The purified proteins were crystallized using the hanging-drop vapour-diffusion method at 293 K. Crystals of RlArDHT that diffracted to 2.40 Å resolution were obtained using sodium formate as a precipitating agent. They belonged to space group P21, with unit-cell parameters a = 106.07, b = 208.61, c = 147.09 Å, β = 90.43°. Eight RlArDHT molecules (two tetramers) in the asymmetric unit give a V M value of 3.2 Å3 Da−1 and a solvent content of 62%. Crystals of CcXyDHT that diffracted to 2.66 Å resolution were obtained using sodium formate and polyethylene glycol 3350. They belonged to space group C2, with unit-cell parameters a = 270.42, b = 236.13, c = 65.17 Å, β = 97.38°. Four CcXyDHT molecules (a tetramer) in the asymmetric unit give a V M value of 4.0 Å3 Da−1 and a solvent content of 69%. PMID:27487924

  16. Crystallization and X-ray diffraction analysis of an L-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii and a D-xylonate dehydratase from Caulobacter crescentus.

    PubMed

    Rahman, Mohammad Mubinur; Andberg, Martina; Koivula, Anu; Rouvinen, Juha; Hakulinen, Nina

    2016-08-01

    L-Arabinonate dehydratase (EC 4.2.1.25) and D-xylonate dehydratase (EC 4.2.1.82) are two enzymes that are involved in a nonphosphorylative oxidation pathway of pentose sugars. L-Arabinonate dehydratase converts L-arabinonate into 2-dehydro-3-deoxy-L-arabinonate, and D-xylonate dehydratase catalyzes the dehydration of D-xylonate to 2-dehydro-3-deoxy-D-xylonate. L-Arabinonate and D-xylonate dehydratases belong to the IlvD/EDD family, together with 6-phosphogluconate dehydratases and dihydroxyacid dehydratases. No crystal structure of any L-arabinonate or D-xylonate dehydratase is available in the PDB. In this study, recombinant L-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii (RlArDHT) and D-xylonate dehydratase from Caulobacter crescentus (CcXyDHT) were heterologously expressed in Escherichia coli and purified by the use of affinity chromatography followed by gel-filtration chromatography. The purified proteins were crystallized using the hanging-drop vapour-diffusion method at 293 K. Crystals of RlArDHT that diffracted to 2.40 Å resolution were obtained using sodium formate as a precipitating agent. They belonged to space group P21, with unit-cell parameters a = 106.07, b = 208.61, c = 147.09 Å, β = 90.43°. Eight RlArDHT molecules (two tetramers) in the asymmetric unit give a VM value of 3.2 Å(3) Da(-1) and a solvent content of 62%. Crystals of CcXyDHT that diffracted to 2.66 Å resolution were obtained using sodium formate and polyethylene glycol 3350. They belonged to space group C2, with unit-cell parameters a = 270.42, b = 236.13, c = 65.17 Å, β = 97.38°. Four CcXyDHT molecules (a tetramer) in the asymmetric unit give a VM value of 4.0 Å(3) Da(-1) and a solvent content of 69%.

  17. RidA Proteins Prevent Metabolic Damage Inflicted by PLP-Dependent Dehydratases in All Domains of Life

    PubMed Central

    Lambrecht, Jennifer A.; Schmitz, George E.; Downs, Diana M.

    2013-01-01

    ABSTRACT Pyridoxal 5′-phosphate (PLP) is a coenzyme synthesized by all forms of life. Relevant to the work reported here is the mechanism of the PLP-dependent threonine/serine dehydratases, which generate reactive enamine/imine intermediates that are converted to keto acids by members of the RidA family of enzymes. The RidA protein of Salmonella enterica serovar Typhimurium LT2 is the founding member of this broadly conserved family of proteins (formerly known as YjgF/YER057c/UK114). RidA proteins were recently shown to be enamine deaminases. Here we demonstrate the damaging potential of enamines in the absence of RidA proteins. Notably, S. enterica strains lacking RidA have decreased activity of the PLP-dependent transaminase B enzyme IlvE, an enzyme involved in branched-chain amino acid biosynthesis. We reconstituted the threonine/serine dehydratase (IlvA)-dependent inhibition of IlvE in vitro, show that the in vitro system reflects the mechanism of RidA function in vivo, and show that IlvE inhibition is prevented by RidA proteins from all domains of life. We conclude that 2-aminoacrylate (2AA) inhibition represents a new type of metabolic damage, and this finding provides an important physiological context for the role of the ubiquitous RidA family of enamine deaminases in preventing damage by 2AA. PMID:23386433

  18. Purification, crystallization and preliminary X-ray diffraction analysis of a novel keto-deoxy-d-galactarate (KDG) dehydratase from Agrobacterium tumefaciens

    PubMed Central

    Taberman, Helena; Andberg, Martina; Parkkinen, Tarja; Richard, Peter; Hakulinen, Nina; Koivula, Anu; Rouvinen, Juha

    2014-01-01

    d-Galacturonic acid is the main component of pectin. It could be used to produce affordable renewable fuels, chemicals and materials through biotechnical conversion. Keto-deoxy-d-galactarate (KDG) dehydratase is an enzyme in the oxidative pathway of d-galacturonic acid in Agrobacterium tumefaciens (At). It converts 3-deoxy-2-keto-l-threo-hexarate to α-ketoglutaric semialdehyde. At KDG dehydratase was crystallized by the hanging-drop vapour-diffusion method. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 169.1, b = 117.8, c = 74.3 Å, β = 112.4° and an asymmetric unit of four monomers. X-ray diffraction data were collected to 1.9 Å resolution using synchrotron radiation. The three-dimensional structure of At KDG dehydratase will provide valuable information on the function of the enzyme and will allow it to be engineered for biorefinery-based applications. PMID:24419616

  19. Dehydratase mediated 1-propanol production in metabolically engineered Escherichia coli

    PubMed Central

    2011-01-01

    Background With the increasing consumption of fossil fuels, the question of meeting the global energy demand is of great importance in the near future. As an effective solution, production of higher alcohols from renewable sources by microorganisms has been proposed to address both energy crisis and environmental concerns. Higher alcohols contain more than two carbon atoms and have better physiochemical properties than ethanol as fuel substitutes. Results We designed a novel 1-propanol metabolic pathway by expanding the well-known 1,2-propanediol pathway with two more enzymatic steps catalyzed by a 1,2-propanediol dehydratase and an alcohol dehydrogenase. In order to engineer the pathway into E. coli, we evaluated the activities of eight different methylglyoxal synthases which play crucial roles in shunting carbon flux from glycolysis towards 1-propanol biosynthesis, as well as two secondary alcohol dehydrogenases of different origins that reduce both methylglyoxal and hydroxyacetone. It is evident from our results that the most active enzymes are the methylglyoxal synthase from Bacillus subtilis and the secondary alcohol dehydrogenase from Klebsiella pneumoniae, encoded by mgsA and budC respectively. With the expression of these two genes and the E. coli ydjG encoding methylglyoxal reductase, we achieved the production of 1,2-propanediol at 0.8 g/L in shake flask experiments. We then characterized the catalytic efficiency of three different diol dehydratases on 1,2-propanediol and identified the optimal one as the 1,2-propanediol dehydratase from Klebsiella oxytoca, encoded by the operon ppdABC. Co-expressing this enzyme with the above 1,2-propanediol pathway in wild type E. coli resulted in the production of 1-propanol at a titer of 0.25 g/L. Conclusions We have successfully established a new pathway for 1-propanol production by shunting the carbon flux from glycolysis. To our knowledge, it is the first time that this pathway has been utilized to produce 1

  20. Crystallization and preliminary X-ray diffraction analysis of prephenate dehydratase from Mycobacterium tuberculosis H37Rv

    SciTech Connect

    Vivan, Ana Luiza; Dias, Márcio Vinícius Bertacini; Azevedo, Walter Filgueira Jr de; Basso, Luiz Augusto Santos, Diógenes Santiago

    2006-04-01

    The M. tuberculosis prephenate dehydratase was cloned, expressed, purified, crystallized by the hanging-drop vapour-diffusion method, and a complete data set collected to 3.2 Å resolution using synchrotron radiation. These results should pave the way for the three-dimensional structure determination of the enzyme and provide a framework on which to base the rational design of chemotherapeutic agents to treat tuberculosis. Tuberculosis remains the leading cause of mortality arising from a bacterial pathogen (Mycobacterium tuberculosis). There is an urgent need for the development of new antimycobacterial agents. The aromatic amino-acid pathway is essential for the survival of this pathogen and represents a target for structure-based drug design. Accordingly, the M. tuberculosis prephenate dehydratase has been cloned, expressed, purified and crystallized by the hanging-drop vapour-diffusion method using PEG 400 as a precipitant. The crystal belongs to the orthorhombic space group I222 or I2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 98.26, b = 133.22, c = 225.01 Å, and contains four molecules in the asymmetric unit. A complete data set was collected to 3.2 Å resolution using a synchrotron-radiation source.

  1. Structural investigation of inhibitor designs targeting 3-dehydroquinate dehydratase from the shikimate pathway of Mycobacterium tuberculosis

    SciTech Connect

    Dias, Marcio V.B.; Snee, William C.; Bromfield, Karen M.; Payne, Richard J.; Palaninathan, Satheesh K.; Ciulli, Alessio; Howard, Nigel I.; Abell, Chris; Sacchettini, James C.; Blundell, Tom L.

    2011-09-06

    The shikimate pathway is essential in Mycobacterium tuberculosis and its absence from humans makes the enzymes of this pathway potential drug targets. In the present paper, we provide structural insights into ligand and inhibitor binding to 3-dehydroquinate dehydratase (dehydroquinase) from M. tuberculosis (MtDHQase), the third enzyme of the shikimate pathway. The enzyme has been crystallized in complex with its reaction product, 3-dehydroshikimate, and with six different competitive inhibitors. The inhibitor 2,3-anhydroquinate mimics the flattened enol/enolate reaction intermediate and serves as an anchor molecule for four of the inhibitors investigated. MtDHQase also forms a complex with citrazinic acid, a planar analogue of the reaction product. The structure of MtDHQase in complex with a 2,3-anhydroquinate moiety attached to a biaryl group shows that this group extends to an active-site subpocket inducing significant structural rearrangement. The flexible extensions of inhibitors designed to form {pi}-stacking interactions with the catalytic Tyr{sup 24} have been investigated. The high-resolution crystal structures of the MtDHQase complexes provide structural evidence for the role of the loop residues 19-24 in MtDHQase ligand binding and catalytic mechanism and provide a rationale for the design and efficacy of inhibitors.

  2. Characterization of D-xylonate dehydratase YjhG from Escherichia coli

    PubMed Central

    Jiang, Yudong; Liu, Wei; Cheng, Tao; Cao, Yujin; Zhang, Rubing; Xian, Mo

    2015-01-01

    D-xylonate dehydratase YjhG from Escherichia coli can convert D-xylonate into 2-keto-3-deoxy- D-xylonate (KDX), and is a key enzyme in the biosynthesis of 1,2,4-butanetriol and other chemicals. However, the biochemical properties of YjhG still remain unknown. In this study, the activity assay method for YjhG was established based on semicarbazide method, in which KDX reacts with semicarbazide reagent, and is further quantified by high-resolution mass spectrometry. The effect of reaction conditions on YjhG activity was determined in vitro using purified His-tagged YjhG protein. This enzyme showed maximal activity at 30°C and pH 8.0. Bivalent metal ions such as Mg2+ and Mn2+ activated, whereas Ni2+ and Zn2+ inhibited the activity of YjhG. Under optimal conditions, the Km and Vmax values were 4.88 mM and 78.62 μM l−1h−1, respectively, when using D-xylonate as a substrate. Amino acids sequence alignments and catalytic properties analysis revealed that YjhG might be a member of IlvD/EDD family. Results obtained in this study may lay a foundation for further investigation on YjhG and will benefit its application in biosynthesis of related chemicals. PMID:26083940

  3. Covalent Modification of the Mycobacterium tuberculosis FAS-II Dehydratase by Isoxyl and Thiacetazone

    PubMed Central

    2015-01-01

    Isoxyl (ISO) and thiacetazone (TAC) are two antitubercular prodrugs formerly used in the clinical treatment of tuberculosis. Although both prodrugs have recently been shown to kill Mycobacterium tuberculosis through the inhibition of the dehydration step of the type II fatty acid synthase pathway, their detailed mechanism of inhibition, the precise number of enzymes involved in their activation, and the nature of their activated forms remained unknown. This paper demonstrates that both ISO and TAC specifically and covalently react with a cysteine residue (Cys61) of the HadA subunit of the dehydratase, thereby inhibiting HadAB activity. The results unveil for the first time the nature of the active forms of ISO and TAC and explain the basis for the structure–activity relationship of and resistance to these thiourea prodrugs. The results further indicate that the flavin-containing monooxygenase EthA is most likely the only enzyme required for the activation of ISO and TAC in mycobacteria. PMID:25897434

  4. Oxidative Profile and δ-Aminolevulinate Dehydratase Activity in Healthy Pregnant Women with Iron Supplementation.

    PubMed

    De Lucca, Leidiane; Rodrigues, Fabiane; Jantsch, Letícia B; Neme, Walter S; Gallarreta, Francisco M P; Gonçalves, Thissiane L

    2016-05-03

    An oxidative burst occurs during pregnancy due to the large consumption of oxygen in the tissues and an increase in metabolic demands in response to maternal physiological changes and fetal growth. This study aimed to determine the oxidative profile and activity of δ-aminolevulinate dehydratase (δ-ALA-D) in pregnant women who received iron supplementation. Oxidative stress parameters were evaluated in 25 pregnant women with iron supplementation, 25 pregnant women without supplementation and 25 non-pregnant women. The following oxidative stress parameters were evaluated: thiobarbituric acid reactive substances (TBARS), protein thiol groups (P-SH), non-protein thiol levels (NP-SH), vitamin C levels, catalase and δ-ALA-D activity. Markers of oxidative stress and cell damage, such as TBARS in plasma were significantly higher in pregnant women without supplementation. Levels of P-SH, NP-SH and δ-ALA-D activity were significantly lower in pregnant women without supplementation compared to non-pregnant and pregnant women with supplementation, while vitamin C levels were significantly lower in pregnant women without supplementation when compared to non-pregnant women. The increase in the generation of oxidative species and decrease of antioxidants suggest the loss of physiological oxidative balance during normal pregnancy, which was not observed in pregnant women with iron supplementation, suggesting a protective effect of iron against oxidative damage.

  5. Insights into the Mechanism of Type I Dehydroquinate Dehydratases from Structures of Reaction Intermediates

    SciTech Connect

    Light, Samuel H.; Minasov, George; Shuvalova, Ludmilla; Duban, Mark-Eugene; Caffrey, Michael; Anderson, Wayne F.; Lavie, Arnon

    2012-02-27

    The biosynthetic shikimate pathway consists of seven enzymes that catalyze sequential reactions to generate chorismate, a critical branch point in the synthesis of the aromatic amino acids. The third enzyme in the pathway, dehydroquinate dehydratase (DHQD), catalyzes the dehydration of 3-dehydroquinate to 3-dehydroshikimate. We present three crystal structures of the type I DHQD from the intestinal pathogens Clostridium difficile and Salmonella enterica. Structures of the enzyme with substrate and covalent pre- and post-dehydration reaction intermediates provide snapshots of successive steps along the type I DHQD-catalyzed reaction coordinate. These structures reveal that the position of the substrate within the active site does not appreciably change upon Schiff base formation. The intermediate state structures reveal a reaction state-dependent behavior of His-143 in which the residue adopts a conformation proximal to the site of catalytic dehydration only when the leaving group is present. We speculate that His-143 is likely to assume differing catalytic roles in each of its observed conformations. One conformation of His-143 positions the residue for the formation/hydrolysis of the covalent Schiff base intermediates, whereas the other conformation positions the residue for a role in the catalytic dehydration event. The fact that the shikimate pathway is absent from humans makes the enzymes of the pathway potential targets for the development of non-toxic antimicrobials. The structures and mechanistic insight presented here may inform the design of type I DHQD enzyme inhibitors.

  6. Oxidative Profile and δ-Aminolevulinate Dehydratase Activity in Healthy Pregnant Women with Iron Supplementation

    PubMed Central

    De Lucca, Leidiane; Rodrigues, Fabiane; Jantsch, Letícia B.; Neme, Walter S.; Gallarreta, Francisco M. P.; Gonçalves, Thissiane L.

    2016-01-01

    An oxidative burst occurs during pregnancy due to the large consumption of oxygen in the tissues and an increase in metabolic demands in response to maternal physiological changes and fetal growth. This study aimed to determine the oxidative profile and activity of δ-aminolevulinate dehydratase (δ-ALA-D) in pregnant women who received iron supplementation. Oxidative stress parameters were evaluated in 25 pregnant women with iron supplementation, 25 pregnant women without supplementation and 25 non-pregnant women. The following oxidative stress parameters were evaluated: thiobarbituric acid reactive substances (TBARS), protein thiol groups (P-SH), non-protein thiol levels (NP-SH), vitamin C levels, catalase and δ-ALA-D activity. Markers of oxidative stress and cell damage, such as TBARS in plasma were significantly higher in pregnant women without supplementation. Levels of P-SH, NP-SH and δ-ALA-D activity were significantly lower in pregnant women without supplementation compared to non-pregnant and pregnant women with supplementation, while vitamin C levels were significantly lower in pregnant women without supplementation when compared to non-pregnant women. The increase in the generation of oxidative species and decrease of antioxidants suggest the loss of physiological oxidative balance during normal pregnancy, which was not observed in pregnant women with iron supplementation, suggesting a protective effect of iron against oxidative damage. PMID:27153075

  7. Understanding the mechanism of B(12)-dependent diol dehydratase: a synergistic retro-push--pull proposal.

    PubMed

    Smith, D M; Golding, B T; Radom, L

    2001-02-28

    Ab initio molecular orbital theory is used to investigate the coenzyme B(12)-dependent reactions catalyzed by diol dehydratase. The key step in such reactions is believed to be a 1,2-hydroxyl migration, which occurs within free-radical intermediates. The barrier for this migration, if unassisted, is calculated to be too high to be consistent with the observed reaction rate. However, we find that "pushing" the migrating hydroxyl, through interaction with a suitable acid, is able to provide significant catalysis. This is denoted retro-push catalysis, the retro prefix signifying that the motion of the migrating group is in the direction opposite to the electron motion. Similarly, the "pulling" of the migrating group, through interaction of the spectator hydroxyl with an appropriate base, is found to substantially reduce the rearrangement barrier. Importantly, the combination of these two effects results in a barrier reduction that is notably greater than additive. This synergistic interplay of the push and the pull provides an attractive means of catalysis. Our proposed retro-push--pull mechanism leads to results that are consistent with isotope-labeling experiments, with experimental rate data, and with the crystal structure of the enzyme.

  8. The structure of (3R)-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from Pseudomonas aeruginosa.

    PubMed

    Kimber, Matthew S; Martin, Fernando; Lu, Yingjie; Houston, Simon; Vedadi, Masoud; Dharamsi, Akil; Fiebig, Klaus M; Schmid, Molly; Rock, Charles O

    2004-12-10

    Type II fatty acid biosynthesis systems are essential for membrane formation in bacteria, making the constituent proteins of this pathway attractive targets for antibacterial drug discovery. The third step in the elongation cycle of the type II fatty acid biosynthesis is catalyzed by beta-hydroxyacyl-(acyl carrier protein) (ACP) dehydratase. There are two isoforms. FabZ, which catalyzes the dehydration of (3R)-hydroxyacyl-ACP to trans-2-acyl-ACP, is a universally expressed component of the bacterial type II system. FabA, the second isoform, as has more limited distribution in nature and, in addition to dehydration, also carries out the isomerization of trans-2- to cis-3-decenoyl-ACP as an essential step in unsaturated fatty acid biosynthesis. We report the structure of FabZ from the important human pathogen Pseudomonas aeruginosa at 2.5 A of resolution. PaFabZ is a hexamer (trimer of dimers) with the His/Glu catalytic dyad located within a deep, narrow tunnel formed at the dimer interface. Site-directed mutagenesis experiments showed that the obvious differences in the active site residues that distinguish the FabA and FabZ subfamilies of dehydratases do not account for the unique ability of FabA to catalyze isomerization. Because the catalytic machinery of the two enzymes is practically indistinguishable, the structural differences observed in the shape of the substrate binding channels of FabA and FabZ lead us to hypothesize that the different shapes of the tunnels control the conformation and positioning of the bound substrate, allowing FabA, but not FabZ, to catalyze the isomerization reaction.

  9. Evolution of Enzymatic Activities int he Enolase Superfamily: L-Talarate/Galactarate Dehydratase from Salmonella typhimurium LT2

    SciTech Connect

    Yew,W.; Fedorov, A.; Fedorov, E.; Almo, S.; Gerlt, J.

    2007-01-01

    We assigned L-talarate dehydratase (TalrD) and galactarate dehydratase (GalrD) functions to a group of orthologous proteins in the mechanistically diverse enolase superfamily, focusing our characterization on the protein encoded by the Salmonella typhimurium LT2 genome (GI:16766982; STM3697). Like the homologous mandelate racemase, L-fuconate dehydratase, and D-tartrate dehydratase, the active site of TalrD/GalrD contains a general acid/base Lys 197 at the end of the second {beta}-strand in the ({beta}/{alpha}){sub 7}{beta}-barrel domain, Asp 226, Glu 252, and Glu 278 as ligands for the essential Mg{sup 2+} at the ends of the third, fourth, and fifth {sup {beta}}-strands, a general acid/base His 328-Asp 301 dyad at the ends of the seventh and sixth {beta}-strands, and an electrophilic Glu 348 at the end of the eighth {beta}-strand. We discovered the function of STM3697 by screening a library of acid sugars; it catalyzes the efficient dehydration of both L-talarate (k{sub cat} = 2.1 s{sup -1}, k{sub cat}/K{sub m} = 9.1 x 10{sup 3} M{sup -1} s{sup -1}) and galactarate (k{sub cat} = 3.5 s{sup -1}, k{sub cat}/K{sub m} = 1.1 x 10{sup 4} M{sup -1} s{sup -1}). Because L-talarate is a previously unknown metabolite, we demonstrated that S. typhimurium LT2 can utilize L-talarate as carbon source. Insertional disruption of the gene encoding STM3697 abolishes this phenotype; this disruption also diminishes, but does not eliminate, the ability of the organism to utilize galactarate as carbon source. The dehydration of L-talarate is accompanied by competing epimerization to galactarate; little epimerization to L-talarate is observed in the dehydration of galactarate. On the basis of (1) structures of the wild type enzyme complexed with L-lyxarohydroxamate, an analogue of the enolate intermediate, and of the K197A mutant complexed with L-glucarate, a substrate for exchange of the {alpha}-proton, and (2) incorporation of solvent deuterium into galactarate in competition with

  10. Discovery of Selective Inhibitors of the Clostridium difficile Dehydroquinate Dehydratase

    PubMed Central

    Anderson, Wayne F.; Caffrey, Michael; Lavie, Arnon

    2014-01-01

    A vibrant and healthy gut flora is essential for preventing the proliferation of Clostridium difficile, a pathogenic bacterium that causes severe gastrointestinal symptoms. In fact, most C. difficile infections (CDIs) occur after broad-spectrum antibiotic treatment, which, by eradicating the commensal gut bacteria, allows its spores to proliferate. Hence, a C. difficile specific antibiotic that spares the gut flora would be highly beneficial in treating CDI. Towards this goal, we set out to discover small molecule inhibitors of the C. difficile enzyme dehydroquinate dehydratase (DHQD). DHQD is the 3rd of seven enzymes that compose the shikimate pathway, a metabolic pathway absent in humans, and is present in bacteria as two phylogenetically and mechanistically distinct types. Using a high-throughput screen we identified three compounds that inhibited the type I C. difficile DHQD but not the type II DHQD from Bacteroides thetaiotaomicron, a highly represented commensal gut bacterial species. Kinetic analysis revealed that the compounds inhibit the C. difficile enzyme with Ki values ranging from 10 to 20 µM. Unexpectedly, kinetic and biophysical studies demonstrate that inhibitors also exhibit selectivity between type I DHQDs, inhibiting the C. difficile but not the highly homologous Salmonella enterica DHQD. Therefore, the three identified compounds seem to be promising lead compounds for the development of C. difficile specific antibiotics. PMID:24586713

  11. A Conserved Surface Loop in Type I Dehydroquinate Dehydratases Positions an Active Site Arginine and Functions in Substrate Binding

    SciTech Connect

    Light, Samuel H.; Minasov, George; Shuvalova, Ludmilla; Peterson, Scott N.; Caffrey, Michael; Anderson, Wayne F.; Lavie, Arnon

    2012-04-18

    Dehydroquinate dehydratase (DHQD) catalyzes the third step in the biosynthetic shikimate pathway. We present three crystal structures of the Salmonella enterica type I DHQD that address the functionality of a surface loop that is observed to close over the active site following substrate binding. Two wild-type structures with differing loop conformations and kinetic and structural studies of a mutant provide evidence of both direct and indirect mechanisms of involvement of the loop in substrate binding. In addition to allowing amino acid side chains to establish a direct interaction with the substrate, closure of the loop necessitates a conformational change of a key active site arginine, which in turn positions the substrate productively. The absence of DHQD in humans and its essentiality in many pathogenic bacteria make the enzyme a target for the development of nontoxic antimicrobials. The structures and ligand binding insights presented here may inform the design of novel type I DHQD inhibiting molecules.

  12. LOCALIZATION OF POLYSOME-BOUND ALBUMIN AND SERINE DEHYDRATASE IN RAT LIVER CELL FRACTIONS

    PubMed Central

    Ikehara, Yukio; Pitot, Henry C.

    1973-01-01

    The polysomes involved in albumin and serine dehydratase synthesis were identified and localized by the binding to rat liver polysomes of anti-rat serum albumin and anti-serine dehydratase [125I]Fab dimer and monomer. Techniques were developed for the isolation of undegraded free and membrane-bound polysomes and for the preparation of [125I]Fab monomers and dimers from the IgG obtained from the antisera to the two proteins, rat serum albumin and serine dehydratase. The distribution of anti-rat serum albumin [125I]Fab dimer in the polysome profile is in accordance with the size of polysomes that are expected to be synthesizing albumin. By direct precipitation, it has been demonstrated that nascent chains isolated from the membrane-bound polysomes by puromycin were precipitated by anti-rat serum albumin-IgG at a level of 5–6 times those released from free polysomes. Anti-rat serum albumin-[125I]Fab dimer reacted with membrane-bound polysomes almost exclusively compared to the binding of nonimmune, control [125I]Fab dimer; a significant degree of binding of anti-rat serum albumin-[125I]Fab to free polysomes was also obtained. The [125I]Fab dimer made from normal control rabbit serum does not react with polysomes from liver at all and this preparation will not interact with polysomes extracted from tissues that do not synthesize rat serum albumin. Both anti-serine dehydratase-[125I]Fab monomer and dimer react with free and bound polysomes from livers of animals fed a chow diet or those fed a high 90% protein diet and given glucagon. In the latter instance, however, it is clear that the majority of the binding occurs to the bound polysomes. Furthermore, the specificity of this reaction may be further shown by the use of kidney polysomes that do not normally synthesize serine dehydratase. When these latter polysomes are isolated, even after the addition of crude and purified serine dehydratase, no reaction with anti-serine dehydratase-Fab fragments could be

  13. The Cryptic dsdA Gene Encodes a Functional D-Serine Dehydratase in Pseudomonas aeruginosa PAO1.

    PubMed

    Li, Guoqing; Lu, Chung-Dar

    2016-06-01

    D-Serine, an important neurotransmitter, also contributes to bacterial adaptation and virulence in humans. It was reported that Pseudomonas aeruginosa PAO1 can grow on D-serine as the sole nitrogen source, and growth was severely reduced in the dadA mutant devoid of the D-alanine dehydrogenase with broad substrate specificity. In this study, the dsdA gene (PA3357) encoding a putative D-serine dehydratase was subjected to further characterization. Growth on D-serine as the sole source of nitrogen was retained in the ∆dsdA mutant and was abolished completely in the ∆dadA and ∆dadA-∆dsdA mutants. However, when complemented by dsdA on a plasmid, the double mutant was able to grow on D-serine as the sole source of carbon and nitrogen, supporting the proposed biochemical function of DsdA in the conversion of D-serine into pyruvate and ammonia. Among D- and L-amino acids tested, only D-serine and D-threonine could serve as the substrates of DsdA, and the Km of DsdA with D-serine was calculated to be 330 μM. Comparative genomics revealed that this cryptic dsdA gene was highly conserved in strains of P. aeruginosa, and that most strains of Pseudomonas putida possess putative dsdCAX genes encoding a transcriptional regulator DsdC and a D-serine transporter DsdX as in enteric bacteria. In conclusion, this study supports the presence of a cryptic dsdA gene encoding a functional D-serine dehydratase in P. aeruginosa, and the absence of dsdA expression in response to exogenous D-serine might be due to the loss of regulatory elements for gene activation during evolution. PMID:26957519

  14. The Cryptic dsdA Gene Encodes a Functional D-Serine Dehydratase in Pseudomonas aeruginosa PAO1.

    PubMed

    Li, Guoqing; Lu, Chung-Dar

    2016-06-01

    D-Serine, an important neurotransmitter, also contributes to bacterial adaptation and virulence in humans. It was reported that Pseudomonas aeruginosa PAO1 can grow on D-serine as the sole nitrogen source, and growth was severely reduced in the dadA mutant devoid of the D-alanine dehydrogenase with broad substrate specificity. In this study, the dsdA gene (PA3357) encoding a putative D-serine dehydratase was subjected to further characterization. Growth on D-serine as the sole source of nitrogen was retained in the ∆dsdA mutant and was abolished completely in the ∆dadA and ∆dadA-∆dsdA mutants. However, when complemented by dsdA on a plasmid, the double mutant was able to grow on D-serine as the sole source of carbon and nitrogen, supporting the proposed biochemical function of DsdA in the conversion of D-serine into pyruvate and ammonia. Among D- and L-amino acids tested, only D-serine and D-threonine could serve as the substrates of DsdA, and the Km of DsdA with D-serine was calculated to be 330 μM. Comparative genomics revealed that this cryptic dsdA gene was highly conserved in strains of P. aeruginosa, and that most strains of Pseudomonas putida possess putative dsdCAX genes encoding a transcriptional regulator DsdC and a D-serine transporter DsdX as in enteric bacteria. In conclusion, this study supports the presence of a cryptic dsdA gene encoding a functional D-serine dehydratase in P. aeruginosa, and the absence of dsdA expression in response to exogenous D-serine might be due to the loss of regulatory elements for gene activation during evolution.

  15. Delta aminolevulinate dehydratase (ALA-D) activity in human and experimental diabetes mellitus.

    PubMed

    Fernández-Cuartero, B; Rebollar, J L; Batlle, A; Enriquez de Salamanca, R

    1999-01-01

    The haem pathway is impaired in porphyrias and a frequent coexistence of diabetes mellitus and porphyria disease has been reported. We have therefore decided to investigate delta-aminolevulinate dehydratase, one of the more sensitive enzymes in the haem pathway, in both human diabetic patients and diabetic rats. We have studied 131 diabetes mellitus patients, 32 insulin dependent and 99 non-insulin dependent. The latter group was further subdivided according to treatment: diet alone (n = 24), diet plus oral hypoglycemic agents (n = 28) and diet plus insulin (n = 47). We have also performed similar studies in the rat model of diabetes mellitus, induced in 11 Wistar rats by streptozotocin. Control groups of both humans and animals were used. Erythrocytic aminolevulinate dehydratase activity was reduced in both insulin dependent and non-insulin dependent diabetic patients as compared to their controls (p < 0.001). This activity was only partially restored by addition of zinc and thiols to the incubation media. In insulin-dependent diabetes mellitus, reduction of enzyme activity was related to the glycosilated hemoglobin concentration (p < 0.05) and in non-insulin dependent diabetes mellitus to the glycemia (p < 0.01). In the diabetic rat, aminolevulinate dehydratase activity was diminished on both erythrocytes (p < 0.01) and hepatic tissue (p < 0.01) when compared to the control group. The decrease in activity of erythrocyte aminolevulinate dehydratase observed in diabetic patients, may represent an additional and useful parameter for the assessment of the severity of carbohydrate metabolism impairment.

  16. The N-Terminal Region of the Medium Subunit (PduD) Packages Adenosylcobalamin-Dependent Diol Dehydratase (PduCDE) into the Pdu Microcompartment ▿

    PubMed Central

    Fan, Chenguang; Bobik, Thomas A.

    2011-01-01

    Salmonella enterica produces a proteinaceous microcompartment for B12-dependent 1,2-propanediol utilization (Pdu MCP). The Pdu MCP consists of catabolic enzymes encased within a protein shell, and its function is to sequester propionaldehyde, a toxic intermediate of 1,2-propanediol degradation. We report here that a short N-terminal region of the medium subunit (PduD) is required for packaging the coenzyme B12-dependent diol dehydratase (PduCDE) into the lumen of the Pdu MCP. Analysis of soluble cell extracts and purified MCPs by Western blotting showed that the PduD subunit mediated packaging of itself and other subunits of diol dehydratase (PduC and PduE) into the Pdu MCP. Deletion of 35 amino acids from the N terminus of PduD significantly impaired the packaging of PduCDE with minimal effects on its enzyme activity. Western blotting showed that fusing the 18 N-terminal amino acids of PduD to green fluorescent protein or glutathione S-transferase resulted in the association of these fusion proteins with the MCP. Immunoprecipitation tests indicated that the fusion proteins were encapsulated inside the MCP shell. PMID:21821773

  17. Evolution of Enzymatic Activities in the Enolase Superfamily: L-Fuconate Dehydratase from Xanthomonas campestris

    SciTech Connect

    Yew,W.; Fedorov, A.; Fedorov, E.; Rakus, J.; Pierce, R.; Almo, S.; Gerlt, J.

    2006-01-01

    Many members of the mechanistically diverse enolase superfamily have unknown functions. In this report the authors use both genome (operon) context and screening of a library of acid sugars to assign the L-fuconate dehydratase (FucD) function to a member of the mandelate racemase (MR) subgroup of the superfamily encoded by the Xanthomonas campestris pv. campestris str. ATCC 33913 genome (GI: 21233491). Orthologues of FucD are found in both bacteria and eukaryotes, the latter including the rTS beta protein in Homo sapiens that has been implicated in regulating thymidylate synthase activity. As suggested by sequence alignments and confirmed by high-resolution structures in the presence of active site ligands, FucD and MR share the same active site motif of functional groups: three carboxylate ligands for the essential Mg2+ located at the ends of th third, fourth, and fifth-strands in the (/)7-barrel domain (Asp 248, Glu 274, and Glu 301, respectively), a Lys-x-Lys motif at the end of the second-strand (Lys 218 and Lys 220), a His-Asp dyad at the end of the seventh and sixth-strands (His 351 and Asp 324, respectively), and a Glue at the end of the eighth-strand (Glu 382). The mechanism of the FucD reaction involves initial abstraction of the 2-proton by Lys 220, acid catalysis of the vinylogous-elimination of the 3-OH group by His 351, and stereospecific ketonization of the resulting 2-keto-3-deoxy-L-fuconate product. Screening of the library of acid sugars revealed substrate and functional promiscuity: In addition to L-fuconate, FucD also catalyzes the dehydration of L-galactonate, D-arabinonate, D-altronate, L-talonate, and D-ribonate. The dehydrations of L-fuconate, L-galactonate, and D-arabinonate are initiated by abstraction of the 2-protons by Lys 220. The dehydrations of L-talonate and D-ribonate are initiated by abstraction of the 2-protons by His 351; however, protonation of the enediolate intermediates by the conjugate acid of Lys 220 yields L

  18. Evolution of Enzymatic Activities in the Enolase Superfamily: D-Tartrate Dehydratase from Bradyrhizobium japonicum

    SciTech Connect

    Yew,W.; Fedorov, A.; Fedorov, E.; Wood, B.; Almo, S.; Gerlt, J.

    2006-01-01

    We focus on the assignment of function to and elucidation of structure-function relationships for a member of the mechanistically diverse enolase superfamily encoded by the Bradyrhizobium japonicum genome (bll6730; GI:27381841). As suggested by sequence alignments, the active site contains the same functional groups found in the active site of mandelate racemase (MR) that catalyzes a 1,1-proton transfer reaction: two acid/base catalysts, Lys 184 at the end of the second {beta}-strand, and a His 322-Asp 292 dyad at the ends of the seventh and sixth -strands, respectively, as well as ligands for an essential Mg{sup 2+}, Asp 213, Glu 239, and Glu 265 at the ends of the third, fourth, and fifth {beta}-strands, respectively. We screened a library of 46 acid sugars and discovered that only D-tartrate is dehydrated, yielding oxaloacetate as product. The kinetic constants (k{sub cat} = 7.3 s{sup -1}; k{sub cat}/K{sub M} = 8.5 x 10{sup 4} M{sup -1} s{sup -1}) are consistent with assignment of the D-tartrate dehydratase (TarD) function. The kinetic phenotypes of mutants as well as the structures of liganded complexes are consistent with a mechanism in which Lys 184 initiates the reaction by abstraction of the {alpha}-proton to generate a Mg{sup 2+}-stabilized enediolate intermediate, and the vinylogous -elimination of the 3-OH group is general acid-catalyzed by the His 322, accomplishing the anti-elimination of water. The replacement of the leaving group by solvent-derived hydrogen is stereorandom, suggesting that the enol tautomer of oxaloacetate is the product; this expectation was confirmed by its observation by {sup 1}H NMR spectroscopy. Thus, the TarD-catalyzed reaction is a 'simple' extension of the two-step reaction catalyzed by MR: base-catalyzed proton abstraction to generate a Mg{sup 2+}-stabilized enediolate intermediate followed by acid-catalyzed decomposition of that intermediate to yield the product.

  19. Evolution of Enzymatic Activities in the Enolase Superfamily: L-Rhamnonate Dehydratase

    SciTech Connect

    Rakus,J.; Fedorov, A.; Fedorov, E.; Glaner, M.; Hubbard, B.; Delli, J.; Babbitt, P.; Almo, S.; Gerlt, J.

    2008-01-01

    The l-rhamnonate dehydratase (RhamD) function was assigned to a previously uncharacterized family in the mechanistically diverse enolase superfamily that is encoded by the genome of Escherichia coli K-12. We screened a library of acid sugars to discover that the enzyme displays a promiscuous substrate specificity: l-rhamnonate (6-deoxy-l-mannonate) has the 'best' kinetic constants, with l-mannonate, l-lyxonate, and d-gulonate dehydrated less efficiently. Crystal structures of the RhamDs from both E. coli K-12 and Salmonella typhimurium LT2 (95% sequence identity) were obtained in the presence of Mg2+; the structure of the RhamD from S. typhimurium was also obtained in the presence of 3-deoxy-l-rhamnonate (obtained by reduction of the product with NaBH4). Like other members of the enolase superfamily, RhamD contains an N-terminal a + {beta} capping domain and a C-terminal ({beta}/a)7{beta}-barrel (modified TIM-barrel) catalytic domain with the active site located at the interface between the two domains. In contrast to other members, the specificity-determining '20s loop' in the capping domain is extended in length and the '50s loop' is truncated. The ligands for the Mg2+ are Asp 226, Glu 252 and Glu 280 located at the ends of the third, fourth and fifth {beta}-strands, respectively. The active site of RhamD contains a His 329-Asp 302 dyad at the ends of the seventh and sixth {beta}-strands, respectively, with His 329 positioned to function as the general base responsible for abstraction of the C2 proton of l-rhamnonate to form a Mg2+-stabilized enediolate intermediate. However, the active site does not contain other acid/base catalysts that have been implicated in the reactions catalyzed by other members of the MR subgroup of the enolase superfamily. Based on the structure of the liganded complex, His 329 also is expected to function as the general acid that both facilitates departure of the 3-OH group in a syn-dehydration reaction and delivers a proton to carbon-3

  20. RNAi Suppression of Arogenate Dehydratase1 Reveals That Phenylalanine Is Synthesized Predominantly via the Arogenate Pathway in Petunia Petals[C][W

    PubMed Central

    Maeda, Hiroshi; Shasany, Ajit K; Schnepp, Jennifer; Orlova, Irina; Taguchi, Goro; Cooper, Bruce R.; Rhodes, David; Pichersky, Eran; Dudareva, Natalia

    2010-01-01

    l-Phe, a protein building block and precursor of numerous phenolic compounds, is synthesized from prephenate via an arogenate and/or phenylpyruvate route in which arogenate dehydratase (ADT) or prephenate dehydratase, respectively, plays a key role. Here, we used Petunia hybrida flowers, which are rich in Phe-derived volatiles, to determine the biosynthetic routes involved in Phe formation in planta. Of the three identified petunia ADTs, expression of ADT1 was the highest in petunia petals and positively correlated with endogenous Phe levels throughout flower development. ADT1 showed strict substrate specificity toward arogenate, although with the lowest catalytic efficiency among the three ADTs. ADT1 suppression via RNA interference in petunia petals significantly reduced ADT activity, levels of Phe, and downstream phenylpropanoid/benzenoid volatiles. Unexpectedly, arogenate levels were unaltered, while shikimate and Trp levels were decreased in transgenic petals. Stable isotope labeling experiments showed that ADT1 suppression led to downregulation of carbon flux toward shikimic acid. However, an exogenous supply of shikimate bypassed this negative regulation and resulted in elevated arogenate accumulation. Feeding with shikimate also led to prephenate and phenylpyruvate accumulation and a partial recovery of the reduced Phe level in transgenic petals, suggesting that the phenylpyruvate route can also operate in planta. These results provide genetic evidence that Phe is synthesized predominantly via arogenate in petunia petals and uncover a novel posttranscriptional regulation of the shikimate pathway. PMID:20215586

  1. Computation-Facilitated Assignment of the Function in the Enolase Superfamily: A Regiochemically Distinct Galactarate Dehydratase from Oceanobacillus iheyensis

    SciTech Connect

    Rakus, J.; Kalyanaraman, C; Fedorov, A; Fedorov, E; Mills-Groninger, F; Burley, S; Almo, S; Jacobson, M; Gerlt, J; et. al.

    2009-01-01

    The structure of an uncharacterized member of the enolase superfamily from Oceanobacillus iheyensis (GI 23100298, IMG locus tag Ob2843, PDB entry 2OQY) was determined by the New York SGX Research Center for Structural Genomics (NYSGXRC). The structure contained two Mg{sup 2+} ions located 10.4 {angstrom} from one another, with one located in the canonical position in the ({beta}/{alpha})7{beta}-barrel domain (although the ligand at the end of the fifth {beta}-strand is His, unprecedented in structurally characterized members of the superfamily); the second is located in a novel site within the capping domain. In silico docking of a library of mono- and diacid sugars to the active site predicted a diacid sugar as a likely substrate. Activity screening of a physical library of acid sugars identified galactarate as the substrate (k{sub cat} = 6.8 s{sup -1}, K{sub M} = 620 {micro}M, k{sub cat}/K{sub M} = 1.1 x 10{sup 4} M{sup -1} s{sup -1}), allowing functional assignment of Ob2843 as galactarate dehydratase (GalrD-II). The structure of a complex of the catalytically impaired Y90F mutant with Mg{sup 2+} and galactarate allowed identification of a Tyr 164-Arg 162 dyad as the base that initiates the reaction by abstraction of the {alpha}-proton and Tyr 90 as the acid that facilitates departure of the {beta}-OH leaving group. The enzyme product is 2-keto-d-threo-4,5-dihydroxyadipate, the enantiomer of the product obtained in the GalrD reaction catalyzed by a previously characterized bifunctional L-talarate/galactarate dehydratase (TalrD/GalrD). On the basis of the different active site structures and different regiochemistries, we recognize that these functions represent an example of apparent, not actual, convergent evolution of function. The structure of GalrD-II and its active site architecture allow identification of the seventh functionally and structurally characterized subgroup in the enolase superfamily. This study provides an additional example in which an

  2. Structural Studies of FlaA1 from Helicobacter Pylori Reveal the Mechanism for Inverting 4,6-dehydratase Activity

    SciTech Connect

    Ishiyama,N.; Creuzenet, C.; Miller, W.; Demendi, M.; Anderson, E.; Harauz, G.; Lam, J.; Berghuis, A.

    2006-01-01

    FlaA1 from the human pathogen Helicobacter pylori is an enzyme involved in saccharide biosynthesis that has been shown to be essential for pathogenicity. Here we present five crystal structures of FlaA1 in the presence of substrate, inhibitors, and bound cofactor, with resolutions ranging from 2.8 to 1.9 {angstrom}. These structures reveal that the enzyme is a novel member of the short-chain dehydrogenase/reductase superfamily. Additional electron microscopy studies show the enzyme to possess a hexameric doughnut-shaped quaternary structure. NMR analyses of 'real time' enzyme-substrate reactions indicate that FlaA1 is a UDP-GlcNAc-inverting 4,6-dehydratase, suggesting that the enzyme catalyzes the first step in the biosynthetic pathway of a pseudaminic acid derivative, which is implicated in protein glycosylation. Guided by evidence from site-directed mutagenesis and computational simulations, a three-step reaction mechanism is proposed that involves Lys-133 functioning as both a catalytic acid and base.

  3. Structure of Glycerol Dehydratase Reactivase: A New Type of Molecular Chaperone

    SciTech Connect

    Liao, Der-Ing; Reiss, Lisa; Turner, Jr., Ivan; Dotson, Garry

    2010-03-08

    The function of glycerol dehydratase (GDH) reactivase is to remove damaged coenzyme B{sub 12} from GDH that has suffered mechanism-based inactivation. The structure of GDH reactivase from Klebsiella pneumoniae was determined at 2.4 {angstrom} resolution by the single isomorphous replacement with anomalous signal (SIR/AS) method. Each tetramer contains two elongated 63 kDa {alpha} subunits and two globular 14 kDa {beta} subunits. The {alpha} subunit contains structural features resembling both GroEL and Hsp70 groups of chaperones, and it appears chaperone like in its interactions with ATP. The fold of the {beta} subunit resembles that of the {beta} subunit of glycerol dehydratase, except that it lacks some coenzyme B12 binding elements. A hypothesis for the reactivation mechanism of reactivase is proposed based on these structural features.

  4. Identification of a small protein domain present in all plant lineages that confers high prephenate dehydratase activity.

    PubMed

    El-Azaz, Jorge; de la Torre, Fernando; Ávila, Concepción; Cánovas, Francisco M

    2016-07-01

    l-Phenylalanine serves as a building block for the biosynthesis of proteins, but also as a precursor for a wide range of plant-derived compounds essential for plants and animals. Plants can synthesize Phe within the plastids using arogenate as a precursor; however, an alternative pathway using phenylpyruvate as an intermediate, described for most microorganisms, has recently been proposed. The functionality of this pathway requires the existence of enzymes with prephenate dehydratase (PDT) activity (EC 4.2.1.51) in plants. Using phylogenetic studies, functional complementation assays in yeast and biochemical analysis, we have identified the enzymes displaying PDT activity in Pinus pinaster. Through sequence alignment comparisons and site-directed mutagenesis we have identified a 22-amino acid region conferring PDT activity (PAC domain) and a single Ala314 residue critical to trigger this activity. Our results demonstrate that all plant clades include PAC domain-containing ADTs, suggesting that the PDT activity, and thus the ability to synthesize Phe using phenylpyruvate as an intermediate, has been preserved throughout the evolution of plants. Moreover, this pathway together with the arogenate pathway gives plants a broad and versatile capacity to synthesize Phe and its derived compounds. PAC domain-containing enzymes are also present in green and red algae, and glaucophytes, the three emerging clades following the primary endosymbiont event resulting in the acquisition of plastids in eukaryotes. The evolutionary prokaryotic origin of this domain is discussed. PMID:27125254

  5. Isolation and characterization of cDNAs encoding imidazoleglycerolphosphate dehydratase from Arabidopsis thaliana.

    PubMed Central

    Tada, S; Volrath, S; Guyer, D; Scheidegger, A; Ryals, J; Ohta, D; Ward, E

    1994-01-01

    cDNA clones encoding imidazoleglycerolphosphate dehydratase (IGPD; EC 4.2.1.19) from Arabidopsis thaliana were isolated by complementation of a bacterial auxotroph. The predicted primary translation product shared significant identity with the corresponding sequences from bacteria and fungi. As in yeast, the plant enzyme is monofunctional, lacking the histidinol phosphatase activity present in the Escherichia coli protein. IGPD mRNA was present in major organs at all developmental stages assayed. The Arabidopsis genome appears to contain two genes encoding this enzyme, based on DNA gel blot and polymerase chain reaction analysis. PMID:8066131

  6. The reaction mechanism for dehydration process catalyzed by type I dehydroquinate dehydratase from Gram-negative Salmonella enterica

    NASA Astrophysics Data System (ADS)

    Yao, Yuan; Li, Ze-Sheng

    2012-01-01

    The fundamental reaction mechanism for the dehydration process catalyzed by type I dehydroquinate dehydratase from Gram-negative Salmonella enterica has been studied by density functional theory calculations. The results indicate that the dehydration process undergoes a two-step cis-elimination mechanism, which is different from the previously proposed one. The catalytic roles of both the highly conserved residue His143 and the Schiff base formed between the substrate and Lys170 have also been elucidated. The structural and mechanistic insight presented here may direct the design of type I dehydroquinate dehydratase enzyme inhibitors as non-toxic antimicrobials, anti-fungals, and herbicides.

  7. Modulation of chlorophyll biosynthesis by water stress in rice seedlings during chloroplast biogenesis.

    PubMed

    Dalal, Vijay K; Tripathy, Baishnab C

    2012-09-01

    To understand the impact of water stress on the greening process, water stress was applied to 6-day-old etiolated seedlings of a drought-sensitive cultivar of rice (Oryza sativa), Pusa Basmati-1 by immersing their roots in 40 mm polyethylene glycol (PEG) 6000 (-0.69 MPa) or 50 mm PEG 6000 (-1.03 MPa) dissolved in half-strength Murashige and Skoog (MS)-nutrient-solution, 16 h prior to transfer to cool-white-fluorescent + incandescent light. Chlorophyll (Chl) accumulation substantially declined in developing water-stressed seedlings. Reduced Chl synthesis was due to decreased accumulation of chlorophyll biosynthetic intermediates, that is, glutamate-1-semialdehyde (GSA), 5-aminolevulinic acid, Mg-protoporphyrin IX monomethylester and protochlorophyllide. Although 5-aminolevulinic acid synthesis decreased, the gene expression and protein abundance of the enzyme responsible for its synthesis, GSA aminotransferase, increased, suggesting its crucial role in the greening process in stressful environment. The biochemical activities of Chl biosynthetic enzymes, that is, 5-aminolevulinic acid dehydratase, porphobilinogen deaminase, coproporphyrinogen III oxidase, porphyrinogen IX oxidase, Mg-chelatase and protochlorophyllide oxidoreductase, were down-regulated due to their reduced protein abundance/gene expression in water-stressed seedlings. Down-regulation of protochlorophyllide oxidoreductase resulted in impaired Shibata shift. Our results demonstrate that reduced synthesis of early intermediates, that is, GSA and 5-aminolevulinic acid, could modulate the gene expression of later enzymes of Chl biosynthesis pathway.

  8. Purification, crystallization and preliminary crystallographic analysis of Arabidopsis thaliana imidazoleglycerol-phosphate dehydratase

    SciTech Connect

    Glynn, Steven E.; Baker, Patrick J.; Sedelnikova, Svetlana E.; Levy, Colin W.; Rodgers, H. Fiona; Blank, Jutta; Hawkes, Timothy R.; Rice, David W.

    2005-08-01

    Imidazoleglycerol-phosphate dehydratase from A. thaliana has been overexpressed, purified and crystallized and data have been collected to 3 Å resolution. Imidazoleglycerol-phosphate dehydratase catalyses the sixth step of the histidine-biosynthesis pathway in plants and microorganisms and has been identified as a possible target for the development of novel herbicides. Arabidopsis thaliana IGPD has been cloned and overexpressed in Escherichia coli, purified and subsequently crystallized in the presence of manganese. Under these conditions, the inactive trimeric form of the metal-free enzyme is assembled into a fully active species consisting of a 24-mer exhibiting 432 symmetry. X-ray diffraction data have been collected to 3.0 Å resolution from a single crystal at 293 K. The crystal belongs to space group R3, with approximate unit-cell parameters a = b = 157.9, c = 480.0 Å, α = β = 90, γ = 120° and with either 16 or 24 subunits in the asymmetric unit. A full structure determination is under way in order to provide insights into the mode of subunit assembly and to initiate a programme of rational herbicide design.

  9. Dual Fatty Acid Elongase Complex Interactions in Arabidopsis.

    PubMed

    Morineau, Céline; Gissot, Lionel; Bellec, Yannick; Hematy, Kian; Tellier, Frédérique; Renne, Charlotte; Haslam, Richard; Beaudoin, Frédéric; Napier, Johnathan; Faure, Jean-Denis

    2016-01-01

    Very long chain fatty acids (VLCFAs) are involved in plant development and particularly in several cellular processes such as membrane trafficking, cell division and cell differentiation. However, the precise role of VLCFAs in these different cellular processes is still poorly understood in plants. In order to identify new factors associated with the biosynthesis or function of VLCFAs, a yeast multicopy suppressor screen was carried out in a yeast mutant strain defective for fatty acid elongation. Loss of function of the elongase 3 hydroxyacyl-CoA dehydratase PHS1 in yeast and PASTICCINO2 in plants prevents growth and induces cytokinesis defects. PROTEIN TYROSIN PHOSPHATASE-LIKE (PTPLA) previously characterized as an inactive dehydratase was able to restore yeast phs1 growth and VLCFAs elongation but not the plant pas2-1 defects. PTPLA interacted with elongase subunits in the Endoplasmic Reticulum (ER) and its absence induced the accumulation of 3-hydroxyacyl-CoA as expected from a dehydratase involved in fatty acid (FA) elongation. However, loss of PTPLA function increased VLCFA levels, an effect that was dependent on the presence of PAS2 indicating that PTPLA activity repressed FA elongation. The two dehydratases have specific expression profiles in the root with PAS2, mostly restricted to the endodermis, while PTPLA was confined in the vascular tissue and pericycle cells. Comparative ectopic expression of PTPLA and PAS2 in their respective domains confirmed the existence of two independent elongase complexes based on PAS2 or PTPLA dehydratase that are functionally interacting. PMID:27583779

  10. Dual Fatty Acid Elongase Complex Interactions in Arabidopsis

    PubMed Central

    Morineau, Céline; Gissot, Lionel; Bellec, Yannick; Hematy, Kian; Tellier, Frédérique; Renne, Charlotte; Haslam, Richard; Beaudoin, Frédéric; Napier, Johnathan; Faure, Jean-Denis

    2016-01-01

    Very long chain fatty acids (VLCFAs) are involved in plant development and particularly in several cellular processes such as membrane trafficking, cell division and cell differentiation. However, the precise role of VLCFAs in these different cellular processes is still poorly understood in plants. In order to identify new factors associated with the biosynthesis or function of VLCFAs, a yeast multicopy suppressor screen was carried out in a yeast mutant strain defective for fatty acid elongation. Loss of function of the elongase 3 hydroxyacyl-CoA dehydratase PHS1 in yeast and PASTICCINO2 in plants prevents growth and induces cytokinesis defects. PROTEIN TYROSIN PHOSPHATASE-LIKE (PTPLA) previously characterized as an inactive dehydratase was able to restore yeast phs1 growth and VLCFAs elongation but not the plant pas2-1 defects. PTPLA interacted with elongase subunits in the Endoplasmic Reticulum (ER) and its absence induced the accumulation of 3-hydroxyacyl-CoA as expected from a dehydratase involved in fatty acid (FA) elongation. However, loss of PTPLA function increased VLCFA levels, an effect that was dependent on the presence of PAS2 indicating that PTPLA activity repressed FA elongation. The two dehydratases have specific expression profiles in the root with PAS2, mostly restricted to the endodermis, while PTPLA was confined in the vascular tissue and pericycle cells. Comparative ectopic expression of PTPLA and PAS2 in their respective domains confirmed the existence of two independent elongase complexes based on PAS2 or PTPLA dehydratase that are functionally interacting. PMID:27583779

  11. Substrate-Induced Radical Formation in 4-Hydroxybutyryl Coenzyme A Dehydratase from Clostridium aminobutyricum

    PubMed Central

    Zhang, Jin; Friedrich, Peter; Pierik, Antonio J.; Martins, Berta M.

    2014-01-01

    4-Hydroxybutyryl-coenzyme A (CoA) dehydratase (4HBD) from Clostridium aminobutyricum catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA and the irreversible isomerization of vinylacetyl-CoA to crotonyl-CoA. 4HBD is an oxygen-sensitive homotetrameric enzyme with one [4Fe-4S]2+ cluster and one flavin adenine dinucleotide (FAD) in each subunit. Upon the addition of crotonyl-CoA or the analogues butyryl-CoA, acetyl-CoA, and CoA, UV-visible light and electron paramagnetic resonance (EPR) spectroscopy revealed an internal one-electron transfer to FAD and the [4Fe-4S]2+ cluster prior to hydration. We describe an active recombinant 4HBD and variants produced in Escherichia coli. The variants of the cluster ligands (H292C [histidine at position 292 is replaced by cysteine], H292E, C99A, C103A, and C299A) had no measurable dehydratase activity and were composed of monomers, dimers, and tetramers. Variants of other potential catalytic residues were composed only of tetramers and exhibited either no measurable (E257Q, E455Q, and Y296W) hydratase activity or <1% (Y296F and T190V) dehydratase activity. The E455Q variant but not the Y296F or E257Q variant displayed the same spectral changes as the wild-type enzyme after the addition of crotonyl-CoA but at a much lower rate. The results suggest that upon the addition of a substrate, Y296 is deprotonated by E455 and reduces FAD to FADH·, aided by protonation from E257 via T190. In contrast to FADH·, the tyrosyl radical could not be detected by EPR spectroscopy. FADH· appears to initiate the radical dehydration via an allylic ketyl radical that was proposed 19 years ago. The mode of radical generation in 4HBD is without precedent in anaerobic radical chemistry. It differs largely from that in enzymes, which use coenzyme B12, S-adenosylmethionine, ATP-driven electron transfer, or flavin-based electron bifurcation for this purpose. PMID:25452282

  12. Substrate-induced radical formation in 4-hydroxybutyryl coenzyme A dehydratase from Clostridium aminobutyricum.

    PubMed

    Zhang, Jin; Friedrich, Peter; Pierik, Antonio J; Martins, Berta M; Buckel, Wolfgang

    2015-02-01

    4-Hydroxybutyryl-coenzyme A (CoA) dehydratase (4HBD) from Clostridium aminobutyricum catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA and the irreversible isomerization of vinylacetyl-CoA to crotonyl-CoA. 4HBD is an oxygen-sensitive homotetrameric enzyme with one [4Fe-4S](2+) cluster and one flavin adenine dinucleotide (FAD) in each subunit. Upon the addition of crotonyl-CoA or the analogues butyryl-CoA, acetyl-CoA, and CoA, UV-visible light and electron paramagnetic resonance (EPR) spectroscopy revealed an internal one-electron transfer to FAD and the [4Fe-4S](2+) cluster prior to hydration. We describe an active recombinant 4HBD and variants produced in Escherichia coli. The variants of the cluster ligands (H292C [histidine at position 292 is replaced by cysteine], H292E, C99A, C103A, and C299A) had no measurable dehydratase activity and were composed of monomers, dimers, and tetramers. Variants of other potential catalytic residues were composed only of tetramers and exhibited either no measurable (E257Q, E455Q, and Y296W) hydratase activity or <1% (Y296F and T190V) dehydratase activity. The E455Q variant but not the Y296F or E257Q variant displayed the same spectral changes as the wild-type enzyme after the addition of crotonyl-CoA but at a much lower rate. The results suggest that upon the addition of a substrate, Y296 is deprotonated by E455 and reduces FAD to FADH·, aided by protonation from E257 via T190. In contrast to FADH·, the tyrosyl radical could not be detected by EPR spectroscopy. FADH· appears to initiate the radical dehydration via an allylic ketyl radical that was proposed 19 years ago. The mode of radical generation in 4HBD is without precedent in anaerobic radical chemistry. It differs largely from that in enzymes, which use coenzyme B12, S-adenosylmethionine, ATP-driven electron transfer, or flavin-based electron bifurcation for this purpose.

  13. Structure of EvaA: a paradigm for sugar 2,3-dehydratases.

    PubMed

    Kubiak, Rachel L; Thoden, James B; Holden, Hazel M

    2013-03-26

    Unusual deoxysugars found appended to natural products often provide or enhance the pharmacokinetic activities of the parent compound. The preferred carbohydrate donors for the biosynthesis of such glycosylated natural products are the dTDP-linked sugars. Many of the biologically relevant dTDP-deoxysugars are constructed around the 2,6-dideoxyhexoses or the 2,3(4),6-trideoxyhexoses. A key step in the biosynthesis of these sugars is the removal of the hexose C-2' hydroxyl group and the oxidation of the C-3' hydroxyl group to a carbonyl moiety. Enzymes that catalyze these reactions are referred to as 2,3-dehydratases and have been, for the most part, largely uncharacterized. Here we report the first structural analysis of a sugar 2,3-dehydratase. For this investigation, the enzyme, EvaA, was cloned from Amycolatopsis orientalis, and the structure was solved and refined to a nominal resolution of 1.7 Å. On the basis of the resulting model, it is clear that EvaA belongs to the large Nudix hydrolase superfamily and is most similar to GDP-mannose hydrolase. Each subunit of the EvaA dimer folds into two domains that clearly arose via gene duplication. Two dTDP-sugar binding pockets, A and B, are present in each EvaA subunit. On the basis of site-directed mutagenesis experiments and activity assays, it appears that pocket A functions as the active site and pocket B is simply a remnant left behind from the gene duplication event. As 2,3-dehydration is crucial for the biosynthesis of many unusual deoxysugars, this investigation provides key structural insight into this widely conserved reaction. PMID:23473392

  14. [Certain properties of "biosynthetic" L-threonine dehydratase from subcellular structures of brewers' yeast Saccharomyces carlsbergensis].

    PubMed

    Kovaleva, S V; Korozhko, A I; Beliaeva, N F; Kagan, Z S

    1981-01-01

    The paper is concerned with kinetic properties of the "biosynthetic" L-threonine dehydratase (EC 4.2.1.16) solubilized from subcellular structures of brewers' yeast Saccharomyces carlsbergensis in the absence and presence of the allosteric inhibitor, L-isoleucine, at three pH-values (pH 6.5, 7.8 and 9.5). The curve of the initial reaction rate versus initial substrate concentration in the absence of L-isoleucine at pH 6.5 was of hyperbolic character (Km = 5.5.10(-2) M), and at pH 7.8 and 9.5 the kinetic curve had a weakly sigmoidal pattern with a sharp going into the saturation plateaux; the values of [S] 0.5 are 1.10(-2) and 8.7.10(-3) M, respectively. An addition of L-isoleucine to the reaction mixture led to the appearance (at pH 6.5) or to an increase (at pH 7.8 and 9.5) of the sigmoidality of these kinetic curves and to a decrease in values of the maximum reaction rate V. The enzyme sensibility to the inhibitory effect of L-isoleucine decreased with an increase in pH values. Low L-isoleucine concentrations at low substrate concentrations activated the enzyme. The pH optimum for L-threonine dehydratase under study was 9.5-10.0. The enzyme molecular weight is about 300 000.

  15. β-Hydroxyacyl-acyl Carrier Protein Dehydratase (FabZ) from Francisella tularensis and Yersinia pestis: Structure Determination, Enzymatic Characterization, and Cross-Inhibition Studies.

    PubMed

    McGillick, Brian E; Kumaran, Desigan; Vieni, Casey; Swaminathan, Subramanyam

    2016-02-23

    The bacterial system for fatty acid biosynthesis (FAS) contains several enzymes whose sequence and structure are highly conserved across a vast array of pathogens. This, coupled with their low homology and difference in organization compared to the equivalent system in humans, makes the FAS pathway an excellent target for antimicrobial drug development. To this end, we have cloned, expressed, and purified the β-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from both Francisella tularensis (FtFabZ) and Yersinia pestis (YpFabZ). We also solved the crystal structures and performed an enzymatic characterization of both enzymes and several mutant forms of YpFabZ. Additionally, we have discovered two novel inhibitors of FabZ, mangostin and stictic acid, which show similar potencies against both YpFabZ and FtFabZ. Lastly, we selected several compounds from the literature that have been shown to be active against single homologues of FabZ and tested them against both YpFabZ and FtFabZ. These results have revealed clues as to which scaffolds are likely to lead to broad-spectrum antimicrobials targeted against FabZ as well as modifications to existing FabZ inhibitors that may improve potency. PMID:26818694

  16. Molecular cloning, co-expression, and characterization of glycerol dehydratase and 1,3-propanediol dehydrogenase from Citrobacter freundii.

    PubMed

    Qi, Xianghui; Deng, Wenying; Wang, Fei; Guo, Qi; Chen, Huayou; Wang, Liang; He, Xiang; Huang, Ribo

    2013-06-01

    1,3-Propanediol (1,3-PD), an important material for chemical industry, is biologically synthesized by glycerol dehydratase (GDHt) and 1,3-propanediol dehydrogenase (PDOR). In present study, the dhaBCE and dhaT genes encoding glycerol dehydratase and 1,3-propanediol dehydrogenase respectively were cloned from Citrobacter freundii and co-expressed in E. coli. Sequence analysis revealed that the cloned genes were 85 and 77 % identical to corresponding gene of C. freundii DSM 30040 (GenBank No. U09771), respectively. The over-expressed recombinant enzymes were purified by nickel-chelate chromatography combined with gel filtration, and recombinant GDHt and PDOR were characterized by activity assay, kinetic analysis, pH, and temperature optimization. This research may form a basis for the future work on biological synthesis of 1,3-PD.

  17. Mechanism and Stereospecificity of a Fully Saturating Polyketide Synthase Module: Nanchangmycin Synthase Module 2 and Its Dehydratase Domain

    PubMed Central

    Guo, Xun; Liu, Tiangang; Valenzano, Chiara R.; Deng, Zixin; Cane, David E.

    2010-01-01

    Recombinant nanchangmycin synthase module 2 (NANS module 2), with the thioesterase domain from the 6-deoxyerythronolide B synthase (DEBS TE) appended to the C-terminus, was cloned and expressed in Escherichia coli. Incubation of NANS module 2+TE with (±)-2-methyl-3-keto-butyryl-N-acetylcysteamine thioester (1), the -SNAC analog of the natural ACP-bound substrate, with methylmalonyl-CoA (MM-CoA) in the absence of NADPH gave 3,5,6-trimethyl-4-hydroxylpyrone (2), identified by direct comparison with synthetic 2 by radio-TLC-phosphorimaging and LC-ESI(+)-MS-MS. The reaction showed kcat 0.5±0.1 min−1 and Km(1) 19±5 mM at 0.5 mM MM-CoA and kcat(app) 0.26±0.02 min−1 and Km(MM-CoA) 0.11 ±0.02 mM at 8 mM 1. Incubation in the presence of NADPH generated the fully saturated triketide chain elongation product as a 5:3 mixture of (2S,4R)-2,4-dimethyl-5-ketohexanoic acid (3a) and the diastereomeric (2S,4S)-3b. The structure and stereochemistry of each product was established by comparison with synthetic 3a and 3b by a combination of radio-TLC-phosphorimaging and LC-ESI(−)-MS-MS, as well as chiral capillary GC-MS analysis of the corresponding methyl esters 3a-Me and 3b-Me. The recombinant dehydratase domain from NANS module 2, NANS DH2, was shown to catalyze the formation of an (E)-double bond by syn-dehydration of the ACP-bound substrate anti-(2R,3R,4S,5R)-2,4-dimethyl-3,5-dihydroxyheptanoyl-ACP6 (4), generated in situ by incubation of (2S,3R)-2-methyl-3-hydroxypentanoyl-SNAC (5), methylmalonyl-CoA, and NADPH with recombinant [KS6][AT6] didomain and ACP6 from DEBS module 6 along with the ketoreductase from the tylactone synthase module 1 (TYLS KR1). These results also indirectly establish the stereochemistry of the reactions catalyzed by the KR and enoylreductase (ER) domains of NANS module 2. PMID:20925339

  18. GDP-Mannose-4,6-Dehydratase Is a Cytosolic Partner of Tankyrase 1 That Inhibits Its Poly(ADP-Ribose) Polymerase Activity

    PubMed Central

    Bisht, Kamlesh K.; Dudognon, Charles; Chang, William G.; Sokol, Ethan S.; Ramirez, Alejandro

    2012-01-01

    Tankyrase 1 is a poly(ADP-ribose) polymerase (PARP) that participates in a broad range of cellular activities due to interaction with multiple binding partners. Tankyrase 1 recognizes a linear six-amino-acid degenerate motif and, hence, has hundreds of potential target proteins. Binding of partner proteins to tankyrase 1 usually results in their poly(ADP-ribosyl)ation (PARsylation) and can lead to ubiquitylation and proteasomal degradation. However, it is not known how tankyrase 1 PARP activity is regulated. Here we identify GDP-mannose 4,6-dehydratase (GMD) as a binding partner of tankyrase 1. GMD is a cytosolic protein required for the first step of fucose synthesis. We show that GMD is complexed to tankyrase 1 in the cytosol throughout interphase, but its association with tankyrase 1 is reduced upon entry into mitosis, when tankyrase 1 binds to its other partners TRF1 (at telomeres) and NuMA (at spindle poles). In contrast to other binding partners, GMD is not PARsylated by tankyrase 1. Indeed, we show that GMD inhibits tankyrase 1 PARP activity in vitro, dependent on the GMD tankyrase 1 binding motif. In vivo, depletion of GMD led to degradation of tankyrase 1, dependent on the catalytic PARP activity of tankyrase 1. We speculate that association of tankyrase 1 with GMD in the cytosol sequesters tankyrase 1 in an inactive stable form that can be tapped by other target proteins as needed. PMID:22645305

  19. The Effect of Multiple Sequential Light Sources to Activate Aminolevulinic Acid in the Treatment of Actinic Keratoses: A Retrospective Study

    PubMed Central

    Goldman, Mitchel P.; Fabi, Sabrina G.; Guiha, Isabella

    2014-01-01

    There is a lack of research regarding the sequential use of multiple light sources for topical 5-aminolevulinic acid activation in photodynamic therapy for actinic keratosis. This study evaluated 5-aminolevulinic acid-photodynamic therapy for actinic keratosis using blue light combined with red light, pulsed dye laser, and/or intense pulsed light in a retrospective fashion. Field-directed 5-aminolevulinic acid-photodynamic therapy was performed with blue light only, blue light + pulsed dye laser, blue light + intense pulsed light, blue light + pulsed dye laser + intense pulsed light, or blue light + red light + pulsed dye laser + intense pulsed light for nonhyperkeratotic actinic keratoses of face, scalp, or upper trunk. Blue light + intense pulsed light + pulsed dye laser produced greater patient-reported improvement in actinic keratoses than blue light or blue light + intense pulsed light and greater subject-reported improvement in overall skin quality than blue light + intense pulsed light. The addition of red light led to no further benefit in either outcome measure. Photodynamic therapy with multiple, sequential laser and light sources led to greater patient-graded improvement in actinic keratoses than that with a single light source (blue light), without significant differences in post-treatment adverse events. However, the small, widely disparate number of patients between groups and follow-up times between patients, as well as retrospective assessments based on subjective patient recall, severely limit the significance of these findings. Nevertheless, the results raise interesting questions regarding the use of multiple light and laser sources for photodynamic therapy of actinic keratoses and warrant further research with a prospective, randomized, controlled study. PMID:25276272

  20. Liver δ-aminolevulinate dehydratase activity is inhibited by neonicotinoids and restored by antioxidant agents.

    PubMed

    Sauer, Elisa; Moro, Angela M; Brucker, Natália; Nascimento, Sabrina; Gauer, Bruna; Fracasso, Rafael; Gioda, Adriana; Beck, Ruy; Moreira, José C F; Eifler-Lima, Vera Lucia; Garcia, Solange Cristina

    2014-11-01

    Neonicotinoids represent the most used class of insecticides worldwide, and their precursor, imidacloprid, is the most widely marketed. The aim of this study was to evaluate the effect of imidacloprid on the activity of hepatic δ-aminolevulinate dehydratase (δ-ALA-D), protective effect of potential antioxidants against this potential effect and presence of chemical elements in the constitution of this pesticide. We observed that δ-ALA-D activity was significantly inhibited by imidacloprid at all concentrations tested in a dose-dependent manner. The IC50 value was obtained and used to evaluate the restoration of the enzymatic activity. δ-ALA-D inhibition was completely restored by addition of dithiotreitol (DTT) and partly by ZnCl2, demonstrating that the inhibition occurs by oxidation of thiol groups and by displacement of the Zn (II), which can be explained by the presence of chemical elements found in the constitution of pesticides. Reduced glutathione (GSH) had the best antioxidant effect against to δ-ALA-D inhibition caused by imidacloprid, followed by curcumin and resveratrol. It is well known that inhibition of the enzyme δ-ALA-D may result in accumulation of its neurotoxic substrate (δ-ALA), in this line, our results suggest that further studies are needed to investigate the possible neurotoxicity induced by neonicotinoids and the involvement of antioxidants in cases of poisoning by neonicotinoids. PMID:25402564

  1. The Dehydratase ADT3 Affects ROS Homeostasis and Cotyledon Development1[OPEN

    PubMed Central

    Para, Alessia; Muhammad, DurreShahwar; Naldrett, Michael J.; Warpeha, Katherine M.

    2016-01-01

    During the transition from seed to seedling, emerging embryos strategically balance available resources between building up defenses against environmental threats and initiating the developmental program that promotes the switch to autotrophy. We present evidence of a critical role for the phenylalanine (Phe) biosynthetic activity of AROGENATE DEHYDRATASE3 (ADT3) in coordinating reactive oxygen species (ROS) homeostasis and cotyledon development in etiolated Arabidopsis (Arabidopsis thaliana) seedlings. We show that ADT3 is expressed in the cotyledon and shoot apical meristem, mainly in the cytosol, and that the epidermis of adt3 cotyledons contains higher levels of ROS. Genome-wide proteomics of the adt3 mutant revealed a general down-regulation of plastidic proteins and ROS-scavenging enzymes, corroborating the hypothesis that the ADT3 supply of Phe is required to control ROS concentration and distribution to protect cellular components. In addition, loss of ADT3 disrupts cotyledon epidermal patterning by affecting the number and expansion of pavement cells and stomata cell fate specification; we also observed severe alterations in mesophyll cells, which lack oil bodies and normal plastids. Interestingly, up-regulation of the pathway leading to cuticle production is accompanied by an abnormal cuticle structure and/or deposition in the adt3 mutant. Such impairment results in an increase in cell permeability and provides a link to understand the cell defects in the adt3 cotyledon epidermis. We suggest an additional role of Phe in supplying nutrients to the young seedling. PMID:27540109

  2. Identification of Polyketide Inhibitors Targeting 3-Dehydroquinate Dehydratase in the Shikimate Pathway of Enterococcus faecalis

    PubMed Central

    Hernandez-Valladares, Maria; Go, Maybelle Kho; Tung, Alvin; Aguda, Adeleke H.; Robinson, Robert C.; Yew, Wen Shan

    2014-01-01

    Due to the emergence of resistance toward current antibiotics, there is a pressing need to develop the next generation of antibiotics as therapeutics against infectious and opportunistic diseases of microbial origins. The shikimate pathway is exclusive to microbes, plants and fungi, and hence is an attractive and logical target for development of antimicrobial therapeutics. The Gram-positive commensal microbe, Enterococcus faecalis, is a major human pathogen associated with nosocomial infections and resistance to vancomycin, the “drug of last resort”. Here, we report the identification of several polyketide-based inhibitors against the E. faecalis shikimate pathway enzyme, 3-dehydroquinate dehydratase (DHQase). In particular, marein, a flavonoid polyketide, both inhibited DHQase and retarded the growth of Enterococcus faecalis. The purification, crystallization and structural resolution of recombinant DHQase from E. faecalis (at 2.2 Å resolution) are also reported. This study provides a route in the development of polyketide-based antimicrobial inhibitors targeting the shikimate pathway of the human pathogen E. faecalis. PMID:25072253

  3. Serine dehydratase expression decreases in rat livers injured by chronic thioacetamide ingestion.

    PubMed

    López-Flores, Inmaculada; Barroso, Juan B; Valderrama, Raquel; Esteban, Francisco J; Martínez-Lara, Esther; Luque, Francisco; Peinado, M Angeles; Ogawa, Hirofumi; Lupiáñez, José A; Peragón, Juan

    2005-01-01

    Serine dehydratase (SerDH) is a gluconeogenic enzyme involved in the catabolism of serine, which is regulated by the composition of their diet and their hormonal status in rats. This study examines how chronic injury caused to the liver of rats by the ingestion of thioacetamide (TAA) affects SerDH protein, mRNA levels, enzyme kinetics and its tissue location. After 97 days' oral intake of TAA, the activity of SerDH at all substrate concentrations assayed was about 60% lower than in controls. No significant differences in Km values were found between the treated group and controls. Immunoblotting and immunohistochemistry revealed a significant reduction in the level of SerDH protein in the livers of the treated rats. SerDH was detected specifically in the periportal zone of the hepatic acinus and this location did not change in response to TAA treatment. The level of SerDH mRNA, quantified by reverse transcription and polymerase chain reaction, was significantly lower in treated rats than in the controls. The present findings suggest that the SerDH expression is rendered to be down regulatory during chronic liver injury induced by TAA. These results enhance our understanding about the biochemical mechanisms implied in the control and integration of serine catabolism during liver injury in rat.

  4. Effects of dietary glycerol on the expression of pterin carbinolamine dehydratase in the rat.

    PubMed Central

    Connolly, Edward; Donlon, John

    2003-01-01

    Earlier studies have shown that the abundance of hepatic phenyl-alanine hydroxylase (PAH) diminishes to 60% of control values in rats fed with a diet composed of 40% (w/w) glycerol [Guerin, Walsh, Donlon and Kaufman (1998) Int. J. Biochem. Cell Biol. 30, 1047-1054]. In this experimental model, there are corresponding decreases in the hepatic concentrations of both the hydroxylase cofactor, tetrahydrobiopterin, and the nucleotide guanosine triphosphate. We now show that the cytoplasmic activities of hepatic pterin-4a-carbinolamine dehydratase (PCD) are also lower in these animals, by approx. 50% compared with control values. Immunoblotting confirmed a diminution of protein abundance in vivo. PCD also functions as a dimerization cofactor (DCoH) for the hepatocyte nuclear factor 1alpha (HNF1alpha) and the relative abundance of PCD/DCoH in the nucleus is also decreased. There is a small reduction in the mRNA levels for PAH and for PCD/DCoH in the glycerol-fed animals. In the kidney, there is also a diminution in the abundance of both PAH and PCD proteins. Hepatic GTP cyclohydrolase I activity was not altered and the abundance of hepatic HNF1alpha remained unchanged. HNF1alpha is required for the expression of PAH in the liver and our results support a role for PCD/DCoH, through its interaction with HNF1alpha, in regulating the expression of PAH. PMID:12683953

  5. Liver δ-Aminolevulinate Dehydratase Activity is Inhibited by Neonicotinoids and Restored by Antioxidant Agents

    PubMed Central

    Sauer, Elisa; Moro, Angela M.; Brucker, Natália; Nascimento, Sabrina; Gauer, Bruna; Fracasso, Rafael; Gioda, Adriana; Beck, Ruy; Moreira, José C. F.; Eifler-Lima, Vera Lucia; Garcia, Solange Cristina

    2014-01-01

    Neonicotinoids represent the most used class of insecticides worldwide, and their precursor, imidacloprid, is the most widely marketed. The aim of this study was to evaluate the effect of imidacloprid on the activity of hepatic δ-aminolevulinate dehydratase (δ-ALA-D), protective effect of potential antioxidants against this potential effect and presence of chemical elements in the constitution of this pesticide. We observed that δ-ALA-D activity was significantly inhibited by imidacloprid at all concentrations tested in a dose-dependent manner. The IC50 value was obtained and used to evaluate the restoration of the enzymatic activity. δ-ALA-D inhibition was completely restored by addition of dithiotreitol (DTT) and partly by ZnCl2, demonstrating that the inhibition occurs by oxidation of thiol groups and by displacement of the Zn (II), which can be explained by the presence of chemical elements found in the constitution of pesticides. Reduced glutathione (GSH) had the best antioxidant effect against to δ-ALA-D inhibition caused by imidacloprid, followed by curcumin and resveratrol. It is well known that inhibition of the enzyme δ-ALA-D may result in accumulation of its neurotoxic substrate (δ-ALA), in this line, our results suggest that further studies are needed to investigate the possible neurotoxicity induced by neonicotinoids and the involvement of antioxidants in cases of poisoning by neonicotinoids. PMID:25402564

  6. Chromosomal location of two human genes encoding tetrahydrobiopterin-metabolizing enzymes: 6-pyruvoyl-tetrahydropterin synthase maps to 11q22. 3-q23. 3, and pterin-4[alpha]-carbinolamine dehydratase maps to 10q22

    SciTech Connect

    Thoeny, B.; Heizmann, C.W. ); Mattei, M.G. )

    1994-01-15

    Tetrahydrobiopterin (BH[sub 4]) is the redox cofactor for the aromatic amino acid hydroxylases such as phenylalanine hydroxylase. At least five enzymes are known to be involved in BH[sub 4] biosynthesis and regeneration. A deficiency in several of the BH[sub 4] metabolic enzymes causes variant types of hyperphenylalaninemias in man. Recently, the authors cloned and expressed the human cDNAs for two of the BH[sub 4] enzymes, the 6-pyruvoyl-tetrahydropterin synthase and the pterin-4[alpha]-carbinolamine dehydratase (gene symbols PTS and PCD/DCOH, respectively). In this report, they localized the two genes on the human chromsomes by in situ hybridization. The PTS gene was mapped to the chromosomal region 11q22.3-q23.3, and the PCD/DCOH gene was mapped to the 10q22 band of the genome. 18 refs., 2 figs.

  7. Functional Characterization of a Dehydratase Domain from the Pikromycin Polyketide Synthase

    PubMed Central

    Li, Yang; Dodge, Greg J.; Fiers, William D.; Fecik, Robert A.; Smith, Janet L.; Aldrich, Courtney C.

    2015-01-01

    Metabolic engineering of polyketide synthase (PKS) pathways represents a promising approach to natural products discovery. The dehydratase (DH) domains of PKSs, which generate an α,β-unsaturated bond through a dehydration reaction, have been poorly studied compared to other domains, likely due to the simple nature of the chemical reaction they catalyze and lack of a convenient assay to measure substrate turnover. Herein we report the first steady-state kinetic analysis of a PKS DH domain employing LC-MS/MS analysis for product quantitation. PikDH2 was selected as a model DH domain. Its substrate specificity and mechanism was interrogated with a systematic series of synthetic triketide substrates containing a nonhydrolyzable thioether linkage as well as by site-directed mutagenesis, evaluation of the pH dependence of catalytic efficiency (Vmax/KM), and through kinetic characterization of a mechanism-based inhibitor. These studies revealed PikDH2 converts d-alcohol substrates to trans-olefin products. The reaction was reversible with equilibrium constants ranging from 1.2–2. Moreover, the enzyme activity was robust and PikDH2 was used on a preparative scale for the chemoenzymatic synthesis of unsaturated triketide products. PikDH2 was shown to possess remarkably strict substrate specificity and was unable to turnover substrates epimeric at the β, γ or δ-positions. We also demonstrated PikDH2 has a key ionizable group with a pKa of 7.0 and can be irreversibly inactivated through covalent modification by a mechanism-based inhibitor, which provides a foundation for future structural studies to elucidate substrate–protein interactions. PMID:26027428

  8. Expression Levels of ALA Dehydratase as a Marker of ALA-PDT Efficacy

    NASA Astrophysics Data System (ADS)

    Avital, Schauder; Tamar, Feuerstein; Zvi, Malik

    2010-05-01

    Accelerated synthesis of protoporphyrinIX (PpIX) following ALA pre-treatment followed by light irradiation is the principle of ALA-PDT. Several limiting enzymes were suggested to control PpIX accumulation and PDT efficacy, among them porphobilinogen deaminase (PBGD) and ferrochelatase. Here we reveal the centrality of ALA dehydratase (ALAD) activity in predicting ALA-PDT efficacy. Silencing of ALAD expression and activity was carried out in leukemic cells using shRNA plasmid transfection or Pb2+ intoxication. ALAD activity, porphyrin synthesis and mitochondrial activity were determined versus PDT efficacy. In K562 ALAD-silenced cells, ALAD activity and expression were reduced and as a result, PpIX synthesis was almost abolished. Following ALA treatment and irradiation, ALAD-silenced cells depicted normal mitochondrial activity, in contrast to control and non-silencing transfected cells where accumulated PpIX and irradiation caused ROS formation and mitochondrial damage. Morphological analysis by scanning electron microscopy (SEM) of ALA-PDT treated cells showed no morphological changes in ALAD-silenced cells, while controls exhibited cell deformations and lysis. Annexin V-FITC/PI staining as well as LDH-L leakage testing showed that membrane integrity was undamaged following ALA-PDT in ALAD silenced cells. Pb2+ treatment in MEL cells impaired ALAD activity and reduced PpIX synthesis but to a lesser extent. In conclusion, we show that a dramatic reduction in PpIX accumulation following down regulation of ALAD expression prevents an efficient PDT. Thus, ALAD has a major role in regulating PpIX synthesis and ALA-PDT therapeutic outcome. Monitoring ALAD expression or activity in various tumors may be useful as prognostic tool to predict PDT efficacy.

  9. Arogenate Dehydratase Isoenzymes Profoundly and Differentially Modulate Carbon Flux into Lignins*

    PubMed Central

    Corea, Oliver R. A.; Ki, Chanyoung; Cardenas, Claudia L.; Kim, Sung-Jin; Brewer, Sarah E.; Patten, Ann M.; Davin, Laurence B.; Lewis, Norman G.

    2012-01-01

    How carbon flux differentially occurs in vascular plants following photosynthesis for protein formation, phenylpropanoid metabolism (i.e. lignins), and other metabolic processes is not well understood. Our previous discovery/deduction that a six-membered arogenate dehydratase (ADT1–6) gene family encodes the final step in Phe biosynthesis in Arabidopsis thaliana raised the fascinating question whether individual ADT isoenzymes (or combinations thereof) differentially modulated carbon flux to lignins, proteins, etc. If so, unlike all other lignin pathway manipulations that target cell wall/cytosolic processes, this would be the first example of a plastid (chloroplast)-associated metabolic process influencing cell wall formation. Homozygous T-DNA insertion lines were thus obtained for five of the six ADTs and used to generate double, triple, and quadruple knockouts (KOs) in different combinations. The various mutants so obtained gave phenotypes with profound but distinct reductions in lignin amounts, encompassing a range spanning from near wild type levels to reductions of up to ∼68%. In the various KOs, there were also marked changes in guaiacyl:syringyl ratios ranging from ∼3:1 to 1:1, respectively; these changes were attributed to differential carbon flux into vascular bundles versus that into fiber cells. Laser microscope dissection/pyrolysis GC/MS, histochemical staining/lignin analyses, and pADT::GUS localization indicated that ADT5 preferentially affects carbon flux into the vascular bundles, whereas the adt3456 knock-out additionally greatly reduced carbon flux into fiber cells. This plastid-localized metabolic step can thus profoundly differentially affect carbon flux into lignins in distinct anatomical regions and provides incisive new insight into different factors affecting guaiacyl:syringyl ratios and lignin primary structure. PMID:22311980

  10. Preliminary crystallographic analysis of l-2-keto-3-deoxyarabonate dehydratase, an enzyme involved in an alternative bacterial pathway of L-arabinose metabolism

    SciTech Connect

    Shimada, Naoko; Mikami, Bunzo; Watanabe, Seiya; Makino, Keisuke

    2007-05-01

    l-2-Keto-3-deoxyarabonate dehydratase was overexpressed, purified and crystallized at 291 K using the hanging-drop vapour-diffusion method. l-2-Keto-3-deoxyarabonate (l-KDA) dehydratase is a novel member of the dihydrodipicolinate synthase (DHDPS)/N-acetylneuraminate lyase (NAL) protein family and catalyzes the hydration of l-KDA to α-ketoglutaric semialdehyde. l-KDA dehydratase was overexpressed, purified and crystallized at 291 K using the hanging-drop vapour-diffusion method. The crystal diffracts to 2.0 Å resolution using synchrotron radiation and belongs to the trigonal space group P3{sub 1}21 or its enantiomorph P3{sub 2}21, with unit-cell parameters a = b = 78.91, c = 207.71 Å.

  11. Overproduction, crystallization and preliminary X-ray crystallographic analysis of Escherichia coli tRNA N 6-threonylcarbamoyladenosine dehydratase

    PubMed Central

    Kim, Sunmin; Kim, Keon Young; Park, Jeong Kuk; Lee, Byung Il; Kim, Yun-Gon; Park, SangYoun

    2014-01-01

    Escherichia coli tRNA N 6-threonylcarbamoyladenosine dehydratase (TcdA), previously called CsdL or YgdL, was overproduced and purified from E. coli and crystallized using polyethylene glycol 3350 as a crystallizing agent. X-ray diffraction data were collected to 2.70 Å resolution under cryoconditions using synchrotron X-rays. The crystals belonged to space group P21, with unit-cell parameters a = 65.4, b = 96.8, c = 83.3 Å, β = 111.7°. According to the Matthews coefficient, the asymmetric unit may contain up to four subunits of the monomeric protein, with a crystal volume per protein mass (V M) of 2.12 Å3 Da−1 and 42.1% solvent content. PMID:25372820

  12. LCAA, a Novel Factor Required for Magnesium Protoporphyrin Monomethylester Cyclase Accumulation and Feedback Control of Aminolevulinic Acid Biosynthesis in Tobacco1[W][OA

    PubMed Central

    Albus, Christin Anne; Salinas, Annabel; Czarnecki, Olaf; Kahlau, Sabine; Rothbart, Maxi; Thiele, Wolfram; Lein, Wolfgang; Bock, Ralph; Grimm, Bernhard; Schöttler, Mark Aurel

    2012-01-01

    Low Chlorophyll Accumulation A (LCAA) antisense plants were obtained from a screen for genes whose partial down-regulation results in a strong chlorophyll deficiency in tobacco (Nicotiana tabacum). The LCAA mutants are affected in a plastid-localized protein of unknown function, which is conserved in cyanobacteria and all photosynthetic eukaryotes. They suffer from drastically reduced light-harvesting complex (LHC) contents, while the accumulation of all other photosynthetic complexes per leaf area is less affected. As the disturbed accumulation of LHC proteins could be either attributable to a defect in LHC biogenesis itself or to a bottleneck in chlorophyll biosynthesis, chlorophyll synthesis rates and chlorophyll synthesis intermediates were measured. LCAA antisense plants accumulate magnesium (Mg) protoporphyrin monomethylester and contain reduced protochlorophyllide levels and a reduced content of CHL27, a subunit of the Mg protoporphyrin monomethylester cyclase. Bimolecular fluorescence complementation assays confirm a direct interaction between LCAA and CHL27. 5-Aminolevulinic acid synthesis rates are increased and correlate with an increased content of glutamyl-transfer RNA reductase. We suggest that LCAA encodes an additional subunit of the Mg protoporphyrin monomethylester cyclase, is required for the stability of CHL27, and contributes to feedback-control of 5-aminolevulinic acid biosynthesis, the rate-limiting step of chlorophyll biosynthesis. PMID:23085838

  13. Crystallization and preliminary X-ray analysis of molecular chaperone-like diol dehydratase-reactivating factor in ADP-bound and nucleotide-free forms

    SciTech Connect

    Mori, Koichi; Hieda, Naoki; Yamanishi, Mamoru; Shibata, Naoki; Toraya, Tetsuo

    2005-06-01

    The molecular chaperone-like reactivating factor for adenosylcobalamin (coenzyme B{sub 12}) dependent diol dehydratase was crystallized in ADP-bound and nucleotide-free forms. Preliminary X-ray analysis indicated that crystals are orthorhombic and diffract to 2.0 Å. Adenosylcobalamin (coenzyme B{sub 12}) dependent diol dehydratase (EC 4.2.1.28) catalyzes the conversion of 1,2-diols and glycerol to the corresponding aldehydes. It undergoes mechanism-based inactivation by glycerol. The diol dehydratase-reactivating factor (DDR) reactivates the inactivated holoenzymes in the presence of adenosylcobalamin, ATP and Mg{sup 2+} by mediating the release of a damaged cofactor. This molecular chaperone-like factor was overexpressed in Escherichia coli, purified and crystallized in the ADP-bound and nucleotide-free forms by the sandwich-drop vapour-diffusion method. The crystals of the ADP-bound form belong to the orthorhombic system, with space group P2{sub 1}2{sub 1}2{sub 1} and unit-cell parameters a = 83.26, b = 84.60, c = 280.09 Å, and diffract to 2.0 Å. In the absence of nucleotide, DDR crystals were orthorhombic, with space group P2{sub 1}2{sub 1}2{sub 1} and unit-cell parameters a = 81.92, b = 85.37, c = 296.99 Å and diffract to 3.0 Å. Crystals of both forms were suitable for structural analysis.

  14. Mutagenic and chemical analyses provide new insight into enzyme activation and mechanism of the type 2 iron-sulfur l-serine dehydratase from Legionella pneumophila.

    PubMed

    Xu, Xiao Lan; Grant, Gregory A

    2016-04-15

    The crystal structure of the Type 2 l-serine dehydratase from Legionella pneumophila (lpLSD), revealed a "tail-in-mouth" configuration where the C-terminal residue acts as an intrinsic competitive inhibitor. This pre-catalytic structure undergoes an activation step prior to catalytic turnover. Mutagenic analysis of residues at or near the active site cleft is consistent with stabilization of substrate binding by many of the same residues that interact with the C-terminal cysteine and highlight the critical role of certain tail residues in activity. pH-rate profiles show that a residue with pK of 5.9 must be deprotonated and a residue with a pK of 8.5 must be protonated for activity. This supports an earlier suggestion that His 61 is the likely catalytic base. An additional residue with a pK of 8.5-9 increases cooperativity when it is deprotonated. This investigation also demonstrates that the Fe-S dehydratases convert the enamine/imine intermediates of the catalytic reaction to products on the enzyme prior to release. This is in contrast to pyridoxyl 5' phosphate based dehydratases that release an enamine/imine intermediate into solution, which then hydrolyzes to produce the ketoamine product.

  15. Effects of lead shot ingestion on delta-aminolevulinic acid dehydratase activity, hemoglobin concentration, and serum chemistry in bald eagles

    USGS Publications Warehouse

    Hoffman, D.J.; Pattee, O.H.; Wiemeyer, Stanley N.; Mulhern, B.

    1981-01-01

    Lead shot ingestion by bald eagles (Haliaeetus leucocephalus) is considered to be widespread and has been implicated in the death of eagles in nature. It was recently demonstrated under experimental conditions that ingestion of as few as 10 lead shot resulted in death within 12 to 20 days. In the present study hematological responses to lead toxicity including red blood cell ALAD activity, hemoglobin concentration and 23 different blood serum chemistries were examined in five captive bald eagles that were unsuitable for rehabilitation and release. Eagles were dosed by force-feeding with 10 lead shot; they were redosed if regurgitation occurred. Red blood cell ALAD activity was inhibited by nearly 80% within 24 hours when mean blood lead concentration had increased to 0.8 parts per million (ppm). By the end of 1 week there was a significant decrease (20-25%) in hematocrit and hemoglobin, and the mean blood lead concentration was over 3 ppm. Within as little as 1-2 weeks after dosing, significant elevations in serum creatinine and serum alanine aminotransferase occurred, as well as a significant decrease in the ratio of serum aspartic aminotransferase to serum alanine aminotransferase. The mean blood lead concentration was over 5 ppm by the end of 2 weeks. These changes in serum chemistry may be indicative of kidney and liver alterations.

  16. Whole blood lead concentration and erythrocyte delta-aminolevulinic acid dehydratase (ALAD) activity in selected canine populations in Greece.

    PubMed

    Polizopoulou, Z S; Kontos, V S; Koutinas, A F; Papasteriades, A

    1994-12-01

    In a total number of 275 dogs of various ages, sex and breed, blood lead concentrations (BLC) and erythrocyte ALAD activity were measured. Sixty-six of the dogs were living in lead mining areas (Group A), 157 in urban areas (Group B) and 52 in rural areas (Group C) of Greece. Mean BLC differed significantly (P < 0.05) between locations and were 326,97 and 68 micrograms/L, respectively. Mean ALAD activity was significantly different (P < 0.05) only between Groups A and B as between groups A and C. A significant (P < 0.05) negative correlation existed between BLC and ALAD activity. A normal range of erythrocyte ALAD activity of 807-992 mumol/PBG/LRBC/h was established for dogs. None of the 33 Group A dogs and 2 of the Group B dogs that had a BLC of 350 micrograms/L presented clinical signs indicating acute or chronic lead intoxication. No erythrocyte basophilic stippling or large number of nucleated red blood cells were seen in the 30 dogs of Group A with BLC > 350 micrograms/L.

  17. Structure-based virtual screening as a tool for the identification of novel inhibitors against Mycobacterium tuberculosis 3-dehydroquinate dehydratase.

    PubMed

    Petersen, Guilherme O; Saxena, Shalini; Renuka, Janupally; Soni, Vijay; Yogeeswari, Perumal; Santos, Diogenes S; Bizarro, Cristiano V; Sriram, Dharmarajan

    2015-07-01

    3-Dehydroquinate dehydratase (DHQase), the third enzyme of the shikimate pathway, catalyzes the reversible reaction of 3-dehydroquinate into 3-dehydroshikimate. The aim of the present study was to identify new drug-like molecules as inhibitors for Mycobacterium tuberculosis DHQase employing structure-based pharmacophore modeling technique using an in house database consisting of about 2500 small molecules. Further the pharmacophore models were validated using enrichment calculations, and finally three models were employed for high-throughput virtual screening and docking to identify novel small molecules as DHQase inhibitors. Five compounds were identified, out of which, one molecule (Lead 1) showed 58% inhibition at 50μ M concentration in the Mtb DHQase assay. Chemical derivatives of the Lead 1 when tested evolved top two hits with IC50s of 17.1 and 31.5 μM as well as MIC values of 25 and 6.25 μg/mL respectively and no cytotoxicity up to 100 μM concentration. PMID:26043661

  18. Crystal Structures Reveal that the Reaction Mechanism of Imidazoleglycerol-Phosphate Dehydratase Is Controlled by Switching Mn(II) Coordination.

    PubMed

    Bisson, Claudine; Britton, K Linda; Sedelnikova, Svetlana E; Rodgers, H Fiona; Eadsforth, Thomas C; Viner, Russell C; Hawkes, Tim R; Baker, Patrick J; Rice, David W

    2015-07-01

    Imidazoleglycerol-phosphate dehydratase (IGPD) catalyzes the Mn(II)-dependent dehydration of imidazoleglycerol phosphate (IGP) to 3-(1H-imidazol-4-yl)-2-oxopropyl dihydrogen phosphate during biosynthesis of histidine. As part of a program of herbicide design, we have determined a series of high-resolution crystal structures of an inactive mutant of IGPD2 from Arabidopsis thaliana in complex with IGP. The structures represent snapshots of the enzyme trapped at different stages of the catalytic cycle and show how substrate binding triggers a switch in the coordination state of an active site Mn(II) between six- and five-coordinate species. This switch is critical to prime the active site for catalysis, by facilitating the formation of a high-energy imidazolate intermediate. This work not only provides evidence for the molecular processes that dominate catalysis in IGPD, but also describes how the manipulation of metal coordination can be linked to discrete steps in catalysis, demonstrating one way that metalloenzymes exploit the unique properties of metal ions to diversify their chemistry.

  19. Anaemia, Serum Iron Concentrations and δ-Aminolevulinate Dehydratase Activity in Laying Hens Infected Naturally by Salmonella Gallinarum.

    PubMed

    Machado, A C; Boiago, M M; do Carmo, G M; Bottari, N B; Araujo, D N; Giuriatti, J; Morsch, V M; Schetinger, M R C; Casagrande, R A; Wisser, C S; Stefani, L M; Alves, M S; Da Silva, A S

    2016-07-01

    The aim of this study was to evaluate anaemia, serum iron concentrations and δ-aminolevulinate dehydratase (ALA-D) activity in laying hens infected naturally by Salmonella Gallinarum and having severe hepatic lesions. Liver and serum samples were collected from 27 laying hens (20 infected and seven uninfected). The δ-ALA-D activity, haematocrit and serum iron concentrations were evaluated. There were significant decreases in δ-ALA-D activity, haematocrit and serum iron concentrations (P <0.01) in birds infected by S. Gallinarum when compared with uninfected birds. There was a positive correlation (P <0.001) between serum iron concentration, haematocrit (r(2) = 0.82) and δ-ALA-D activity (r(2) = 0.75). A positive correlation was also observed between δ-ALA-D activity and haematocrit (r(2) = 0.78; P <0.01). Liver samples showed moderate focal coagulative necrosis associated with infiltration of lymphoplasmacytic cells, macrophages and heterophils. The anaemia in the infected hens may be related to reduction in δ-ALA-D activity and serum iron concentrations, since both are important for haemopoiesis.

  20. Engineering Filamentous Fungi for Conversion of d-Galacturonic Acid to l-Galactonic Acid

    PubMed Central

    Kuivanen, Joosu; Mojzita, Dominik; Wang, Yanming; Hilditch, Satu; Penttilä, Merja; Richard, Peter

    2012-01-01

    d-Galacturonic acid, the main monomer of pectin, is an attractive substrate for bioconversions, since pectin-rich biomass is abundantly available and pectin is easily hydrolyzed. l-Galactonic acid is an intermediate in the eukaryotic pathway for d-galacturonic acid catabolism, but extracellular accumulation of l-galactonic acid has not been reported. By deleting the gene encoding l-galactonic acid dehydratase (lgd1 or gaaB) in two filamentous fungi, strains were obtained that converted d-galacturonic acid to l-galactonic acid. Both Trichoderma reesei Δlgd1 and Aspergillus niger ΔgaaB strains produced l-galactonate at yields of 0.6 to 0.9 g per g of substrate consumed. Although T. reesei Δlgd1 could produce l-galactonate at pH 5.5, a lower pH was necessary for A. niger ΔgaaB. Provision of a cosubstrate improved the production rate and titer in both strains. Intracellular accumulation of l-galactonate (40 to 70 mg g biomass−1) suggested that export may be limiting. Deletion of the l-galactonate dehydratase from A. niger was found to delay induction of d-galacturonate reductase and overexpression of the reductase improved initial production rates. Deletion of the l-galactonate dehydratase from A. niger also delayed or prevented induction of the putative d-galacturonate transporter An14g04280. In addition, A. niger ΔgaaB produced l-galactonate from polygalacturonate as efficiently as from the monomer. PMID:23042175

  1. Structure and Identification of a Pterin Dehydratase-like Protein as a Ribulose-bisphosphate Carboxylase/Oxygenase (RuBisCO) Assembly Factor in the α-Carboxysome*

    PubMed Central

    Wheatley, Nicole M.; Sundberg, Christopher D.; Gidaniyan, Soheil D.; Cascio, Duilio; Yeates, Todd O.

    2014-01-01

    Carboxysomes are proteinaceous bacterial microcompartments that increase the efficiency of the rate-limiting step in carbon fixation by sequestering reaction substrates. Typically, α-carboxysomes are genetically encoded as a single operon expressing the structural proteins and the encapsulated enzymes of the microcompartment. In addition, depending on phylogeny, as many as 13 other genes are found to co-occur near or within α-carboxysome operons. One of these genes codes for a protein with distant homology to pterin-4α-carbinolamine dehydratase (PCD) enzymes. It is present in all α-carboxysome containing bacteria and has homologs in algae and higher plants. Canonical PCDs play an important role in amino acid hydroxylation, a reaction not associated with carbon fixation. We determined the crystal structure of an α-carboxysome PCD-like protein from the chemoautotrophic bacterium Thiomonas intermedia K12, at 1.3-Å resolution. The protein retains a three-dimensional fold similar to canonical PCDs, although the prominent active site cleft present in PCD enzymes is disrupted in the α-carboxysome PCD-like protein. Using a cell-based complementation assay, we tested the PCD-like proteins from T. intermedia and two additional bacteria, and found no evidence for PCD enzymatic activity. However, we discovered that heterologous co-expression of the PCD-like protein from Halothiobacillus neapolitanus with RuBisCO and GroELS in Escherichia coli increased the amount of soluble, assembled RuBisCO recovered from cell lysates compared with co-expression of RuBisCO with GroELS alone. We conclude that this conserved PCD-like protein, renamed here α-carboxysome RuBisCO assembly factor (or acRAF), is a novel RuBisCO chaperone integral to α-carboxysome function. PMID:24459150

  2. Alternative fermentation pathway of cinnamic acid production via phenyllactic acid.

    PubMed

    Masuo, Shunsuke; Kobayashi, Yuta; Oinuma, Ken-Ichi; Takaya, Naoki

    2016-10-01

    Cinnamic acid (CA) is the chemical basis for bulk production of flavoring reagents and chemical intermediates, and it can be fermented from biomass. Phenylalanine ammonia lyase (PAL) has been used exclusively in the bacterial fermentation of sugar biomass in which the fermentation intermediate phenylalanine is deaminated to CA. Here, we designed an alternative metabolic pathway for fermenting glucose to CA. An Escherichia coli strain that generates phenylalanine in this pathway also produces Wickerhamia fluorescens phenylpyruvate reductase and ferments glucose to D-phenyllactate (D-PhLA) (Fujita et al. Appl Microbiol Biotechnol 97: 8887-8894, 2013). Thereafter, phenyllactate dehydratase encoded by fldABCI genes in Clostridium sporogenes converts the resulting D-PhLA into CA. The phenyllactate dehydratase expressed by fldABCI in the D-PhLA-producing bacterium fermented glucose to CA, but D-PhLA fermentation and phenyllactate dehydration were aerobic and anaerobic processes, respectively, which disrupted high-yield CA fermentation in single batch cultures. We overcame this disruption by sequentially culturing the two strains under aerobic and anaerobic conditions. We optimized the incubation periods of the respective aeration steps to produce 1.7 g/L CA from glucose, which exceeded the yield from PAL-dependent glucose fermentation to CA 11-fold. This process is a novel, efficient alternative to conventional PAL-dependent CA production.

  3. Arabidopsis and Maize RidA Proteins Preempt Reactive Enamine/Imine Damage to Branched-Chain Amino Acid Biosynthesis in Plastids[C][W][OPEN

    PubMed Central

    Niehaus, Thomas D.; Nguyen, Thuy N.D.; Gidda, Satinder K.; ElBadawi-Sidhu, Mona; Lambrecht, Jennifer A.; McCarty, Donald R.; Downs, Diana M.; Cooper, Arthur J.L.; Fiehn, Oliver; Mullen, Robert T.; Hanson, Andrew D.

    2014-01-01

    RidA (for Reactive Intermediate Deaminase A) proteins are ubiquitous, yet their function in eukaryotes is unclear. It is known that deleting Salmonella enterica ridA causes Ser sensitivity and that S. enterica RidA and its homologs from other organisms hydrolyze the enamine/imine intermediates that Thr dehydratase forms from Ser or Thr. In S. enterica, the Ser-derived enamine/imine inactivates a branched-chain aminotransferase; RidA prevents this damage. Arabidopsis thaliana and maize (Zea mays) have a RidA homolog that is predicted to be plastidial. Expression of either homolog complemented the Ser sensitivity of the S. enterica ridA mutant. The purified proteins hydrolyzed the enamines/imines formed by Thr dehydratase from Ser or Thr and protected the Arabidopsis plastidial branched-chain aminotransferase BCAT3 from inactivation by the Ser-derived enamine/imine. In vitro chloroplast import assays and in vivo localization of green fluorescent protein fusions showed that Arabidopsis RidA and Thr dehydratase are chloroplast targeted. Disrupting Arabidopsis RidA reduced root growth and raised the root and shoot levels of the branched-chain amino acid biosynthesis intermediate 2-oxobutanoate; Ser treatment exacerbated these effects in roots. Supplying Ile reversed the root growth defect. These results indicate that plastidial RidA proteins can preempt damage to BCAT3 and Ile biosynthesis by hydrolyzing the Ser-derived enamine/imine product of Thr dehydratase. PMID:25070638

  4. Arabidopsis and maize RidA proteins preempt reactive enamine/imine damage to branched-chain amino acid biosynthesis in plastids.

    PubMed

    Niehaus, Thomas D; Nguyen, Thuy N D; Gidda, Satinder K; ElBadawi-Sidhu, Mona; Lambrecht, Jennifer A; McCarty, Donald R; Downs, Diana M; Cooper, Arthur J L; Fiehn, Oliver; Mullen, Robert T; Hanson, Andrew D

    2014-07-01

    RidA (for Reactive Intermediate Deaminase A) proteins are ubiquitous, yet their function in eukaryotes is unclear. It is known that deleting Salmonella enterica ridA causes Ser sensitivity and that S. enterica RidA and its homologs from other organisms hydrolyze the enamine/imine intermediates that Thr dehydratase forms from Ser or Thr. In S. enterica, the Ser-derived enamine/imine inactivates a branched-chain aminotransferase; RidA prevents this damage. Arabidopsis thaliana and maize (Zea mays) have a RidA homolog that is predicted to be plastidial. Expression of either homolog complemented the Ser sensitivity of the S. enterica ridA mutant. The purified proteins hydrolyzed the enamines/imines formed by Thr dehydratase from Ser or Thr and protected the Arabidopsis plastidial branched-chain aminotransferase BCAT3 from inactivation by the Ser-derived enamine/imine. In vitro chloroplast import assays and in vivo localization of green fluorescent protein fusions showed that Arabidopsis RidA and Thr dehydratase are chloroplast targeted. Disrupting Arabidopsis RidA reduced root growth and raised the root and shoot levels of the branched-chain amino acid biosynthesis intermediate 2-oxobutanoate; Ser treatment exacerbated these effects in roots. Supplying Ile reversed the root growth defect. These results indicate that plastidial RidA proteins can preempt damage to BCAT3 and Ile biosynthesis by hydrolyzing the Ser-derived enamine/imine product of Thr dehydratase.

  5. Identification of the in vivo function of the high-efficiency D-mannonate dehydratase in Caulobacter crescentus NA1000 from the enolase superfamily.

    PubMed

    Wichelecki, Daniel J; Graff, Dylan C; Al-Obaidi, Nawar; Almo, Steven C; Gerlt, John A

    2014-07-01

    The d-mannonate dehydratase (ManD) subgroup of the enolase superfamily contains members with varying catalytic activities (high-efficiency, low-efficiency, or no activity) that dehydrate d-mannonate and/or d-gluconate to 2-keto-3-deoxy-d-gluconate [Wichelecki, D. J., et al. (2014) Biochemistry 53, 2722-2731]. Despite extensive in vitro characterization, the in vivo physiological role of a ManD has yet to be established. In this study, we report the in vivo functional characterization of a high-efficiency ManD from Caulobacter crescentus NA1000 (UniProt entry B8GZZ7) by in vivo discovery of its essential role in d-glucuronate metabolism. This in vivo functional annotation may be extended to ~50 additional proteins.

  6. Identification and Characterization of FabA from the Type II Fatty Acid Synthase of Streptomyces coelicolor.

    PubMed

    Singh, Renu; Reynolds, Kevin A

    2016-01-22

    FabA is proposed to catalyze the dehydration step of chain elongation in fatty acid and undecylprodiginine biosynthesis in Streptomyces coelicolor. Analysis of the S. coelicolor genome has revealed a fabA gene (SCO4636-SCO4637, encoding a heterodimer 3-hydroxyacyl-ACP dehydratase). Herein, we report the identification and characterization of the corresponding gene products. Kinetic analysis has demonstrated that FabA is capable of utilizing various chain lengths of straight- and branched-chain 3-hydroxyacyl-NAC substrates. Additionally, FabA does not discriminate between acyl carrier proteins (ACPs) from primary and secondary metabolism. These data provide the first experimental evidence that FabA has 3-hydroxyacyl-ACP dehydratase activity and processes intermediates for both biosynthetic pathways.

  7. Production of 3-hydroxypropionic acid from glycerol by recombinant Pseudomonas denitrificans.

    PubMed

    Zhou, Shengfang; Catherine, Christy; Rathnasingh, Chelladurai; Somasundar, Ashok; Park, Sunghoon

    2013-12-01

    3-Hydroxypropionic acid (3-HP) can be produced from glycerol through two sequential enzymatic reactions that are catalyzed by a coenzyme B12 -dependent glycerol dehydratase and an NAD(P)(+) -dependent aldehyde dehydrogenase (ALDH), respectively. Pseudomonas denitrificans synthesizes coenzyme B12 under aerobic conditions, where NAD(P)(+) is regenerated efficiently. Hence, it is considered an ideal host for the production of 3-HP from glycerol under aerobic conditions. In this study, recombinant strains of P. denitrificans were developed and their potential for the production of 3-HP from glycerol was evaluated. When the enzymes, glycerol dehydratase (DhaB) and glycerol dehydratase reactivase (GdrAB), of Klebsiella pneumoniae were expressed heterologously, P. denitrificans could produce 3-HP at 37.7 mmol/L with 62% (mol/mol) yield on glycerol. Glucose was required as the carbon and energy sources for cell growth. The overexpression of heterologous ALDH was not essential; however, the titer and yield of 3-HP were improved to 54.7 mmol/L and 67% (mol/mol), respectively, when an ALDH gene (puuC) from K. pneumoniae was overexpressed. One serious drawback hindering the use of P. denitrificans as a recombinant host for 3-HP production is that it oxidizes 3-HP to malonate and utilizes 3-HP as a carbon source for growth. This is the first report on the development and use of recombinant P. denitrificans for 3-HP production from glycerol.

  8. Primers and a specific DNA probe for detecting lactic acid bacteria producing 3-hydroxypropionaldehyde from glycerol in spoiled ciders.

    PubMed

    Claisse, O; Lonvaud-Funel, A

    2001-06-01

    Of the 40 strains isolated from several spoiled ciders where glycerol was degraded, 36 were identified as Lactobacillus collinoides, three were Lactobacillus hilgardii, and one was Lactobacillus mali. However, only 30 L. collinoides and two L. hilgardii could degrade glycerol. The glycerol dehydratase activity was shown. The main product of the transformation was 1.3 propanediol. Two DNA primers GD1 and GD2 were chosen in the region encoding one of the subunits of glycerol dehydratase of Citrobacter freundii, Klebsiella pneumoniae, Klebsiella oxytoca, Salmonella Typhimurium, and Clostridium pasteurianum. A 279-bp amplicon in polymerase chain reaction amplification was obtained with the genomic L. collinoides IOEB 9527 DNA as template. The amino acid sequence deduced from the amplicon DNA sequence showed a very high similarity and identity with the gene of gram-negative and C. pasteurianum species. After labeling, the amplicon was used as DNA probe in dot-blot hybridization with the genomic DNA of all the tested strains. Only strains that could degrade glycerol hybridized. Moreover, polymerase chain reactions using GDI and GD2 revealed only glycerol dehydratase genes of positive L. collinoides and L. hilgardii strains. The primers and the amplicon proved to be suitable and reliable tools to detect the lactic acid bacteria involved in the deterioration of cider. PMID:11403134

  9. Crystal structures of type I dehydroquinate dehydratase in complex with quinate and shikimate suggest a novel mechanism of Schiff base formation.

    PubMed

    Light, Samuel H; Antanasijevic, Aleksandar; Krishna, Sankar N; Caffrey, Michael; Anderson, Wayne F; Lavie, Arnon

    2014-02-11

    A component of the shikimate biosynthetic pathway, dehydroquinate dehydratase (DHQD) catalyzes the dehydration of 3-dehydroquniate (DHQ) to 3-dehydroshikimate. In the type I DHQD reaction mechanism a lysine forms a Schiff base intermediate with DHQ. The Schiff base acts as an electron sink to facilitate the catalytic dehydration. To address the mechanism of Schiff base formation, we determined structures of the Salmonella enterica wild-type DHQD in complex with the substrate analogue quinate and the product analogue shikimate. In addition, we determined the structure of the K170M mutant (Lys170 being the Schiff base forming residue) in complex with quinate. Combined with nuclear magnetic resonance and isothermal titration calorimetry data that revealed altered binding of the analogue to the K170M mutant, these structures suggest a model of Schiff base formation characterized by the dynamic interplay of opposing forces acting on either side of the substrate. On the side distant from the substrate 3-carbonyl group, closure of the enzyme's β8-α8 loop is proposed to guide DHQ into the proximity of the Schiff base-forming Lys170. On the 3-carbonyl side of the substrate, Lys170 sterically alters the position of DHQ's reactive ketone, aligning it at an angle conducive for nucleophilic attack. This study of a type I DHQD reveals the interplay between the enzyme and substrate required for the correct orientation of a functional group constrained within a cyclic substrate.

  10. 2-Butanol and Butanone Production in Saccharomyces cerevisiae through Combination of a B12 Dependent Dehydratase and a Secondary Alcohol Dehydrogenase Using a TEV-Based Expression System

    PubMed Central

    Ghiaci, Payam; Norbeck, Joakim; Larsson, Christer

    2014-01-01

    2-Butanol and its chemical precursor butanone (methyl ethyl ketone – MEK) are chemicals with potential uses as biofuels and biocommodity chemicals. In order to produce 2-butanol, we have demonstrated the utility of using a TEV-protease based expression system to achieve equimolar expression of the individual subunits of the two protein complexes involved in the B12-dependent dehydratase step (from the pdu-operon of Lactobacillus reuterii), which catalyze the conversion of meso-2,3-butanediol to butanone. We have furthermore identified a NADH dependent secondary alcohol dehydrogenase (Sadh from Gordonia sp.) able to catalyze the subsequent conversion of butanone to 2-butanol. A final concentration of 4±0.2 mg/L 2-butanol and 2±0.1 mg/L of butanone was found. A key factor for the production of 2-butanol was the availability of NADH, which was achieved by growing cells lacking the GPD1 and GPD2 isogenes under anaerobic conditions. PMID:25054226

  11. Chemical Modification of a Dehydratase Enzyme Involved in Bacterial Virulence by an Ammonium Derivative: Evidence of its Active Site Covalent Adduct.

    PubMed

    González-Bello, Concepción; Tizón, Lorena; Lence, Emilio; Otero, José M; van Raaij, Mark J; Martinez-Guitian, Marta; Beceiro, Alejandro; Thompson, Paul; Hawkins, Alastair R

    2015-07-29

    The first example of an ammonium derivative that causes a specific modification of the active site of type I dehydroquinase (DHQ1), a dehydratase enzyme that is a promising target for antivirulence drug discovery, is described. The resolution at 1.35 Å of the crystal structure of DHQ1 from Salmonella typhi chemically modified by this ammonium derivative revealed that the ligand is covalently attached to the essential Lys170 through the formation of an amine. The detection by mass spectroscopy of the reaction intermediates, in conjunction with the results of molecular dynamics simulations, allowed us to explain the inhibition mechanism and the experimentally observed differences between S. typhi and Staphylococcus aureus enzymes. The results presented here reveal that the replacement of Phe225 in St-DHQ1 by Tyr214 in Sa-DHQ1 and its hydrogen bonding interaction with the conserved water molecule observed in several crystal structures protects the amino adduct against further dehydration/aromatization reactions. In contrast, for the St-DHQ1 enzyme, the carboxylate group of Asp114, with the assistance of this water molecule, would trigger the formation of a Schiff base that can undergo further dehydration reactions until full aromatization of the cyclohexane ring is achieved. Moreover, in vitro antivirulence studies showed that the reported compound is able to reduce the ability of Salmonella Enteritidis to kill A459 respiratory cells. These studies have identified a good scaffold for the design of irreversible inhibitors that can be used as drugs and has opened up new opportunities for the development of novel antivirulence agents by targeting the DHQ1 enzyme.

  12. New insights into the mechanism of the Schiff base hydrolysis catalyzed by type I dehydroquinate dehydratase from S. enterica: a theoretical study.

    PubMed

    Yao, Yuan; Li, Ze-Sheng

    2012-09-21

    The reaction pathway of Schiff base hydrolysis catalyzed by type I dehydroquinate dehydratase (DHQD) from S. enterica has been studied by performing molecular dynamics (MD) simulations and density functional theory (DFT) calculations and the corresponding potential energy profile has also been identified. On the basis of the results, the catalytic hydrolysis process for the wild-type enzyme consists of three major reaction steps, including nucleophilic attack on the carbon atom involved in the carbon-nitrogen double bond of the Schiff base intermediate by a water molecule, deprotonation of the His143 residue, and dissociation between the product and the Lys170 residue of the enzyme. The remarkable difference between this and the previously proposed reaction mechanism is that the second step here, absent in the previously proposed reaction mechanism, plays an important role in facilitating the reaction through a key proton transfer by the His143 residue, resulting in a lower energy barrier. Comparison with our recently reported results on the Schiff base formation and dehydration processes clearly shows that the Schiff base hydrolysis is rate-determining in the overall reaction catalyzed by type I DHQD, consistent with the experimental prediction, and the calculated energy barrier of ∼16.0 kcal mol(-1) is in good agreement with the experimentally derived activation free energy of ∼14.3 kcal mol(-1). When the imidazole group of His143 residue is missing, the Schiff base hydrolysis is initiated by a hydroxide ion in the solution, rather than a water molecule, and both the reaction mechanism and the kinetics of Schiff base hydrolysis have been remarkably changed, clearly elucidating the catalytic role of the His143 residue in the reaction. The new mechanistic insights obtained here will be valuable for the rational design of high-activity inhibitors of type I DHQD as non-toxic antimicrobials, anti-fungals, and herbicides.

  13. Diphenyl diselenide [(PhSe)2] inhibits Drosophila melanogaster delta-aminolevulinate dehydratase (delta-ALA-D) gene transcription and enzyme activity.

    PubMed

    Golombieski, R M; Graichen, D A S; Pivetta, L A; Nogueira, C W; Loreto, E L S; Rocha, J B T

    2008-03-01

    The main objective of the present study was to compare the inhibitory effect of diphenyl diselenide (PhSe)(2) and Pb(2+) on mice and fruit fly delta-Aminolevulinate dehydratase (delta-ALA-D). Optimum pH was quite different for mice (pH 6.5) and flies (pH 8.5). At pH 8.5, the inhibitory potency of (PhSe)(2) was higher for the fruit flies (IC(50) 8.2 micromol/l) than for mice (IC(50) 19.5 micromol/l). Pb(2+) inhibited mice delta-ALA-D at pH 6.5 (IC(50) 6.2 micromol/l) and 8.5 (IC(50) 5.6 micromol/l) with higher potency than the fly enzyme (IC(50) 43.7 micromol/l). delta-ALA-D transcription was reduced by 15% in flies exposed to 0.3 mmol/kg (PhSe)(2), which is similar to the reduction observed in activity measured in the presence of dithiothreitol. The three-dimensional prediction by SWISS-PROT mouse and fly delta-ALA-D revealed differences in the number of hydrogen bonds and turns for the 2 enzymes. Sulfhydryl groups (-SH) that could be oxidized by (PhSe)(2) are conserved in the two sources of enzyme. Distinct responsiveness to pH, (PhSe)(2) and Pb(2+) of these enzymes may be related to subtle differences in tertiary or quaternary structure of mouse and fly delta-ALA-D. Furthermore, mechanism underlying enzyme inhibition after in vivo exposure seems to be different for Drosophila melanogaster and rodent enzymes. PMID:17936691

  14. Comparison of in vivo effect of inorganic lead and cadmium on glutathione reductase system and delta-aminolevulinate dehydratase in human erythrocytes.

    PubMed Central

    Roels, H A; Buchet, J P; Lauwerys, R R; Sonnet, J

    1975-01-01

    The activity of delta-aminolevulinate dehydratase (ALAD) of erythrocytes, the lead (Pb-B) and cadmium (Cd-B) concentration in whole blood, the content of reduced glutathion (GSH) in erythrocytes, and the regeneration rate of GSH by intact erythrocytes were measured during an epidemiological survey of 84 men employed in a Belgian cadmium and lead producing plant. A control group of 26 persons (students and laboratory staff) was also examined. The logarithm of the ALAD activity is highly inversely correlated with log Pb-B (r = -0.760) but no correlation was found with log Cd-B. There exists a significant negative correlation between GSH and log Pb-B (r = -0.423) but not between GSH AND LOG Cd-B. The apparently good relationship between log ALAD and GSH disappeared completely by holding log Pb-B constant, but log ALAD remained highly inversely correlated with log Pb-B when standardized for GSH concentration (r = -0.748). In vivo investigation of the GSH regeneration rate of intact erythrocytes demonstrated clearly that the overall activity of the glutathione oxidation-reduction pathways is not impaired in Pb and Cd-exposed workers with significantly increased Pb-B and Cd-B, since their initial GSH regeneration rate (first 15 minutes) was identical with that of the control group. Results of similar in vitro experiments in which control whole blood was incubated before-hand with Pb2+ or Cd2+, or both, reinforce this conclusion. Since increased Cd-B and Pb-B do not influence the glutathione reductase system of erythrocytes, and since endogenous erythrocyte GSH is not correlated with Cd-B, the moderate decrease in endogenous erythrocyte Gsh found in Pb-exposed workers might result from a Pb-induced impairment for the erythrocyte mechanism for glutathione synthesis. PMID:1156566

  15. Crystal structure of a tetrameric GDP-D-mannose 4,6-dehydratase from a bacterial GDP-D-rhamnose biosynthetic pathway

    SciTech Connect

    Webb, N.A.; Mulichak, A.M.; Lam, J.S.; Rocchetta, H.L.; Garavito, R.M.

    2010-03-08

    D-Rhamnose is a rare 6-deoxy monosaccharide primarily found in the lipopolysaccharide of pathogenic bacteria, where it is involved in host-bacterium interactions and the establishment of infection. The biosynthesis of D-rhamnose proceeds through the conversion of GDP-D-mannose by GDP-D-mannose 4,6-dehydratase (GMD) to GDP-4-keto-6-deoxymannose, which is subsequently reduced to GDP-D-rhamnose by a reductase. We have determined the crystal structure of GMD from Pseudomonas aeruginosa in complex with NADPH and GDP. GMD belongs to the NDP-sugar modifying subfamily of the short-chain dehydrogenase/reductase (SDR) enzymes, all of which exhibit bidomain structures and a conserved catalytic triad (Tyr-XXX-Lys and Ser/Thr). Although most members of this enzyme subfamily display homodimeric structures, this bacterial GMD forms a tetramer in the same fashion as the plant MUR1 from Arabidopsis thaliana. The cofactor binding sites are adjoined across the tetramer interface, which brings the adenosyl phosphate moieties of the adjacent NADPH molecules to within 7 {angstrom} of each other. A short peptide segment (Arg35-Arg43) stretches into the neighboring monomer, making not only protein-protein interactions but also hydrogen bonding interactions with the neighboring cofactor. The interface hydrogen bonds made by the Arg35-Arg43 segment are generally conserved in GMD and MUR1, and the interacting residues are highly conserved among the sequences of bacterial and eukaryotic GMDs. Outside of the Arg35-Arg43 segment, residues involved in tetrameric contacts are also quite conserved across different species. These observations suggest that a tetramer is the preferred, and perhaps functionally relevant, oligomeric state for most bacterial and eukaryotic GMDs.

  16. Delta-aminolevulinic acid dehydratase (δALAD) activity in four free-living bird species exposed to different levels of lead under natural conditions.

    PubMed

    Espín, Silvia; Martínez-López, Emma; Jiménez, Pedro; María-Mojica, Pedro; García-Fernández, Antonio J

    2015-02-01

    The purposes of this study were: (1) to determine the δALAD activity and δALAD ratio in blood of four free-living bird species (Griffon vulture, Eagle owl, Slender-billed gull and Audouin's gull); (2) and to investigate the correlations between δALAD activity/ratio and Pb concentrations in blood samples. A decrease was observed in δALAD activity in Griffon vultures and Eagle owls exposed to Pb. In addition, negative relationships were found between δALAD ratio or δALAD activity and Log blood Pb levels in Griffon vultures and Eagle owls, and these relationships were stronger in areas with the highest Pb exposure. We provide equations that may be helpful to estimate δALAD activity and δALAD ratio using blood Pb concentrations. Regarding gull species, δALAD activity found in the present study may be considered the normal activity in Slender-billed gull and Audouin's gull species, since very low blood Pb concentrations and no correlations were found in these species. Although both δALAD activity and δALAD ratio are sensitive biomarkers of Pb exposure and effect in birds, the use of δALAD ratio may improve the results. Besides, this study provides blood threshold concentrations at which Pb bears effects on δALAD enzyme (5µg/dl in Eagle owl; 8µg/dl in Griffon vulture; and probably >2µg/dl in Slender-billed gull and Audouin's gull). Our findings show that Eagle owl seems to be more sensitive to δALAD enzymatic inhibition by Pb than Griffon vultures. Eagle owls and Griffon vultures exhibited up to 79% and 94% decrease in δALAD activity when blood Pb concentrations exceeded 19 and 30µg/dl, respectively. Regarding the effects related with δALAD inhibition, significant negative correlations were found between δALAD activity and hematocrit in Eagle owls and Griffon vultures, which may be related to compensatory response associated with a decrease in δALAD activity. In addition, an effect on creatine kinase activity and total proteins in plasma was found in Griffon vultures. The significant negative correlations found between δALAD activity and tGSH in Griffon vulture, and between δALAD activity and Log CAT activity in Eagle owls, may be related to a protective response of antioxidant system against reactive oxygen species (ROS). The negative relationship found between δALAD activity and TBARS levels in Griffon vulture is probably related to an induction of lipid peroxidation by ROS that may be generated by δALA accumulation when δALAD activity is depressed. δALAD activity and δALAD ratio in blood are suggested as important nondestructive biomarkers for Pb exposure and effect for future biomonitoring studies in Griffon vulture and Eagle owl. Further studies are recommended to provide new data on Pb concentrations at which δALAD activity is affected in different wild bird species and to elucidate why different species tolerate Pb in different ways. PMID:25569843

  17. Delta-aminolevulinic acid dehydratase (δALAD) activity in four free-living bird species exposed to different levels of lead under natural conditions.

    PubMed

    Espín, Silvia; Martínez-López, Emma; Jiménez, Pedro; María-Mojica, Pedro; García-Fernández, Antonio J

    2015-02-01

    The purposes of this study were: (1) to determine the δALAD activity and δALAD ratio in blood of four free-living bird species (Griffon vulture, Eagle owl, Slender-billed gull and Audouin's gull); (2) and to investigate the correlations between δALAD activity/ratio and Pb concentrations in blood samples. A decrease was observed in δALAD activity in Griffon vultures and Eagle owls exposed to Pb. In addition, negative relationships were found between δALAD ratio or δALAD activity and Log blood Pb levels in Griffon vultures and Eagle owls, and these relationships were stronger in areas with the highest Pb exposure. We provide equations that may be helpful to estimate δALAD activity and δALAD ratio using blood Pb concentrations. Regarding gull species, δALAD activity found in the present study may be considered the normal activity in Slender-billed gull and Audouin's gull species, since very low blood Pb concentrations and no correlations were found in these species. Although both δALAD activity and δALAD ratio are sensitive biomarkers of Pb exposure and effect in birds, the use of δALAD ratio may improve the results. Besides, this study provides blood threshold concentrations at which Pb bears effects on δALAD enzyme (5µg/dl in Eagle owl; 8µg/dl in Griffon vulture; and probably >2µg/dl in Slender-billed gull and Audouin's gull). Our findings show that Eagle owl seems to be more sensitive to δALAD enzymatic inhibition by Pb than Griffon vultures. Eagle owls and Griffon vultures exhibited up to 79% and 94% decrease in δALAD activity when blood Pb concentrations exceeded 19 and 30µg/dl, respectively. Regarding the effects related with δALAD inhibition, significant negative correlations were found between δALAD activity and hematocrit in Eagle owls and Griffon vultures, which may be related to compensatory response associated with a decrease in δALAD activity. In addition, an effect on creatine kinase activity and total proteins in plasma was found in Griffon vultures. The significant negative correlations found between δALAD activity and tGSH in Griffon vulture, and between δALAD activity and Log CAT activity in Eagle owls, may be related to a protective response of antioxidant system against reactive oxygen species (ROS). The negative relationship found between δALAD activity and TBARS levels in Griffon vulture is probably related to an induction of lipid peroxidation by ROS that may be generated by δALA accumulation when δALAD activity is depressed. δALAD activity and δALAD ratio in blood are suggested as important nondestructive biomarkers for Pb exposure and effect for future biomonitoring studies in Griffon vulture and Eagle owl. Further studies are recommended to provide new data on Pb concentrations at which δALAD activity is affected in different wild bird species and to elucidate why different species tolerate Pb in different ways.

  18. Dexamethasone alone and in combination with desipramine, phenytoin, valproic acid or levetiracetam interferes with 5-ALA-mediated PpIX production and cellular retention in glioblastoma cells.

    PubMed

    Lawrence, Johnathan E; Steele, Christopher J; Rovin, Richard A; Belton, Robert J; Winn, Robert J

    2016-03-01

    Extent of resection of glioblastoma (GBM) correlates with overall survival. Fluorescence-guided resection (FGR) using 5-aminolevulinic acid (5-ALA) can improve the extent of resection. Unfortunately not all patients given 5-ALA accumulate sufficient quantities of protoporphyrin IX (PpIX) for successful FGR. In this study, we investigated the effects of dexamethasone, desipramine, phenytoin, valproic acid, and levetiracetam on the production and accumulation of PpIX in U87MG cells. All of these drugs, except levetiracetam, reduce the total amount of PpIX produced by GBM cells (p < 0.05). When dexamethasone is mixed with another drug (desipramine, phenytoin, valproic acid or levetiracetam) the amount of PpIX produced is further decreased (p < 0.01). However, when cells are analyzed for PpIX cellular retention, dexamethasone accumulated significantly more PpIX than the vehicle control (p < 0.05). Cellular retention of PpIX was not different from controls in cells treated with dexamethasone plus desipramine, valproic acid or levetiracetam, but was significantly less for dexamethasone plus phenytoin (p < 0.01). These data suggest that medications given before and during surgery may interfere with PpIX accumulation in malignant cells. At this time, levetiracetam appears to be the best medication in its class (anticonvulsants) for patients undergoing 5-ALA-mediated FGR.

  19. Regulation of ion homeostasis by aminolevulinic acid in salt-stressed wheat seedlings

    NASA Astrophysics Data System (ADS)

    Türk, Hülya; Genişel, Mucip; Erdal, Serkan

    2016-04-01

    Salinity is regarded as a worldwide agricultural threat, as it seriously limits plant development and productivity. Salt stress reduces water uptake in plants by disrupting the osmotic balance of soil solution. In addition, it creates a damaged metabolic process by causing ion imbalance in cells. In this study, we aim to examine the negative effects of 5-aminolevulinic acid (ALA) (20 mg/l) on the ion balance in wheat seedling leaves exposed to salt stress (150 mM). Sodium is known to be highly toxic for plant cells at high concentrations, and is significantly increased by salt stress. However, it can be reduced by combined application of ALA and salt, compared to salt application alone. On the other hand, while the K+/Na+ ratio was reduced by salt stress, ALA application changed this ratio in favor of K+. Manganese, iron, and copper were also able to reduce stress. However, ALA pre-treatment resulted in mineral level increments. Conversely, the stress-induced rise in magnesium, potassium, calcium, phosphorus, zinc, and molybdenum were further improved by ALA application. These data clearly show that ALA has an important regulatory effect of ion balance in wheat leaves.

  20. Delineating Normal from Diseased Brain by Aminolevulinic Acid-Induced Fluorescence

    NASA Astrophysics Data System (ADS)

    Stepp, Herbert; Stummer, Walter

    5-Aminolevulinic acid (5-ALA) as a precursor of protoporphyrin IX (PpIX) has been established as an orally applied drug to guide surgical resection of malignant brain tumors by exciting the red fluorescence of PpIX. The accumulation of PpIX in glioblastoma multiforme (GBM) is highly selective and provides excellent contrast to normal brain when using surgical microscopes with appropriately filtered light sources and cameras. The positive predictive value of fluorescent tissue is very high, enabling safe gross total resection of GBM and other brain tumors and improving prognosis of patients. Compared to other intraoperative techniques that have been developed with the aim of increasing the rate of safe gross total resections of malignant gliomas, PpIX fluorescence is considerably simpler, more cost effective, and comparably reliable. We present the basics of 5-ALA-based fluorescence-guided resection, and discuss the clinical results obtained for GBM and the experience with the fluorescence staining of other primary brain tumors and metastases as well as the results for spinal cord tumors. The phototoxicity of PpIX, increasingly used for photodynamic therapy of brain tumors, is mentioned briefly in this chapter.

  1. Photodynamic therapy with 5-aminoolevulinic acid-induced porphyrins and DMSO/EDTA for basal cell carcinoma

    NASA Astrophysics Data System (ADS)

    Warloe, Trond; Peng, Qian; Heyerdahl, Helen; Moan, Johan; Steen, Harald B.; Giercksky, Karl-Erik

    1995-03-01

    Seven hundred sixty three basal cell carcinomas (BCCs) in 122 patients were treated by photodynamic therapy by 5-aminolevulinic acid (ALA) in cream topically applied, either alone, in combination with dimethyl sulphoxide (DMSO) and ethylenediaminetetraacetic acid disodium salt (EDTA), or with DMSO as a pretreatment. After 3 hours cream exposure 40 - 200 Joules/cm2 of 630 nm laser light was given. Fluorescence imaging of biopsies showed highly improved ALA penetration depth and doubled ALA-induced porphyrin production using DMSO/EDTA. Treatment response was recorded after 3 months. After a single treatment 90% of 393 superficial lesions responded completely, independent of using DMSO/EDTA. In 363 nodulo-ulcerative lesions the complete response rate increased from 67% to above 90% with DMSO/EDTA for lesions less than 2 mm thickness and from 34% to about 50% for lesions thicker than 2 mm. Recurrence rate observed during a follow-up period longer than 12 months was 2 - 5%. PDT of superficial thin BCCs with ALA-induced porphyrins and DMSO/EDTA equals surgery and radiotherapy with respect to cure rate and recurrence. Cosmetic results of ALA-based PDT seemed to be better than those after other therapies. In patients with the nevoid BCC syndrome the complete response rate after PDT was far lower.

  2. Mechanisms of 5-aminolevulic acid ester uptake in mammalian cells

    PubMed Central

    Rodriguez, Lorena; Batlle, Alcira; Di Venosa, Gabriela; Battah, Sinan; Dobbin, Paul; MacRobert, Alexander J; Casas, Adriana

    2006-01-01

    The porphyrin precursor 5-aminolevulinic acid (ALA) is being widely used in photodynamic therapy of cancer. Improvement in ALA delivery has been sought through the use of ALA derivatives, in particular the esterification of ALA with aliphatic alcohols, which in certain cases can improve cellular penetration and selectivity. ALA uptake systems appear to be distinctive for each cell type. The LM3 mammary adenocarcinoma cell line takes ALA up by BETA transporters. In this work, we investigated ALA derivative transport systems through the inhibition of radiolabelled ALA uptake in the LM3 cells. We also performed inhibition studies of γ-aminobutyric acid (GABA) uptake. The more lipohilic ALA derivatives hexyl-ALA and undecanoyl-ALA inhibit ALA uptake, whereas methyl-ALA, R, S-ALA-2-(hydroxymethyl)tetrahydropyranyl ester and the dendron aminomethane tris methyl 5-ALA does not inhibit ALA uptake. A similar pattern was found for GABA, except that the dendron inhibited GABA uptake. However, hexyl-ALA and undecanoyl-ALA are not taken up by BETA transporters, but by simple diffusion, although they still inhibit ALA uptake by binding to the cell membrane. These results show that different modifications to the ALA molecule lead to different uptake mechanisms. Whereas ALA is taken up by BETA transporters, none of the ALA derivatives shares the same mechanism. Knowledge of the mechanisms of ALA derivatives entry into the cells is essential to understand and improve ALA-mediated PDT and to the design of new ALA derivatives that may be taken up at a higher rate than ALA. PMID:16432502

  3. Glycerol metabolism and bitterness producing lactic acid bacteria in cidermaking.

    PubMed

    Garai-Ibabe, G; Ibarburu, I; Berregi, I; Claisse, O; Lonvaud-Funel, A; Irastorza, A; Dueñas, M T

    2008-02-10

    Several lactic acid bacteria were isolated from bitter tasting ciders in which glycerol was partially removed. The degradation of glycerol via glycerol dehydratase pathway was found in 22 out of 67 isolates. The confirmation of glycerol degradation by this pathway was twofold: showing their glycerol dehydratase activity and detecting the presence of the corresponding gene by a PCR method. 1,3-propanediol (1,3-PDL) and 3-hydroxypropionic acid (3-HP) were the metabolic end-products of glycerol utilization, and the accumulation of the acrolein precursor 3-hydroxypropionaldehyde (3-HPA) was also detected in most of them. The strain identification by PCR-DGGE rpoB showed that Lactobacillus collinoides was the predominant species and only 2 belonged to Lactobacillus diolivorans. Environmental conditions conducting to 3-HPA accumulation in cidermaking were studied by varying the fructose concentration, pH and incubation temperature in L. collinoides 17. This strain failed to grow with glycerol as sole carbon source and the addition of fructose enhanced both growth and glycerol degradation. Regarding end-products of glycerol metabolism, 1,3-PDL was always the main end-product in all environmental conditions assayed, the only exception being the culture with 5.55 mM fructose, where equimolar amounts of 1,3-PDL and 3-HP were found. The 3-HPA was transitorily accumulated in the culture medium under almost all culture conditions, the degradation rate being notably slower at 15 degrees C. However, no disappearance of 3-HPA was found at pH 3.6, a usual value in cider making. After sugar exhaustion, L. collinoides 17 oxidated lactic acid and/or mannitol to obtain energy and these oxidations were accompanied by the removal of the toxic 3-HPA increasing the 1,3-PDL, 3-HP and acetic acid contents. PMID:18180066

  4. Gene-Enzyme Relationships of Aromatic Amino Acid Biosynthesis in Higher Plants

    SciTech Connect

    2002-08-12

    Inhibition studies of amino acids in Nicotiana silvestris suspension cells gave clues to the difficulties for obtaining mutants deficient in post prephenate pathway proteins of aromatic amino acid biosynthesis (prephenate aminotransferase, arogenate dehydrogenase and arogenate dehydratase). Such mutants, if successfully obtained, would allow gene-enzyme relationships of aromatic amino acid proteins to be studied. We found that amino acids were inhibitory toward plant cell growth, and thus were unable to rescue analog resistant mutants. Toxicity of all amino acids toward exponentially dividing Nicotiana silvestris suspension cultured cells was monitored by following growth rates. Except for L-glutamine, all 19 protein amino acids inhibited cell growth. Inhibition of growth progressed to cell deterioration. Electron microscopy showed that amino acids triggered a state of cell shrinkage that eventually degenerated to total cellular disorganization. L-glutamine was not only an effective agent for prevention of amino acid toxicity, but enhanced the final growth yield. L-glutamine also was able to completely reverse inhibition effects in cells that had been in the slowed exponential phase. Two types of inhibition occurred and we have proposed that any amino acid inhibition that can be completely antagonized by L-glutamine be called ''general amino acid inhibition''. ''Specific amino acid inhibition'' resulting from particular pathway imbalances caused by certain exogenous amino acids, can be recognized and studied in the presence of L-glutamine which can abolishes the complication effects of general amino acid inhibition.

  5. Derepression of certain aromatic amino acid biosynthetic enzymes of Escherichia coli K-12 by growth in Fe3+-deficient medium.

    PubMed Central

    McCray, J W; Herrmann, K M

    1976-01-01

    3-Deoxy-arabino-heptulosonic acid 7-phosphate synthase, prephenate dehydratase, tryptophan synthase, and 2,3-dihydroxybenzoylserine synthase enzyme activities are derepressed in wild-type Escherichia coli K-12 cells grown on Fe3+-deficient medium. This derepression is reversed when FeSO4 is added to the growth medium. Addition of shikimic acid to the Fe3+-deficient growth medium caused repression of the first three enzyme activities but not of 2,3-dihydroxybenzoylserine synthase activity. Addition of 2,3-dihydroxybenzoic acid to the Fe3+-deficient growth medium has no effect on any of the above-mentioned enzyme activities. The Fe3+ deficiency-mediated derepression of 3-deoxyarabino-heptulosonic acid 7-phosphate synthase activity is due to an elevation of the tyrosine-sensitive isoenzyme; the phenylalanine-sensitive isoenzyme is not derepressed under these conditions. PMID:1383

  6. 5-Aminolevolinic acid mitigates the cadmium-induced changes in Brassica napus as revealed by the biochemical and ultra-structural evaluation of roots.

    PubMed

    Ali, Basharat; Tao, Qiaojing; Zhou, Yuanfei; Gill, Rafaqat A; Ali, Shafaqat; Rafiq, Muhammad T; Xu, Ling; Zhou, Weijun

    2013-06-01

    In the present study, the ameliorating effects of 5-aminolevulinic acid (ALA) under cadmium (Cd) stress conditions were studied with special emphasis on root morphology and ultra-structure in oilseed rape. For this purpose, plants were treated hydroponically at three different Cd levels (0, 100, 500μM) and foliar spray of ALA with three concentrations (0, 12.5, 25mg/l) simultaneously. The results showed that foliar application of ALA improved the plant growth, root morphology and reduced the reactive oxygen species and malondialdehyde contents in roots under Cd stress conditions. The higher concentration of Cd (500μM) decreased the activities of antioxidants enzymes like catalase (CAT), superoxide dismutase (SOD), peroxidase (POD) and glutathione reductase (GR) and also reduced the oxidized glutathione and total glutathione contents in roots. Application of ALA at 25mg/l dosage significantly enhanced the antioxidant activities e.g. APX, SOD, POD, and GSH contents under Cd stress. The microscopic micrographs showed that application of exogenous ALA improved the cell structure under Cd toxicity. A whole cell with developed nucleus, nuclear membrane, smooth cell wall, continuous endoplasmic reticulum, and well shaped mitochondria was observed under the combine application of ALA and Cd. These results suggest that, application of ALA helped the plants to improve root growth, root antioxidant enzymes, and ultra-structural changes in root tip cells under fifteen days Cd-induced stress. PMID:23490193

  7. Adaptational modification of serine and threonine metabolism in the liver to essential amino acid deficiency in rats.

    PubMed

    Nagao, Kenji; Bannai, Makoto; Seki, Shinobu; Mori, Masato; Takahashi, Michio

    2009-03-01

    It is known that plasma serine and threonine concentrations are elevated in rats chronically fed an essential amino acid deficient diet, but the underlying mechanisms including related gene expressions or serine and threonine concentrations in liver remained to be elucidated. We fed rats lysine or valine deficient diet for 4 weeks and examined the mRNA expressions of serine synthesising (3-phosphoglycerate dehydrogenase, PHGDH) and serine/threonine degrading enzymes (serine dehydratase, SDS) in the liver. Dietary deficiency induced marked elevation of hepatic serine and threonine levels associated with enhancement of PHGDH mRNA expression and repression of SDS mRNA expression. Increases in plasma serine and threonine levels due to essential amino acid deficiency in diet were caused by marked increases in hepatic serine and threonine levels. Proteolytic responses to the amino acid deficiency may be lessened by storing amino radicals as serine and inducing anorexia through elevation of threonine. PMID:18584286

  8. Isolation and Characterization of Unsaturated Fatty Acid Auxotrophs of Streptococcus pneumoniae and Streptococcus mutans▿

    PubMed Central

    Altabe, Silvia; Lopez, Paloma; de Mendoza, Diego

    2007-01-01

    Unsaturated fatty acid (UFA) biosynthesis is essential for the maintenance of membrane structure and function in many groups of anaerobic bacteria. Like Escherichia coli, the human pathogen Streptococcus pneumoniae produces straight-chain saturated fatty acids (SFA) and monounsaturated fatty acids. In E. coli UFA synthesis requires the action of two gene products, the essential isomerase/dehydratase encoded by fabA and an elongation condensing enzyme encoded by fabB. S. pneumoniae lacks both genes and instead employs a single enzyme with only an isomerase function encoded by the fabM gene. In this paper we report the construction and characterization of an S. pneumoniae 708 fabM mutant. This mutant failed to grow in complex medium, and the defect was overcome by addition of UFAs to the growth medium. S. pneumoniae fabM mutants did not produce detectable levels of monounsaturated fatty acids as determined by gas chromatography-mass spectrometry and thin-layer chromatography analysis of the radiolabeled phospholipids. We also demonstrate that a fabM null mutant of the cariogenic organism Streptococcus mutants is a UFA auxotroph, indicating that FabM is the only enzyme involved in the control of membrane fluidity in streptococci. Finally we report that the fabN gene of Enterococcus faecalis, coding for a dehydratase/isomerase, complements the growth of S. pneumoniae fabM mutants. Taken together, these results suggest that FabM is a potential target for chemotherapeutic agents against streptococci and that S. pneumoniae UFA auxotrophs could help identify novel genes encoding enzymes involved in UFA biosynthesis. PMID:17827283

  9. Combinatorial Effects of Fatty Acid Elongase Enzymes on Nervonic Acid Production in Camelina sativa

    PubMed Central

    Huai, Dongxin; Zhang, Yuanyuan; Zhang, Chunyu; Cahoon, Edgar B.; Zhou, Yongming

    2015-01-01

    Very long chain fatty acids (VLCFAs) with chain lengths of 20 carbons and longer provide feedstocks for various applications; therefore, improvement of VLCFA contents in seeds has become an important goal for oilseed enhancement. VLCFA biosynthesis is controlled by a multi-enzyme protein complex referred to as fatty acid elongase, which is composed of β-ketoacyl-CoA synthase (KCS), β-ketoacyl-CoA reductase (KCR), β-hydroxyacyl-CoA dehydratase (HCD) and enoyl reductase (ECR). KCS has been identified as the rate-limiting enzyme, but little is known about the involvement of other three enzymes in VLCFA production. Here, the combinatorial effects of fatty acid elongase enzymes on VLCFA production were assessed by evaluating the changes in nervonic acid content. A KCS gene from Lunaria annua (LaKCS) and the other three elongase genes from Arabidopsis thaliana were used for the assessment. Five seed-specific expressing constructs, including LaKCS alone, LaKCS with AtKCR, LaKCS with AtHCD, LaKCS with AtECR, and LaKCS with AtKCR and AtHCD, were transformed into Camelina sativa. The nervonic acid content in seed oil increased from null in wild type camelina to 6-12% in LaKCS-expressing lines. However, compared with that from the LaKCS-expressing lines, nervonic acid content in mature seeds from the co-expressing lines with one or two extra elongase genes did not show further increases. Nervonic acid content from LaKCS, AtKCR and AtHCD co-expressing line was significantly higher than that in LaKCS-expressing line during early seed development stage, while the ultimate nervonic acid content was not significantly altered. The results from this study thus provide useful information for future engineering of oilseed crops for higher VLCFA production. PMID:26121034

  10. Stress response of lead-exposed rainbow trout (Oncorhynchus mykiss) during swimming performance and hypoxia challenges

    SciTech Connect

    Phillips, K.A. |; Caldwell, C.A.; Sandheinrich, M.B.

    1995-12-31

    Contaminants often invoke a stress response in aquatic organisms, and may compromise their capacity to respond to secondary stressors. This may reduce growth, reproduction and survival. The authors objectives were to assess the effects of lead and secondary stressors on hematology and blood chemistry of rainbow trout. After a 7 to 8-week aqueous exposure to Pb(100{micro}g/L), rainbow trout were challenged with forced swimming or hypoxia. Lead significantly reduced concentrations of 5-aminolevulinic acid dehydratase (ALAD), but not other constituents in the blood. Lead did not affect the swimming endurance of the fish. Hematocrit, mean cell hemoglobin content, and mean cell volume were significantly lower in Pb-exposed trout following the swimming challenge. Although hypoxia resulted in increased hematocrit and plasma glucose concentrations, there were no significant differences between the Pb and control groups. Hypoxia did not affect plasma chloride concentrations, although concentrations increased in Pb-exposed trout. There was no difference in lactic acid concentrations between Pb-exposed and control fish after forced swimming or hypoxia.

  11. A pair of light signaling factors FHY3 and FAR1 regulates plant immunity by modulating chlorophyll biosynthesis.

    PubMed

    Wang, Wanqing; Tang, Weijiang; Ma, Tingting; Niu, De; Jin, Jing Bo; Wang, Haiyang; Lin, Rongcheng

    2016-01-01

    Light and chloroplast function is known to affect the plant immune response; however, the underlying mechanism remains elusive. We previously demonstrated that two light signaling factors, FAR-RED ELONGATED HYPOCOTYL 3 (FHY3) and FAR-RED IMPAIRED RESPONSE 1 (FAR1), regulate chlorophyll biosynthesis and seedling growth via controlling HEMB1 expression in Arabidopsis thaliana. In this study, we reveal that FHY3 and FAR1 are involved in modulating plant immunity. We showed that the fhy3 far1 double null mutant displayed high levels of reactive oxygen species and salicylic acid (SA) and increased resistance to Pseudomonas syringae pathogen infection. Microarray analysis revealed that a large proportion of pathogen-related genes, particularly genes encoding nucleotide-binding and leucine-rich repeat domain resistant proteins, are highly induced in fhy3 far1. Genetic studies indicated that the defects of fhy3 far1 can be largely rescued by reducing SA signaling or blocking SA accumulation, and by overexpression of HEMB1, which encodes a 5-aminolevulinic acid dehydratase in the chlorophyll biosynthetic pathway. Furthermore, we found that transgenic plants with reduced expression of HEMB1 exhibit a phenotype similar to fhy3 far1. Taken together, this study demonstrates an important role of FHY3 and FAR1 in regulating plant immunity, through integrating chlorophyll biosynthesis and the SA signaling pathway.

  12. Investigating the physiological roles of low-efficiency D-mannonate and D-gluconate dehydratases in the enolase superfamily: pathways for the catabolism of L-gulonate and L-idonate.

    PubMed

    Wichelecki, Daniel J; Vendiola, Jean Alyxa Ferolin; Jones, Amy M; Al-Obaidi, Nawar; Almo, Steven C; Gerlt, John A

    2014-09-01

    The sequence/function space in the D-mannonate dehydratase subgroup (ManD) of the enolase superfamily was investigated to determine how enzymatic function diverges as sequence identity decreases [Wichelecki, D. J., et al. (2014) Biochemistry 53, 2722-2731]. That study revealed that members of the ManD subgroup vary in substrate specificity and catalytic efficiency: high-efficiency (kcat/KM = 10(3)-10(4) M(-1) s(-1)) for dehydration of D-mannonate, low-efficiency (kcat/KM = 10-10(2) M(-1) s(-1)) for dehydration of D-mannonate and/or D-gluconate, and no activity. Characterization of high-efficiency members revealed that these are ManDs in the D-glucuronate catabolic pathway {analogues of UxuA [Wichelecki, D. J., et al. (2014) Biochemistry 53, 4087-4089]}. However, the genomes of organisms that encode low-efficiency members of the ManDs subgroup encode UxuAs; therefore, these must have divergent physiological functions. In this study, we investigated the physiological functions of three low-efficiency members of the ManD subgroup and identified a novel physiologically relevant pathway for L-gulonate catabolism in Chromohalobacter salexigens DSM3043 as well as cryptic pathways for L-gulonate catabolism in Escherichia coli CFT073 and L-idonate catabolism in Salmonella enterica subsp. enterica serovar Enteritidis str. P125109. However, we could not identify physiological roles for the low-efficiency members of the ManD subgroup, allowing the suggestion that these pathways may be either evolutionary relics or the starting points for new metabolic potential.

  13. Deletion of fucose residues in plant N-glycans by repression of the GDP-mannose 4,6-dehydratase gene using virus-induced gene silencing and RNA interference.

    PubMed

    Matsuo, Kouki; Matsumura, Takeshi

    2011-02-01

    Production of pharmaceutical glycoproteins in plants has many advantages in terms of safety and reduced costs. However, plant-produced glycoproteins have N-glycans with plant-specific sugar residues (core β-1,2-xylose and α-1,3-fucose) and a Lewis a (Le(a) ) epitope, i.e., Galβ(1-3)[Fucα(1-4)]GlcNAc. Because these sugar residues and glycan structures seemed to be immunogenic, several attempts have been made to delete them by repressing their respective glycosyltransferase genes. However, until date, such deletions have not been successful in completely eliminating the fucose residues. In this study, we simultaneously reduced the plant-specific core α-1,3-fucose and α-1,4-fucose residues in the Le(a) epitopes by repressing the Guanosine 5'-diphosphate (GDP)-D-mannose 4,6-dehydratase (GMD) gene, which is associated with GDP-L-fucose biosynthesis, in Nicotiana benthamiana plants. Repression of GMD was achieved using virus-induced gene silencing (VIGS) and RNA interference (RNAi). The proportion of fucose-free N-glycans found in total soluble protein from GMD gene-repressed plants increased by 80% and 95% following VIGS and RNAi, respectively, compared to wild-type plants. A small amount of putative galactose substitution in N-glycans from the NbGMD gene-repressed plants was observed, similar to what has been previously reported GMD-knockout Arabidopsis mutant. On the other hand, the recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) with fucose-deleted N-glycans was successfully produced in NbGMD-RNAi transgenic N. benthamiana plants. Thus, repression of the GMD gene is thus very useful for deleting immunogenic total fucose residues and facilitating the production of pharmaceutical glycoproteins in plants.

  14. Deletion of plant-specific sugar residues in plant N-glycans by repression of GDP-D-mannose 4,6-dehydratase and β-1,2-xylosyltransferase genes.

    PubMed

    Matsuo, Kouki; Kagaya, Uiko; Itchoda, Noriko; Tabayashi, Noriko; Matsumura, Takeshi

    2014-10-01

    Production of pharmaceutical glycoproteins, such as therapeutic antibodies and cytokines, in plants has many advantages in safety and reduced costs. However, plant-made glycoproteins have N-glycans with plant-specific sugar residues (core β-1,2-xylose and α-1,3-fucose) and a Lewis a (Le(a)) epitope, Galβ(1-3)[Fucα(1-4)]GlcNAc. Because it is likely that these sugar residues and glycan structures are immunogenic, many attempts have been made to delete them. Previously, we reported the simultaneous deletion of the plant-specific core α-1,3-fucose and α-1,4-fucose residues in Le(a) epitopes by repressing the GDP-D-mannose 4,6-dehydratase (GMD) gene, which is associated with GDP-L-fucose biosynthesis, in Nicotiana benthamiana plants (rGMD plants, renamed to ΔGMD plants) (Matsuo and Matsumura, Plant Biotechnol. J., 9, 264-281, 2011). In the present study, we generated a core β-1,2-xylose residue-repressed transgenic N. benthamiana plant by co-suppression of β-1,2-xylosyltransferase (ΔXylT plant). By crossing ΔGMD and ΔXylT plants, we successfully generated plants in which plant-specific sugar residues were repressed (ΔGMDΔXylT plants). The proportion of N-glycans with deleted plant-specific sugar residues found in total soluble protein from ΔGMDΔXylT plants increased by 82.41%. Recombinant mouse granulocyte/macrophage-colony stimulating factor (mGM-CSF) and human monoclonal immunoglobulin G (hIgG) harboring N-glycans with deleted plant-specific sugar residues were successfully produced in ΔGMDΔXylT plants. Simultaneous repression of the GMD and XylT genes in N. benthamiana is thus very useful for deleting plant-specific sugar residues.

  15. Clinically important features of porphyrin and heme metabolism and the porphyrias.

    PubMed

    Besur, Siddesh; Hou, Wehong; Schmeltzer, Paul; Bonkovsky, Herbert L

    2014-11-03

    Heme, like chlorophyll, is a primordial molecule and is one of the fundamental pigments of life. Disorders of normal heme synthesis may cause human diseases, including certain anemias (X-linked sideroblastic anemias) and porphyrias. Porphyrias are classified as hepatic and erythropoietic porphyrias based on the organ system in which heme precursors (5-aminolevulinic acid (ALA), porphobilinogen and porphyrins) are chiefly overproduced. The hepatic porphyrias are further subdivided into acute porphyrias and chronic hepatic porphyrias. The acute porphyrias include acute intermittent, hereditary copro-, variegate and ALA dehydratase deficiency porphyria. Chronic hepatic porphyrias include porphyria cutanea tarda and hepatoerythropoietic porphyria. The erythropoietic porphyrias include congenital erythropoietic porphyria (Gűnther's disease) and erythropoietic protoporphyria. In this review, we summarize the key features of normal heme synthesis and its differing regulation in liver versus bone marrow. In both organs, principal regulation is exerted at the level of the first and rate-controlling enzyme, but by different molecules (heme in the liver and iron in the bone marrow). We also describe salient clinical, laboratory and genetic features of the eight types of porphyria.

  16. Clinically Important Features of Porphyrin and Heme Metabolism and the Porphyrias

    PubMed Central

    Besur, Siddesh; Hou, Weihong; Schmeltzer, Paul; Bonkovsky, Herbert L.

    2014-01-01

    Heme, like chlorophyll, is a primordial molecule and is one of the fundamental pigments of life. Disorders of normal heme synthesis may cause human diseases, including certain anemias (X-linked sideroblastic anemias) and porphyrias. Porphyrias are classified as hepatic and erythropoietic porphyrias based on the organ system in which heme precursors (5-aminolevulinic acid (ALA), porphobilinogen and porphyrins) are chiefly overproduced. The hepatic porphyrias are further subdivided into acute porphyrias and chronic hepatic porphyrias. The acute porphyrias include acute intermittent, hereditary copro-, variegate and ALA dehydratase deficiency porphyria. Chronic hepatic porphyrias include porphyria cutanea tarda and hepatoerythropoietic porphyria. The erythropoietic porphyrias include congenital erythropoietic porphyria (Gűnther’s disease) and erythropoietic protoporphyria. In this review, we summarize the key features of normal heme synthesis and its differing regulation in liver versus bone marrow. In both organs, principal regulation is exerted at the level of the first and rate-controlling enzyme, but by different molecules (heme in the liver and iron in the bone marrow). We also describe salient clinical, laboratory and genetic features of the eight types of porphyria. PMID:25372274

  17. High yield production of D-xylonic acid from D-xylose using engineered Escherichia coli.

    PubMed

    Liu, Huaiwei; Valdehuesa, Kris Niño G; Nisola, Grace M; Ramos, Kristine Rose M; Chung, Wook-Jin

    2012-07-01

    An engineered Escherichia coli was constructed to produce D-xylonic acid, one of the top 30 high-value chemicals identified by US Department of Energy. The native pathway for D-xylose catabolism in E. coli W3110 was blocked by disrupting xylose isomerase (XI) and xylulose kinase (XK) genes. The native pathway for xylonic acid catabolism was also blocked by disrupting two genes both encoding xylonic acid dehydratase (yagE and yjhG). Through the introduction of a D-xylose dehydrogenase from Caulobacter crescentus, a D-xylonic acid producing E. coli was constructed. The recombinant E. coli produced up to 39.2 g L(-1) D-xylonic acid from 40 g L(-1) D-xylose in M9 minimal medium. The average productivity was as high as 1.09 g L(-1) h(-1) and no gluconic acid byproduct was produced. These results suggest that the engineered E. coli has a promising application for the industrial-scale production of D-xylonic acid.

  18. High yield production of D-xylonic acid from D-xylose using engineered Escherichia coli.

    PubMed

    Liu, Huaiwei; Valdehuesa, Kris Niño G; Nisola, Grace M; Ramos, Kristine Rose M; Chung, Wook-Jin

    2012-07-01

    An engineered Escherichia coli was constructed to produce D-xylonic acid, one of the top 30 high-value chemicals identified by US Department of Energy. The native pathway for D-xylose catabolism in E. coli W3110 was blocked by disrupting xylose isomerase (XI) and xylulose kinase (XK) genes. The native pathway for xylonic acid catabolism was also blocked by disrupting two genes both encoding xylonic acid dehydratase (yagE and yjhG). Through the introduction of a D-xylose dehydrogenase from Caulobacter crescentus, a D-xylonic acid producing E. coli was constructed. The recombinant E. coli produced up to 39.2 g L(-1) D-xylonic acid from 40 g L(-1) D-xylose in M9 minimal medium. The average productivity was as high as 1.09 g L(-1) h(-1) and no gluconic acid byproduct was produced. These results suggest that the engineered E. coli has a promising application for the industrial-scale production of D-xylonic acid. PMID:21917451

  19. Efficient production of free fatty acids from soybean meal carbohydrates.

    PubMed

    Wang, Dan; Thakker, Chandresh; Liu, Ping; Bennett, George N; San, Ka-Yiu

    2015-11-01

    Conversion of biomass feedstock to chemicals and fuels has attracted increasing attention recently. Soybean meal, containing significant quantities of carbohydrates, is an inexpensive renewable feedstock. Glucose, galactose, and fructose can be obtained by enzymatic hydrolysis of soluble carbohydrates of soybean meal. Free fatty acids (FFAs) are valuable molecules that can be used as precursors for the production of fuels and other value-added chemicals. In this study, free fatty acids were produced by mutant Escherichia coli strains with plasmid pXZ18Z (carrying acyl-ACP thioesterase (TE) and (3R)-hydroxyacyl-ACP dehydratase) using individual sugars, sugar mixtures, and enzymatic hydrolyzed soybean meal extract. For individual sugar fermentations, strain ML211 (MG1655 fadD(-) fabR(-) )/pXZ18Z showed the best performance, which produced 4.22, 3.79, 3.49 g/L free fatty acids on glucose, fructose, and galactose, respectively. While the strain ML211/pXZ18Z performed the best with individual sugars, however, for sugar mixture fermentation, the triple mutant strain XZK211 (MG1655 fadD(-) fabR(-) ptsG(-) )/pXZ18Z with an additional deletion of ptsG encoding the glucose-specific transporter, functioned the best due to relieved catabolite repression. This strain produced approximately 3.18 g/L of fatty acids with a yield of 0.22 g fatty acids/g total sugar. Maximum free fatty acids production of 2.78 g/L with a high yield of 0.21 g/g was achieved using soybean meal extract hydrolysate. The results suggested that soybean meal carbohydrates after enzymatic treatment could serve as an inexpensive feedstock for the efficient production of free fatty acids.

  20. Recent advances in inhibitors of bacterial fatty acid synthesis type II (FASII) system enzymes as potential antibacterial agents.

    PubMed

    Wang, Yi; Ma, Shutao

    2013-10-01

    Bacterial infections are a constant and serious threat to human health. With the increase of multidrug resistance of clinically pathogenic bacteria, common antibiotic therapies have been less effective. Fatty acid synthesis type II (FASII) system enzymes are essential for bacterial membrane lipid biosynthesis and represent increasingly promising targets for the discovery of antibacterial agents with new mechanisms of action. This review highlights recent advances in inhibitors of bacterial FASII as potential antibacterial agents, paying special attention to the activities, mechanisms, and structure-activity relationships of those inhibitors that mainly target β-ketoacyl-ACP synthase, β-ketoacyl-ACP reductase, β-hydroxyacyl-ACP dehydratase, and enoyl-ACP reductase. Although inhibitors with low nanomolar and selective activity against various bacterial FASII have entered clinical trials, further research is needed to expand upon both available and yet unknown scaffolds to identify new FASII inhibitors that may have antibacterial potential, particularly against resistant bacterial strains.

  1. The intracellular parasite Toxoplasma gondii depends on the synthesis of long chain and very long-chain unsaturated fatty acids not supplied by the host cell

    PubMed Central

    Ramakrishnan, Srinivasan; Docampo, Melissa D.; MacRae, James I.; Ralton, Julie E.; Rupasinghe, Thusitha; McConville, Malcolm J.; Striepen, Boris

    2015-01-01

    SUMMARY Apicomplexa are parasitic protozoa that cause important human diseases including malaria, cryptosporidiosis and toxoplasmosis. The replication of these parasites within their target host cell is dependent on both salvage as well as de novo synthesis of fatty acids. In T. gondii, fatty acid synthesis via the apicoplast-localized FASII is essential for pathogenesis, while the role of two other fatty acid biosynthetic complexes remains unclear. Here we demonstrate that the ER-localized fatty acid elongation (ELO) is essential for parasite growth. Conditional knock-down of the non-redundant hydroxyacyl-CoA dehydratase and enoyl-CoA reductase enzymes in the ELO pathway severely repressed intracellular parasite growth. 13C-glucose and 13C-acetate labeling and comprehensive lipidomic analyses of these mutants showed a selective defect in synthesis of unsaturated long and very long chain fatty acids (LCFAs and VLCFAs) and depletion of phosphatidylinositol and phosphatidylethanolamine species containing unsaturated LCFAs and VLCFAs. This requirement for ELO pathway was by-passed by supplementing the media with specific fatty acids, indicating active, but inefficient import of host fatty acids. Our experiments highlight a gap between the fatty acid needs of the parasite and availability of specific fatty acids in the host cell that the parasite has to close using a dedicated synthesis and modification pathway. PMID:25825226

  2. Involvement of a universal amino acid synthesis impediment in cytoplasmic male sterility in pepper

    PubMed Central

    Fang, Xianping; Fu, Hong-Fei; Gong, Zhen-Hui; Chai, Wei-Guo

    2016-01-01

    To explore the mechanisms of pepper (Capsicum annuum L.) cytoplasmic male sterility (CMS), we studied the different maturation processes of sterile and fertile pepper anthers. A paraffin section analysis of the sterile anthers indicated an abnormality of the tapetal layer and an over-vacuolization of the cells. The quantitative proteomics results showed that the expression of histidinol dehydrogenase (HDH), dihydroxy-acid dehydratase (DAD), aspartate aminotransferase (ATAAT), cysteine synthase (CS), delta-1-pyrroline-5-carboxylate synthase (P5CS), and glutamate synthetase (GS) in the amino acid synthesis pathway decreased by more than 1.5-fold. Furthermore, the mRNA and protein expression levels of DAD, ATAAT, CS and P5CS showed a 2- to 16-fold increase in the maintainer line anthers. We also found that most of the amino acid content levels decreased to varying degrees during the anther tapetum period of the sterile line, whereas these levels increased in the maintainer line. The results of our study indicate that during pepper anther development, changes in amino acid synthesis are significant and accompany abnormal tapetum maturity, which is most likely an important cause of male sterility in pepper. PMID:26987793

  3. Structural elucidation and genomic scrutiny of the C60-C100 mycolic acids of Segniliparus rotundus.

    PubMed

    Lanéelle, Marie-Antoinette; Eynard, Nathalie; Spina, Lucie; Lemassu, Anne; Laval, Françoise; Huc, Emilie; Etienne, Gilles; Marrakchi, Hedia; Daffé, Mamadou

    2013-01-01

    Mycolic acids, very long-chain α-alkyl, β-hydroxylated fatty acids, occur in the members of the order Corynebacteriales where their chain lengths (C(26)-C(88)) and structural features (oxygen functions, cis or trans double bonds, cyclopropane rings and methyl branches) are genus- and species-specific. The molecular composition and structures of the mycolic acids of two species belonging to the genus Segniliparus were determined by a combination of modern analytical chemical techniques, which include MS and NMR. They consist of mono-ethylenic C(62-)C(64) (α'), di-ethylenic C(77)-C(79) (α) and extremely long-chain mycolic acids (α(+)) ranging from 92 to 98 carbon atoms and containing three unsaturations, cis and/or trans double bonds and/or cyclopropanes. The double bonds in each class of mycolic acids were positioned by oxidative cleavage and exhibit locations similar to those of α- and α'-mycolic acids of mycobacteria. For the ultralong chain α-mycolic acids, the three double bonds were located at equally spaced carbon intervals (C(13)-C(16)), with the methyl branches adjacent to the proximal and distal trans double bonds. Examination of the Segniliparus rotundus genome compared with those of other members of the Corynebacteriales indicated two obvious differences in genes encoding the elongation fatty acid (FAS-II) enzymes involved in the biosynthesis of mycolic acids: the organization of 3-ketoacyl-ACP synthases (KasA and KasB) and (3R)-hydroxyacyl-ACP dehydratases (HadAB/BC), on one hand, and the presence of two copies of the hadB gene encoding the catalytic domain of the latter enzyme type, on the other. This observation is discussed in light of the most recent data accumulated on the biosynthesis of this hallmark of Corynebacteriales. PMID:23154972

  4. Metabolic Flux Between Unsaturated and Saturated Fatty Acids is Controlled by the FabA:FabB Ratio in the Fully Reconstituted Fatty Acid Biosynthetic Pathway of E. coli#

    PubMed Central

    Xiao, Xirui; Yu, Xingye; Khosla, Chaitan

    2013-01-01

    The entire fatty acid biosynthetic pathway from Escherichia coli, starting from the acetyl-CoA carboxylase, has been reconstituted in vitro from fourteen purified protein components. Radiotracer analysis verified stoichiometric conversion of acetyl-CoA and NAD(P)H into the free fatty acid product, allowing implementation of a facile spectrophotometric assay for kinetic analysis of this multi-enzyme system. At steady state, a maximum turnover rate of 0.5 s−1 was achieved. Under optimal turnover conditions, the predominant products were C16 and C18 saturated as well as monounsaturated fatty acids. The reconstituted system allowed us to quantitatively interrogate the factors that influence metabolic flux toward unsaturated versus saturated fatty acids. In particular, the concentrations of the dehydratase FabA and the β-ketoacyl synthase FabB were found to be crucial for controlling this property. By altering these variables, the percentage of unsaturated fatty acid produced could be adjusted between 10 and 50% without significantly affecting the maximum turnover rate of the pathway. Our reconstituted system provides a powerful tool to understand and engineer rate-limiting and regulatory steps in this complex and practically significant metabolic pathway. PMID:24147979

  5. Oral co-administration of α-lipoic acid, quercetin and captopril prevents gallium arsenide toxicity in rats.

    PubMed

    Bhatt, Kapil; Flora, S J S

    2009-07-01

    Gallium arsenide (GaAs), an inter-metallic semiconductor, known to exhibit superior optical and electronic properties compared to silicon, promotes its use in semiconductor industries. Extensive use of GaAs will inevitably lead to an increase in the exposure of workers manufacturing these products. Antioxidants are exogenous or endogenous compounds acting in several ways, including scavenging reactive oxygen species (ROS) or their precursors, inhibiting ROS formation, and binding metal ions needed for the catalysis of ROS generation. In the present study we investigated the protective efficacy of α-lipoic acid, quercetin and captopril individually against gallium arsenide exposure. Co-administration of α-lipoic acid with GaAs was most effective in reducing GaAs induced inhibition of blood δ-aminolevulinic acid dehydratase (ALAD) activity, liver, kidney and brain reduced glutathione (GSH) level and elevation of oxidized glutathione (GSSG). Captopril, on the other hand was effective in reducing thiobarbituric acid reactive substance (TBARS) levels, while quercetin reduced ROS in liver and kidney. The results suggest comparatively better preventive efficacy of concomitant α-lipoic acid administration during Gallium arsenide exposure compared to quercetin and captopril in preventing GaAs induced oxidative stress.

  6. The efficacy of monoisoamyl ester of dimercaptosuccinic acid in chronic experimental arsenic poisoning in mice.

    PubMed

    Flora, S J S; Kannan, G M; Pant, B P; Jaiswal, D K

    2003-01-01

    The therapeutic efficacy of monoisoamyl meso-2,3-dimercaptosuccinic acid (MiADMSA), a new monoester of 2,3-dimercaptosuccinic acid on arsenic induced oxidative stress in liver and kidneys, alterations in hematopoietic system and depletion of arsenic burden was assessed, in mice. Three different doses of MiADMSA (25, 50 or 100 mg/kg) for five consecutive days were administered in chronically arsenic exposed mice (10 ppm in drinking water for six months). Oral administration of MiADMSA particularly at a dose of 50 mg/kg, produced relatively more pronounced beneficial effects on the inhibited blood delta-aminolevulinic acid dehydratase (ALAD), biochemical variables indicative of hepatic and renal oxidative stress and depletion of arsenic concentration in blood, liver and kidneys, compared with intraperitoneal administration of the drug. The treatment with MiADMSA although, produced essential metals imbalance which could be a restrictive factor for the possible therapeutic use of this compound in chronic arsenic poisoning and thus require further exploration.

  7. Alanylclavam Biosynthetic Genes Are Clustered Together with One Group of Clavulanic Acid Biosynthetic Genes in Streptomyces clavuligerus▿ §

    PubMed Central

    Zelyas, Nathan J.; Cai, Hui; Kwong, Thomas; Jensen, Susan E.

    2008-01-01

    Streptomyces clavuligerus produces at least five different clavam metabolites, including clavulanic acid and the methionine antimetabolite, alanylclavam. In vitro transposon mutagenesis was used to analyze a 13-kb region upstream of the known paralogue gene cluster. The paralogue cluster includes one group of clavulanic acid biosynthetic genes in S. clavuligerus. Twelve open reading frames (ORFs) were found in this area, and mutants were generated in each using either in vitro transposon or PCR-targeted mutagenesis. Mutants with defects in any of the genes orfA, orfB, orfC, or orfD were unable to produce alanylclavam but could produce all of the other clavams, including clavulanic acid. orfA encodes a predicted hydroxymethyltransferase, orfB encodes a YjgF/YER057c/UK114-family regulatory protein, orfC encodes an aminotransferase, and orfD encodes a dehydratase. All of these types of proteins are normally involved in amino acid metabolism. Mutants in orfC or orfD also accumulated a novel clavam metabolite instead of alanylclavam, and a complemented orfC mutant was able to produce trace amounts of alanylclavam while still producing the novel clavam. Mass spectrometric analyses, together with consideration of the enzymes involved in its production, led to tentative identification of the novel clavam as 8-OH-alanylclavam, an intermediate in the proposed alanylclavam biosynthetic pathway. PMID:18931110

  8. Modulation of biosynthesis of photosynthetic pigments and light-harvesting complex in wild-type and gun5 mutant of Arabidopsis thaliana during impaired chloroplast development.

    PubMed

    Pattanayak, Gopal K; Tripathy, Baishnab C

    2016-05-01

    Plants in response to different environmental cues need to modulate the expression of nuclear and chloroplast genomes that are in constant communication. To understand the signals that are responsible for inter-organellar communication, levulinic acid (LA), an inhibitor of 5-aminolevulinic acid dehydratase, was used to suppress the synthesis of pyrrole-derived tetrapyrroles chlorophylls. Although, it does not specifically inhibit carotenoid biosynthesis enzymes, LA reduced the carotenoid contents during photomorphogenesis of etiolated Arabidopsis seedlings. The expression of nuclear genes involved in carotenoid biosynthesis, i.e., geranylgeranyl diphosphate synthase, phytoene synthase, and phytoene desaturase, was downregulated in LA-treated seedlings. Similarly, the transcript abundance of nuclear genes, i.e., Lhcb1, PsbO, and RcbS, coding for chloroplastic proteins was severely attenuated in LA-treated samples. In contrast, LA treatment did not affect the transcript abundance of chalcone synthase, a marker gene for cytoplasm, and β-ATP synthase, a marker gene for mitochondria. This demonstrates the retrograde signaling from chloroplast to nucleus to suppress chloroplastic proteins during impaired chloroplast development. However, under identical conditions in LA-treated tetrapyrrole-deficient gun5 mutant, retrograde signal continued. The tetrapyrrole biosynthesis inhibitor LA suppressed formation of all tetrapyrroles both in WT and gun5. This rules out the role of tetrapyrroles as signaling molecules in WT and gun5. The removal of LA from the Arabidopsis seedlings restored the chlorophyll and carotenoid contents and expression of nuclear genes coding for chloroplastic proteins involved in chloroplast biogenesis. Therefore, LA could be used to modulate chloroplast biogenesis at a desired phase of chloroplast development. PMID:27001427

  9. The use of photodynamic therapy for treatment of acne vulgaris.

    PubMed

    Nestor, Mark S

    2007-01-01

    Current topical and most oral therapies for acne vulgaris have limited efficacy, especially in moderate to severe cases. Photodynamic therapy with 5-aminolevulinic acid and recently methyl aminolevulinate has been shown to be a safe and effective modality for the treatment of acne vulgaris. Consensus guidelines suggest that 30 to 60 minutes is sufficient 5-aminolevulinic acid contact time before photoactivation with blue light, red light, yellow light, broadband light, halogen, or pulsed dye laser devices. An average of three treatment can yield significant long-term improvement. PMID:17126741

  10. Engineering a Polyketide Synthase for In Vitro Production of Adipic Acid.

    PubMed

    Hagen, Andrew; Poust, Sean; Rond, Tristan de; Fortman, Jeffrey L; Katz, Leonard; Petzold, Christopher J; Keasling, Jay D

    2016-01-15

    Polyketides have enormous structural diversity, yet polyketide synthases (PKSs) have thus far been engineered to produce only drug candidates or derivatives thereof. Thousands of other molecules, including commodity and specialty chemicals, could be synthesized using PKSs if composing hybrid PKSs from well-characterized parts derived from natural PKSs was more efficient. Here, using modern mass spectrometry techniques as an essential part of the design-build-test cycle, we engineered a chimeric PKS to enable production one of the most widely used commodity chemicals, adipic acid. To accomplish this, we introduced heterologous reductive domains from various PKS clusters into the borrelidin PKS' first extension module, which we previously showed produces a 3-hydroxy-adipoyl intermediate when coincubated with the loading module and a succinyl-CoA starter unit. Acyl-ACP intermediate analysis revealed an unexpected bottleneck at the dehydration step, which was overcome by introduction of a carboxyacyl-processing dehydratase domain. Appending a thioesterase to the hybrid PKS enabled the production of free adipic acid. Using acyl-intermediate based techniques to "debug" PKSs as described here, it should one day be possible to engineer chimeric PKSs to produce a variety of existing commodity and specialty chemicals, as well as thousands of chemicals that are difficult to produce from petroleum feedstocks using traditional synthetic chemistry.

  11. Engineering a Polyketide Synthase for In Vitro Production of Adipic Acid.

    PubMed

    Hagen, Andrew; Poust, Sean; Rond, Tristan de; Fortman, Jeffrey L; Katz, Leonard; Petzold, Christopher J; Keasling, Jay D

    2016-01-15

    Polyketides have enormous structural diversity, yet polyketide synthases (PKSs) have thus far been engineered to produce only drug candidates or derivatives thereof. Thousands of other molecules, including commodity and specialty chemicals, could be synthesized using PKSs if composing hybrid PKSs from well-characterized parts derived from natural PKSs was more efficient. Here, using modern mass spectrometry techniques as an essential part of the design-build-test cycle, we engineered a chimeric PKS to enable production one of the most widely used commodity chemicals, adipic acid. To accomplish this, we introduced heterologous reductive domains from various PKS clusters into the borrelidin PKS' first extension module, which we previously showed produces a 3-hydroxy-adipoyl intermediate when coincubated with the loading module and a succinyl-CoA starter unit. Acyl-ACP intermediate analysis revealed an unexpected bottleneck at the dehydration step, which was overcome by introduction of a carboxyacyl-processing dehydratase domain. Appending a thioesterase to the hybrid PKS enabled the production of free adipic acid. Using acyl-intermediate based techniques to "debug" PKSs as described here, it should one day be possible to engineer chimeric PKSs to produce a variety of existing commodity and specialty chemicals, as well as thousands of chemicals that are difficult to produce from petroleum feedstocks using traditional synthetic chemistry. PMID:26501439

  12. Folic Acid

    MedlinePlus

    Folic acid is a B vitamin. It helps the body make healthy new cells. Everyone needs folic acid. For women who may get pregnant, it is really important. Getting enough folic acid before and during pregnancy can prevent major birth ...

  13. Folic Acid

    MedlinePlus

    Folic acid is used to treat or prevent folic acid deficiency. It is a B-complex vitamin needed by ... Folic acid comes in tablets. It usually is taken once a day. Follow the directions on your prescription label ...

  14. Amino acids

    MedlinePlus

    ... amino acids are: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan , and valine. Nonessential amino acids "Nonessential" means that our bodies produce an amino ...

  15. Protective effect of rosmarinic acid against oxidative stress biomarkers in liver and kidney of strepotozotocin-induced diabetic rats.

    PubMed

    Mushtaq, Nadia; Schmatz, Roberta; Ahmed, Mushtaq; Pereira, Luciane Belmonte; da Costa, Pauline; Reichert, Karine Paula; Dalenogare, Diéssica; Pelinson, Luana Paula; Vieira, Juliano Marchi; Stefanello, Naiara; de Oliveira, Lizielle Souza; Mulinacci, Nadia; Bellumori, Maria; Morsch, Vera Maria; Schetinger, Maria Rosa

    2015-12-01

    In the present study, we investigated the efficiency of rosmarinic acid (RA) in preventing the alteration of oxidative parameters in the liver and kidney of diabetic rats induced by streptozotocin (STZ). The animals were divided into six groups (n = 8): control, ethanol, RA 10 mg/kg, diabetic, diabetic/ethanol, and diabetic/RA 10 mg/kg. After 3 weeks of treatment, we found that TBARS levels in liver and kidney were significantly increased in the diabetic/saline group and the administration of RA prevented this increase in the liver and kidney (P < 0.05). Diabetes caused a significant decrease in the activity of superoxide dismutase (SOD) and catalase (CAT) in the diabetes/saline group (P < 0.05). However, the treatment with 10 mg/kg RA (antioxidant) prevented this alteration in SOD and CAT activity in the diabetic RA group (P < 0.05). In addition, RA reverses the decrease in ascorbic acid and non-protein-thiol (NPSH) levels in diabetic rats. The treatment with RA also prevented the decrease in the Delta-aminolevulinic acid dehydratase (ALA-D) activity in the liver and kidney of diabetic rats. Furthermore, RA did not have any effect on glycemic levels. These results indicate that RA effectively reduced the oxidative stress induced by STZ, suggesting that RA is a potential candidate for the prevention and treatment of pathological conditions in diabetic models.

  16. Meso 2,3-dimercaptosuccinic acid (DMSA) and monoisoamyl DMSA effect on gallium arsenide induced pathological liver injury in rats.

    PubMed

    Flora, S J S; Dubey, Rupa; Kannan, G M; Chauhan, R S; Pant, B P; Jaiswal, D K

    2002-06-01

    The effect of meso 2,3-dimercaptosuccinic acid (DMSA) and monoisoamyl DMSA (MiADMSA) on gallium arsenide (GaAs) induced liver damage was studied. The oral feeding rat model was used in this study. The animals were exposed to 10 mg/kg GaAs, orally, once daily, 5 days a week for 24 weeks and treated thereafter with single oral daily dose of either 0.3 mmol/kg DMSA or MiADMSA for two course of 5 days treatment. The animals were sacrificed thereafter. Lipid peroxidation was assessed by measuring liver thiobarbituric acid reactive substance (TBARS). Liver damage was assessed by number of biochemical variables and by light microscopy. The activity of superoxide dismutase (SOD) and delta-aminolevulinic acid dehydratase (ALAD) beside reduced glutathione (GSH) concentration was measured in blood. Exposure to GaAs produced a significant reduction in GSH while, increased the oxidized glutathione (GSSG) concentration. Hepatic glutathione peroxidase (GPx) and catalase activity increased significantly while level of serum transaminase increased moderately. Gallium arsenide exposure also produced marked hepatic histopathological lesions. Overall, treatment with MiADMSA proved to be better than DMSA in the mobilization of arsenic and in the turnover of some of the above mentioned GaAs sensitive biochemical alterations. Histopathological lesions also, responded more favorably to chelation treatment with MiADMSA than DMSA.

  17. Reversal of Lead-Induced Acute Toxicity by Lipoic Acid with Nutritional Supplements in Male Wistar Rats.

    PubMed

    Shukla, Sangeeta; Sharma, Yamini; Shrivastava, Sadhana

    2016-01-01

    Lead (Pb) is a pleiotropic toxicant. The potential role of oxidative stress injury that is associated with Pb poisoning suggests that antioxidants may enhance the efficacy of treatment designed to mitigate Pb-induced toxicity. The aim of this study is to investigate the comparative ameliorative potential of lipoic acid (LA) alone or in combination with calcium (Ca) and zinc (Zn). Pb acetate (50 mg/kg, intraperitoneally) was administered for 3 d. After 24 h of the last toxicant dose, LA (100 mg/kg, orally [po]) alone or in conjuction with Ca (50 mg/kg, po) and Zn (10 mg/kg, po) was administered for 3 d. Significant alterations in the concentration of urea, uric acid, triglycerides, cholesterol, aspartate amino transferase, alanine amino transferase, lipid peroxidation, and reduced glutathione as well as alterations in enzyme activity of δ-aminolevulinic acid (ALA) dehydratase were observed following acute Pb exposure. These findings were also supported by elevated mean DNA damage and Pb body burden in blood and soft tissues compared to controls (p ≤ 0.05). Three d posttreatment with LA along with Zn and Ca could significantly restore the biochemical parameters and Pb body burden to near-normal status through antioxidant activity or by preventing bioaccumulation of Pb within the blood and tissues of experimental rats. PMID:27481494

  18. Protective effect of rosmarinic acid against oxidative stress biomarkers in liver and kidney of strepotozotocin-induced diabetic rats.

    PubMed

    Mushtaq, Nadia; Schmatz, Roberta; Ahmed, Mushtaq; Pereira, Luciane Belmonte; da Costa, Pauline; Reichert, Karine Paula; Dalenogare, Diéssica; Pelinson, Luana Paula; Vieira, Juliano Marchi; Stefanello, Naiara; de Oliveira, Lizielle Souza; Mulinacci, Nadia; Bellumori, Maria; Morsch, Vera Maria; Schetinger, Maria Rosa

    2015-12-01

    In the present study, we investigated the efficiency of rosmarinic acid (RA) in preventing the alteration of oxidative parameters in the liver and kidney of diabetic rats induced by streptozotocin (STZ). The animals were divided into six groups (n = 8): control, ethanol, RA 10 mg/kg, diabetic, diabetic/ethanol, and diabetic/RA 10 mg/kg. After 3 weeks of treatment, we found that TBARS levels in liver and kidney were significantly increased in the diabetic/saline group and the administration of RA prevented this increase in the liver and kidney (P < 0.05). Diabetes caused a significant decrease in the activity of superoxide dismutase (SOD) and catalase (CAT) in the diabetes/saline group (P < 0.05). However, the treatment with 10 mg/kg RA (antioxidant) prevented this alteration in SOD and CAT activity in the diabetic RA group (P < 0.05). In addition, RA reverses the decrease in ascorbic acid and non-protein-thiol (NPSH) levels in diabetic rats. The treatment with RA also prevented the decrease in the Delta-aminolevulinic acid dehydratase (ALA-D) activity in the liver and kidney of diabetic rats. Furthermore, RA did not have any effect on glycemic levels. These results indicate that RA effectively reduced the oxidative stress induced by STZ, suggesting that RA is a potential candidate for the prevention and treatment of pathological conditions in diabetic models. PMID:26452500

  19. Acid Rain.

    ERIC Educational Resources Information Center

    Openshaw, Peter

    1987-01-01

    Provides some background information on acid deposition. Includes a historical perspective, describes some effects of acid precipitation, and discusses acid rain in the United Kingdom. Contains several experiments that deal with the effects of acid rain on water quality and soil. (TW)

  20. Acid rain

    SciTech Connect

    Not Available

    1985-01-01

    This report has four parts: they discuss acid rain in relation to acid soils, agriculture, forests, and aquatic ecosystems. Among findings: modern sources of acid deposition from the atmosphere for all the acid soils in the world, nor even chiefly responsible for those of northern U.S. Agriculture has its problems, but acid precipitation is probably not one of them. More research is needed to determine to what extent acid precipitation is responsible for forest declines and for smaller detrimental effects on forest growth where no damage to the foliage is evident. Many lakes and streams are extremely sensitive to added acids.

  1. Evaluation of the pharmacological properties of salicylic acid-derivative organoselenium: 2-hydroxy-5-selenocyanatobenzoic acid as an anti-inflammatory and antinociceptive compound.

    PubMed

    Chagas, Pietro Maria; Rosa, Suzan Gonçalves; Sari, Marcel Henrique Marcondes; Oliveira, Carla Elena Sartori; Canto, Rômulo Faria Santos; da Luz, Sônia Cristina Almeida; Braga, Antonio Luiz; Nogueira, Cristina Wayne

    2014-03-01

    The present study evaluated the antinociceptive and anti-inflammatory effects of per oral (p.o.) administration of salicylic acid-derivative organoselenium compounds in chemical models of nociception in mice. The compounds (50 mg/kg; p.o.) were administered 30 and 60 min before the nociceptive behavior and compared to the positive-control, acetylsalicylic acid (ASA; 200 mg/kg; p.o.). In addition, a dose-response curve (25-100 mg/kg) for compounds was carried out in the formalin test. When assessed in the chemical models, acetic acid-induced writhing behavior, formalin and glutamate tests, the compounds showed the following antinociceptive profile 1B>2B>1A>2A, suggesting a chemical structure-dependent relationship. Then, the anti-inflammatory properties and toxicological potential of compound 1B were investigated. Compound 1B, similar to the positive-control, ASA, diminished the edema formation and decreased the myeloperoxidase activity induced by croton oil (2.5%) in the ear tissue. The results also indicate that a single oral administration of 1B caused neither signs of acute toxicity nor those of gastrointestinal injury. The administration of 1B did not alter the water and food intakes, plasma alanine and aspartate aminotransferase activities or urea levels and cerebral or hepatic δ-aminolevulinate dehydratase activity. Salicylic acid-derivative organoseleniums, mainly compound 1B, have been found to be novel compounds with antinociceptive/anti-inflammatory properties; nevertheless, more studies are required to examine their therapeutic potential for pain treatment.

  2. Trapping the dynamic acyl carrier protein in fatty acid biosynthesis

    PubMed Central

    Nguyen, Chi; Haushalter, Robert W.; Lee, D. John; Markwick, Phineus R. L.; Bruegger, Joel; Caldara-Festin, Grace; Finzel, Kara; Jackson, David R.; Ishikawa, Fumihiro; O’Dowd, Bing; McCammon, J. Andrew; Opella, Stanley J.; Tsai, Shiou-Chuan; Burkart, Michael D.

    2015-01-01

    Acyl carrier protein (ACP) transports the growing fatty acid chain between enzyme domains of fatty acid synthase (FAS) during biosynthesis.1 Because FAS enzymes operate upon ACP-bound acyl groups, ACP must stabilize and transport the growing lipid chain.2 The transient nature of ACP-enzyme interactions imposes a major obstacle to gaining high-resolution structural information about fatty acid biosynthesis, and a new strategy is required to properly study protein-protein interactions. In this work, we describe the application of a mechanism-based probe that allows site-selective covalent crosslinking of AcpP to FabA, the E. coli ACP and fatty acid 3-hydroxyacyl-ACP dehydratase. We report the 1.9 Å crystal structure of the crosslinked AcpP=FabA complex as a homo-dimer, in which AcpP exhibits two different conformations likely representing snapshots of ACP in action: the 4′-phosphopantetheine (PPant) group of AcpP first binds an arginine-rich groove of FabA, followed by an AcpP helical conformational change that locks the AcpP and FabA in place. Residues at the interface of AcpP and FabA are identified and validated by solution NMR techniques, including chemical shift perturbations and RDC measurements. These not only support our interpretation of the crystal structures but also provide an animated view of ACP in action during fatty acid dehydration. Combined with molecular dynamics simulations, we show for the first time that FabA extrudes the sequestered acyl chain from the ACP binding pocket before dehydration by repositioning helix III. Extensive sequence conservation among carrier proteins suggests that the mechanistic insights gleaned from our studies will prove general for fatty acid, polyketide and non-ribosomal biosyntheses. Here the foundation is laid for defining the dynamic action of carrier protein activity in primary and secondary metabolism, providing insight into pathways that can play major roles in the treatment of cancer, obesity and infectious

  3. Aminocaproic Acid

    MedlinePlus

    Aminocaproic acid is used to control bleeding that occurs when blood clots are broken down too quickly. This type ... the baby is ready to be born). Aminocaproic acid is also used to control bleeding in the ...

  4. Ethacrynic Acid

    MedlinePlus

    Ethacrynic acid, a 'water pill,' is used to treat swelling and fluid retention caused by various medical problems. It ... Ethacrynic acid comes as a tablet to take by mouth. It is usually taken once or twice a day ...

  5. Aristolochic Acids

    MedlinePlus

    ... Sciences NIH-HHS www.niehs.nih.gov Aristolochic Acids Key Points Report on Carcinogens Status Known to be human carcinogens Aristolochia Clematitis Aristolochic Acids n Known human carcinogens n Found in certain ...

  6. Obeticholic Acid

    MedlinePlus

    Obeticholic acid is used alone or in combination with ursodiol (Actigall, Urso) to treat primary biliary cholangitis (PBC; a ... were not treated successfully with ursodiol alone. Obeticholic acid is in a class of medications called farnesoid ...

  7. Acid mucopolysaccharides

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003368.htm Acid mucopolysaccharides To use the sharing features on this page, please enable JavaScript. Acid mucopolysaccharides is a test that measures the amount ...

  8. Efficacy of 2,3-dimercapto-1-propanesulfonic acid (DMPS) and diphenyl diselenide on cadmium induced testicular damage in mice.

    PubMed

    Santos, Francielli W; Zeni, Gilson; Rocha, Joao B T; do Nascimento, Paulo C; Marques, Marieli S; Nogueira, Cristina W

    2005-12-01

    The deleterious effect of acute cadmium-intoxication in mice testes was evaluated. Animals received a single dose of CdCl2 (2.5 or 5 mg/kg, intraperitoneally) and a number of toxicological parameters in mice testes were examined, such as delta-aminolevulinic acid dehydratase (delta-ALA-D) activity, lipid peroxidation, hemoglobin and ascorbic acid contents. Furthermore, the parameters that indicate tissue damage such as plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were also determined. Thus, a possible protective effect of 2,3-dimercapto-1-propane-sulfonic acid (DMPS) and diphenyl diselenide (PhSe)2 were studied. The results demonstrated an inhibition of delta-ALA-D activity, a reduction of ascorbic acid and an increase of lipid peroxidation induced by cadmium, indicating testes damage. Furthermore, we observed an increase of plasma LDH, AST and ALT activities. DMPS (400 mol/kg) and (PhSe)2 (100 micromol/kg) partially protected from the inhibitory effect of 2.5 mg/kg CdCl2 on delta-ALA-D and from the increase of TBARS (thiobarbituric acid reactive species) levels. (PhSe)2 therapy was effective in ameliorate ascorbic acid content when the cadmium dose was 2.5 mg/kg. Treatment with DMPS and (PhSe)2, individually or combined, was inefficient in reducing cadmium-induced plasma LDH and ALT activity increase. The use of combined therapy (DMPS plus (PhSe)2) proved to be efficient in decreasing cadmium levels in testes and in ameliorating plasma AST activity from animals that received the highest dose of cadmium. PMID:16000234

  9. Fatty acids - trans fatty acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The data supporting a negative effect of dietary trans fatty acids on cardiovascular disease risk is consistent. The primary dietary sources of trans fatty acids include partially hydrogenated fat and rudiment fat. The adverse effect of trans fatty acids on plasma lipoprotein profiles is consisten...

  10. Therapeutic potentials of combined use of DMSA with calcium and ascorbic acid in the treatment of mild to moderately lead intoxicated mice.

    PubMed

    Liao, Yingjun; Zhang, Jun; Jin, Yaping; Lu, Chunwei; Li, Gexin; Yu, Fei; Zhi, Xuping; An, Li; Yang, Jun

    2008-02-01

    The aim of this study was to explore the therapeutic efficacies of combined use of meso-2,3-dimercaptosuccinic acid (DMSA) with calcium and ascorbic acid in the treatment of mild to moderately lead-intoxicated mice. Female albino mice were exposed to lead by drinking water contaminated with 0.1% (moderate lead exposure) or 0.05% (mild lead exposure) lead acetate. After the cessation of lead exposure, mice were supplemented by gavage with saline solution, 50 mg/kg body weight (b.w) DMSA, 100 mg/kg b.w DMSA, calcium and ascorbic acid, or 50 mg/kg b.w DMSA and calcium as well as ascorbic acid, respectively. Atomic absorption spectrophotometric method was used to analyze lead levels in blood, bone, liver, kidney and brain. Activities of blood delta-aminolevulinic acid dehydratase (ALAD) were determined by colorimetric method. DMSA supplemented alone could reduce lead levels in both soft tissues and bone and reverse lead-inhibited activities of blood ALAD in mild to moderately lead-intoxicated mice. On the other hand, combined use of DMSA with calcium and ascorbic acid achieved better therapeutic efficacies in mobilizing lead in blood, liver and kidney, and reversing lead-inhibited activities of blood ALAD in moderately lead intoxicated mice than DMSA supplemented alone. Moreover, the better therapeutic efficacies were also found in mildly lead intoxicated mice in mobilizing lead in blood and bone achieved by combined use of DMSA with calcium and ascorbic acid. Combined use of DMSA with calcium and ascorbic acid seems to be the better choice in the treatment of mild to moderate lead-intoxication.

  11. Heme precursor injection is effective for Arthromyces ramosus peroxidase fusion protein production by a silkworm expression system.

    PubMed

    Hayashi, Kounosuke; Lee, Jae Man; Tomozoe, Yusuke; Kusakabe, Takahiro; Kamiya, Noriho

    2015-10-01

    Recombinant peroxidase from Arthromyces ramosus, fused with domains of antibody-binding proteins, was successfully obtained by a silkworm larvae expression system. The catalytic activity of the fusion peroxidase was increased 6-fold with the injection of 5-aminolevulinic acid into silkworm larvae as a heme precursor.

  12. Mass Spectrometry-Based Systems Approach for Identification of Inhibitors of Plasmodium falciparum Fatty Acid Synthase▿

    PubMed Central

    Sharma, Shilpi; Sharma, Shailendra Kumar; Modak, Rahul; Karmodiya, Krishanpal; Surolia, Namita; Surolia, Avadhesha

    2007-01-01

    The emergence of strains of Plasmodium falciparum resistant to the commonly used antimalarials warrants the development of new antimalarial agents. The discovery of type II fatty acid synthase (FAS) in Plasmodium distinct from the FAS in its human host (type I FAS) opened up new avenues for the development of novel antimalarials. The process of fatty acid synthesis takes place by iterative elongation of butyryl-acyl carrier protein (butyryl-ACP) by two carbon units, with the successive action of four enzymes constituting the elongation module of FAS until the desired acyl length is obtained. The study of the fatty acid synthesis machinery of the parasite inside the red blood cell culture has always been a challenging task. Here, we report the in vitro reconstitution of the elongation module of the FAS of malaria parasite involving all four enzymes, FabB/F (β-ketoacyl-ACP synthase), FabG (β-ketoacyl-ACP reductase), FabZ (β-ketoacyl-ACP dehydratase), and FabI (enoyl-ACP reductase), and its analysis by matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS). That this in vitro systems approach completely mimics the in vivo machinery is confirmed by the distribution of acyl products. Using known inhibitors of the enzymes of the elongation module, cerulenin, triclosan, NAS-21/91, and (−)-catechin gallate, we demonstrate that accumulation of intermediates resulting from the inhibition of any of the enzymes can be unambiguously followed by MALDI-TOF MS. Thus, this work not only offers a powerful tool for easier and faster throughput screening of inhibitors but also allows for the study of the biochemical properties of the FAS pathway of the malaria parasite. PMID:17485508

  13. Therapeutic efficiency of succimer used with calcium and ascorbic acid in the treatment of mild lead-poisoning.

    PubMed

    Jin, Yaping; Yu, Fei; Liao, Yingjun; Liu, Shaoxia; Liu, Meimei; Xu, Jianhong; Yang, Jun

    2011-01-01

    The aim of this study was to explore therapeutic efficiency of succimer used with calcium and ascorbic acid in the treatment of mildly lead-poisoned mice and preschool children. Mice were exposed to lead by drinking water, and then treated with saline solution, 50mg/kg body weight (b.w.) succimer, 100mg/kg b.w. succimer, or 50mg/kg b.w. succimer plus calcium and ascorbic acid by gavage. Seventy-two children aged 48-72 months were randomly assigned into combined treatment or nutritional intervention group. Lead levels in blood and bone were analyzed by atomic absorption spectrophotometry. Activities of aminolevulinic acid dehydratase (ALAD) in blood were determined by colorimetric method. Results of animal experiment showed that succimer used alone could reduce lead levels in blood and bone and reverse activities of ALAD in blood, however, a better therapeutic efficiency in mobilizing bone lead could be achieved by succimer used with calcium and ascorbic acid. Findings from the clinical study showed that reduction of blood lead levels (BLLs) between the end and initiation of therapy in the combined treatment group was significantly greater than that in the nutritional intervention group. Percentage of children with BLLs less than 10μg/dL at the end of therapy and the eighth week after therapy in the combined treatment group was significantly higher than that in the nutritional intervention group. In conclusion, combined use of succimer with calcium and ascorbic acid seemed to be a choice in the treatment of mildly lead poisoned children. PMID:21787678

  14. Therapeutic efficiency of succimer used with calcium and ascorbic acid in the treatment of mild lead-poisoning.

    PubMed

    Jin, Yaping; Yu, Fei; Liao, Yingjun; Liu, Shaoxia; Liu, Meimei; Xu, Jianhong; Yang, Jun

    2011-01-01

    The aim of this study was to explore therapeutic efficiency of succimer used with calcium and ascorbic acid in the treatment of mildly lead-poisoned mice and preschool children. Mice were exposed to lead by drinking water, and then treated with saline solution, 50mg/kg body weight (b.w.) succimer, 100mg/kg b.w. succimer, or 50mg/kg b.w. succimer plus calcium and ascorbic acid by gavage. Seventy-two children aged 48-72 months were randomly assigned into combined treatment or nutritional intervention group. Lead levels in blood and bone were analyzed by atomic absorption spectrophotometry. Activities of aminolevulinic acid dehydratase (ALAD) in blood were determined by colorimetric method. Results of animal experiment showed that succimer used alone could reduce lead levels in blood and bone and reverse activities of ALAD in blood, however, a better therapeutic efficiency in mobilizing bone lead could be achieved by succimer used with calcium and ascorbic acid. Findings from the clinical study showed that reduction of blood lead levels (BLLs) between the end and initiation of therapy in the combined treatment group was significantly greater than that in the nutritional intervention group. Percentage of children with BLLs less than 10μg/dL at the end of therapy and the eighth week after therapy in the combined treatment group was significantly higher than that in the nutritional intervention group. In conclusion, combined use of succimer with calcium and ascorbic acid seemed to be a choice in the treatment of mildly lead poisoned children.

  15. The Structure of LiuC, a 3-Hydroxy-3-Methylglutaconyl CoA Dehydratase Involved in Isovaleryl-CoA Biosynthesis in Myxococcus xanthus, Reveals Insights into Specificity and Catalysis.

    PubMed

    Bock, Tobias; Reichelt, Joachim; Müller, Rolf; Blankenfeldt, Wulf

    2016-09-01

    Myxobacteria are able to produce the important metabolite isovaleryl coenzyme A by a route other than leucine degradation. The first step into this pathway is mediated by LiuC, a member of the 3-methylglutaconyl CoA hydratases (MGCH). Here we present crystal structures refined to 2.05 and 1.1 Å of LiuC in the apo form and bound to coenzyme A, respectively. By using simulated annealing we modeled the enzyme substrate complex and identified residues potentially involved in substrate binding, specificity, and catalysis. The dehydration of 3-hydroxy-3-methylglutaconyl CoA to 3-methylglutaconyl CoA catalyzed by LiuC involves Glu112 and Glu132 and likely employs the typical crotonase acid-base mechanism. In this, Tyr231 and Arg69 are key players in positioning the substrate to enable catalysis. Surprisingly, LiuC shows higher sequence and structural similarity to human MGCH than to bacterial forms, although they convert the same substrate. This study provides structural insights into the alternative isovaleryl coenzyme A biosynthesis pathway and might open a path for biofuel research, as isovaleryl-CoA is a source for isobutene, a precursor for renewable fuels and chemicals. PMID:27271456

  16. Aspartic acid

    MedlinePlus

    ... Hormone production and release Normal nervous system function Plant sources of aspartic acid include: Legumes such as soybeans, garbanzo beans, and lentils Peanuts, almonds, walnuts, and flaxseeds Animal ...

  17. Amino acid-dependent growth of Campylobacter jejuni: key roles for aspartase (AspA) under microaerobic and oxygen-limited conditions and identification of AspB (Cj0762), essential for growth on glutamate.

    PubMed

    Guccione, Edward; Leon-Kempis, Maria del Rocio; Pearson, Bruce M; Hitchin, Edward; Mulholland, Francis; van Diemen, Pauline M; Stevens, Mark P; Kelly, David J

    2008-07-01

    Amino acids are key carbon and energy sources for the asaccharolytic food-borne human pathogen Campylobacter jejuni. During microaerobic growth in amino acid rich complex media, aspartate, glutamate, proline and serine are the only amino acids significantly utilized by strain NCTC 11168. The catabolism of aspartate and glutamate was investigated. An aspartase (aspA) mutant (unable to utilize any amino acid except serine) and a Cj0762c (aspB) mutant lacking aspartate:glutamate aminotransferase (unable to utilize glutamate), were severely growth impaired in complex media, and an aspA sdaA mutant (also lacking serine dehydratase) failed to grow in complex media unless supplemented with pyruvate and fumarate. Aspartase was shown by activity and proteomic analyses to be upregulated by oxygen limitation, and aspartate enhanced oxygen-limited growth of C. jejuni in an aspA-dependent manner. Stoichiometric aspartate uptake and succinate excretion involving the redundant DcuA and DcuB transporters indicated that in addition to a catabolic role, AspA can provide fumarate for respiration. Significantly, an aspA mutant of C. jejuni 81-176 was impaired in its ability to persist in the intestines of outbred chickens relative to the parent strain. Together, our data highlight the dual function of aspartase in C. jejuni and suggest a role during growth in the avian gut.

  18. Usnic acid.

    PubMed

    Ingólfsdóttir, K

    2002-12-01

    Since its first isolation in 1844, usnic acid [2,6-diacetyl-7,9-dihydroxy-8,9b-dimethyl-1,3(2H,9bH)-dibenzo-furandione] has become the most extensively studied lichen metabolite and one of the few that is commercially available. Usnic acid is uniquely found in lichens, and is especially abundant in genera such as Alectoria, Cladonia, Usnea, Lecanora, Ramalina and Evernia. Many lichens and extracts containing usnic acid have been utilized for medicinal, perfumery, cosmetic as well as ecological applications. Usnic acid as a pure substance has been formulated in creams, toothpaste, mouthwash, deodorants and sunscreen products, in some cases as an active principle, in others as a preservative. In addition to antimicrobial activity against human and plant pathogens, usnic acid has been shown to exhibit antiviral, antiprotozoal, antiproliferative, anti-inflammatory and analgesic activity. Ecological effects, such as antigrowth, antiherbivore and anti-insect properties, have also been demonstrated. A difference in biological activity has in some cases been observed between the two enantiomeric forms of usnic acid. Recently health food supplements containing usnic acid have been promoted for use in weight reduction, with little scientific support. The emphasis of the current review is on the chemistry and biological activity of usnic acid and its derivatives in addition to rational and ecologically acceptable methods for provision of this natural compound on a large scale.

  19. Acid rain

    SciTech Connect

    Elsworth, S.

    1985-01-01

    This book was written in a concise and readable style for the lay public. It's purpose was to make the public aware of the damage caused by acid rain and to mobilize public opinion to favor the elimination of the causes of acid rain.

  20. Acid rain

    SciTech Connect

    White, J.C. )

    1988-01-01

    This book presents the proceedings of the third annual conference sponsored by the Acid Rain Information Clearinghouse (ARIC). Topics covered include: Legal aspects of the source-receptor relationship: an energy perspective; Scientific uncertainty, agency inaction, and the courts; and Acid rain: the emerging legal framework.

  1. How Acidic Is Carbonic Acid?

    PubMed

    Pines, Dina; Ditkovich, Julia; Mukra, Tzach; Miller, Yifat; Kiefer, Philip M; Daschakraborty, Snehasis; Hynes, James T; Pines, Ehud

    2016-03-10

    Carbonic, lactic, and pyruvic acids have been generated in aqueous solution by the transient protonation of their corresponding conjugate bases by a tailor-made photoacid, the 6-hydroxy-1-sulfonate pyrene sodium salt molecule. A particular goal is to establish the pK(a) of carbonic acid H2CO3. The on-contact proton transfer (PT) reaction rate from the optically excited photoacid to the carboxylic bases was derived, with unprecedented precision, from time-correlated single-photon-counting measurements of the fluorescence lifetime of the photoacid in the presence of the proton acceptors. The time-dependent diffusion-assisted PT rate was analyzed using the Szabo-Collins-Kimball equation with a radiation boundary condition. The on-contact PT rates were found to follow the acidity order of the carboxylic acids: the stronger was the acid, the slower was the PT reaction to its conjugate base. The pK(a) of carbonic acid was found to be 3.49 ± 0.05 using both the Marcus and Kiefer-Hynes free energy correlations. This establishes H2CO3 as being 0.37 pK(a) units stronger and about 1 pK(a) unit weaker, respectively, than the physiologically important lactic and pyruvic acids. The considerable acid strength of intact carbonic acid indicates that it is an important protonation agent under physiological conditions. PMID:26862781

  2. Acid rain

    SciTech Connect

    Sweet, W.

    1980-06-20

    Acid precipitation includes not only rain but also acidified snow, hail and frost, as well as sulfur and nitrogen dust. The principal source of acid precipitation is pollution emitted by power plants and smelters. Sulfur and nitrogen compounds contained in the emissions combine with moisture to form droplets with a high acid content - sometimes as acidic as vinegar. When sufficiently concentrated, these acids can kill fish and damage material structures. Under certain circumstances they may reduce crop and forest yields and cause or aggravate respiratory diseases in humans. During the summer, especially, pollutants tend to collect over the Great Lakes in high pressure systems. Since winds typically are westerly and rotate clockwise around high pressure systems, the pollutants gradually are dispersed throughout the eastern part of the continent.

  3. Asparagusic acid.

    PubMed

    Mitchell, Stephen C; Waring, Rosemary H

    2014-01-01

    Asparagusic acid (1,2-dithiolane-4-carboxylic acid) is a simple sulphur-containing 5-membered heterocyclic compound that appears unique to asparagus, though other dithiolane derivatives have been identified in non-food species. This molecule, apparently innocuous toxicologically to man, is the most probable culprit responsible for the curious excretion of odorous urine following asparagus ingestion. The presence of the two adjacent sulphur atoms leads to an enhanced chemical reactivity, endowing it with biological properties including the ability to substitute potentially for α-lipoic acid in α-keto-acid oxidation systems. This brief review collects the scattered data available in the literature concerning asparagusic acid and highlights its properties, intermediary metabolism and exploratory applications.

  4. Acid rain

    SciTech Connect

    Bess, F.D.

    1980-01-01

    The acid rain problem in the northeastern U.S. has been growing in severity and geographical areas affected. Acid rain has damaged, or will result in damage to visibility, physical structures and materials, aquatic life, timber, crops, and soils. The principal causes of acid rain in the northeastern U.S. are sulfur oxide and nitrogen oxide emissions from large power plants and smelters in the Ohio River Valley. Immediate corrective action and appropriate research are needed to reduce acid precipitation. Short-term programs that will define the rate of environmental deterioration, remaining environmental capacity to resist sudden deterioration, mechanisms of acid rain formation, and costs of various control options must be developed. (3 maps, 13 references, 1 table)

  5. Asparagusic acid.

    PubMed

    Mitchell, Stephen C; Waring, Rosemary H

    2014-01-01

    Asparagusic acid (1,2-dithiolane-4-carboxylic acid) is a simple sulphur-containing 5-membered heterocyclic compound that appears unique to asparagus, though other dithiolane derivatives have been identified in non-food species. This molecule, apparently innocuous toxicologically to man, is the most probable culprit responsible for the curious excretion of odorous urine following asparagus ingestion. The presence of the two adjacent sulphur atoms leads to an enhanced chemical reactivity, endowing it with biological properties including the ability to substitute potentially for α-lipoic acid in α-keto-acid oxidation systems. This brief review collects the scattered data available in the literature concerning asparagusic acid and highlights its properties, intermediary metabolism and exploratory applications. PMID:24099657

  6. Aluminum-induced oxidative stress in rat brain: response to combined administration of citric acid and HEDTA.

    PubMed

    Flora, Swaran J S; Mehta, Ashish; Satsangi, Kiran; Kannan, Gurusamy M; Gupta, Manju

    2003-03-01

    Aluminum, a known neurotoxic substance, has been suggested as a contributing factor in the pathogenesis of Alzheimer's disease. Therapeutic efficacy of combined administration of citric acid (CA) and N-(2-hydroxyethyl) ethylenediaminetriacetic acid (HEDTA) was evaluated in decreasing blood and brain aluminum concentration and parameters indicative of hematological disorders and brain oxidative stress. Adult male wistar rats were exposed to drinking water containing 0.2% aluminum nitrate for 8 months and treated once daily for 5 consecutive days with CA (50 mg/kg, orally) or HEDTA (50 mg/kg, intraperitoneally) either individually or in combination. Aluminum exposure significantly inhibited blood delta-aminolevulinic acid dehydratase while increased zinc protoporphyrin confirming changed heme biosynthesis. Significant decrease in the level of glutathione S-transferase in various brain regions and an increase in whole brain thiobarbituric acid reactive substance, and oxidized glutathione (GSSG) levels were also observed. Glutathione peroxidase activity showed a significant increase in cerebellum of aluminum exposed rats. Most of the above parameters responded moderately to the individual treatment with CA and HEDTA, but significantly reduced blood and brain aluminum burden. However, more pronounced beneficial effects on some of the above described parameters were observed when CA and HEDTA were administered concomitantly. Blood and brain aluminum concentration however, showed no further decline on combined treatment over the individual effect with HEDTA or CA. We conclude that in order to achieve an optimum effect of chelation, combined administration of CA and HEDTA might be preferred. However, further work is needed before a final recommendation could be made.

  7. [Biochemical evaluation of damage due to lead: importance and significance of erythrocyte zinc-protoporhyrin IX and urinary amino acid determination].

    PubMed

    Sanguinetti, F; Dompè, M; Ronca, G

    1977-01-01

    The early detection of lead intoxitation needs practical, simple, reproducible and diagnostically valid screening test. The determination of ALA-D (delta-amino-levulinic acid-dehydratase) in erythrocytes is one of the most reliable test for the evaluation of the occupational exposure to lead. However this test is difficult to standardize, sensible to lead contamination of laboratory glassware and the activity of enzyme decreases rapidly if stored. The determination of erythrocytes ZPP (zinco-protoporphyrin IX) was proposed as useful, alternative test. The protoporphyrin IX is a metabolic intermediate in heme biosynthesis; in erythrocytes is present as free form and zinc-boundend compound. The ZPP give high values only in lead intoxication and sideropenic anemia. The ALA-D and ZPP in erythrocytes were measured and compared in a group of workers exposed to lead. We have shown a good correlation between these two biochemical parameters. Aminoacid excretion in urine from workers exposed to lead was measured and compared with other biochemical parameters of intoxication. All lead workers examined had excessive urinary CP (coproporphyrin) and ALA (delta-amino-levulinic acid) excretion. An abnormal excretion of glycine was present in eight workers (32%), whereas in other four (15%) the glycinuria was at limit of normal values. An abnormal excretion of lysine was present in six workers (21%). The last data appear very interesting because the action of lead in lysine metabolism was not known. PMID:603137

  8. The Non-Essential Mycolic Acid Biosynthesis Genes hadA and hadC Contribute to the Physiology and Fitness of Mycobacterium smegmatis

    PubMed Central

    Jamet, Stevie; Slama, Nawel; Domingues, Joana; Laval, Françoise; Texier, Pauline; Eynard, Nathalie; Quémard, Annaik; Peixoto, Antonio; Lemassu, Anne; Daffé, Mamadou; Cam, Kaymeuang

    2015-01-01

    Gram positive mycobacteria with a high GC content, such as the etiological agent of tuberculosis Mycobacterium tuberculosis, possess an outer membrane mainly composed of mycolic acids (MAs), the so-called mycomembrane, which is essential for the cell. About thirty genes are involved in the biosynthesis of MAs, which include the hadA, hadB and hadC genes that encode the dehydratases Fatty Acid Synthase type II (FAS-II) known to function as the heterodimers HadA-HadB and HadB-HadC. The present study shows that M. smegmatis cells remain viable in the absence of either HadA and HadC or both. Inactivation of HadC has a dramatic effect on the physiology and fitness of the mutant strains whereas that of HadA exacerbates the phenotype of a hadC deletion. The hadC mutants exhibit a novel MA profile, display a distinct colony morphology, are less aggregated, are impaired for sliding motility and biofilm development and are more resistant to detergent. Conversely, the hadC mutants are significantly more susceptible to low- and high-temperature and to selective toxic compounds, including several current anti-tubercular drugs. PMID:26701652

  9. Acid fog

    SciTech Connect

    Hileman, B.

    1983-03-01

    Fog in areas of southern California previously thought to be pollution-free has been shown to have a pH as low as 1.69. It has been found to be most acidic after smoggy days, suggesting that it forms on the aerosol associated with the previously exiting smog. Studies on Whiteface Mountain in the Adirondacks show that fog water is often 10 times as acidic as rainwater. As a result of their studies, California plans to spend $4 million on acid deposition research in the coming year. (JMT)

  10. Tranexamic Acid

    MedlinePlus

    ... is used to treat heavy bleeding during the menstrual cycle (monthly periods) in women. Tranexamic acid is in ... tablets for more than 5 days in a menstrual cycle or take more than 6 tablets in a ...

  11. Mefenamic Acid

    MedlinePlus

    ... as mefenamic acid may cause ulcers, bleeding, or holes in the stomach or intestine. These problems may ... like coffee grounds, blood in the stool, or black and tarry stools.Keep all appointments with your ...

  12. Acid Precipitation

    ERIC Educational Resources Information Center

    Likens, Gene E.

    1976-01-01

    Discusses the fact that the acidity of rain and snow falling on parts of the U.S. and Europe has been rising. The reasons are still not entirely clear and the consequences have yet to be well evaluated. (MLH)

  13. Acidic precipitation

    SciTech Connect

    Martin, H.C.

    1987-01-01

    At the International Symposium on Acidic Precipitation, over 400 papers were presented, and nearly 200 of them are included here. They provide an overview of the present state of the art of acid rain research. The Conference focused on atmospheric science (monitoring, source-receptor relationships), aquatic effects (marine eutrophication, lake acidification, impacts on plant and fish populations), and terrestrial effects (forest decline, soil acidification, etc.).

  14. The Crystal Structure of Burkholderia cenocepacia DfsA Provides Insights into Substrate Recognition and Quorum Sensing Fatty Acid Biosynthesis.

    PubMed

    Spadaro, Francesca; Scoffone, Viola C; Chiarelli, Laurent R; Fumagalli, Marco; Buroni, Silvia; Riccardi, Giovanna; Forneris, Federico

    2016-06-14

    Burkholderia cenocepacia is a major concern among respiratory tract infections in cystic fibrosis patients. This pathogen is particularly difficult to treat because of its high level of resistance to the clinically relevant antimicrobial agents. In B. cenocepacia, the quorum sensing cell-cell communication system is involved in different processes that are important for bacterial virulence, such as biofilm formation and protease and siderophore production. Targeting the enzymes involved in this process represents a promising therapeutic approach. With the aim of finding effective quorum sensing inhibitors, we have determined the three-dimensional structure of B. cenocepacia diffusible factor synthase A, DfsA. This bifunctional crotonase (dehydratase/thioesterase) produces the characteristic quorum sensing molecule of B. cenocepacia, cis-2-dodecenoic acid or BDSF, starting from 3-hydroxydodecanoyl-acyl carrier protein. Unexpectedly, the crystal structure revealed the presence of a lipid molecule in the catalytic site of the enzyme, which was identified as dodecanoic acid. Our biochemical characterization shows that DfsA is able to use dodecanoyl-acyl carrier protein as a substrate, demonstrating that dodecanoic acid, the product of this reaction, is released very slowly from the DfsA active site, therefore acting as a DfsA inhibitor. This molecule shows an unprecedented conformational arrangement inside the DfsA active site. In contrast with previous hypotheses, our data illustrate how DfsA and closely related homologous enzymes can recognize long hydrophobic substrates without large conformational changes or assistance by additional regulator molecules. The elucidation of the substrate binding mode in DfsA provides the starting point for structure-based drug discovery studies targeting B. cenocepacia quorum sensing-assisted virulence.

  15. The Crystal Structure of Burkholderia cenocepacia DfsA Provides Insights into Substrate Recognition and Quorum Sensing Fatty Acid Biosynthesis.

    PubMed

    Spadaro, Francesca; Scoffone, Viola C; Chiarelli, Laurent R; Fumagalli, Marco; Buroni, Silvia; Riccardi, Giovanna; Forneris, Federico

    2016-06-14

    Burkholderia cenocepacia is a major concern among respiratory tract infections in cystic fibrosis patients. This pathogen is particularly difficult to treat because of its high level of resistance to the clinically relevant antimicrobial agents. In B. cenocepacia, the quorum sensing cell-cell communication system is involved in different processes that are important for bacterial virulence, such as biofilm formation and protease and siderophore production. Targeting the enzymes involved in this process represents a promising therapeutic approach. With the aim of finding effective quorum sensing inhibitors, we have determined the three-dimensional structure of B. cenocepacia diffusible factor synthase A, DfsA. This bifunctional crotonase (dehydratase/thioesterase) produces the characteristic quorum sensing molecule of B. cenocepacia, cis-2-dodecenoic acid or BDSF, starting from 3-hydroxydodecanoyl-acyl carrier protein. Unexpectedly, the crystal structure revealed the presence of a lipid molecule in the catalytic site of the enzyme, which was identified as dodecanoic acid. Our biochemical characterization shows that DfsA is able to use dodecanoyl-acyl carrier protein as a substrate, demonstrating that dodecanoic acid, the product of this reaction, is released very slowly from the DfsA active site, therefore acting as a DfsA inhibitor. This molecule shows an unprecedented conformational arrangement inside the DfsA active site. In contrast with previous hypotheses, our data illustrate how DfsA and closely related homologous enzymes can recognize long hydrophobic substrates without large conformational changes or assistance by additional regulator molecules. The elucidation of the substrate binding mode in DfsA provides the starting point for structure-based drug discovery studies targeting B. cenocepacia quorum sensing-assisted virulence. PMID:27198181

  16. Salicylic acids

    PubMed Central

    Hayat, Shamsul; Irfan, Mohd; Wani, Arif; Nasser, Alyemeni; Ahmad, Aqil

    2012-01-01

    Salicylic acid is well known phytohormone, emerging recently as a new paradigm of an array of manifestations of growth regulators. The area unleashed yet encompassed the applied agriculture sector to find the roles to strengthen the crops against plethora of abiotic and biotic stresses. The skipped part of integrated picture, however, was the evolutionary insight of salicylic acid to either allow or discard the microbial invasion depending upon various internal factors of two interactants under the prevailing external conditions. The metabolic status that allows the host invasion either as pathogenesis or symbiosis with possible intermediary stages in close systems has been tried to underpin here. PMID:22301975

  17. Stearic Acid

    ERIC Educational Resources Information Center

    Young, Jay A.

    2004-01-01

    A chemical laboratory information profile (CLIP) is presented for the chemical, stearic acid. The profile lists the chemical's physical and harmful characteristics, exposure limits, and symptoms of major exposure, for the benefit of teachers and students, who use the chemical in the laboratory.

  18. Trichloroacetic acid

    Integrated Risk Information System (IRIS)

    Trichloroacetic acid ( TCA ) ; CASRN 76 - 03 - 9 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Nonca

  19. Acrylic acid

    Integrated Risk Information System (IRIS)

    Acrylic acid ( CASRN 79 - 10 - 7 ) Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Eff

  20. Selenious acid

    Integrated Risk Information System (IRIS)

    Selenious acid ; CASRN 7783 - 00 - 8 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic E

  1. Dichloroacetic acid

    Integrated Risk Information System (IRIS)

    Dichloroacetic acid ; CASRN 79 - 43 - 6 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogeni

  2. Cacodylic acid

    Integrated Risk Information System (IRIS)

    Cacodylic acid ; CASRN 75 - 60 - 5 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Eff

  3. Phosphoric acid

    Integrated Risk Information System (IRIS)

    Phosphoric acid ; CASRN 7664 - 38 - 2 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic

  4. Benzoic acid

    Integrated Risk Information System (IRIS)

    Benzoic acid ; CASRN 65 - 85 - 0 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Effec

  5. Formic acid

    Integrated Risk Information System (IRIS)

    Formic acid ; CASRN 64 - 18 - 6 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Effect

  6. [Hyaluronic acid].

    PubMed

    Pomarede, N

    2008-01-01

    Hyaluronic Acid (HA) is now a leader product in esthetic procedures for the treatment of wrinkles and volumes. The structure of HA, its metabolism, its physiological function are foremost breaking down then its use in aesthetic dermatology: steps of injection, possible side effects, benefits and downsides of the use of HA in aesthetic dermatology.

  7. Effects of chlorogenic acid, caffeine, and coffee on behavioral and biochemical parameters of diabetic rats.

    PubMed

    Stefanello, Naiara; Schmatz, Roberta; Pereira, Luciane Belmonte; Rubin, Maribel A; da Rocha, João Batista Teixeira; Facco, Graziela; Pereira, Maria Ester; Mazzanti, Cinthia Melazzo de Andrade; Passamonti, Sabina; Rodrigues, Marília Valvassori; Carvalho, Fabiano Barbosa; da Rosa, Michelle Melgarejo; Gutierres, Jessie Martins; Cardoso, Andréia Machado; Morsch, Vera Maria; Schetinger, Maria Rosa Chitolina

    2014-03-01

    Diabetes mellitus (DM) is associated with brain alterations that may contribute to cognitive dysfunctions. Chlorogenic acid (CGA) and caffeine (CA), abundant in coffee (CF), are natural compounds that have showed important actions in the brain. The present study aimed to evaluate the effect of CGA, CA, and CF on acetylcholinesterase (AChE), Na(+), K(+)-ATPase, aminolevulinate dehydratase (δ-ALA-D) activities and TBARS levels from cerebral cortex, as well as memory and anxiety in streptozotocin-induced diabetic rats. Animals were divided into eight groups (n = 5-10): control; control/CGA 5 mg/kg; control/CA 15 mg/kg; control/CF 0.5 g/kg; diabetic; diabetic/CGA 5 mg/kg; diabetic/CA 15 mg/kg; and diabetic/CF 0.5 g/kg. Our results demonstrated an increase in AChE activity and TBARS levels in cerebral cortex, while δ-ALA-D and Na(+), K(+)-ATPase activities were decreased in the diabetic rats when compared to control water group. Furthermore, a memory deficit and an increase in anxiety in diabetic rats were observed. The treatment with CGA and CA prevented the increase in AChE activity in diabetic rats when compared to the diabetic water group. CGA, CA, and CF intake partially prevented cerebral δ-ALA-D and Na(+), K(+)-ATPase activity decrease due to diabetes. Moreover, CGA prevented diabetes-induced TBARS production, improved memory, and decreased anxiety. In conclusion, among the compounds studied CGA proved to be a compound which acts better in the prevention of brain disorders promoted by DM. PMID:24370728

  8. Hydroxycarboxylic acids and salts

    DOEpatents

    Kiely, Donald E; Hash, Kirk R; Kramer-Presta, Kylie; Smith, Tyler N

    2015-02-24

    Compositions which inhibit corrosion and alter the physical properties of concrete (admixtures) are prepared from salt mixtures of hydroxycarboxylic acids, carboxylic acids, and nitric acid. The salt mixtures are prepared by neutralizing acid product mixtures from the oxidation of polyols using nitric acid and oxygen as the oxidizing agents. Nitric acid is removed from the hydroxycarboxylic acids by evaporation and diffusion dialysis.

  9. Methylmalonic acid blood test

    MedlinePlus

    ... acid is a substance produced when proteins, called amino acids, in the body break down. The health care ... Cederbaum S, Berry GT. Inborn errors of carbohydrate, ammonia, amino acid, and organic acid metabolism. In: Gleason CA, Devaskar ...

  10. Folic Acid and Pregnancy

    MedlinePlus

    ... 5 Things to Know About Zika & Pregnancy Folic Acid and Pregnancy KidsHealth > For Parents > Folic Acid and ... before conception and during early pregnancy . About Folic Acid Folic acid, sometimes called folate, is a B ...

  11. Understanding Acid Rain

    ERIC Educational Resources Information Center

    Damonte, Kathleen

    2004-01-01

    The term acid rain describes rain, snow, or fog that is more acidic than normal precipitation. To understand what acid rain is, it is first necessary to know what an acid is. Acids can be defined as substances that produce hydrogen ions (H+), when dissolved in water. Scientists indicate how acidic a substance is by a set of numbers called the pH…

  12. Responses in whole-body amino acid kinetics to an acute, sub-clinical endotoxin challenge in lambs.

    PubMed

    Hoskin, S O; Bremner, D M; Holtrop, G; Lobley, G E

    2016-02-28

    Some effects of parasitism, endotoxaemia or sepsis can be mitigated by provision of extra protein. Supplemented protein may encompass a metabolic requirement for specific amino acids (AA). The current study investigates a method to identify and quantify the amounts of AA required during inflammation induced by an endotoxin challenge. One of each pair of six twin sheep was infused in the jugular vein for 20 h with either saline (control) or lipopolysaccharide (LPS, 2 ng/kg body weight per min) from Escherichia coli. Between 12 and 20 h a mixture of stable isotope-labelled AA was infused to measure irreversible loss rates. From 16 to 20 h all sheep were supplemented with a mixture of unlabelled AA infused intravenously. Blood samples were taken before the start of infusions, and then continuously over intervals between 14 and 20 h. At 20 h the sheep were euthanised, and liver and kidney samples were taken for measurement of serine-threonine dehydratase (SDH) activity. LPS infusion decreased plasma concentrations of most AA (P<0·05; P<0·10 for leucine and tryptophan), except for phenylalanine (which increased P=0·022) and tyrosine. On the basis of the incremental response to the supplemental AA, arginine, aspartate, cysteine, glutamate, lysine (tendency only), glycine, methionine, proline, serine and threonine were important in the metabolic response to the endotoxaemia. The AA infusion between 16 and 20 h restored the plasma concentrations in the LPS-treated sheep for the majority of AA, except for glutamine, isoleucine, methionine, serine and valine. LPS treatment increased (P<0·02) SDH activity in both liver and kidney. The approach allows quantification of key AA required during challenge situations. PMID:26652711

  13. Production of 3-Hydroxypropionic Acid via the Propionyl-CoA Pathway Using Recombinant Escherichia coli Strains.

    PubMed

    Luo, Hui; Zhou, Dafeng; Liu, Xiaohui; Nie, Zhihua; Quiroga-Sánchez, Diego Leandro; Chang, Yanhong

    2016-01-01

    Our study aimed to produce the commercially promising platform chemical 3-hydroxypropionic acid (3-HP) via the propionyl-CoA pathway in genetically engineered Escherichia coli. Recombinant E. coli Ec-P overexpressing propionyl-CoA dehydrogenase (PACD, encoded by the pacd gene from Candida rugosa) under the T7 promoter produced 1.33 mM of 3-HP in a shake flask culture supplemented with 0.5% propionate. When propionate CoA-transferase (PCT, encoded by the pct gene from Megasphaera elsdenii) and 3-hydroxypropionyl-CoA dehydratase (HPCD, encoded by the hpcd gene from Chloroflexus aurantiacus) were expressed along with PACD, the 3-HP titer of the resulting E. coli Ec-PPH strain was improved by 6-fold. The effect of the cultivation conditions on the 3-HP yield from propionate in the Ec-PPH strain was also investigated. When cultured at 30°C with 1% glucose in addition to propionate, 3-HP production by Ec-PPH increased 2-fold and 12-fold compared to the cultivation at 37°C (4.23 mM) or without glucose (0.68 mM). Deletion of the ygfH gene encoding propionyl-CoA: succinate CoA-transferase from Ec-PPH (resulting in the strain Ec-△Y-PPH) led to increase of 3-HP production in shake flask experiments (15.04 mM), whereas the strain Ec-△Y-PPH with deletion of the prpC gene (encoding methylcitrate synthase in the methylcitrate cycle) produced 17.76 mM of 3-HP. The strain Ec-△Y-△P-PPH with both ygfH and prpC genes deleted produced 24.14 mM of 3-HP, thus showing an 18-fold increase in the 3-HP titer in compare to the strain Ec-P. PMID:27227837

  14. Production of 3-Hydroxypropionic Acid via the Propionyl-CoA Pathway Using Recombinant Escherichia coli Strains

    PubMed Central

    Luo, Hui; Zhou, Dafeng; Liu, Xiaohui; Nie, Zhihua; Quiroga-Sánchez, Diego Leandro; Chang, Yanhong

    2016-01-01

    Our study aimed to produce the commercially promising platform chemical 3-hydroxypropionic acid (3-HP) via the propionyl-CoA pathway in genetically engineered Escherichia coli. Recombinant E. coli Ec-P overexpressing propionyl-CoA dehydrogenase (PACD, encoded by the pacd gene from Candida rugosa) under the T7 promoter produced 1.33 mM of 3-HP in a shake flask culture supplemented with 0.5% propionate. When propionate CoA-transferase (PCT, encoded by the pct gene from Megasphaera elsdenii) and 3-hydroxypropionyl-CoA dehydratase (HPCD, encoded by the hpcd gene from Chloroflexus aurantiacus) were expressed along with PACD, the 3-HP titer of the resulting E. coli Ec-PPH strain was improved by 6-fold. The effect of the cultivation conditions on the 3-HP yield from propionate in the Ec-PPH strain was also investigated. When cultured at 30°C with 1% glucose in addition to propionate, 3-HP production by Ec-PPH increased 2-fold and 12-fold compared to the cultivation at 37°C (4.23 mM) or without glucose (0.68 mM). Deletion of the ygfH gene encoding propionyl-CoA: succinate CoA-transferase from Ec-PPH (resulting in the strain Ec-△Y-PPH) led to increase of 3-HP production in shake flask experiments (15.04 mM), whereas the strain Ec-△Y-PPH with deletion of the prpC gene (encoding methylcitrate synthase in the methylcitrate cycle) produced 17.76 mM of 3-HP. The strain Ec-△Y-△P-PPH with both ygfH and prpC genes deleted produced 24.14 mM of 3-HP, thus showing an 18-fold increase in the 3-HP titer in compare to the strain Ec-P. PMID:27227837

  15. Precipitation: its acidic nature.

    PubMed

    Frohliger, J O; Kane, R

    1975-08-01

    A comparison of the free hydrogen ion concentration and the total hydrogen ion concentration of rain samples shows that rain is a weak acid. The weak acid nature of rain casts doubt on the concepts that the acidity of rain is increasing and that these increases are due to strong acids such as sulfuric acid.

  16. ZIP8 Zinc Transporter: Indispensable Role for Both Multiple-Organ Organogenesis and Hematopoiesis In Utero

    PubMed Central

    Gálvez-Peralta, Marina; He, Lei; Jorge-Nebert, Lucia F.; Wang, Bin; Miller, Marian L.; Eppert, Bryan L.; Afton, Scott; Nebert, Daniel W.

    2012-01-01

    Previously this laboratory characterized Slc39a8-encoded ZIP8 as a Zn2+/(HCO3–)2 symporter; yet, the overall physiological importance of ZIP8 at the whole-organism level remains unclear. Herein we describe the phenotype of the hypomorphic Slc39a8(neo/neo) mouse which has retained the neomycin-resistance gene in intron 3, hence causing significantly decreased ZIP8 mRNA and protein levels in embryo, fetus, placenta, yolk sac, and several tissues of neonates. The Slc39a8(neo) allele is associated with diminished zinc and iron uptake in mouse fetal fibroblast and liver-derived cultures; consequently, Slc39a8(neo/neo) newborns exhibit diminished zinc and iron levels in several tissues. Slc39a8(neo/neo) homozygotes from gestational day(GD)-11.5 onward are pale, growth-stunted, and die between GD18.5 and 48 h postnatally. Defects include: severely hypoplastic spleen; hypoplasia of liver, kidney, lung, and lower limbs. Histologically, Slc39a8(neo/neo) neonates show decreased numbers of hematopoietic islands in yolk sac and liver. Low hemoglobin, hematocrit, red cell count, serum iron, and total iron-binding capacity confirmed severe anemia. Flow cytometry of fetal liver cells revealed the erythroid series strikingly affected in the hypomorph. Zinc-dependent 5-aminolevulinic acid dehydratase, required for heme synthesis, was not different between Slc39a8(+/+) and Slc39a8(neo/neo) offspring. To demonstrate further that the mouse phenotype is due to ZIP8 deficiency, we bred Slc39a8(+/neo) with BAC-transgenic BTZIP8-3 line (carrying three extra copies of the Slc39a8 allele); this cross generated viable Slc39a8(neo/neo)_BTZIP8-3(+/+) pups showing none of the above-mentioned congenital defects–proving Slc39a8(neo/neo) causes the described phenotype. Our study demonstrates that ZIP8-mediated zinc transport plays an unappreciated critical role during in utero and neonatal growth, organ morphogenesis, and hematopoiesis. PMID:22563477

  17. Amino Acid Metabolism Disorders

    MedlinePlus

    ... defects & other health conditions > Amino acid metabolism disorders Amino acid metabolism disorders E-mail to a friend Please ... baby’s newborn screening may include testing for certain amino acid metabolism disorders. These are rare health conditions that ...

  18. Carbolic acid poisoning

    MedlinePlus

    Phenol poisoning; Phenylic acid poisoning; Hydroxybenzene poisoning; Phenic acid poisoning; Benzenol poisoning ... Below are symptoms of carbolic acid poisoning in different parts of the ... urine Decreased urine output No urine output EYES, EARS, ...

  19. Azelaic Acid Topical

    MedlinePlus

    Azelaic acid gel is used to clear the bumps, lesions, and swelling caused by rosacea (a skin disease that ... redness, flushing, and pimples on the face). Azelaic acid cream is used to treat acne. Azelaic acid ...

  20. Uric acid test (image)

    MedlinePlus

    Uric acid urine test is performed to check for the amount of uric acid in urine. Urine is collected over a 24 ... testing. The most common reason for measuring uric acid levels is in the diagnosis or treatment of ...

  1. Facts about Folic Acid

    MedlinePlus

    ... Information For... Media Policy Makers Facts About Folic Acid Language: English Español (Spanish) Recommend on Facebook Tweet ... of the baby's brain and spine. About folic acid Folic acid is a B vitamin. Our bodies ...

  2. Acid Lipase Disease

    MedlinePlus

    ... Awards Enhancing Diversity Find People About NINDS NINDS Acid Lipase Disease Information Page Synonym(s): Cholesterol Ester Storage ... Trials Related NINDS Publications and Information What is Acid Lipase Disease ? Acid lipase disease or deficiency occurs ...

  3. Co-administration of monoisoamyl dimercaptosuccinic acid and Moringa oleifera seed powder protects arsenic-induced oxidative stress and metal distribution in mice.

    PubMed

    Mishra, Deepshikha; Gupta, Richa; Pant, S C; Kushwah, Pramod; Satish, H T; Flora, S J S

    2009-02-01

    Arsenic contamination of groundwater in the West Bengal basin in India is unfolding as one of the worst natural geo-environmental disasters to date. Chelation therapy with chelating agents is considered to be the best known treatment against arsenic poisoning; however, they are compromised with certain serious drawbacks/side-effects. Efficacy of combined administration of Moringa oleifera (M. oleifera) (English: Drumstick tree) seed powder, a herbal extract, with a thiol chelator monoisoamyl DMSA (MiADMSA) post-arsenic exposure in mice was studied. Mice were exposed to 100 ppm arsenic in drinking water for 6 months, followed by 10-days treatment with M. oleifera seed powder (500 mg/kg, orally through gastric gavage, once daily), MiADMSA (50 mg/kg, intraperitoneally, once daily) either individually or in combination. Arsenic exposure caused significant decrease in blood glutathione, delta-aminolevulinic acid dehydratase (ALAD), accompanied by increased production of reactive oxygen species in blood and soft tissues. Significant inhibition of superoxide dismutase, catalase, and glutathione peroxidase activities in tissues (liver in particular) along with significant increase in thiobarbituric acid reactive substances and metallothionein levels in arsenic intoxicated mice was also noted. Combined administration of MiADMSA with M. oleifera proved better than all other treatments in the recovery of most of the above parameters accompanied by more pronounced depletion of arsenic. The results suggest that concomitant administration of M. oleifera during chelation treatment with MiADMSA might be a better treatment option than monotherapy with the thiol chelator in chronic arsenic toxicity.

  4. Structural and Functional Characterization of PseC, an Aminotransferase Involved in the Biosynthesis of Pseudaminic Acid, an Essential Flagellar Modification in Helicobacter Pylori

    SciTech Connect

    Schoenhofen,I.; Lunin, V.; Julien, J.; Li, Y.; Ajamian, E.; Matte, A.; Cygler, M.; Brisson, J.; Aubry, A.; et al.

    2006-01-01

    Helicobacter pylori flagellin is heavily glycosylated with the novel sialic acid-like nonulosonate, pseudaminic acid (Pse). The glycosylation process is essential for assembly of functional flagellar filaments and consequent bacterial motility. As motility is a key virulence factor for this and other important pathogens, the Pse biosynthetic pathway offers potential for novel therapeutic targets. From recent NMR analyses, we determined that the conversion of UDP-a-D-GlcNAc to the central intermediate in the pathway, UDP-4-amino-4,6-dideoxy-{beta}-L-AltNAc, proceeds by formation of UDP-2-acetamido-2,6-dideoxy-{beta}-L-arabino-4-hexulose by the dehydratase/epimerase PseB (HP0840) followed with amino transfer by the aminotransferase, PseC (HP0366). The central role of PseC in the H. pylori Pse biosynthetic pathway prompted us to determine crystal structures of the native protein, its complexes with pyridoxal phosphate alone and in combination with the UDP-4-amino-4,6-dideoxy-{beta}-L-AltNAc product, the latter being converted to the external aldimine form in the enzyme's active site. In the binding site, the AltNAc sugar ring adopts a 4C1 chair conformation which is different from the predominant 1C4 form found in solution. The enzyme forms a homodimer where each monomer contributes to the active site, and these structures have permitted the identification of key residues involved in stabilization, and possibly catalysis, of the {beta}-L-arabino intermediate during the amino transfer reaction. The essential role of Lys183 in the catalytic event was confirmed by site-directed mutagenesis. This work presents for the first time a nucleotide-sugar aminotransferase co-crystallized with its natural ligand, and in conjunction with the recent functional characterization of this enzyme, will assist in elucidating the aminotransferase reaction mechanism within the Pse biosynthetic pathway.

  5. Combined administration of oxalic acid, succimer and its analogue for the reversal of gallium arsenide-induced oxidative stress in rats.

    PubMed

    Flora, Swaran J S; Kannan, Gurusamy M; Pant, Bhagwat P; Jaiswal, Devendra K

    2002-06-01

    Gallium arsenide (GaAs), a group III-VA intermetallic semiconductor, possesses superior electronic and optical properties and has a wide application in the electronics industry. Exposure to GaAs in the semiconductor industry is a potential occupational hazard because cleaning and slicing GaAs ingots to yield the desired wafer could generate GaAs particles. The ability of GaAs to induce oxidative stress has not yet been reported. The present study reports the role of oxidative stress in GaAs-induced haematological and liver disorders and its possible reversal overturn by administration of meso-2,3-dimercaptosuccinic acid (DMSA) and one of its analogue, monoisoamyl DMSA (MiADMSA), either individually or in combination with oxalic acid. While DMSA and MiADMSA are potential arsenic chelators, oxalic acid is reported to be an effective gallium chelator. Male rats were exposed to 10 mg/kg GaAs orally, 5 days a week for 8 weeks. GaAs exposure was then stopped and rats were given a 0.5 mmol/kg dose of succimers (DMSA or MiADMSA), oxalic acid or a combination of the two, intraperitoneally once daily for 5 consecutive days. We found a significant fall in blood delta-aminolevulinic acid dehydratase (ALAD) activity and blood glutathione (GSH) level, and an increased urinary excretion of delta-aminolevulinic acid (ALA) and an increased malondialdehyde (MDA) level in erythrocytes of rats exposed to GaAs. Hepatic GSH levels decreased, whereas there was an increase in GSSG and MDA levels. The results suggest a role of oxidative stress in GaAs-induced haematological and hepatic damage. Administration of DMSA and MiADMSA produced effective recovery in most of the above variables. However, a greater effectiveness of the chelation treatment (i.e. removal of both gallium and arsenic from body organs) could be achieved by combined administration of succimer (DMSA) with oxalic acid since, after MiADMSA administration, a marked loss of essential metals (copper and zinc) is of concern.

  6. Acid distribution in phosphoric acid fuel cells

    SciTech Connect

    Okae, I.; Seya, A.; Umemoto, M.

    1996-12-31

    Electrolyte acid distribution among each component of a cell is determined by capillary force when the cell is not in operation, but the distribution under the current load conditions had not been clear so far. Since the loss of electrolyte acid during operation is inevitable, it is necessary to store enough amount of acid in every cell. But it must be under the level of which the acid disturbs the diffusion of reactive gases. Accordingly to know the actual acid distribution during operation in a cell is very important. In this report, we carried out experiments to clarify the distribution using small single cells.

  7. Evolution of Mycolic Acid Biosynthesis Genes and Their Regulation during Starvation in Mycobacterium tuberculosis

    PubMed Central

    Jamet, Stevie; Quentin, Yves; Coudray, Coralie; Texier, Pauline; Laval, Françoise; Daffé, Mamadou

    2015-01-01

    ABSTRACT Mycobacterium tuberculosis, the etiological agent of tuberculosis, is a Gram-positive bacterium with a unique cell envelope composed of an essential outer membrane. Mycolic acids, which are very-long-chain (up to C100) fatty acids, are the major components of this mycomembrane. The enzymatic pathways involved in the biosynthesis and transport of mycolates are fairly well documented and are the targets of the major antituberculous drugs. In contrast, only fragmented information is available on the expression and regulation of the biosynthesis genes. In this study, we report that the hadA, hadB, and hadC genes, which code for the mycolate biosynthesis dehydratase enzymes, are coexpressed with three genes that encode proteins of the translational apparatus. Consistent with the well-established control of the translation potential by nutrient availability, starvation leads to downregulation of the hadABC genes along with most of the genes required for the synthesis, modification, and transport of mycolates. The downregulation of a subset of the biosynthesis genes is partially dependent on RelMtb, the key enzyme of the stringent response. We also report the phylogenetic evolution scenario that has shaped the current genetic organization, characterized by the coregulation of the hadABC operon with genes of the translational apparatus and with genes required for the modification of the mycolates. IMPORTANCE Mycobacterium tuberculosis infects one-third of the human population worldwide, and despite the available therapeutic arsenal, it continues to kill millions of people each year. There is therefore an urgent need to identify new targets and develop a better understanding of how the bacterium is adapting itself to host defenses during infection. A prerequisite of this understanding is knowledge of how this adaptive skill has been implanted by evolution. Nutrient scarcity is an environmental condition the bacterium has to cope with during infection. In many

  8. Beneficial role of monoesters of meso-2,3-dimercaptosuccinic acid in the mobilization of lead and recovery of tissue oxidative injury in rats.

    PubMed

    Saxena, Geetu; Pathak, Uma; Flora, S J S

    2005-10-15

    We investigated the therapeutic efficacy of meso-2,3-dimercaptosuccinic acid (DMSA) and two of its analogues, monomethyl dimercaptosuccinic acid (MmDMSA) and mono-cyclohexyl dimercaptosuccinic acid (MchDMSA) in reducing lead concentration in blood and soft tissues, and in recovering lead induced oxidative stress in rats. Male wistar rats were exposed to lead acetate in drinking water for 20 weeks, followed by 5 days of oral treatment with DMSA (100mg/kg, oral, once daily), MmDMSA or MchDMSA (50 and 100mg/kg). Biochemical variables indicative of oxidative stress along with lead, zinc and copper concentration were evaluated in blood and other soft tissues. Exposure to lead caused a significant decrease in blood delta-aminolevulinic acid dehydratase (ALAD) activity and glutathione (GSH) level. These changes were accompanied by inhibition of kidney ALAD and an increase in delta-aminolevulinic acid synthatase (ALAS) activity in liver and kidneys. Also seen were a pronounced depletion of brain GSH, glutathione peroxidase (GPx), glutathione-S-transferase (GST) and decreased superoxide dismutase (SOD) activity and an increase in thiobarbituric acid reactive substances (TBARS) and reactive oxygen species (ROS) levels. These biochemical changes were correlated with an increased uptake of lead in blood and soft tissues. Blood and kidneys zinc concentration decreased significantly following lead exposure while, copper concentration remained unchanged. No effect of chelation on hepatic zinc concentration was noted, only liver copper concentration showed significant depletion on treatment with DMSA and MmDMSA (100mg/kg). Treatment with DMSA, MmDMSA and MchDMSA provided significant recovery in altered biochemical variables and brain DNA damage besides significant depletion of tissue lead burden. Among the chelating agents used, MchDMSA and MmDMSA provided better recovery in altered biochemical variables and depletion of lead concentration in tissues compared to DMSA. The above

  9. Acid tolerance in amphibians

    SciTech Connect

    Pierce, B.A.

    1985-04-01

    Studies of amphibian acid tolerance provide information about the potential effects of acid deposition on amphibian communities. Amphibians as a group appear to be relatively acid tolerant, with many species suffering increased mortality only below pH 4. However, amphibians exhibit much intraspecific variation in acid tolerance, and some species are sensitive to even low levels of acidity. Furthermore, nonlethal effects, including depression of growth rates and increases in developmental abnormalities, can occur at higher pH.

  10. Bioconversions of ferulic acid, an hydroxycinnamic acid.

    PubMed

    Mathew, Sindhu; Abraham, T Emilia

    2006-01-01

    Ferulic acid is the most abundant hydroxycinnamic acid in the plant world and is ester linked to arabinose, in various plant polysaccharides such as arabinoxylans and pectins. It is a precursor to vanillin, one of the most important aromatic flavor compound used in foods, beverages, pharmaceuticals, and perfumes. This article presents an overview of the various biocatalytic routes, focusing on the relevant biotransformations of ferulic acid using plant sources, microorganisms, and enzymes.

  11. Photodynamic therapy using light-emitting diodes for the treatment of viral warts.

    PubMed

    Ohtsuki, Akiko; Hasegawa, Toshio; Hirasawa, Yusuke; Tsuchihashi, Hitoshi; Ikeda, Shigaku

    2009-10-01

    Photodynamic therapy with topical 5-aminolevulinic acid is an effective and safe treatment for actinic keratosis and superficial non-melanoma skin cancer. Further, some studies have reported good efficacy when using photodynamic therapy to treat viral warts. The light-emitting diode is an incoherent, narrow-spectrum light source. The purpose of this study is to evaluate the efficacy of photodynamic therapy using a light-emitting diode for viral warts. Six patients with a total of 41 foot and hand warts were recruited in this study. They were treated with 20% 5-aminolevulinic acid cream under occlusion for 5 h. Thereafter, the treated area was irradiated with the light from a red light-emitting diode (633 +/- 6 nm) with a dose of 126 J/cm(2). This treatment was repeated at 2- or 3-week intervals. The rate of improvement observed in patients was 68.3%. The adverse effects included mild to moderate pain and erythema, which was well-tolerated by all six patients. No patients withdrew from the study due to the adverse effects. Photodynamic therapy with topical 5-aminolevulinic acid using the light from a red light-emitting diode has the advantage of non-invasiveness, minimal associated adverse reactions, and production of good results in a significant proportion of cases: therefore, it is an alternative treatment for recalcitrant viral warts.

  12. Acid Thunder: Acid Rain and Ancient Mesoamerica

    ERIC Educational Resources Information Center

    Kahl, Jonathan D. W.; Berg, Craig A.

    2006-01-01

    Much of Mesoamerica's rich cultural heritage is slowly eroding because of acid rain. Just as water dissolves an Alka-Seltzer tablet, acid rain erodes the limestone surfaces of Mexican archaeological sites at a rate of about one-half millimeter per century (Bravo et al. 2003). A half-millimeter may not seem like much, but at this pace, a few…

  13. Quantity of acid in acid fog

    SciTech Connect

    Deal, W.J.

    1983-07-01

    This communication notes the actual magnitude of the acidity in acidic fog particles and suggests a possible line of inquiry into the health effects of such fog so that it can be determined whether a typical fog is detrimental or beneficial relative to dry air.

  14. Increased 3-hydroxypropionic acid production from glycerol, by modification of central metabolism in Escherichia coli

    PubMed Central

    2014-01-01

    Background 3-hydroxypropionic acid (3HP) is an important chemical precursor for the production of bioplastics. Microbial production of 3HP from glycerol has previously been developed through the optimization of culture conditions and the 3HP biosynthesis pathway. In this study, a novel strategy for improving 3HP production in Escherichia coli was investigated by the modification of central metabolism based on a genome-scale metabolic model and experimental validation. Results Metabolic simulation identified the double knockout of tpiA and zwf as a candidate for improving 3HP production. A 3HP-producing strain was constructed by the expression of glycerol dehydratase and aldehyde dehydrogenase. The double knockout of tpiA and zwf increased the percentage carbon-molar yield (C-mol%) of 3HP on consumed glycerol 4.4-fold (20.1 ± 9.2 C-mol%), compared to the parental strain. Increased extracellular methylglyoxal concentrations in the ΔtpiA Δzwf strain indicated that glycerol catabolism was occurring through the methylglyoxal pathway, which converts dihydroxyacetone phosphate to pyruvate, as predicted by the metabolic model. Since the ΔtpiA Δzwf strain produced abundant 1,3-propanediol as a major byproduct (37.7 ± 13.2 C-mol%), yqhD, which encodes an enzyme involved in the production of 1,3-propanediol, was disrupted in the ΔtpiA Δzwf strain. The 3HP yield of the ΔtpiA Δzwf ΔyqhD strain (33.9 ± 1.2 C-mol%) was increased 1.7-fold further compared to the ΔtpiA Δzwf strain and by 7.4-fold compared to the parental strain. Conclusion This study successfully increased 3HP production by 7.4-fold in the ΔtpiA Δzwf ΔyqhD E. coli strain by the modification of the central metabolism, based on metabolic simulation and experimental validation of engineered strains. PMID:24885133

  15. Lactic acid test

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003507.htm Lactic acid test To use the sharing features on this page, please enable JavaScript. Lactic acid is mainly produced in muscle cells and red ...

  16. Omega-6 Fatty Acids

    MedlinePlus

    ... types of fats. Some types are found in vegetable oils, including corn, evening primrose seed, safflower, and soybean ... from studying specific omega-6 fatty acids or plant oils containing omega-6 fatty acids. See the separate ...

  17. Fatty acid analogs

    DOEpatents

    Elmaleh, David R.; Livni, Eli

    1985-01-01

    In one aspect, a radioactively labeled analog of a fatty acid which is capable of being taken up by mammalian tissue and which exhibits an in vivo beta-oxidation rate below that with a corresponding radioactively labeled fatty acid.

  18. Deoxycholic Acid Injection

    MedlinePlus

    Deoxycholic acid injection is used to improve the appearance and profile of moderate to severe submental fat ('double chin'; fatty tissue located under the chin). Deoxycholic acid injection is in a class of medications called ...

  19. Aminocaproic Acid Injection

    MedlinePlus

    Aminocaproic acid injection is used to control bleeding that occurs when blood clots are broken down too quickly. This ... the baby is ready to be born). Aminocaproic acid injection is also used to control bleeding in ...

  20. Zoledronic Acid Injection

    MedlinePlus

    ... acid (Reclast) is used to prevent or treat osteoporosis (condition in which the bones become thin and ... Zoledronic acid (Reclast) is also used to treat osteoporosis in men, and to prevent or treat osteoporosis ...

  1. Uric Acid Test

    MedlinePlus

    ... limited. Home Visit Global Sites Search Help? Uric Acid Share this page: Was this page helpful? Also known as: Serum Urate; UA Formal name: Uric Acid Related tests: Synovial Fluid Analysis , Kidney Stone Analysis , ...

  2. Methylmalonic Acid Test

    MedlinePlus

    ... limited. Home Visit Global Sites Search Help? Methylmalonic Acid Share this page: Was this page helpful? Also known as: MMA Formal name: Methylmalonic Acid Related tests: Vitamin B12 and Folate , Homocysteine , Intrinsic ...

  3. Hydrochloric acid poisoning

    MedlinePlus

    Hydrochloric acid is a clear, poisonous liquid. It is highly corrosive, which means it immediately causes severe ... discusses poisoning due to swallowing or breathing in hydrochloric acid. This article is for information only. Do ...

  4. Mixed Acid Oxidation

    SciTech Connect

    Pierce, R.A.

    1999-10-26

    Several non-thermal processes have been developed to destroy organic waste compounds using chemicals with high oxidation potentials. These efforts have focused on developing technologies that work at low temperatures, relative to incineration, to overcome many of the regulatory issues associated with obtaining permits for waste incinerators. One such technique with great flexibility is mixed acid oxidation. Mixed acid oxidation, developed at the Savannah River Site, uses a mixture of an oxidant (nitric acid) and a carrier acid (phosphoric acid). The carrier acid acts as a non-volatile holding medium for the somewhat volatile oxidant. The combination of acids allows appreciable amounts of the concentrated oxidant to remain in the carrier acid well above the oxidant''s normal boiling point.

  5. Plant fatty acid hydroxylases

    DOEpatents

    Somerville, Chris; Broun, Pierre; van de Loo, Frank

    2001-01-01

    This invention relates to plant fatty acyl hydroxylases. Methods to use conserved amino acid or nucleotide sequences to obtain plant fatty acyl hydroxylases are described. Also described is the use of cDNA clones encoding a plant hydroxylase to produce a family of hydroxylated fatty acids in transgenic plants. In addition, the use of genes encoding fatty acid hydroxylases or desaturases to alter the level of lipid fatty acid unsaturation in transgenic plants is described.

  6. PRODUCTION OF TRIFLUOROACETIC ACID

    DOEpatents

    Haworth, W.N.; Stacey, M.

    1949-07-19

    A method is given for the production of improved yields of trifluoroacetic acid. The compound is prepared by oxidizing m-aminobenzotrifluoride with an alkali metal or alkaline earth metal permanganate at a temperature in the range of 80 deg C to 100 deg C while dissolved ln a mixture of water with glacial acetic acid and/or trifluoroacetic acid. Preferably a mixture of water and trifluoroacetic acid ls used as the solvent.

  7. Quantity of acid in acid fog

    SciTech Connect

    Deal, W.J.

    1983-07-01

    The chemical composition of fog particles has become of considerable interest, because of both the possibility of interpreting atmospheric- chemistry processes in fog particles in terms of the principles of aqueous chemistry and the potential health effects of species present in fog particles. The acidity of fog particles has received wide attention. This communication noted the actual magnitude of the excess acidity in acidic fog particles and suggested a possible line of inquiry into the health effects of such fog so that it can be determined whether a typical fog is detrimental or beneficial relative to dry air. (DP)

  8. Acid Rain Study Guide.

    ERIC Educational Resources Information Center

    Hunger, Carolyn; And Others

    Acid rain is a complex, worldwide environmental problem. This study guide is intended to aid teachers of grades 4-12 to help their students understand what acid rain is, why it is a problem, and what possible solutions exist. The document contains specific sections on: (1) the various terms used in conjunction with acid rain (such as acid…

  9. The Acid Rain Reader.

    ERIC Educational Resources Information Center

    Stubbs, Harriett S.; And Others

    A topic which is often not sufficiently dealt with in elementary school textbooks is acid rain. This student text is designed to supplement classroom materials on the topic. Discussed are: (1) "Rain"; (2) "Water Cycle"; (3) "Fossil Fuels"; (4) "Air Pollution"; (5) "Superstacks"; (6) "Acid/Neutral/Bases"; (7) "pH Scale"; (8) "Acid Rain"; (9)…

  10. What Is Acid Rain?

    ERIC Educational Resources Information Center

    Likens, Gene E.

    2004-01-01

    Acid rain is the collective term for any type of acidified precipitation: rain, snow, sleet, and hail, as well as the presence of acidifying gases, particles, cloud water, and fog in the atmosphere. The increased acidity, primarily from sulfuric and nitric acids, is generated as a by-product of the combustion of fossil fuels such as coal and oil.…

  11. [alpha]-Oxocarboxylic Acids

    ERIC Educational Resources Information Center

    Kerber, Robert C.; Fernando, Marian S.

    2010-01-01

    Several [alpha]-oxocarboxylic acids play key roles in metabolism in plants and animals. However, there are inconsistencies between the structures as commonly portrayed and the reported acid ionization constants, which result because the acids are predominantly hydrated in aqueous solution; that is, the predominant form is RC(OH)[subscript 2]COOH…

  12. Nucleic acid detection compositions

    DOEpatents

    Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow, Mary Ann; Dahlberg, James L.

    2008-08-05

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

  13. Cleavage of nucleic acids

    DOEpatents

    Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow, Mary Ann D.; Dahlberg, James E.

    2000-01-01

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

  14. Nucleic acid detection assays

    DOEpatents

    Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow, Mary Ann; Dahlberg, James E.

    2005-04-05

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

  15. Cleavage of nucleic acids

    DOEpatents

    Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor L.; Brow, Mary Ann D.; Dahlberg, James E.

    2007-12-11

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

  16. Cleavage of nucleic acids

    SciTech Connect

    Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow; Mary Ann D.; Dahlberg, James E.

    2010-11-09

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

  17. Editorial: Acid precipitation

    SciTech Connect

    1995-09-01

    This editorial focuses on acid rain and the history of public and governmental response to acid rain. Comments on a book by Gwineth Howell `Acid Rain and Acid Waters` are included. The editor feels that Howells has provide a service to the environmental scientific community, with a textbook useful to a range of people, as well as a call for decision makers to learn from the acid rain issue and use it as a model for more sweeping global environmental issues. A balance is needed among several parameters such as level of evidence, probability that the evidence will lead to a specific direction and the cost to the global community. 1 tab.

  18. [Safety of folic acid].

    PubMed

    Ströhle, Alexander; Wolters, Maike; Hahn, Andreas

    2015-08-01

    Improving dietary folate intake is a central public health goal. However, critical voices have become louder warning of too high intake of folic acid. Safety concerns of a high folic acid exposure are usually limited to synthetic folic acid contained in drugs and food supplements. Against this background, the present article focuses on two matters: (a) How do the absorption and metabolism of synthetic folic acid differ from that of other folates? (b) How has the longterm safety of folic acid to be judged, especially regarding the risk of colorectal cancer, autism, asthma, impaired immune defence, masking vitamin B12 deficiency and interactions with the methotrexate metabolism?

  19. Amino acid analysis

    NASA Technical Reports Server (NTRS)

    Winitz, M.; Graff, J. (Inventor)

    1974-01-01

    The process and apparatus for qualitative and quantitative analysis of the amino acid content of a biological sample are presented. The sample is deposited on a cation exchange resin and then is washed with suitable solvents. The amino acids and various cations and organic material with a basic function remain on the resin. The resin is eluted with an acid eluant, and the eluate containing the amino acids is transferred to a reaction vessel where the eluant is removed. Final analysis of the purified acylated amino acid esters is accomplished by gas-liquid chromatographic techniques.

  20. Acidic Ionic Liquids.

    PubMed

    Amarasekara, Ananda S

    2016-05-25

    Ionic liquid with acidic properties is an important branch in the wide ionic liquid field and the aim of this article is to cover all aspects of these acidic ionic liquids, especially focusing on the developments in the last four years. The structural diversity and synthesis of acidic ionic liquids are discussed in the introduction sections of this review. In addition, an unambiguous classification system for various types of acidic ionic liquids is presented in the introduction. The physical properties including acidity, thermo-physical properties, ionic conductivity, spectroscopy, and computational studies on acidic ionic liquids are covered in the next sections. The final section provides a comprehensive review on applications of acidic ionic liquids in a wide array of fields including catalysis, CO2 fixation, ionogel, electrolyte, fuel-cell, membrane, biomass processing, biodiesel synthesis, desulfurization of gasoline/diesel, metal processing, and metal electrodeposition.

  1. Nucleic acid detection kits

    DOEpatents

    Hall, Jeff G.; Lyamichev, Victor I.; Mast, Andrea L.; Brow, Mary Ann; Kwiatkowski, Robert W.; Vavra, Stephanie H.

    2005-03-29

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of nucleic acid from various viruses in a sample.

  2. Acidic Ionic Liquids.

    PubMed

    Amarasekara, Ananda S

    2016-05-25

    Ionic liquid with acidic properties is an important branch in the wide ionic liquid field and the aim of this article is to cover all aspects of these acidic ionic liquids, especially focusing on the developments in the last four years. The structural diversity and synthesis of acidic ionic liquids are discussed in the introduction sections of this review. In addition, an unambiguous classification system for various types of acidic ionic liquids is presented in the introduction. The physical properties including acidity, thermo-physical properties, ionic conductivity, spectroscopy, and computational studies on acidic ionic liquids are covered in the next sections. The final section provides a comprehensive review on applications of acidic ionic liquids in a wide array of fields including catalysis, CO2 fixation, ionogel, electrolyte, fuel-cell, membrane, biomass processing, biodiesel synthesis, desulfurization of gasoline/diesel, metal processing, and metal electrodeposition. PMID:27175515

  3. Therapeutic potential of monoisoamyl and monomethyl esters of meso 2,3-dimercaptosuccinic acid in gallium arsenide intoxicated rats.

    PubMed

    Flora, Swaran J S; Mehta, Ashish; Rao, P V Lakshmana; Kannan, Gurusamy M; Bhaskar, A S B; Dube, Shashi N; Pant, Bhagwat P

    2004-02-15

    The dose dependent effects of monoisoamyl and monomethyl esters of meso 2,3-dimercaptosuccinic acid (DMSA) (0.1, 0.3 and 0.5 mmol kg(-1), intraperitoneally (i.p.) once daily for 5 days) to offset the characteristic biochemical, immunological, oxidative stress consequences and DNA damage (based on DNA fragmentation and comet assay) following sub-chronic administration of gallium arsenide and the mobilization of gallium and arsenic were examined. The effects of these chelators alone in normal animals too were examined on above-mentioned variables. Male Wistar rats were exposed to 10 mg kg(-1), GaAs, orally once daily for 12 weeks and were administered DMSA or two of its monoesters (monoisoamyl or monomethyl) for 5 consecutive days. DMSA was used as a positive control. DMSA and its derivatives, when given alone, generally have no adverse effects on various parameters. After 5 days of chelation therapy in GaAs pre-exposed rats, MiADMSA was most effective in the reduction of inhibited blood delta-aminolevulinic acid dehydratase (ALAD) activity and zinc protoporphyrin level while, all three chelators effectively reduced urinary ALA excretion, compared to GaAs alone exposed rats. MiADMSA was also effective, particularly at a dose of 0.3 mmol kg(-1), in enhancing the inhibited hepatic transaminase activities. Parameters indicative of oxidative stress responded less favorably to the chelation therapy, however, three chelators significantly restored the altered immunological variables. MiADMSA was relatively more effective than the other two chelators. GaAs produced significant DNA damage in the liver and kidneys and the chelation treatment had moderate but significant influence in reducing DNA damage. All three chelators significantly reduced arsenic concentration and, however, MiADMSA was more effective than the other two chelators in depleting arsenic concentration from blood and other soft tissues. A dose of 0.3 mmol kg(-1) was found to be relatively better than the

  4. Boric acid and boronic acids inhibition of pigeonpea urease.

    PubMed

    Reddy, K Ravi Charan; Kayastha, Arvind M

    2006-08-01

    Urease from the seeds of pigeonpea was competitively inhibited by boric acid, butylboronic acid, phenylboronic acid, and 4-bromophenylboronic acid; 4-bromophenylboronic acid being the strongest inhibitor, followed by boric acid > butylboronic acid > phenylboronic acid, respectively. Urease inhibition by boric acid is maximal at acidic pH (5.0) and minimal at alkaline pH (10.0), i.e., the trigonal planar B(OH)3 form is a more effective inhibitor than the tetrahedral B(OH)4 -anionic form. Similarly, the anionic form of phenylboronic acid was least inhibiting in nature.

  5. Biotransformation of cinnamic acid, p-coumaric acid, caffeic acid, and ferulic acid by plant cell cultures of Eucalyptus perriniana.

    PubMed

    Katsuragi, Hisashi; Shimoda, Kei; Kubota, Naoji; Nakajima, Nobuyoshi; Hamada, Hatsuyuki; Hamada, Hiroki

    2010-01-01

    Biotransformations of phenylpropanoids such as cinnamic acid, p-coumaric acid, caffeic acid, and ferulic acid were investigated with plant-cultured cells of Eucalyptus perriniana. The plant-cultured cells of E. perriniana converted cinnamic acid into cinnamic acid β-D-glucopyranosyl ester, p-coumaric acid, and 4-O-β-D-glucopyranosylcoumaric acid. p-Coumaric acid was converted into 4-O-β-D-glucopyranosylcoumaric acid, p-coumaric acid β-D-glucopyranosyl ester, 4-O-β-D-glucopyranosylcoumaric acid β-D-glucopyranosyl ester, a new compound, caffeic acid, and 3-O-β-D-glucopyranosylcaffeic acid. On the other hand, incubation of caffeic acid with cultured E. perriniana cells gave 3-O-β-D-glucopyranosylcaffeic acid, 3-O-(6-O-β-D-glucopyranosyl)-β-D-glucopyranosylcaffeic acid, a new compound, 3-O-β-D-glucopyranosylcaffeic acid β-D-glucopyranosyl ester, 4-O-β-D-glucopyranosylcaffeic acid, 4-O-β-D-glucopyranosylcaffeic acid β-D-glucopyranosyl ester, ferulic acid, and 4-O-β-D-glucopyranosylferulic acid. 4-O-β-D-Glucopyranosylferulic acid, ferulic acid β-D-glucopyranosyl ester, and 4-O-β-D-glucopyranosylferulic acid β-D-glucopyranosyl ester were isolated from E. perriniana cells treated with ferulic acid.

  6. Process for the preparation of lactic acid and glyceric acid

    DOEpatents

    Jackson, James E [Haslett, MI; Miller, Dennis J [Okemos, MI; Marincean, Simona [Dewitt, MI

    2008-12-02

    Hexose and pentose monosaccharides are degraded to lactic acid and glyceric acid in an aqueous solution in the presence of an excess of a strongly anionic exchange resin, such as AMBERLITE IRN78 and AMBERLITE IRA400. The glyceric acid and lactic acid can be separated from the aqueous solution. Lactic acid and glyceric acid are staple articles of commerce.

  7. Well acidizing compositions and methods

    SciTech Connect

    Swanson, B. L.

    1980-12-23

    Gelled acidic compositions suitable for matrix acidizing or fracture acidizing of subterranean formations are provided comprising water, a water-dispersible polymeric viscosifier such as a polymer of acrylamide, an acid, and a polyphenolic material such as lignite.

  8. Bile acids but not acidic acids induce Barrett's esophagus.

    PubMed

    Sun, Dongfeng; Wang, Xiao; Gai, Zhibo; Song, Xiaoming; Jia, Xinyong; Tian, Hui

    2015-01-01

    Barrett's esophagus (BE) is associated with the development of esophageal adenocarcinoma (EAC). Bile acids (BAs) refluxing into the esophagus contribute to esophageal injury, which results in BE and subsequent EAC. We developed two animal models to test the role of BAs in the pathogenesis of BE. We surgically generated BA reflux, with or without gastric acid, in rats. In a second experiment, we fed animals separately with BAs and gastric acid. Pathologic changes were examined and the expression of Muc2 and Cdx2 in BE tissue was tested by immunostaining. Inflammatory factors in the plasma, as well as differentiation genes in BE were examined through highly sensitive ELISA and semi-quantitative RT-PCR techniques. We found that BAs are sufficient for the induction of esophagitis and Barrett's-like metaplasia in the esophagus. Overexpression of inflammatory cells, IL-6, and TNF-α was observed both in animals fed with BAs and surgically generated BA reflux. Furthermore, elevated levels of Cdx2, Muc2, Bmp4, Kit19, and Tff2 (differentiation genes in BE) were found in BA-treated rats. In conclusion, BAs, but not gastric acid, are a major causative factor for BE. We confirmed that BAs contribute to the development of BE by inducing the inflammatory response in the esophagus. Inhibiting BAs may be a promising therapy for BE.

  9. Microorganisms for producing organic acids

    DOEpatents

    Pfleger, Brian Frederick; Begemann, Matthew Brett

    2014-09-30

    Organic acid-producing microorganisms and methods of using same. The organic acid-producing microorganisms comprise modifications that reduce or ablate AcsA activity or AcsA homolog activity. The modifications increase tolerance of the microorganisms to such organic acids as 3-hydroxypropionic acid, acrylic acid, propionic acid, lactic acid, and others. Further modifications to the microorganisms increase production of such organic acids as 3-hydroxypropionic acid, lactate, and others. Methods of producing such organic acids as 3-hydroxypropionic acid, lactate, and others with the modified microorganisms are provided. Methods of using acsA or homologs thereof as counter-selectable markers are also provided.

  10. Recent advances in the prevention and treatment of skin cancer using photodynamic therapy

    PubMed Central

    Zhao, Baozhong; He, Yu-Ying

    2011-01-01

    Photodynamic therapy (PDT) is a noninvasive procedure that involves a photosensitizing drug and its subsequent activation by light to produce reactive oxygen species that specifically destroy target cells. Recently, PDT has been widely used in treating non-melanoma skin malignancies, the most common cancer in the USA, with superior cosmetic outcomes compared with conventional therapies. The topical ‘photosensitizers’ commonly used are 5-aminolevulinic acid (ALA) and its esterified derivative methyl 5-aminolevulinate, which are precursors of the endogenous photosensitizer protoporphyrin IX. After treatment with ALA or methyl 5-aminolevulinate, protoporphyrin IX preferentially accumulates in the lesion area of various skin diseases, which allows not only PDT treatment but also fluorescence diagnosis with ALA-induced porphyrins. Susceptible lesions include various forms of non-melanoma skin cancer such as actinic keratosis, basal cell carcinoma and squamous cell carcinoma. The most recent and promising developments in PDT include the discovery of new photosensitizers, the exploitation of new drug delivery systems and the combination of other modalities, which will all contribute to increasing PDT therapeutic efficacy and improving outcome. This article summarizes the main principles of PDT and its current clinical use in the management of non-melanoma skin cancers, as well as recent developments and possible future research directions. PMID:21080805

  11. Acid-Base Homeostasis.

    PubMed

    Hamm, L Lee; Nakhoul, Nazih; Hering-Smith, Kathleen S

    2015-12-01

    Acid-base homeostasis and pH regulation are critical for both normal physiology and cell metabolism and function. The importance of this regulation is evidenced by a variety of physiologic derangements that occur when plasma pH is either high or low. The kidneys have the predominant role in regulating the systemic bicarbonate concentration and hence, the metabolic component of acid-base balance. This function of the kidneys has two components: reabsorption of virtually all of the filtered HCO3(-) and production of new bicarbonate to replace that consumed by normal or pathologic acids. This production or generation of new HCO3(-) is done by net acid excretion. Under normal conditions, approximately one-third to one-half of net acid excretion by the kidneys is in the form of titratable acid. The other one-half to two-thirds is the excretion of ammonium. The capacity to excrete ammonium under conditions of acid loads is quantitatively much greater than the capacity to increase titratable acid. Multiple, often redundant pathways and processes exist to regulate these renal functions. Derangements in acid-base homeostasis, however, are common in clinical medicine and can often be related to the systems involved in acid-base transport in the kidneys.

  12. Citric Acid Alternative to Nitric Acid Passivation

    NASA Technical Reports Server (NTRS)

    Lewis, Pattie L. (Compiler)

    2013-01-01

    The Ground Systems Development and Operations GSDO) Program at NASA John F. Kennedy Space Center (KSC) has the primary objective of modernizing and transforming the launch and range complex at KSC to benefit current and future NASA programs along with other emerging users. Described as the launch support and infrastructure modernization program in the NASA Authorization Act of 2010, the GSDO Program will develop and implement shared infrastructure and process improvements to provide more flexible, affordable, and responsive capabilities to a multi-user community. In support of the GSDO Program, the purpose of this project is to demonstratevalidate citric acid as a passivation agent for stainless steel. Successful completion of this project will result in citric acid being qualified for use as an environmentally preferable alternative to nitric acid for passivation of stainless steel alloys in NASA and DoD applications.

  13. Enzymatic gallic acid esterification.

    PubMed

    Weetal, H H

    1985-02-01

    Gallic acid esters of n-propyl and amyl alcohols have been produced by enzymatic synthesis in organic solvents using immobilized tannase. Studies indicate that maximum esterification of gallic acid occurs with amyl alcohol. The enzyme shows broad alcohol specificity. However, the enzyme exhibits absolute specificity for the acid portion of the ester. Studies were carried out on K(m), V(max), pH, and temperature optima.

  14. Amino acids and proteins.

    PubMed

    van Goudoever, Johannes B; Vlaardingerbroek, Hester; van den Akker, Chris H; de Groof, Femke; van der Schoor, Sophie R D

    2014-01-01

    Amino acids and protein are key factors for growth. The neonatal period requires the highest intake in life to meet the demands. Those demands include amino acids for growth, but proteins and amino acids also function as signalling molecules and function as neurotransmitters. Often the nutritional requirements are not met, resulting in a postnatal growth restriction. However, current knowledge on adequate levels of both amino acid as well as protein intake can avoid under nutrition in the direct postnatal phase, avoid the need for subsequent catch-up growth and improve later outcome.

  15. USGS Tracks Acid Rain

    USGS Publications Warehouse

    Gordon, John D.; Nilles, Mark A.; Schroder, LeRoy J.

    1995-01-01

    The U.S. Geological Survey (USGS) has been actively studying acid rain for the past 15 years. When scientists learned that acid rain could harm fish, fear of damage to our natural environment from acid rain concerned the American public. Research by USGS scientists and other groups began to show that the processes resulting in acid rain are very complex. Scientists were puzzled by the fact that in some cases it was difficult to demonstrate that the pollution from automobiles and factories was causing streams or lakes to become more acidic. Further experiments showed how the natural ability of many soils to neutralize acids would reduce the effects of acid rain in some locations--at least as long as the neutralizing ability lasted (Young, 1991). The USGS has played a key role in establishing and maintaining the only nationwide network of acid rain monitoring stations. This program is called the National Atmospheric Deposition Program/National Trends Network (NADP/NTN). Each week, at approximately 220 NADP/NTN sites across the country, rain and snow samples are collected for analysis. NADP/NTN site in Montana. The USGS supports about 72 of these sites. The information gained from monitoring the chemistry of our nation's rain and snow is important for testing the results of pollution control laws on acid rain.

  16. Recovery of organic acids

    DOEpatents

    Verser, Dan W.; Eggeman, Timothy J.

    2011-11-01

    A method is disclosed for the recovery of an organic acid from a dilute salt solution in which the cation of the salt forms an insoluble carbonate salt. A tertiary amine and CO.sub.2 are introduced to the solution to form the insoluble carbonate salt and a complex between the acid and an amine. A water immiscible solvent, such as an alcohol, is added to extract the acid/amine complex from the dilute salt solution to a reaction phase. The reaction phase is continuously dried and a product between the acid and the solvent, such as an ester, is formed.

  17. Recovery of organic acids

    DOEpatents

    Verser, Dan W.; Eggeman, Timothy J.

    2009-10-13

    A method is disclosed for the recovery of an organic acid from a dilute salt solution in which the cation of the salt forms an insoluble carbonate salt. A tertiary amine and CO.sub.2 are introduced to the solution to form the insoluble carbonate salt and a complex between the acid and an amine. A water immiscible solvent, such as an alcohol, is added to extract the acid/amine complex from the dilute salt solution to a reaction phase. The reaction phase is continuously dried and a product between the acid and the solvent, such as an ester, is formed.

  18. /sup 13/C NMR studies of porphobilinogen synthase: observation of intermediates bound to a 280,000-dalton protein

    SciTech Connect

    Jaffe, E.K.; Markham, G.D.

    1987-07-14

    /sup 13/C NMR has been used to observe the equilibrium complex of (4-/sup 13/C)-5-aminolevulinate ((4-/sup 13/C)ALA) bound to porphobilinogen (PBG) synthase (5-aminolevulinate dehydratase), a 280,000-dalton protein. (4-/sup 13/C)ALA (chemical shift = 205.9 ppm) forms (3,5-/sup 13/C)PBG (chemical shifts = 121.0 and 123.0 ppm). PBG prepared from a mixture of (4-/sup 13/C)ALA and (/sup 15/N)ALA was used to assign the 121.0 and 123.0 ppm resonances to C/sub 5/ and C/sub 3/, respectively. For the enzyme-bound equilibrium complex formed from holoenzyme and (4-/sup 13/C)ALA, two peaks of equal area with chemical shifts of 121.5 and 127.2 ppm are observed (line widths approx. 50 Hz), indicating that the predominant species is probably a distorted form of PBG. When excess free PBG is present, it is in slow exchange with bound PBG, indicating an exchange rate of < 10 s/sup -1/, which is consistent with the turnover rate of the enzyme. For the complex formed from (4-/sup 13/C)ALA and methyl methanethiosulfonate (MMTS) modified PBG synthase, which does not catalyze PBG formation, the predominant species is a Schiff base adduct (chemical shift = 166.5 ppm, line width approx. 50 Hz). Free ALA is in slow exchange with the Schiff base. Activation of the MMTS-modified enzyme-Schiff base complex with /sup 113/Cd and 2-mercaptoethanol results in the loss of the Schiff base signal and the appearance of bound PBG with the same chemical shifts as for the bound equilibrium complex with Zn(II) enzyme. Neither splitting nor broadening from /sup 113/Cd-/sup 13/C coupling was observed.

  19. Mutant fatty acid desaturase

    DOEpatents

    Shanklin, John; Cahoon, Edgar B.

    2004-02-03

    The present invention relates to a method for producing mutants of a fatty acid desaturase having a substantially increased activity towards fatty acid substrates with chains containing fewer than 18 carbons relative to an unmutagenized precursor desaturase having an 18 carbon atom chain length substrate specificity. The method involves inducing one or more mutations in the nucleic acid sequence encoding the precursor desaturase, transforming the mutated sequence into an unsaturated fatty acid auxotroph cell such as MH13 E. coli, culturing the cells in the absence of supplemental unsaturated fatty acids, thereby selecting for recipient cells which have received and which express a mutant fatty acid desaturase with an elevated specificity for fatty acid substrates having chain lengths of less than 18 carbon atoms. A variety of mutants having 16 or fewer carbon atom chain length substrate specificities are produced by this method. Mutant desaturases produced by this method can be introduced via expression vectors into prokaryotic and eukaryotic cells and can also be used in the production of transgenic plants which may be used to produce specific fatty acid products.

  20. Amino Acid Crossword Puzzle

    ERIC Educational Resources Information Center

    Sims, Paul A.

    2011-01-01

    Learning the 20 standard amino acids is an essential component of an introductory course in biochemistry. Later in the course, the students study metabolism and learn about various catabolic and anabolic pathways involving amino acids. Learning new material or concepts often is easier if one can connect the new material to what one already knows;…

  1. Toxicology of Perfluoroalkyl acids

    EPA Science Inventory

    The Perfluoroalkyl acids(PFAAs) area a family of organic chemicals consisting of a perflurinated carbon backbone (4-12in length) and a acidic functional moiety (Carboxylate or sulfonate). These compounds have excellent surface-tension reducing properties and have numerous industr...

  2. Uric acid - blood

    MedlinePlus

    ... High levels of uric acid can sometimes cause gout or kidney disease. You may have this test if you have had or are about to have certain types of chemotherapy. Rapid weight loss, which may occur with such treatments, can increase the amount of uric acid in ...

  3. Bile acid transporters

    PubMed Central

    Dawson, Paul A.; Lan, Tian; Rao, Anuradha

    2009-01-01

    In liver and intestine, transporters play a critical role in maintaining the enterohepatic circulation and bile acid homeostasis. Over the past two decades, there has been significant progress toward identifying the individual membrane transporters and unraveling their complex regulation. In the liver, bile acids are efficiently transported across the sinusoidal membrane by the Na+ taurocholate cotransporting polypeptide with assistance by members of the organic anion transporting polypeptide family. The bile acids are then secreted in an ATP-dependent fashion across the canalicular membrane by the bile salt export pump. Following their movement with bile into the lumen of the small intestine, bile acids are almost quantitatively reclaimed in the ileum by the apical sodium-dependent bile acid transporter. The bile acids are shuttled across the enterocyte to the basolateral membrane and effluxed into the portal circulation by the recently indentified heteromeric organic solute transporter, OSTα-OSTβ. In addition to the hepatocyte and enterocyte, subgroups of these bile acid transporters are expressed by the biliary, renal, and colonic epithelium where they contribute to maintaining bile acid homeostasis and play important cytoprotective roles. This article will review our current understanding of the physiological role and regulation of these important carriers. PMID:19498215

  4. Analysis of Organic Acids.

    ERIC Educational Resources Information Center

    Griswold, John R.; Rauner, Richard A.

    1990-01-01

    Presented are the procedures and a discussion of the results for an experiment in which students select unknown carboxylic acids, determine their melting points, and investigate their solubility behavior in water and ethanol. A table of selected carboxylic acids is included. (CW)

  5. Omega-3 Fatty Acids

    MedlinePlus

    Omega-3 fatty acids are used together with lifestyle changes (diet, weight-loss, exercise) to reduce the amount of triglycerides (a fat-like ... people with very high triglycerides. Omega-3 fatty acids are in a class of medications called antilipemic ...

  6. Toxicology of Perfluoroalkyl Acids*

    EPA Science Inventory

    The perfluoroalkyl acids (PFAAs) are a family of organic chemicals consisting of a perfluorinated carbon backbone (4-12 in length) and an acidic functional moiety (carboxylate or sulfonate). These compounds are chemically stable, have excellent surface-tension reducing properties...

  7. Salicylic Acid Topical

    MedlinePlus

    ... skin blemishes in people who have acne. Topical salicylic acid is also used to treat skin conditions that involve scaling or overgrowth of skin ... water for 15 minutes.Do not apply topical salicylic acid to skin that is broken, red, swollen, irritated, or infected. ...

  8. Uric acid and hypertension.

    PubMed

    Feig, Daniel I

    2011-09-01

    A link between serum uric acid and the development of hypertension was first hypothesized in the 1870s. Although numerous epidemiologic studies in the 1980s and 1990s suggested an association, relatively little attention was paid to it until recently. Animal models have suggested a two-step pathogenesis by which uric acid initially activates the renin angiotensin system and suppresses nitric oxide, leading to uric acid-dependent increase in systemic vascular resistance, followed by a uric acid-mediated vasculopathy, involving renal afferent arterioles, resulting in a late sodium-sensitive hypertension. Initial clinical trials in young patients have supported these mechanisms in young patients but do not yet support pharmacologic reduction of serum uric acid as first-line therapy for hypertension.

  9. Biosynthesis of pulcherriminic acid

    PubMed Central

    MacDonald, J. C.

    1965-01-01

    1. Candida pulcherrima was grown on a complex medium to which various compounds had been added to determine their effect on the biosynthesis of pulcherriminic acid. Most of the pulcherriminic acid synthesized by C. pulcherrima PRL2019 was derived from the l-[1-14C]leucine added to the medium. 2. The cyclic dipeptide of l-leucine (cyclo-l-leucyl-l-leucyl) was shown, by trapping experiments involving cycloleucyl-leucyl isomers, to be synthesized by strain PRL2019. Cyclo-l-leucyl-l-leucyl was derived from l-leucine and was converted into pulcherriminic acid. Cyclo-l-leucyl-l-leucyl was a precursor of pulcherriminic acid in strain PRL2007 also. 3. The results supported the hypothesis that pulcherriminic acid is derived from l-leucine and that cyclo-l-leucyl-l-leucyl is an intermediate in the biosynthesis. PMID:5837792

  10. Total syntheses of cis-cyclopropane fatty acids: dihydromalvalic acid, dihydrosterculic acid, lactobacillic acid, and 9,10-methylenehexadecanoic acid.

    PubMed

    Shah, Sayali; White, Jonathan M; Williams, Spencer J

    2014-12-14

    cis-Cyclopropane fatty acids (cis-CFAs) are widespread constituents of the seed oils of subtropical plants, membrane components of bacteria and protozoa, and the fats and phospholipids of animals. We describe a systematic approach to the synthesis of enantiomeric pairs of four cis-CFAs: cis-9,10-methylenehexadecanoic acid, lactobacillic acid, dihydromalvalic acid, and dihydrosterculic acid. The approach commences with Rh2(OAc)4-catalyzed cyclopropenation of 1-octyne and 1-decyne, and hinges on the preparative scale chromatographic resolution of racemic 2-alkylcycloprop-2-ene-1-carboxylic acids using a homochiral Evan's auxiliary. Saturation of the individual diastereomeric N-cycloprop-2-ene-1-carbonylacyloxazolidines, followed by elaboration to alkylcyclopropylmethylsulfones, allowed Julia-Kocienski olefination with various ω-aldehyde-esters. Finally, saponification and diimide reduction afforded the individual cis-CFA enantiomers. PMID:25321346

  11. Gluconic acid production.

    PubMed

    Anastassiadis, Savas; Morgunov, Igor G

    2007-01-01

    Gluconic acid, the oxidation product of glucose, is a mild neither caustic nor corrosive, non toxic and readily biodegradable organic acid of great interest for many applications. As a multifunctional carbonic acid belonging to the bulk chemicals and due to its physiological and chemical characteristics, gluconic acid itself, its salts (e.g. alkali metal salts, in especially sodium gluconate) and the gluconolactone form have found extensively versatile uses in the chemical, pharmaceutical, food, construction and other industries. Present review article presents the comprehensive information of patent bibliography for the production of gluconic acid and compares the advantages and disadvantages of known processes. Numerous manufacturing processes are described in the international bibliography and patent literature of the last 100 years for the production of gluconic acid from glucose, including chemical and electrochemical catalysis, enzymatic biocatalysis by free or immobilized enzymes in specialized enzyme bioreactors as well as discontinuous and continuous fermentation processes using free growing or immobilized cells of various microorganisms, including bacteria, yeast-like fungi and fungi. Alternatively, new superior fermentation processes have been developed and extensively described for the continuous and discontinuous production of gluconic acid by isolated strains of yeast-like mold Aureobasidium pullulans, offering numerous advantages over the traditional discontinuous fungi processes.

  12. Trans Fatty Acids

    NASA Astrophysics Data System (ADS)

    Doyle, Ellin

    1997-09-01

    Fats and their various fatty acid components seem to be a perennial concern of nutritionists and persons concerned with healthful diets. Advice on the consumption of saturated, polyunsaturated, monounsaturated, and total fat bombards us from magazines and newspapers. One of the newer players in this field is the group of trans fatty acids found predominantly in partially hydrogenated fats such as margarines and cooking fats. The controversy concerning dietary trans fatty acids was recently addressed in an American Heart Association (AHA) science advisory (1) and in a position paper from the American Society of Clinical Nutrition/American Institute of Nutrition (ASCN/AIN) (2). Both reports emphasize that the best preventive strategy for reducing risk for cardiovascular disease and some types of cancer is a reduction in total and saturated fats in the diet, but a reduction in the intake of trans fatty acids was also recommended. Although the actual health effects of trans fatty acids remain uncertain, experimental evidence indicates that consumption of trans fatty acids adversely affects serum lipid levels. Since elevated levels of serum cholesterol and triacylglycerols are associated with increased risk of cardiovascular disease, it follows that intake of trans fatty acids should be minimized.

  13. Sulfuric Acid on Europa

    NASA Technical Reports Server (NTRS)

    1999-01-01

    Frozen sulfuric acid on Jupiter's moon Europa is depicted in this image produced from data gathered by NASA's Galileo spacecraft. The brightest areas, where the yellow is most intense, represent regions of high frozen sulfuric acid concentration. Sulfuric acid is found in battery acid and in Earth's acid rain.

    This image is based on data gathered by Galileo's near infrared mapping spectrometer.

    Europa's leading hemisphere is toward the bottom right, and there are enhanced concentrations of sulfuric acid in the trailing side of Europa (the upper left side of the image). This is the face of Europa that is struck by sulfur ions coming from Jupiter's innermost moon, Io. The long, narrow features that crisscross Europa also show sulfuric acid that may be from sulfurous material extruded in cracks.

    Galileo, launched in 1989, has been orbiting Jupiter and its moons since December 1995. JPL manages the Galileo mission for NASA's Office of Space Science, Washington DC. JPL is a division of the California Institute of Technology, Pasadena, CA.

  14. Strongly Acidic Auxin Indole-3-Methanesulfonic Acid

    PubMed Central

    Cohen, Jerry D.; Baldi, Bruce G.; Bialek, Krystyna

    1985-01-01

    A radiochemical synthesis is described for [14C]indole-3-methanesulfonic acid (IMS), a strongly acidic auxin analog. Techniques were developed for fractionation and purification of IMS using normal and reverse phase chromatography. In addition, the utility of both Fourier transform infrared spectrometry and fast atom bombardment mass spectrometry for analysis of IMS has been demonstrated. IMS was shown to be an active auxin, stimulating soybean hypocotyl elongation, bean first internode curvature, and ethylene production. IMS uptake by thin sections of soybean hypocotyl was essentially independent of solution pH and, when applied at a 100 micromolar concentration, IMS exhibited a basipetal polarity in its transport in both corn coleoptile and soybean hypocotyl sections. [14C]IMS should, therefore, be a useful compound to study fundamental processes related to the movement of auxins in plant tissues and organelles. PMID:16664007

  15. Understanding acid rain

    SciTech Connect

    Budiansky, S.

    1981-06-01

    The complexities of the phenomenon of acid rain are described. Many factors, including meteorology, geology, chemistry, and biology, all play parts. Varying weather, varying soils, the presence of other pollutants and species differences all act to blur the connections between industrial emissions, acid rain, and environmental damage. Some experts believe that the greatest pH shock to lakes occurs during snow melt and runoff in the spring; others believe that much of the plant damage ascribed to acid rain is actually due to the effects of ozone. Much work needs to be done in the area of sampling. Historical data are lacking and sampling methods are not sufficiently accurate. (JMT)

  16. Understanding Acid Base Disorders.

    PubMed

    Gomez, Hernando; Kellum, John A

    2015-10-01

    The concentration of hydrogen ions is regulated in biologic solutions. There are currently 3 recognized approaches to assess changes in acid base status. First is the traditional Henderson-Hasselbalch approach, also called the physiologic approach, which uses the relationship between HCO3(-) and Pco2; the second is the standard base excess approach based on the Van Slyke equation. The third approach is the quantitative or Stewart approach, which uses the strong ion difference and the total weak acids. This article explores the origins of the current concepts framing the existing methods to analyze acid base balance.

  17. Acid rain and soil.

    PubMed

    vanLoon, G W

    1984-08-01

    A summary of important chemical properties of soil is given and the way in which acid rain may affect these properties is discussed. Acid rain may suppress microbiological decomposition and nitrification processes, thus influencing the nutrient status of soils. It has also been found that soil organic matter is less soluble in more acid solutions. Changed nutrient availability patterns are predicted in a low pH environment and enhanced leaching of essential elements from the soil exchange complex has been observed. Increased solubility of potentially toxic elements such as aluminium may also occur from soils which have been exposed to acidified rainfall.

  18. Disorders of Amino Acid Metabolism

    MedlinePlus

    ... Aspiration Syndrome Additional Content Medical News Disorders of Amino Acid Metabolism By Lee M. Sanders, MD, MPH NOTE: ... Metabolic Disorders Disorders of Carbohydrate Metabolism Disorders of Amino Acid Metabolism Disorders of Lipid Metabolism Amino acids are ...

  19. Pantothenic acid and biotin

    MedlinePlus

    ... well as other nutrients, are provided in the Dietary Reference Intakes (DRIs) developed by the Food and Nutrition Board ... level that is thought to ensure enough nutrition. Dietary Reference Intakes for pantothenic acid: Age 0 to 6 months: ...

  20. Amino Acid Metabolism Disorders

    MedlinePlus

    Metabolism is the process your body uses to make energy from the food you eat. Food is ... One group of these disorders is amino acid metabolism disorders. They include phenylketonuria (PKU) and maple syrup ...

  1. [Hydrofluoric acid burns].

    PubMed

    Holla, Robin; Gorter, Ramon R; Tenhagen, Mark; Vloemans, A F P M Jos; Breederveld, Roelf S

    2016-01-01

    Hydrofluoric acid is increasingly used as a rust remover and detergent. Dermal contact with hydrofluoric acid results in a chemical burn characterized by severe pain and deep tissue necrosis. It may cause electrolyte imbalances with lethal consequences. It is important to identify high-risk patients. 'High risk' is defined as a total affected body area > 3% or exposure to hydrofluoric acid in a concentration > 50%. We present the cases of three male patients (26, 31, and 39 years old) with hydrofluoric acid burns of varying severity and describe the subsequent treatments. The application of calcium gluconate 2.5% gel to the skin is the cornerstone of the treatment, reducing pain as well as improving wound healing. Nails should be thoroughly inspected and possibly removed if the nail is involved, to ensure proper healing. In high-risk patients, plasma calcium levels should be evaluated and cardiac monitoring is indicated.

  2. Folic acid - test

    MedlinePlus

    ... folic acid before and during pregnancy helps prevent neural tube defects, such as spina bifida. Women who ... take more if they have a history of neural tube defects in earlier pregnancies. Ask your provider ...

  3. Nitric acid poisoning

    MedlinePlus

    Symptoms from swallowing nitric acid may include: Abdominal pain - severe Burns to skin or mouth Drooling Fever Mouth pain - severe Rapid drop in blood pressure (shock) Throat swelling, which leads to breathing difficulty ...

  4. [Hydrofluoric acid burns].

    PubMed

    Holla, Robin; Gorter, Ramon R; Tenhagen, Mark; Vloemans, A F P M Jos; Breederveld, Roelf S

    2016-01-01

    Hydrofluoric acid is increasingly used as a rust remover and detergent. Dermal contact with hydrofluoric acid results in a chemical burn characterized by severe pain and deep tissue necrosis. It may cause electrolyte imbalances with lethal consequences. It is important to identify high-risk patients. 'High risk' is defined as a total affected body area > 3% or exposure to hydrofluoric acid in a concentration > 50%. We present the cases of three male patients (26, 31, and 39 years old) with hydrofluoric acid burns of varying severity and describe the subsequent treatments. The application of calcium gluconate 2.5% gel to the skin is the cornerstone of the treatment, reducing pain as well as improving wound healing. Nails should be thoroughly inspected and possibly removed if the nail is involved, to ensure proper healing. In high-risk patients, plasma calcium levels should be evaluated and cardiac monitoring is indicated. PMID:27189091

  5. Difficult Decisions: Acid Rain.

    ERIC Educational Resources Information Center

    Miller, John A.; Slesnick, Irwin L.

    1989-01-01

    Discusses some of the contributing factors and chemical reactions involved in the production of acid rain, its effects, and political issues pertaining to who should pay for the clean up. Supplies questions for consideration and discussion. (RT)

  6. Hyaluronic acid fillers.

    PubMed

    Monheit, Gary D; Coleman, Kyle M

    2006-01-01

    Although hyaluronic acids are a relatively new treatment for facial lines and wrinkles, they have provided numerous advances in the area of cosmetic surgery. This article discusses the inherent properties of hyaluronic acid fillers that make them ideal for treatment of facial lines. It encompasses a review of the current literature on U.S. Food and Drug Administration-approved hyaluronic acid fillers and the role that each of these fillers currently has in facial cosmetics. This article also discusses the potential pitfalls and adverse effects that can be associated with using hyaluronic acids for filling facial lines. Finally, it serves as an overview of current techniques for clinical assessment of patients as well as administration and treatment of facial lines and wrinkles.

  7. Boric acid poisoning

    MedlinePlus

    Borax poisoning ... The main symptoms of boric acid poisoning are blue-green vomit, diarrhea, and a bright red rash on the skin. Other symptoms may include: Blisters Collapse Coma Convulsions Drowsiness ...

  8. Stomach acid test

    MedlinePlus

    Gastric acid secretion test ... The test is done after you have not eaten for a while so fluid is all that remains in ... injected into your body. This is done to test the ability of the cells in the stomach ...

  9. Aminolevulinic Acid Topical

    MedlinePlus

    ... under the skin that result from exposure to sunlight and can develop into skin cancer) of the ... acid will make your skin very sensitive to sunlight (likely to get sunburn). Avoid exposure of treated ...

  10. Amino Acids and Chirality

    NASA Technical Reports Server (NTRS)

    Cook, Jamie E.

    2012-01-01

    Amino acids are among the most heavily studied organic compound class in carbonaceous chondrites. The abundance, distributions, enantiomeric compositions, and stable isotopic ratios of amino acids have been determined in carbonaceous chondrites fi'om a range of classes and petrographic types, with interesting correlations observed between these properties and the class and typc of the chondritcs. In particular, isomeric distributions appear to correlate with parent bodies (chondrite class). In addition, certain chiral amino acids are found in enantiomeric excess in some chondrites. The delivery of these enantiomeric excesses to the early Earth may have contributed to the origin of the homochirality that is central to life on Earth today. This talk will explore the amino acids in carbonaceous chondritcs and their relevance to the origin of life.

  11. (Acid rain workshop)

    SciTech Connect

    Turner, R.S.

    1990-12-05

    The traveler presented a paper entitled Susceptibility of Asian Ecosystems to Soil-Mediated Acid Rain Damage'' at the Second Workshop on Acid Rain in Asia. The workshop was organized by the Asian Institute of Technology (Bangkok, Thailand), Argonne National Laboratory (Argonne, Illinois), and Resource Management Associates (Madison, Wisconsin) and was sponsored by the US Department of Energy, the United Nations Environment Program, the United Nations Economic and Social Commission for Asia and the Pacific, and the World Bank. Papers presented on the first day discussed how the experience gained with acid rain in North America and Europe might be applied to the Asian situation. Papers describing energy use projections, sulfur emissions, and effects of acid rain in several Asian countries were presented on the second day. The remaining time was allotted to discussion, planning, and writing plans for a future research program.

  12. Folic acid in diet

    MedlinePlus

    ... a regular supply of the vitamin in the foods you eat. ... vitamins have been added to the food. Many foods are now fortified with folic acid. Some of these are enriched breads, cereals, flours, ...

  13. Valproic Acid and Pregnancy

    MedlinePlus

    ... in the treatment of epilepsy, and to treat bipolar disorder and migraines. I have been taking valproic acid ... that women with seizure disorders and women with bipolar disorder might have menstrual problems and difficulty getting pregnant. ...

  14. Citric acid urine test

    MedlinePlus

    ... The test is used to diagnose renal tubular acidosis and evaluate kidney stone disease. Normal Results The ... level of citric acid may mean renal tubular acidosis and a tendency to form calcium kidney stones. ...

  15. Folic Acid Quiz

    MedlinePlus

    ... more easily than natural food folate. Close × Answer: D CORRECT: Folic acid reduces the risk for spina ... g., orange juice and green vegetables). Close × Answer: D CORRECT: Spina bifida and anencephaly are neural tube ...

  16. Hydrofluoric acid poisoning

    MedlinePlus

    ... your skin or eyes, you may have: Blisters Burns Pain Vision loss Hydrofluoric acid poisoning can have ... urine tests Camera down the throat to see burns in the esophagus and the stomach (endoscopy) Fluids ...

  17. Portable nucleic acid thermocyclers.

    PubMed

    Almassian, David R; Cockrell, Lisa M; Nelson, William M

    2013-11-21

    A nucleic acid thermal cycler is considered to be portable if it is under ten pounds, easily carried by one individual, and battery powered. Nucleic acid amplification includes both polymerase chain reaction (e.g. PCR, RT-PCR) and isothermal amplification (e.g. RPA, HDA, LAMP, NASBA, RCA, ICAN, SMART, SDA). There are valuable applications for portable nucleic acid thermocyclers in fields that include clinical diagnostics, biothreat detection, and veterinary testing. A system that is portable allows for the distributed detection of targets at the point of care and a reduction of the time from sample to answer. The designer of a portable nucleic acid thermocycler must carefully consider both thermal control and the detection of amplification. In addition to thermal control and detection, the designer may consider the integration of a sample preparation subsystem with the nucleic acid thermocycler. There are a variety of technologies that can achieve accurate thermal control and the detection of nucleic acid amplification. Important evaluation criteria for each technology include maturity, power requirements, cost, sensitivity, speed, and manufacturability. Ultimately the needs of a particular market will lead to user requirements that drive the decision between available technologies.

  18. Neutron Nucleic Acid Crystallography.

    PubMed

    Chatake, Toshiyuki

    2016-01-01

    The hydration shells surrounding nucleic acids and hydrogen-bonding networks involving water molecules and nucleic acids are essential interactions for the structural stability and function of nucleic acids. Water molecules in the hydration shells influence various conformations of DNA and RNA by specific hydrogen-bonding networks, which often contribute to the chemical reactivity and molecular recognition of nucleic acids. However, X-ray crystallography could not provide a complete description of structural information with respect to hydrogen bonds. Indeed, X-ray crystallography is a powerful tool for determining the locations of water molecules, i.e., the location of the oxygen atom of H2O; however, it is very difficult to determine the orientation of the water molecules, i.e., the orientation of the two hydrogen atoms of H2O, because X-ray scattering from the hydrogen atom is very small.Neutron crystallography is a specialized tool for determining the positions of hydrogen atoms. Neutrons are not diffracted by electrons, but are diffracted by atomic nuclei; accordingly, neutron scattering lengths of hydrogen and its isotopes are comparable to those of non-hydrogen atoms. Therefore, neutron crystallography can determine both of the locations and orientations of water molecules. This chapter describes the current status of neutron nucleic acid crystallographic research as well as the basic principles of neutron diffraction experiments performed on nucleic acid crystals: materials, crystallization, diffraction experiments, and structure determination. PMID:26227050

  19. Neutron Nucleic Acid Crystallography.

    PubMed

    Chatake, Toshiyuki

    2016-01-01

    The hydration shells surrounding nucleic acids and hydrogen-bonding networks involving water molecules and nucleic acids are essential interactions for the structural stability and function of nucleic acids. Water molecules in the hydration shells influence various conformations of DNA and RNA by specific hydrogen-bonding networks, which often contribute to the chemical reactivity and molecular recognition of nucleic acids. However, X-ray crystallography could not provide a complete description of structural information with respect to hydrogen bonds. Indeed, X-ray crystallography is a powerful tool for determining the locations of water molecules, i.e., the location of the oxygen atom of H2O; however, it is very difficult to determine the orientation of the water molecules, i.e., the orientation of the two hydrogen atoms of H2O, because X-ray scattering from the hydrogen atom is very small.Neutron crystallography is a specialized tool for determining the positions of hydrogen atoms. Neutrons are not diffracted by electrons, but are diffracted by atomic nuclei; accordingly, neutron scattering lengths of hydrogen and its isotopes are comparable to those of non-hydrogen atoms. Therefore, neutron crystallography can determine both of the locations and orientations of water molecules. This chapter describes the current status of neutron nucleic acid crystallographic research as well as the basic principles of neutron diffraction experiments performed on nucleic acid crystals: materials, crystallization, diffraction experiments, and structure determination.

  20. Utilization of acid tars

    SciTech Connect

    Frolov, A.F.; Denisova, T.L.; Aminov, A.N.

    1987-01-01

    Freshly produced acid tar (FPAT), obtained as refinery waste in treating petroleum oils with sulfuric acid and oleum, contains 80% or more sulfuric acid. Of such tars, pond acid tars, which contain up to 80% neutral petroleum products and sulfonated resins, are more stable, and have found applications in the production of binders for paving materials. In this article the authors are presenting results obtained in a study of the composition and reactivity of FPAT and its stability in storage in blends with asphalts obtained in deasphalting operations, and the possibility of using the FPAT in road construction has been examined. In this work, wastes were used which were obtained in treating the oils T-750, KhF-12, I-8A, and MS-14. Data on the change in group chemical composition of FPAT are shown, and the acidity, viscosity, needle penetration, and softening point of acid tars obtained from different grades of oils are plotted as functions of the storage time. It is also shown that the fresh and hardened FPATs differ in their solubilities in various solvents.

  1. Method for isolating nucleic acids

    SciTech Connect

    Hurt, Jr., Richard Ashley; Elias, Dwayne A.

    2015-09-29

    The current disclosure provides methods and kits for isolating nucleic acid from an environmental sample. The current methods and compositions further provide methods for isolating nucleic acids by reducing adsorption of nucleic acids by charged ions and particles within an environmental sample. The methods of the current disclosure provide methods for isolating nucleic acids by releasing adsorbed nucleic acids from charged particles during the nucleic acid isolation process. The current disclosure facilitates the isolation of nucleic acids of sufficient quality and quantity to enable one of ordinary skill in the art to utilize or analyze the isolated nucleic acids for a wide variety of applications including, sequencing or species population analysis.

  2. Acidification and Acid Rain

    NASA Astrophysics Data System (ADS)

    Norton, S. A.; Veselã½, J.

    2003-12-01

    Air pollution by acids has been known as a problem for centuries (Ducros, 1845; Smith, 1872; Camuffo, 1992; Brimblecombe, 1992). Only in the mid-1900s did it become clear that it was a problem for more than just industrially developed areas, and that precipitation quality can affect aquatic resources ( Gorham, 1955). The last three decades of the twentieth century saw tremendous progress in the documentation of the chemistry of the atmosphere, precipitation, and the systems impacted by acid atmospheric deposition. Chronic acidification of ecosystems results in chemical changes to soil and to surface waters and groundwater as a result of reduction of base cation supply or an increase in acid (H+) supply, or both. The most fundamental changes during chronic acidification are an increase in exchangeable H+ or Al3+ (aluminum) in soils, an increase in H+ activity (˜concentration) in water in contact with soil, and a decrease in alkalinity in waters draining watersheds. Water draining from the soil is acidified and has a lower pH (=-log [H+]). As systems acidify, their biotic community changes.Acidic surface waters occur in many parts of the world as a consequence of natural processes and also due to atmospheric deposition of strong acid (e.g., Canada, Jeffries et al. (1986); the United Kingdom, Evans and Monteith (2001); Sweden, Swedish Environmental Protection Board (1986); Finland, Forsius et al. (1990); Norway, Henriksen et al. (1988a); and the United States (USA), Brakke et al. (1988)). Concern over acidification in the temperate regions of the northern hemisphere has been driven by the potential for accelerating natural acidification by pollution of the atmosphere with acidic or acidifying compounds. Atmospheric pollution ( Figure 1) has resulted in an increased flux of acid to and through ecosystems. Depending on the ability of an ecosystem to neutralize the increased flux of acidity, acidification may increase only imperceptibly or be accelerated at a rate that

  3. Discovery of essential fatty acids

    PubMed Central

    Spector, Arthur A.; Kim, Hee-Yong

    2015-01-01

    Dietary fat was recognized as a good source of energy and fat-soluble vitamins by the first part of the 20th century, but fatty acids were not considered to be essential nutrients because they could be synthesized from dietary carbohydrate. This well-established view was challenged in 1929 by George and Mildred Burr who reported that dietary fatty acid was required to prevent a deficiency disease that occurred in rats fed a fat-free diet. They concluded that fatty acids were essential nutrients and showed that linoleic acid prevented the disease and is an essential fatty acid. The Burrs surmised that other unsaturated fatty acids were essential and subsequently demonstrated that linolenic acid, the omega-3 fatty acid analog of linoleic acid, is also an essential fatty acid. The discovery of essential fatty acids was a paradigm-changing finding, and it is now considered to be one of the landmark discoveries in lipid research. PMID:25339684

  4. Boric acid catalyzed chemoselective esterification of alpha-hydroxycarboxylic acids.

    PubMed

    Houston, Todd A; Wilkinson, Brendan L; Blanchfield, Joanne T

    2004-03-01

    Boric acid catalyzes the selective esterification of alpha-hydroxycarboxylic acids without causing significant esterification to occur with other carboxylic acids. The procedure is simple, high-yielding, and applicable to the esterification of alpha-hydroxy carboxylates in the presence of other carboxylic acids including beta-hydroxyacids within the same molecule. [reaction: see text

  5. Acid Rain, pH & Acidity: A Common Misinterpretation.

    ERIC Educational Resources Information Center

    Clark, David B.; Thompson, Ronald E.

    1989-01-01

    Illustrates the basis for misleading statements about the relationship between pH and acid content in acid rain. Explains why pH cannot be used as a measure of acidity for rain or any other solution. Suggests that teachers present acidity and pH as two separate and distinct concepts. (RT)

  6. Amino-acid contamination of aqueous hydrochloric acid.

    NASA Technical Reports Server (NTRS)

    Wolman, Y.; Miller, S. L.

    1971-01-01

    Considerable amino-acid contamination in commercially available analytical grade hydrochloric acid (37% HCl) was found. One bottle contained 8,300 nmol of amino-acids per liter. A bottle from another supplier contained 6,700 nmol per liter. The contaminants were mostly protein amino-acids and several unknowns. Data on the volatility of the amino-acids during HCl distillation were also obtained.

  7. Analysis of Bile Acids

    NASA Astrophysics Data System (ADS)

    Sjövall, Jan; Griffiths, William J.; Setchell, Kenneth D. R.; Mano, Nariyasu; Goto, Junichi

    Bile acids constitute a large family of steroids in vertebrates, normally formed from cholesterol and carrying a carboxyl group in a side-chain of variable length. Bile alcohols, also formed from cholesterol, have similar structures as bile acids, except for the absence of a carboxyl group in the steroid skeleton. The conversion of cholesterol to bile acids and/or bile alcohols is of major importance for maintenance of cholesterol homeostasis, both from quantitative and regulatory points of view (Chiang, 2004; Kalaany and Mangelsdorf, 2006; Moore, Kato, Xie, et al., 2006; Scotti, Gilardi, Godio, et al., 2007). Appropriately conjugated bile acids and bile alcohols (also referred to as bile salts) are secreted in bile and serve vital functions in the absorption of lipids and lipid-soluble compounds (Hofmann, 2007). Reliable analytical methods are required for studies of the functions and pathophysiological importance of the variety of bile acids and bile alcohols present in living organisms. When combined with genetic and proteomic studies, analysis of these small molecules (in today's terminology: metabolomics, steroidomics, sterolomics, cholanoidomics, etc.) will lead to a deeper understanding of the integrated metabolic processes in lipid metabolism.

  8. Optical high acidity sensor

    DOEpatents

    Jorgensen, B.S.; Nekimken, H.L.; Carey, W.P.; O`Rourke, P.E.

    1997-07-22

    An apparatus and method for determining acid concentrations in solutions having acid concentrations of from about 0.1 Molar to about 16 Molar is disclosed. The apparatus includes a chamber for interrogation of the sample solution, a fiber optic light source for passing light transversely through the chamber, a fiber optic collector for receiving the collimated light after transmission through the chamber, a coating of an acid resistant polymeric composition upon at least one fiber end or lens, the polymeric composition in contact with the sample solution within the chamber and having a detectable response to acid concentrations within the range of from about 0.1 Molar to about 16 Molar, a measurer for the response of the polymeric composition in contact with the sample solution, and a comparer of the measured response to predetermined standards whereby the acid molarity of the sample solution within the chamber can be determined. Preferably, a first lens is attached to the end of the fiber optic light source, the first lens adapted to collimate light from the fiber optic light source, and a second lens is attached to the end of the fiber optic collector for focusing the collimated light after transmission through the chamber. 10 figs.

  9. Optical high acidity sensor

    DOEpatents

    Jorgensen, Betty S.; Nekimken, Howard L.; Carey, W. Patrick; O'Rourke, Patrick E.

    1997-01-01

    An apparatus and method for determining acid concentrations in solutions having acid concentrations of from about 0.1 Molar to about 16 Molar is disclosed. The apparatus includes a chamber for interrogation of the sample solution, a fiber optic light source for passing light transversely through the chamber, a fiber optic collector for receiving the collimated light after transmission through the chamber, a coating of an acid resistant polymeric composition upon at least one fiber end or lens, the polymeric composition in contact with the sample solution within the chamber and having a detectable response to acid concentrations within the range of from about 0.1 Molar to about 16 Molar, a measurer for the response of the polymeric composition in contact with the sample solution, and, a comparer of the measured response to predetermined standards whereby the acid molarity of the sample solution within the chamber can be determined. Preferably, a first lens is attached to the end of the fiber optic light source, the first lens adapted to collimate light from the fiber optic light source, and a second lens is attached to the end of the fiber optic collector for focusing the collimated light after transmission through the chamber.

  10. Acid sludge utilization

    SciTech Connect

    Suarez, M.

    1980-09-01

    The Peak Oil Company of Tampa, Florida, in cooperation with the United States Department of Energy, has completed an initial study for the incorporation of acid-sludge derived from the rerefining of used lubricating oil into a useful and salable building material. Both bricks and paving materials have been produced using a formulation developed by Peak. Equipment has been designed and constructed for the specific purpose of preparing emulsions containing the acid-sludge, which is a vital ingredient in the final formulation. Testing of products obtained from these initial efforts shows that the acid in the sludge has been effectively neutralized and that heavy metals are not leached from the bricks or paving material in normal testing. While some properties of the building materials that incorporate the acid-sludge by-product are below standards for clay and shale brick, uses are defined for the product as is, and there is some promise of eventual production of building materials that meet all specifications for competitive materials. Initial cost estimations are encouraging, indicating that a profit can be derived by converting a hazardous and noxious by-product of rerefining to a construction material. Acid-sludge has presented a complex and costly disposal problem to the industry resulting in a serious depletion in the capacity for rerefining used lubricating oil.

  11. Domoic acid epileptic disease.

    PubMed

    Ramsdell, John S; Gulland, Frances M

    2014-03-01

    Domoic acid epileptic disease is characterized by spontaneous recurrent seizures weeks to months after domoic acid exposure. The potential for this disease was first recognized in a human case study of temporal lobe epilepsy after the 1987 amnesic shellfish-poisoning event in Quebec, and was characterized as a chronic epileptic syndrome in California sea lions through investigation of a series of domoic acid poisoning cases between 1998 and 2006. The sea lion study provided a breadth of insight into clinical presentations, unusual behaviors, brain pathology, and epidemiology. A rat model that replicates key observations of the chronic epileptic syndrome in sea lions has been applied to identify the progression of the epileptic disease state, its relationship to behavioral manifestations, and to define the neural systems involved in these behavioral disorders. Here, we present the concept of domoic acid epileptic disease as a delayed manifestation of domoic acid poisoning and review the state of knowledge for this disease state in affected humans and sea lions. We discuss causative mechanisms and neural underpinnings of disease maturation revealed by the rat model to present the concept for olfactory origin of an epileptic disease; triggered in dendodendritic synapases of the olfactory bulb and maturing in the olfactory cortex. We conclude with updated information on populations at risk, medical diagnosis, treatment, and prognosis. PMID:24663110

  12. Domoic Acid Epileptic Disease

    PubMed Central

    Ramsdell, John S.; Gulland, Frances M.

    2014-01-01

    Domoic acid epileptic disease is characterized by spontaneous recurrent seizures weeks to months after domoic acid exposure. The potential for this disease was first recognized in a human case study of temporal lobe epilepsy after the 1987 amnesic shellfish-poisoning event in Quebec, and was characterized as a chronic epileptic syndrome in California sea lions through investigation of a series of domoic acid poisoning cases between 1998 and 2006. The sea lion study provided a breadth of insight into clinical presentations, unusual behaviors, brain pathology, and epidemiology. A rat model that replicates key observations of the chronic epileptic syndrome in sea lions has been applied to identify the progression of the epileptic disease state, its relationship to behavioral manifestations, and to define the neural systems involved in these behavioral disorders. Here, we present the concept of domoic acid epileptic disease as a delayed manifestation of domoic acid poisoning and review the state of knowledge for this disease state in affected humans and sea lions. We discuss causative mechanisms and neural underpinnings of disease maturation revealed by the rat model to present the concept for olfactory origin of an epileptic disease; triggered in dendodendritic synapases of the olfactory bulb and maturing in the olfactory cortex. We conclude with updated information on populations at risk, medical diagnosis, treatment, and prognosis. PMID:24663110

  13. A Demonstration of Acid Rain

    ERIC Educational Resources Information Center

    Fong, Man Wai

    2004-01-01

    A demonstration showing acid rain formation is described. Oxides of sulfur and nitrogen that result from the burning of fossil fuels are the major pollutants of acid rain. In this demonstration, SO[subscript 2] gas is produced by the burning of matches. An acid-base indicator will show that the dissolved gas turns an aqueous solution acidic.

  14. DOCOSAHEXAENOIC ACID AND ARACHIDONIC ACID PREVENT ESSENTIAL FATTY ACID DEFICIENCY AND HEPATIC STEATOSIS

    PubMed Central

    Le, Hau D.; Meisel, Jonathan A.; de Meijer, Vincent E.; Fallon, Erica M.; Gura, Kathleen M.; Nose, Vania; Bistrian, Bruce R.; Puder, Mark

    2012-01-01

    Objectives Essential fatty acids are important for growth, development, and physiologic function. Alpha-linolenic acid and linoleic acid are the precursors of docosahexaenoic and arachidonic acid, respectively, and have traditionally been considered the essential fatty acids. However, we hypothesized that docosahexaenoic acid and arachidonic acid can function as the essential fatty acids. Methods Using a murine model of essential fatty acid deficiency and consequent hepatic steatosis, we provided mice with varying amounts of docosahexaenoic and arachidonic acids to determine whether exclusive supplementation of docosahexaenoic and arachidonic acids could prevent essential fatty acid deficiency and inhibit or attenuate hepatic steatosis. Results Mice supplemented with docosahexaenoic and arachidonic acids at 2.1% or 4.2% of their calories for 19 days had normal liver histology and no biochemical evidence of essential fatty acid deficiency, which persisted when observed after 9 weeks. Conclusion Supplementation of sufficient amounts of docosahexaenoic and arachidonic acids alone without alpha-linolenic and linoleic acids meets essential fatty acid requirements and prevents hepatic steatosis in a murine model. PMID:22038210

  15. Biodegradation of cyanuric acid.

    PubMed

    Saldick, J

    1974-12-01

    Cyanuric acid biodegrades readily under a wide variety of natural conditions, and particularly well in systems of either low or zero dissolved-oxygen level, such as anaerobic activated sludge and sewage, soils, muds, and muddy streams and river waters, as well as ordinary aerated activated sludge systems with typically low (1 to 3 ppm) dissolved-oxygen levels. Degradation also proceeds in 3.5% sodium chloride solution. Consequently, there are degradation pathways widely available for breaking down cyanuric acid discharged in domestic effluents. The overall degradation reaction is merely a hydrolysis; CO(2) and ammonia are the initial hydrolytic breakdown products. Since no net oxidation occurs during this breakdown, biodegradation of cyanuric acid exerts no primary biological oxygen demand. However, eventual nitrification of the ammonia released will exert its usual biological oxygen demand.

  16. Exposures to acidic aerosols.

    PubMed

    Spengler, J D; Keeler, G J; Koutrakis, P; Ryan, P B; Raizenne, M; Franklin, C A

    1989-02-01

    Ambient monitoring of acid aerosols in four U.S. cities and in a rural region of southern Ontario clearly show distinct periods of strong acidity. Measurements made in Kingston, TN, and Steubenville, OH, resulted in 24-hr H+ ion concentrations exceeding 100 nmole/m3 more than 10 times during summer months. Periods of elevated acidic aerosols occur less frequently in winter months. The H+ determined during episodic conditions in southern Ontario indicates that respiratory tract deposition can exceed the effects level reported in clinical studies. Observed 12-hr H+ concentrations exceeded 550 nmole/m3 (approximately 27 micrograms/m3 H2SO4). The maximum estimated 1-hr concentration exceeded 1500 nmole/m3 for H+ ions. At these concentrations, an active child might receive more than 2000 nmole of H+ ion in 12 hr and in excess of 900 nmole during the hour when H2SO4 exceeded 50 micrograms/m3.

  17. Biodegradation of Cyanuric Acid

    PubMed Central

    Saldick, Jerome

    1974-01-01

    Cyanuric acid biodegrades readily under a wide variety of natural conditions, and particularly well in systems of either low or zero dissolved-oxygen level, such as anaerobic activated sludge and sewage, soils, muds, and muddy streams and river waters, as well as ordinary aerated activated sludge systems with typically low (1 to 3 ppm) dissolved-oxygen levels. Degradation also proceeds in 3.5% sodium chloride solution. Consequently, there are degradation pathways widely available for breaking down cyanuric acid discharged in domestic effluents. The overall degradation reaction is merely a hydrolysis; CO2 and ammonia are the initial hydrolytic breakdown products. Since no net oxidation occurs during this breakdown, biodegradation of cyanuric acid exerts no primary biological oxygen demand. However, eventual nitrification of the ammonia released will exert its usual biological oxygen demand. PMID:4451360

  18. Calorimetry of Nucleic Acids.

    PubMed

    Rozners, Eriks; Pilch, Daniel S; Egli, Martin

    2015-12-01

    This unit describes the application of calorimetry to characterize the thermodynamics of nucleic acids, specifically, the two major calorimetric methodologies that are currently employed: differential scanning (DSC) and isothermal titration calorimetry (ITC). DSC is used to study thermally induced order-disorder transitions in nucleic acids. A DSC instrument measures, as a function of temperature (T), the excess heat capacity (C(p)(ex)) of a nucleic acid solution relative to the same amount of buffer solution. From a single curve of C(p)(ex) versus T, one can derive the following information: the transition enthalpy (ΔH), entropy (ΔS), free energy (ΔG), and heat capacity (ΔCp); the state of the transition (two-state versus multistate); and the average size of the molecule that melts as a single thermodynamic entity (e.g., the duplex). ITC is used to study the hybridization of nucleic acid molecules at constant temperature. In an ITC experiment, small aliquots of a titrant nucleic acid solution (strand 1) are added to an analyte nucleic acid solution (strand 2), and the released heat is monitored. ITC yields the stoichiometry of the association reaction (n), the enthalpy of association (ΔH), the equilibrium association constant (K), and thus the free energy of association (ΔG). Once ΔH and ΔG are known, ΔS can also be derived. Repetition of the ITC experiment at a number of different temperatures yields the ΔCp for the association reaction from the temperature dependence of ΔH.

  19. Acid rain in Asia

    NASA Astrophysics Data System (ADS)

    Bhatti, Neeloo; Streets, David G.; Foell, Wesley K.

    1992-07-01

    Acid rain has been an issue of great concern in North America and Europe during the past several decades. However, due to the passage of a number of recent regulations, most notably the Clean Air Act in the United States in 1990, there is an emerging perception that the problem in these Western nations is nearing solution. The situation in the developing world, particularly in Asia, is much bleaker. Given the policies of many Asian nations to achieve levels of development comparable with the industrialized world—which necessitate a significant expansion of energy consumption (most derived from indigenous coal reserves)—the potential for the formation of, and damage from, acid deposition in these developing countries is very high. This article delineates and assesses the emissions patterns, meteorology, physical geology, and biological and cultural resources present in various Asian nations. Based on this analysis and the risk factors to acidification, it is concluded that a number of areas in Asia are currently vulnerable to acid rain. These regions include Japan, North and South Korea, southern China, and the mountainous portions of Southeast Asia and southwestern India. Furthermore, with accelerated development (and its attendant increase in energy use and production of emissions of acid deposition precursors) in many nations of Asia, it is likely that other regions will also be affected by acidification in the near future. Based on the results of this overview, it is clear that acid deposition has significant potential to impact the Asian region. However, empirical evidence is urgently needed to confirm this and to provide early warning of increases in the magnitude and spread of acid deposition and its effects throughout this part of the world.

  20. Acid Precipitation; (USA)

    SciTech Connect

    Rushing, J.W.; Hicks, S.C.

    1991-01-01

    This publication, Acid Precipitation (APC) announces on a monthly basis the current worldwide information on acid precipitation and closely related subjects, including wet and dry deposition, long-range transport, environmental effects, modeling, and socioeconomic factors. Information on the following subjects is included within the scope of this publication, but all subjects may not appear in each issue: Pollution sources and pollution control technology; atmospheric transport and chemistry; terrestrial transport and chemistry; aquatic transport and chemistry; biological effects; corrosive effects; and socioeconomics, policy, and legislation.