Paclobutrazol biodegradation in unsaturated soil in the Semi-Arid ...

African Journals Online (AJOL)

Paclobutrazol (PBZ) is a plant growth regulator, increasing flowe ring and yield that is widely used in mango cultivation in the semi-arid northeastern Brazil. PBZ remains active in the soil for several years. However, it can severely affect the growth and development of subsequent crops, mainly by reducing vegetative vigor.

Isolated etioplasts as test system for inhibitors of fatty acid biosynthesis

International Nuclear Information System (INIS)

Lichtenthaler, H.K.; Kobek, K.

1989-01-01

Isolated intact chloroplasts of mono- and dicotyledonous plants possess the capacity for de novo fatty acid biosynthesis, starting from 14 C-acetate. These can be taken as test system for herbicides affecting fatty acid biosynthesis as shown earlier in our laboratory. The incorporation rates of acetate into the total fatty acids depend on the photosynthetic cofactors ATP and NADPH and amount in the light to 33 kBq (oat) and 39 kBq (pea) per mg chlorophyll x h, whereas in the dark only ca. 10% of these rates are obtained. In order to establish a test system, which is fully independent of light, we isolated and characterized etioplast fractions from oat and pea seedlings with a very high capacity of de novo fatty acid biosynthesis (500 and 400 kBq per mg carotenoids in a 20 min period). This activity was blocked by herbicides such as cycloxydim, sethoxydim and diclofop in a dose-dependent manner. This new test system has the great advantage that one can verify whether inhibitors of photosynthesis affect fatty acid biosynthesis

Crescimento de girassol como flor em vaso em função de doses de paclobutrazol

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Christina da Silva Wanderley

2014-02-01

Full Text Available O girassol (Helianthus annuus L., que é comumente cultivado para produção de sementes e óleo, também apresenta potencial de uso como planta ornamental. Entretanto, seu porte elevado dificulta essa utilização, principalmente como flor em vaso. Objetivou-se neste experimento avaliar os efeitos do regulador de crescimento paclobutrazol sobre o crescimento de plantas de girassol em vaso, cultivadas em sistema hidropônico, dentro de estufa, em vasos preenchidos com sílica moída. Os dois genótipos de girassol utilizados foram BRS Oásis, com capítulo marrom e altura média de 1,85 m, e Helio 358, com capítulo amarelo e altura média de 1,45 m. As doses de paclobutrazol avaliadas foram 0; 0,5; 1; 2; 4; e 6 mg L-1 , aplicadas diretamente no substrato de sílica moída. A solução nutritiva utilizada foi a de Hoagland, e as plantas foram mantidas sob aeração constante. O delineamento experimental foi o de blocos casualizados, em esquema fatorial com cinco repetições, totalizando 60 parcelas. Avaliaram-se a altura das plantas e a massa de matéria seca de raiz, caule, folhas e de capítulos. Houve efeito do paclobutrazol sobre o crescimento das plantas. O uso do paclobutrazol na dose de 2mgL-1 reduz a altura do girassol, porém não afeta a qualidade da inflorescência, o que pode viabilizar o seu uso na produção de flores de girassol como ornamentais. A sensibilidade ao paclobutrazol é maior para o genótipo Helio 358 em relação ao genótipo BRS Oásis.

Comparison of Effect of Brassinosteroid and Gibberellin Biosynthesis Inhibitors on Growth of Rice Seedlings

OpenAIRE

Matusmoto, Tadashi; Yamada, Kazuhiro; Yoshizawa, Yuko; Oh, Keimei

2016-01-01

Brassinosteroid (BR) and gibberellin (GA) are two predominant plant hormones that regulate plant cell elongation. Mutants disrupt the biosynthesis of these hormones and display different degrees of dwarf phenotypes in rice. Although the role of each plant hormone in promoting the longitudinal growth of plants has been extensively studied using genetic methods, their relationship is still poorly understood. In this study, we used two specific inhibitors targeting BR and GA biosynthesis to inve...

Propiconazole is a specific and accessible brassinosteroid (BR) biosynthesis inhibitor for Arabidopsis and maize.

Science.gov (United States)

Hartwig, Thomas; Corvalan, Claudia; Best, Norman B; Budka, Joshua S; Zhu, Jia-Ying; Choe, Sunghwa; Schulz, Burkhard

2012-01-01

Brassinosteroids (BRs) are steroidal hormones that play pivotal roles during plant development. In addition to the characterization of BR deficient mutants, specific BR biosynthesis inhibitors played an essential role in the elucidation of BR function in plants. However, high costs and limited availability of common BR biosynthetic inhibitors constrain their key advantage as a species-independent tool to investigate BR function. We studied propiconazole (Pcz) as an alternative to the BR inhibitor brassinazole (Brz). Arabidopsis seedlings treated with Pcz phenocopied BR biosynthetic mutants. The steady state mRNA levels of BR, but not gibberellic acid (GA), regulated genes increased proportional to the concentrations of Pcz. Moreover, root inhibition and Pcz-induced expression of BR biosynthetic genes were rescued by 24epi-brassinolide, but not by GA(3) co-applications. Maize seedlings treated with Pcz showed impaired mesocotyl, coleoptile, and true leaf elongation. Interestingly, the genetic background strongly impacted the tissue specific sensitivity towards Pcz. Based on these findings we conclude that Pcz is a potent and specific inhibitor of BR biosynthesis and an alternative to Brz. The reduced cost and increased availability of Pcz, compared to Brz, opens new possibilities to study BR function in larger crop species.

Dissipation and enantioselective degradation of plant growth retardants paclobutrazol and uniconazole in open field, greenhouse, and laboratory soils.

Science.gov (United States)

Wu, Chengwang; Sun, Jianqiang; Zhang, Anping; Liu, Weiping

2013-01-15

Greenhouses are increasingly important in human food supply. Pesticides used in greenhouses play important roles in horticulture; however, little is known about their behavior in greenhouse environments. This work investigates the dissipation and enantioselctive degradation of plant growth retardants including paclobutrazol and uniconazole in soils under three conditions (i.e., open field, greenhouse, and laboratory). The dissipation and enantioselective degradation of paclobutrazol and uniconazole in greenhouse were different from those in open field; they were more persistent in greenhouse than in open field soil. Leaching produced by rainfall is responsible for the difference in dissipation. Thus, local environmental impacts may occur more easily inside greenhouses, while groundwater may be more contaminated in open field. Spike concentrations of 5, 10, and 20 times the concentrations of native residues were tested for the enantioselective dissipation of the two pesticides; the most potent enantioselective degradation of paclobutrazol and uniconazole occurred at the 10 times that of the native residues in the greenhouse environments and at 20 times native residues in open field environments. The higher soil activity in greenhouses than in open fields was thought to be responsible for such a difference. The environmental risk and regulation of paclobutrazol and uniconazole should be considered at the enantiomeric level.

Efeitos do paclobutrazol na fertilidade de gemas e no crescimento dos ramos de videiras cv Rubi Effects of paclobutrazol on bud fertility and shoot growth of grapevine cv Rubi

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Renato Vasconcelos Botelho

2004-08-01

Full Text Available A baixa fertilidade de gemas tem sido um dos fatores limitantes da produção em vinhedos do Estado de São Paulo, estando este problema relacionado, em muitos casos, ao excesso de vigor das plantas. Neste contexto, um experimento foi conduzido em vinhedo comercial da cultivar de uva de mesa Rubi, localizado no município de Palmeira D'Oeste (SP. O delineamento experimental foi em blocos casualizados, com seis tratamentos e quatro repetições. Os ramos foram pulverizados com soluções de paclobutrazol nas doses de 0; 500; 1.000; 1.500; 2.000 e 2.500 mg L-1, no estádio de 5ª folha. As variáveis avaliadas foram: porcentagem de gemas férteis, porcentagem de gemas mortas, diâmetro de internódios e massa fresca e comprimento dos ramos. Aplicações de paclobutrazol reduziram o diâmetro dos internódios e a massa e o comprimento dos ramos. A porcentagem de gemas férteis também foi diminuída, possivelmente pela redução dos níveis endógenos de giberelina necessários para o processo de formação do primórdio indiferenciado.The low bud fertility has been one of the most limiting factors in vineyards at São Paulo State, and this problem has been correlated, in many cases, to the excess of plant vigor. In this context, a trial was carried out in a commercial vineyard of 'Rubi' table grape, located at Palmeira D'Oeste (SP, Brazil. The experimental design was in complete randomized blocks with six treatments and four replications. The vine shoots were sprayed with paclobutrazol at 0; 500; 1,000; 1,500; 2,000 and 2,500 mgL-1, at the fifth leaf phenological stage. The variables evaluated were: percentage of fertile buds; percentage of bud necrosis; fresh weight and length of shoots and diameter of internodes. Applications of paclobutrazol reduced the weight and the length of shoots and the internodes diameter. The percentage of fertile buds was also reduced, probably due to the decreasing in endogenous gibberellin levels necessary for

Structure-activity relationships of new cyanothiophene inhibitors of the essential peptidoglycan biosynthesis enzyme MurF.

Science.gov (United States)

Hrast, Martina; Turk, Samo; Sosič, Izidor; Knez, Damijan; Randall, Christopher P; Barreteau, Hélène; Contreras-Martel, Carlos; Dessen, Andréa; O'Neill, Alex J; Mengin-Lecreulx, Dominique; Blanot, Didier; Gobec, Stanislav

2013-08-01

Peptidoglycan is an essential component of the bacterial cell wall, and enzymes involved in its biosynthesis represent validated targets for antibacterial drug discovery. MurF catalyzes the final intracellular peptidoglycan biosynthesis step: the addition of D-Ala-D-Ala to the nucleotide precursor UDP-MurNAc-L-Ala-γ-D-Glu-meso-DAP (or L-Lys). As MurF has no human counterpart, it represents an attractive target for the development of new antibacterial drugs. Using recently published cyanothiophene inhibitors of MurF from Streptococcus pneumoniae as a starting point, we designed and synthesized a series of structurally related derivatives and investigated their inhibition of MurF enzymes from different bacterial species. Systematic structural modifications of the parent compounds resulted in a series of nanomolar inhibitors of MurF from S. pneumoniae and micromolar inhibitors of MurF from Escherichia coli and Staphylococcus aureus. Some of the inhibitors also show antibacterial activity against S. pneumoniae R6. These findings, together with two new co-crystal structures, represent an excellent starting point for further optimization toward effective novel antibacterials. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Essential oil composition of sweet basil (Ocimum basilicum L.) in symbiotic relationship with Piriformospora indica and paclobutrazol application under salt stress.

Science.gov (United States)

Keramati, Sara; Pirdashti, Hemmatollah; Babaeizad, Valliollah; Dehestani, Ali

2016-12-01

Essential oil content and oil composition of paclobutrazol treated sweet basil (Ocimum basilicum L.) plant inoculated with Piriformospora indica under salt stress were investigated by GC-MS. The results show a slight increase in essential oil content when basil plants subjected to moderate salinity stress (3 dS m -1 of NaCl). It decreased signifiicantly with increasing salinity level to 9 dS m -1 . The findings revealed that leaf area, above ground and leaf dry weights, essential oil content and yield were significantly affected by P. indica inoculation, however paclobutrazol application significantly influenced essential oil yield but not content. Fungal symbiosis as well as paclobutrazol application ameliorated the negative effects of salinity on dry matter and essential oil yield. The main constituents found in the volatile oil of O. basilicum in control treatment were Geranial (26.03%), Neral (24.88%) and Estragole (24.78%). The compounds concentrations showed some differences in P. indica and paclobutrazol treatments. The results demonstrate that micorrhiza-like fungi concomitantly increase essential oil production and biomass in sweet basil, a medicinal herb rich in commercially valuable essential oils.

Targeting S-adenosylmethionine biosynthesis with a novel allosteric inhibitor of Mat2A

Energy Technology Data Exchange (ETDEWEB)

Quinlan, Casey L.; Kaiser, Stephen E.; Bolaños, Ben; Nowlin, Dawn; Grantner, Rita; Karlicek-Bryant, Shannon; Feng, Jun Li; Jenkinson, Stephen; Freeman-Cook, Kevin; Dann, Stephen G.; Wang, Xiaoli; Wells, Peter A.; Fantin, Valeria R.; Stewart, Al E.; Grant, Stephan K. (Pfizer)

2017-05-29

S-Adenosyl-L-methionine (SAM) is an enzyme cofactor used in methyl transfer reactions and polyamine biosynthesis. The biosynthesis of SAM from ATP and L-methionine is performed by the methionine adenosyltransferase enzyme family (Mat; EC 2.5.1.6). Human methionine adenosyltransferase 2A (Mat2A), the extrahepatic isoform, is often deregulated in cancer. We identified a Mat2A inhibitor, PF-9366, that binds an allosteric site on Mat2A that overlaps with the binding site for the Mat2A regulator, Mat2B. Studies exploiting PF-9366 suggested a general mode of Mat2A allosteric regulation. Allosteric binding of PF-9366 or Mat2B altered the Mat2A active site, resulting in increased substrate affinity and decreased enzyme turnover. These data support a model whereby Mat2B functions as an inhibitor of Mat2A activity when methionine or SAM levels are high, yet functions as an activator of Mat2A when methionine or SAM levels are low. The ramification of Mat2A activity modulation in cancer cells is also described.

Trypanosoma cruzi response to sterol biosynthesis inhibitors: morphophysiological alterations leading to cell death.

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Rafael Luis Kessler

Full Text Available The protozoan parasite Trypanosoma cruzi displays similarities to fungi in terms of its sterol lipid biosynthesis, as ergosterol and other 24-alkylated sterols are its principal endogenous sterols. The sterol pathway is thus a potential drug target for the treatment of Chagas disease. We describe here a comparative study of the growth inhibition, ultrastructural and physiological changes leading to the death of T. cruzi cells following treatment with the sterol biosynthesis inhibitors (SBIs ketoconazole and lovastatin. We first calculated the drug concentration inhibiting epimastigote growth by 50% (EC(50/72 h or killing all cells within 24 hours (EC(100/24 h. Incubation with inhibitors at the EC(50/72 h resulted in interesting morphological changes: intense proliferation of the inner mitochondrial membrane, which was corroborated by flow cytometry and confocal microscopy of the parasites stained with rhodamine 123, and strong swelling of the reservosomes, which was confirmed by acridine orange staining. These changes to the mitochondria and reservosomes may reflect the involvement of these organelles in ergosterol biosynthesis or the progressive autophagic process culminating in cell lysis after 6 to 7 days of treatment with SBIs at the EC(50/72 h. By contrast, treatment with SBIs at the EC(100/24 h resulted in rapid cell death with a necrotic phenotype: time-dependent cytosolic calcium overload, mitochondrial depolarization and reservosome membrane permeabilization (RMP, culminating in cell lysis after a few hours of drug exposure. We provide the first demonstration that RMP constitutes the "point of no return" in the cell death cascade, and propose a model for the necrotic cell death of T. cruzi. Thus, SBIs trigger cell death by different mechanisms, depending on the dose used, in T. cruzi. These findings shed new light on ergosterol biosynthesis and the mechanisms of programmed cell death in this ancient protozoan parasite.

Glyoxal bis(guanylhydrazone) as an inhibitor of polyamine biosynthesis in tumour cells.

OpenAIRE

Seppänen, P; Fagerström, R; Alhonen-Hongisto, L; Elo, H; Lumme, P; Jänne, J

1984-01-01

Glyoxal bis(guanylhydrazone), the parent compound of methylglyoxal bis(guanylhydrazone), was synthesized and tested for its ability to inhibit the biosynthesis of polyamines. It was found to be a powerful competitive inhibitor of adenosylmethionine decarboxylase (EC 4.1.1.50), yet the lack of the methyl group at the glyoxal portion increased the apparent Ki value for the enzyme by about 30-fold in comparison with methylglyoxal bis(guanylhydrazone). Glyoxal bis(guanylhydrazone) inhibited diami...

Highly efficient detection of paclobutrazol in environmental water and soil samples by time-resolved fluoroimmunoassay

Energy Technology Data Exchange (ETDEWEB)

Liu, Zhenjiang, E-mail: lzj1984@ujs.edu.cn [School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang 212013 (China); Wei, Xi [School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang 212013 (China); The Affiliated First People' s Hospital of Jiangsu University, Zhenjiang 212002 (China); Ren, Kewei; Zhu, Gangbing; Zhang, Zhen; Wang, Jiagao; Du, Daolin [School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang 212013 (China)

2016-11-01

A fast and ultrasensitive indirect competitive time-resolved fluoroimmunoassay (TRFIA) was developed for the analysis of paclobutrazol in environmental water and soil samples. Paclobutrazol hapten was synthesized and conjugated to bovine serum albumin (BSA) for producing polyclonal antibodies. Under optimal conditions, the 50% inhibitory concentration (IC{sub 50} value) and limit of detection (LOD, IC{sub 20} value) were 1.09 μg L{sup −} {sup 1} and 0.067 μg L{sup −} {sup 1}, respectively. The LOD of TRFIA was improved 30-fold compared to the already reported ELISA. There was almost no cross-reactivity of the antibody with the other structural analogues of triazole compounds, indicating that the antibody had high specificity. The average recoveries from spiked samples were in the range from 80.2% to 104.7% with a relative standard deviation of 1.0–9.5%. The TRFIA results for the real samples were in good agreement with that obtained by high-performance liquid chromatography analyses. The results indicate that the established TRFIA has potential application for screening paclobutrazol in environmental samples. - Highlights: • The approach to design and synthesize the PBZ hapten was more straightforward. • A rapid and ultrasensitive TRFIA was developed and applied to the screening of PBZ. • The TRFIA for real soil samples showed reliability and high correlation with HPLC. • The PBZ TRFIA showed high sensitivity, simple operation, a wide range of quantitative analyses and no radioactive hazards.

Identification of novel bacterial histidine biosynthesis inhibitors using docking, ensemble rescoring, and whole-cell assays

DEFF Research Database (Denmark)

Henriksen, Signe Teuber; Liu, J.; Estiu, G.

2010-01-01

histidine biosynthesis pathway, which is predicted to be essential for bacterial biomass productions. Virtual screening of a library of similar to 10(6) compounds identified 49 potential inhibitors of three enzymes of this pathway. Eighteen representative compounds were directly tested on three S. aureus......-and two Escherichia coli strains in standard disk inhibition assays. Thirteen compounds are inhibitors of some or all of the S. aureus strains, while 14 compounds weakly inhibit growth in one or both E. coli strains. The high hit rate obtained from a fast virtual screen demonstrates the applicability...

Global transcriptome profiling analysis of inhibitory effects of paclobutrazol on leaf growth in lily (Lilium Longiflorum-Asiatic hybrid

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Xiaopei eZhu

2016-04-01

Full Text Available As a popular ornamental flower, potted lily is an important object of lily breeding. Paclobutrazol, a chemical growth retardation compound, is often used to dwarf plant in producing potted lilies. However, in recent years, the plants with inherited dwarf traits by using genetic engineer breeding technology are being developed. The studies on molecular basis of lily dwarfism will offer some target genes which have profound dwarf effect for genetic engineer breeding. Here, we confirmed that paclobutrazol inhibited plant height and leaf size in Lilium Longiflorum-Asiatic hybrid, and then RNA-Seq technique was employed to analyze gene transcripts of Lilium Longiflorum-Asiatic hybrid leaves by paclobutrazol treatment in order to get a deeper insight into dwarfism mechanism of lily. Approximately 38.6 Gb data was obtained and assemble into 53,681 unigenes. Annotation, pathways, functional classification and phylogenetic classification of these data were analyzed based on Nr, Nt, Swiss-Prot, KEGG, COG and GO databases. 2,704 differentially expressed genes were screened by comparing paclobutrazol-treated samples with untreated samples and quantitative real-time PCR was performed to validate expression profiles. By analyzing dynamic changes of differentially expressed genes, nine metabolic pathways and signal transduction pathways were significantly enriched and many potentially interesting genes were identified that encoded putative regulators or key components of cell division, cell expansion, GA metabolism and signaling transduction and these genes were highlighted to reveal their importance in regulation of plant size. These results will provide a better understanding of the molecular mechanism on lily dwarfism and some potential genes related to lily organ size, which will lay the foundation for molecular breeding of potted lilies. These transcriptome data will also serve as valuable public genomic resources for other genetic research in lily.

Phenyl thiazolyl urea and carbamate derivatives as new inhibitors of bacterial cell-wall biosynthesis.

Science.gov (United States)

Francisco, Gerardo D; Li, Zhong; Albright, J Donald; Eudy, Nancy H; Katz, Alan H; Petersen, Peter J; Labthavikul, Pornpen; Singh, Guy; Yang, Youjun; Rasmussen, Beth A; Lin, Yang-I; Mansour, Tarek S

2004-01-05

Over 50 phenyl thiazolyl urea and carbamate derivatives were synthesized for evaluation as new inhibitors of bacterial cell-wall biosynthesis. Many of them demonstrated good activity against MurA and MurB and gram-positive bacteria including MRSA, VRE and PRSP. 3,4-Difluorophenyl 5-cyanothiazolylurea (3p) with clog P of 2.64 demonstrated antibacterial activity against both gram-positive and gram-negative bacteria.

Glyoxal bis(guanylhydrazone) as an inhibitor of polyamine biosynthesis in tumour cells.

Science.gov (United States)

Seppänen, P; Fagerström, R; Alhonen-Hongisto, L; Elo, H; Lumme, P; Jänne, J

1984-07-15

Glyoxal bis(guanylhydrazone), the parent compound of methylglyoxal bis(guanylhydrazone), was synthesized and tested for its ability to inhibit the biosynthesis of polyamines. It was found to be a powerful competitive inhibitor of adenosylmethionine decarboxylase (EC 4.1.1.50), yet the lack of the methyl group at the glyoxal portion increased the apparent Ki value for the enzyme by about 30-fold in comparison with methylglyoxal bis(guanylhydrazone). Glyoxal bis(guanylhydrazone) inhibited diamine oxidase (EC 1.4.3.6) activity as effectively as did methylglyoxal bis(guanylhydrazone). The cellular accumulation curves of glyoxal bis(guanylhydrazone) in L1210 cells were practically superimposable with those of methylglyoxal bis(guanylhydrazone), and the uptake of both compounds was distinctly stimulated by a prior treatment with 2-difluoromethylornithine. The drug decreased the concentration of spermidine in a dose-dependent manner and, in contrast with methylglyoxal bis(guanylhydrazone), without a concomitant accumulation of putrescine. The fact that putrescine concentrations were decreased in cells exposed to glyoxal bis(guanylhydrazone) was, at least in part, attributable to an inhibition of ornithine decarboxylase (EC 4.1.1.17) activity in cells treated with the compound. Under these experimental conditions equivalent concentrations of methylglyoxal bis(guanylhydrazone) [1,1'-[(methylethanediylidine)dinitrilo]diguanidine] elicited large increases in the enzyme activity. When combined with difluoromethylornithine, glyoxal bis(guanylhydrazone) potentiated the growth-inhibitory effect of that drug. Taking into consideration the proven anti-leukaemic activity of glyoxal bis(guanylhydrazone), its effectiveness to inhibit spermidine biosynthesis (without raising the concentration of putrescine) as well as its suitability for combined use with inhibitors of ornithine decarboxylase, this drug is apparently worthy of further testing in tumour-bearing animals, especially in

Effects of thidiazuron and paclobutrazol on regeneration potential of tulip flower stalk explants in vitro and subsequent shoot multiplication

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Małgorzata Podwyszyńska

2011-01-01

Full Text Available The effects of TDZ and paclobutrazol on the primary regeneration on tulip flower stalk explants of six cultivars and subsequent shoot multiplication were examined. Explants, flower stalk slices, were excised from cooled and subsequently forced bulbs. The explants were incubated for two months in darkness on medium containing NAA and cytokinins, 2iP and BAP, as control, or TDZ (0.5-4 mg l-1 and paclobutrazol (0.05-0.4 mg l-1. Then, the regenerating explants were subcultured on medium with TDZ and NAA applied at low concentrations. Different regeneration capabilities were found depending on cultivar and growth regulators. The percentage of explants forming leaf-like structures ranged, on the control medium, from 80% in 'Blue Parrot' and 'Prominence' to below 30% in 'Apeldoorn' and 'Mirjoran'. TDZ, applied at optimum for each cultivar concentration, greatly increased the regeneration potential up to 70-100%. Paclobutrazol, added to the TDZ-containing medium, significantly enhanced the response of explants, resulting in high numbers of leaf-like structures formed per explant (13.7-22.8. The structures developed gradually into characteristic forms: the growing up cotyledonary leaf, the probable root primordium formed at its base, the growing downwards stolon and the shoot meristem developed finely on its tip. It is suggested that such primary regeneration may have a nature of somatic embryogenesis. Then, the adventitious shoots developed and formed clusters, which were divided into 2-3 smaller ones every two months. The growth regulators, used at initial stage, markedly influenced subsequent shoot multiplication. Thus, the most intensive shoot formation was noted with TDZ at concentrations of 0.5-2 mg l-1 and paclobutrazol of 0.05-0.1 mg l-1.

Modelagem da cinética de biodegradação de paclobutrazol em dois solos do semiárido do nordeste brasileiro

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Fernanda Leitão Vaz

2012-01-01

Full Text Available Mathematical models can help to prevent high levels of toxic substances in soil or fruits of plants treated with pesticides and indicate that such substances should be systematically monitored. The aim of this research was to study the kinetics of paclobutrazol biodegradation by soil native bacteria using mathematical models. Three models were used to assess the kinetics of paclobutrazol biodegradation obtained experimentally. Excellent fits were obtained using dual kinetic and logistic models. The use of glycerol as additional carbon source increased the biodegradation of PBZ and consequently decreased the time required for a given PBZ initial concentration be halved.

Amelioration in secretion of hyperthermostable and Ca2+ -independent alpha-amylase of Geobacillus thermoleovorans by some polyamines and their biosynthesis inhibitor methylglyoxal-bis-guanylhydrazone.

Science.gov (United States)

Uma Maheswar Rao, J L; Satyanarayana, T

2004-01-01

Effect of polyamines and their biosynthesis inhibitors on the production of hyperthermostable and Ca2+ -independent alpha-amylase by Geobacillus thermoleovorans MTCC 4220. The alpha-amylase was produced in starch-yeast extract-tryptone (SYT) broth with different polyamines (PA) and polyamine biosynthesis inhibitors, methylglyoxal-bis-guanylhydrazone (MGBG) and cyclohexylammonium sulphate (CHA) at 70 degrees C. The bacterial pellets were obtained after growing G. thermoleovorans at different temperatures, and used in determining total PA. The cell-free culture filtrates were used in alpha-amylase assays. During growth, total polyamines in biomass increased till 2 h, and thereafter, decreased gradually. The total polyamine content was very high in the biomass cultivated at 55 degrees C when compared with that of higher temperatures. Enzyme titre enhanced up to 70 degrees C, and thereafter declined. Extracellular enzyme and protein levels declined in the presence of exogenously added PA. The intracellular enzyme titres, however, were higher in putrescine (put) and spermidine (spd) than in spermine (spm). Polyamine biosynthesis inhibitor, MGBG enhanced secretion of alpha-amylase in a laboratory fermentor as well as shake flasks, although CHA did not affect it. The intracellular accumulation of put in the presence of MGBG appeared to enhance synthesis and secretion of alpha-amylase. Extracellular enzyme and protein levels were low in the presence of exogenously added PA, but their intracellular levels, however, were higher in put and spd than in spm. A substantial increase in the synthesis and secretion of alpha-amylase was attained in G. thermoleovorans in the presence of polyamine biosynthesis inhibitor MGBG.

Differential effects of lipid biosynthesis inhibitors on Zika and Semliki Forest viruses.

Science.gov (United States)

Royle, Jamie; Donald, Claire L; Merits, Andres; Kohl, Alain; Varjak, Margus

2017-12-01

The recent outbreak of infection with Zika virus (ZIKV; Flaviviridae) has attracted attention to this previously neglected mosquito-borne pathogen and the need for efficient therapies. Since flavivirus replication is generally known to be dependent on fatty acid biosynthesis, two inhibitors of this pathway, 5-(tetradecyloxyl)-2-furoic acid (TOFA) and cerulenin, were tested for their potentiality to inhibit virus replication. At concentrations previously shown to inhibit the replication of other flaviviruses, neither drug had a significant antiviral affect against ZIKV, but reduced the replication of the non-related mosquito-borne Semliki Forest virus (Togaviridae). Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Fluridone and norflurazon, carotenoid-biosynthesis inhibitors, promote seed conditioning and germination of the holoparasite Orobanche minor.

Science.gov (United States)

Chae, Sang Heon; Yoneyama, Koichi; Takeuchi, Yasutomo; Joel, Daniel M.

2004-02-01

Fluridone and norflurazon, two carotenoid-biosynthesis inhibitors, shortened the conditioning period required by seeds of Orobanche minor in order to respond to the germination stimulant strigol. Neither fluridone nor norflurazon alone induced seed germination of O. minor, they promoted strigol-induced germination. In addition, these compounds restored the conditioning and germination of seeds at a supraoptimal temperature (30 degrees C) as well as in the light. Gibberellic acid (GA(3)) showed similar promotive and protective effects on the conditioning and germination of O. minor seeds. Although fluridone and norflurazon are known to prevent abscisic acid (ABA)-biosynthesis, and stresses such as supraoptimal temperatures have been reported to induce ABA accumulation in plants, the amount of ABA in the seeds or that released from the seeds into the conditioning media was not affected by the fluridone treatment and by exposure to the supraoptimal temperature. These results indicate that the promotive and protective effects of fluridone and norflurazon on the conditioning and germination of O. minor seeds would be attributed to other perturbations rather than the inhibition of ABA-biosynthesis.

Effects of polyamine biosynthesis inhibitors on S-adenosylmethionine synthetase and S-adenosylmethionine decarboxylase activities in carrot cell cultures

Science.gov (United States)

S.C. Minocha; R. Minocha; A. Komamine

1991-01-01

Changes in the activites of S-adcnosylmethionine (SAM) synthetase (methionine adenosyltransferase, EC 2.5.1.6.) and SAM decarboxylase (EC 4.1.1.50) were studied in carrot (Daucus carota) cell cultures in response to 2,4-dichlorophenoxyacetic acid (2,4-D) and several inhibitors of polyamine biosynthesis. Activity of SAM synthetase increased...

Phosphorylated hydroxyethylamines as novel inhibitors of the bacterial cell wall biosynthesis enzymes MurC to MurF.

Science.gov (United States)

Sova, Matej; Kovac, Andreja; Turk, Samo; Hrast, Martina; Blanot, Didier; Gobec, Stanislav

2009-12-01

Enzymes involved in the biosynthesis of bacterial peptidoglycan represent important targets for development of new antibacterial drugs. Among them, Mur ligases (MurC to MurF) catalyze the formation of the final cytoplasmic precursor UDP-N-acetylmuramyl-pentapeptide from UDP-N-acetylmuramic acid. We present the design, synthesis and biological evaluation of a series of phosphorylated hydroxyethylamines as new type of small-molecule inhibitors of Mur ligases. We show that the phosphate group attached to the hydroxyl moiety of the hydroxyethylamine core is essential for good inhibitory activity. The IC(50) values of these inhibitors were in the micromolar range, which makes them a promising starting point for the development of multiple inhibitors of Mur ligases as potential antibacterial agents. In addition, 1-(4-methoxyphenylsulfonamido)-3-morpholinopropan-2-yl dihydrogen phosphate 7a was discovered as one of the best inhibitors of MurE described so far.

Cholesterol biosynthesis inhibitor RO 48-8071 suppresses growth of hormone-dependent and castration-resistant prostate cancer cells

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Liang Y

2016-05-01

growth of aggressive castration-resistant human prostate cancer cell xenografts in vivo without any signs of toxicity to experimental animals. Importantly, RO did not reduce the viability of normal prostate cells in vitro. Our study is the first to demonstrate that the cholesterol biosynthesis inhibitor RO effectively suppresses growth of human prostate cancer cells. Our findings suggest that cholesterol biosynthesis inhibitors such as RO, when used in combination with commonly used chemotherapeutic drugs or ERβ specific ligands, could represent a novel therapeutic approach to prevent the growth of prostate cancer tumors. Keywords: prostate cancer, cholesterol biosynthesis inhibitor, cell viability, xenograft, castration resistant

Exogenous auxin represses soybean seed germination through decreasing the gibberellin/abscisic acid (GA/ABA) ratio.

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Shuai, Haiwei; Meng, Yongjie; Luo, Xiaofeng; Chen, Feng; Zhou, Wenguan; Dai, Yujia; Qi, Ying; Du, Junbo; Yang, Feng; Liu, Jiang; Yang, Wenyu; Shu, Kai

2017-10-03

Auxin is an important phytohormone which mediates diverse development processes in plants. Published research has demonstrated that auxin induces seed dormancy. However, the precise mechanisms underlying the effect of auxin on seed germination need further investigation, especially the relationship between auxins and both abscisic acid (ABA) and gibberellins (GAs), the latter two phytohormones being the key regulators of seed germination. Here we report that exogenous auxin treatment represses soybean seed germination by enhancing ABA biosynthesis, while impairing GA biogenesis, and finally decreasing GA 1 /ABA and GA 4 /ABA ratios. Microscope observation showed that auxin treatment delayed rupture of the soybean seed coat and radicle protrusion. qPCR assay revealed that transcription of the genes involved in ABA biosynthetic pathway was up-regulated by application of auxin, while expression of genes involved in GA biosynthetic pathway was down-regulated. Accordingly, further phytohormone quantification shows that auxin significantly increased ABA content, whereas the active GA 1 and GA 4 levels were decreased, resulting insignificant decreases in the ratiosGA 1 /ABA and GA 4 /ABA.Consistent with this, ABA biosynthesis inhibitor fluridone reversed the delayed-germination phenotype associated with auxin treatment, while paclobutrazol, a GA biosynthesis inhibitor, inhibited soybean seed germination. Altogether, exogenous auxin represses soybean seed germination by mediating ABA and GA biosynthesis.

The mechanism of ethylene signaling induced by endophytic fungus Gilmaniella sp. AL12 mediating sesquiterpenoids biosynthesis in Atractylodes lancea

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Jie eYuan

2016-03-01

Full Text Available Ethylene, the first known gaseous phytohormone, is involved in plant growth, development as well as responses to environmental signals. However, limited information is available on the role of ethylene in endophytic fungi induced secondary metabolites biosynthesis. Atractylodes lancea is a traditional Chinese herb, and its quality depends on the main active compounds sesquiterpenoids. This work showed that the endophytic fungus Gilmaniella sp. AL12 induced ethylene production in Atractylodes lancea. Pre-treatment of plantlets with ethylene inhibiter aminooxyacetic acid (AOA suppressed endophytic fungi induced accumulation of ethylene and sesquiterpenoids. Plantlets were further treated with AOA, salicylic acid (SA biosynthesis inhibitor paclobutrazol (PAC, jasmonic acid inhibitor ibuprofen (IBU, hydrogen peroxide (H2O2 scavenger catalase (CAT, nitric oxide (NO-specific scavenger 2-(4-Carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO. With endophytic fungi inoculation, IBU or PAC did not inhibit ethylene production, and JA and SA generation were suppressed by AOA, showing that ethylene may act as an upstream signal of JA and SA pathway. With endophytic fungi inoculation, CAT or cPTIO suppressed ethylene production, and H2O2 or NO generation was not affected by 1-aminocyclopropane-1-carboxylic acid (ACC, showing that ethylene may act as a downstream signal of H2O2 and NO pathway. Then, plantlets were treated with ethylene donor ACC, JA, SA, H2O2, NO donor sodium nitroprusside (SNP. Exogenous ACC could trigger JA and SA generation, whereas exogenous JA or SA did not affect ethylene production, and the induced sesquiterpenoids accumulation triggered by ACC was partly suppressed by IBU and PAC, showing that ethylene acted as an upstream signal of JA and SA pathway. Exogenous ACC did not affect H2O2 or NO generation, whereas exogenous H2O2 and SNP induced ethylene production, and the induced sesquiterpenoids

Karrikins delay soybean seed germination by mediating abscisic acid and gibberellin biogenesis under shaded conditions

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Meng, Yongjie; Chen, Feng; Shuai, Haiwei; Luo, Xiaofeng; Ding, Jun; Tang, Shengwen; Xu, Shuanshuan; Liu, Jianwei; Liu, Weiguo; Du, Junbo; Liu, Jiang; Yang, Feng; Sun, Xin; Yong, Taiwen; Wang, Xiaochun; Feng, Yuqi; Shu, Kai; Yang, Wenyu

2016-01-01

Karrikins (KAR) are a class of signal compounds, discovered in wildfire smoke, which affect seed germination. Currently, numerous studies have focused on the model plant Arabidopsis in the KAR research field, rather than on crops. Thus the regulatory mechanisms underlying KAR regulation of crop seed germination are largely unknown. Here, we report that KAR delayed soybean seed germination through enhancing abscisic acid (ABA) biosynthesis, while impairing gibberellin (GA) biogenesis. Interestingly, KAR only retarded soybean seed germination under shaded conditions, rather than under dark and white light conditions, which differs from in Arabidopsis. Phytohormone quantification showed that KAR enhanced ABA biogenesis while impairing GA biosynthesis during the seed imbibition process, and subsequently, the ratio of active GA4 to ABA was significantly reduced. Further qRT-PCR analysis showed that the transcription pattern of genes involved in ABA and GA metabolic pathways are consistent with the hormonal measurements. Finally, fluridone, an ABA biogenesis inhibitor, remarkably rescued the delayed-germination phenotype of KAR-treatment; and paclobutrazol, a GA biosynthesis inhibitor, inhibited soybean seed germination. Taken together, these evidences suggest that KAR inhibit soybean seed germination by mediating the ratio between GA and ABA biogenesis. PMID:26902640

Karrikins delay soybean seed germination by mediating abscisic acid and gibberellin biogenesis under shaded conditions.

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Meng, Yongjie; Chen, Feng; Shuai, Haiwei; Luo, Xiaofeng; Ding, Jun; Tang, Shengwen; Xu, Shuanshuan; Liu, Jianwei; Liu, Weiguo; Du, Junbo; Liu, Jiang; Yang, Feng; Sun, Xin; Yong, Taiwen; Wang, Xiaochun; Feng, Yuqi; Shu, Kai; Yang, Wenyu

2016-02-23

Karrikins (KAR) are a class of signal compounds, discovered in wildfire smoke, which affect seed germination. Currently, numerous studies have focused on the model plant Arabidopsis in the KAR research field, rather than on crops. Thus the regulatory mechanisms underlying KAR regulation of crop seed germination are largely unknown. Here, we report that KAR delayed soybean seed germination through enhancing abscisic acid (ABA) biosynthesis, while impairing gibberellin (GA) biogenesis. Interestingly, KAR only retarded soybean seed germination under shaded conditions, rather than under dark and white light conditions, which differs from in Arabidopsis. Phytohormone quantification showed that KAR enhanced ABA biogenesis while impairing GA biosynthesis during the seed imbibition process, and subsequently, the ratio of active GA4 to ABA was significantly reduced. Further qRT-PCR analysis showed that the transcription pattern of genes involved in ABA and GA metabolic pathways are consistent with the hormonal measurements. Finally, fluridone, an ABA biogenesis inhibitor, remarkably rescued the delayed-germination phenotype of KAR-treatment; and paclobutrazol, a GA biosynthesis inhibitor, inhibited soybean seed germination. Taken together, these evidences suggest that KAR inhibit soybean seed germination by mediating the ratio between GA and ABA biogenesis.

Inhibitors of amino acids biosynthesis as antifungal agents.

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Jastrzębowska, Kamila; Gabriel, Iwona

2015-02-01

Fungal microorganisms, including the human pathogenic yeast and filamentous fungi, are able to synthesize all proteinogenic amino acids, including nine that are essential for humans. A number of enzymes catalyzing particular steps of human-essential amino acid biosynthesis are fungi specific. Numerous studies have shown that auxotrophic mutants of human pathogenic fungi impaired in biosynthesis of particular amino acids exhibit growth defect or at least reduced virulence under in vivo conditions. Several chemical compounds inhibiting activity of one of these enzymes exhibit good antifungal in vitro activity in minimal growth media, which is not always confirmed under in vivo conditions. This article provides a comprehensive overview of the present knowledge on pathways of amino acids biosynthesis in fungi, with a special emphasis put on enzymes catalyzing particular steps of these pathways as potential targets for antifungal chemotherapy.

Fenarimol, a Pyrimidine-Type Fungicide, Inhibits Brassinosteroid Biosynthesis

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Keimei Oh

2015-07-01

Full Text Available The plant steroid hormone brassinosteroids (BRs are important signal mediators that regulate broad aspects of plant growth and development. With the discovery of brassinoazole (Brz, the first specific inhibitor of BR biosynthesis, several triazole-type BR biosynthesis inhibitors have been developed. In this article, we report that fenarimol (FM, a pyrimidine-type fungicide, exhibits potent inhibitory activity against BR biosynthesis. FM induces dwarfism and the open cotyledon phenotype of Arabidopsis seedlings in the dark. The IC50 value for FM to inhibit stem elongation of Arabidopsis seedlings grown in the dark was approximately 1.8 ± 0.2 μM. FM-induced dwarfism of Arabidopsis seedlings could be restored by brassinolide (BL but not by gibberellin (GA. Assessment of the target site of FM in BR biosynthesis by feeding BR biosynthesis intermediates indicated that FM interferes with the side chain hydroxylation of BR biosynthesis from campestanol to teasterone. Determination of the binding affinity of FM to purified recombinant CYP90D1 indicated that FM induced a typical type II binding spectrum with a Kd value of approximately 0.79 μM. Quantitative real-time PCR analysis of the expression level of the BR responsive gene in Arabidopsis seedlings indicated that FM induces the BR deficiency in Arabidopsis.

The effect of haem biosynthesis inhibitors and inducers on intestinal iron absorption and liver haem biosynthetic enzyme activities

International Nuclear Information System (INIS)

Laftah, A.H.; Simpson, R.J.; Peters, T.J.; Raja, K.B.

2008-01-01

The relation between haem biosynthesis and intestinal iron absorption is not well understood, we therefore investigated the effect of compounds that alter haem metabolism on duodenal iron absorption. CD1 mice were treated with either an inhibitor (succinyl acetone (SA)) or stimulator (2-allyl-2-isopropylacetamide (AIA)) of haem biosynthesis. 5-Aminolaevulinic acid (ALA) dehydratase and urinary ALA and porphobilinogen (PBG) levels, were determined. Intestinal iron absorption was assayed with in vivo and in vitro techniques. Liver hepcidin (Hamp1) and duodenal iron transporter mRNA levels were measured using RT-PCR. AIA caused increased hepatic ALA synthase (1.6-fold) and ALA dehydratase (1.4-fold, both p < 0.005) activities and increased urinary ALA and PBG excretion (2.1- and 1.4-fold, p < 0.005, p < 0.05, respectively). In vivo intestinal iron absorption was reduced to 49% of control (p < 0.005). Mice treated with SA showed decreased urinary ALA and PBG levels (75 and 55% control, both p < 0.005) and reductions in both ALA synthase and ALA dehydratase activities (77 and 56% control, p < 0.05, p < 0.005, respectively) in the liver. Liver and duodenal haem and cytochrome oxidase levels were not significantly decreased. Iron absorption was enhanced (1.26-fold, p < 0.05) and hepatic Hamp1 mRNA was reduced (53% of control, p < 0.05). In vitro duodenal iron uptake after mice were injected with SA also demonstrated an increase in Fe(III) reduction and uptake (1.27- and 1.41-fold, p < 0.01 respectively). Simultaneous injections of SA and ALA blocked the enhancing effect on iron absorption seen with SA alone. We conclude that alterations in haem biosynthesis can influence iron absorption and in particular, the intermediate ALA seems to be an inhibitor of iron absorption

Distribution of Silicified Microstructures, Regulation of Cinnamyl Alcohol Dehydrogenase and Lodging Resistance in Silicon and Paclobutrazol Mediated Oryza sativa

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Deivaseeno Dorairaj

2017-07-01

Full Text Available Lodging is a phenomenon that affects most of the cereal crops including rice, Oryza sativa. This is due to the fragile nature of herbaceous plants whose stems are non-woody, thus affecting its ability to grow upright. Silicon (Si, a beneficial nutrient is often used to toughen and protect plants from biotic and abiotic stresses. Deposition of Si in plant tissues enhances the rigidity and stiffness of the plant as a whole. Silicified cells provide the much needed strength to the culm to resist breaking. Lignin plays important roles in cell wall structural integrity, stem strength, transport, mechanical support, and plant pathogen defense. The aim of this study is to resolve effects of Si on formation of microstructure and regulation of cinnamyl alcohol dehydrogenase (CAD, a key gene responsible for lignin biosynthesis. Besides evaluating silicon, paclobutrazol (PBZ a plant growth retartdant that reduces internode elongation is also incorporated in this study. Hardness, brittleness and stiffness were improved in presence of silicon thus reducing lodging. Scanning electron micrographs with the aid of energy dispersive x-ray (EDX was used to map silicon distribution. Presence of trichomes, silica cells, and silica bodies were detected in silicon treated plants. Transcripts of CAD gene was also upregulated in these plants. Besides, phloroglucinol staining showed presence of lignified vascular bundles and sclerenchyma band. In conclusion, silicon treated rice plants showed an increase in lignin content, silicon content, and formation of silicified microstructures.

The Arabidopsis MYB96 transcription factor plays a role in seed dormancy.

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Lee, Hong Gil; Lee, Kyounghee; Seo, Pil Joon

2015-03-01

Seed dormancy facilitates to endure environmental disadvantages by confining embryonic growth until the seeds encounter favorable environmental conditions for germination. Abscisic acid (ABA) and gibberellic acid (GA) play a pivotal role in the determination of the seed dormancy state. ABA establishes seed dormancy, while GA triggers seed germination. Here, we demonstrate that MYB96 contributes to the fine-tuning of seed dormancy regulation through the coordination of ABA and GA metabolism. The MYB96-deficient myb96-1 seeds germinated earlier than wild-type seeds, whereas delayed germination was observed in the activation-tagging myb96-1D seeds. The differences in germination rate disappeared after stratification or after-ripening. The MYB96 transcription factor positively regulates ABA biosynthesis genes 9-CIS-EPOXYCAROTENOID DIOXYGENASE 2 (NCED2), NCED5, NCED6, and NCED9, and also affects GA biosynthetic genes GA3ox1 and GA20ox1. Notably, MYB96 directly binds to the promoters of NCED2 and NCED6, primarily modulating ABA biosynthesis, which subsequently influences GA metabolism. In agreement with this, hyperdormancy of myb96-1D seeds was recovered by an ABA biosynthesis inhibitor fluridone, while hypodormancy of myb96-1 seeds was suppressed by a GA biosynthesis inhibitor paclobutrazol (PAC). Taken together, the metabolic balance of ABA and GA underlies MYB96 control of primary seed dormancy.

A Comparative Study of Ethylene Growth Response Kinetics in Eudicots and Monocots Reveals a Role for Gibberellin in Growth Inhibition and Recovery1[W][OA

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Kim, Joonyup; Wilson, Rebecca L.; Case, J. Brett; Binder, Brad M.

2012-01-01

Time-lapse imaging of dark-grown Arabidopsis (Arabidopsis thaliana) hypocotyls has revealed new aspects about ethylene signaling. This study expands upon these results by examining ethylene growth response kinetics of seedlings of several plant species. Although the response kinetics varied between the eudicots studied, all had prolonged growth inhibition for as long as ethylene was present. In contrast, with continued application of ethylene, white millet (Panicum miliaceum) seedlings had a rapid and transient growth inhibition response, rice (Oryza sativa ‘Nipponbare’) seedlings had a slow onset of growth stimulation, and barley (Hordeum vulgare) had a transient growth inhibition response followed, after a delay, by a prolonged inhibition response. Growth stimulation in rice correlated with a decrease in the levels of rice ETHYLENE INSENSTIVE3-LIKE2 (OsEIL2) and an increase in rice F-BOX DOMAIN AND LRR CONTAINING PROTEIN7 transcripts. The gibberellin (GA) biosynthesis inhibitor paclobutrazol caused millet seedlings to have a prolonged growth inhibition response when ethylene was applied. A transient ethylene growth inhibition response has previously been reported for Arabidopsis ethylene insensitive3-1 (ein3-1) eil1-1 double mutants. Paclobutrazol caused these mutants to have a prolonged response to ethylene, whereas constitutive GA signaling in this background eliminated ethylene responses. Sensitivity to paclobutrazol inversely correlated with the levels of EIN3 in Arabidopsis. Wild-type Arabidopsis seedlings treated with paclobutrazol and mutants deficient in GA levels or signaling had a delayed growth recovery after ethylene removal. It is interesting to note that ethylene caused alterations in gene expression that are predicted to increase GA levels in the ein3-1 eil1-1 seedlings. These results indicate that ethylene affects GA levels leading to modulation of ethylene growth inhibition kinetics. PMID:22977279

A comparative study of ethylene growth response kinetics in eudicots and monocots reveals a role for gibberellin in growth inhibition and recovery.

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Kim, Joonyup; Wilson, Rebecca L; Case, J Brett; Binder, Brad M

2012-11-01

Time-lapse imaging of dark-grown Arabidopsis (Arabidopsis thaliana) hypocotyls has revealed new aspects about ethylene signaling. This study expands upon these results by examining ethylene growth response kinetics of seedlings of several plant species. Although the response kinetics varied between the eudicots studied, all had prolonged growth inhibition for as long as ethylene was present. In contrast, with continued application of ethylene, white millet (Panicum miliaceum) seedlings had a rapid and transient growth inhibition response, rice (Oryza sativa 'Nipponbare') seedlings had a slow onset of growth stimulation, and barley (Hordeum vulgare) had a transient growth inhibition response followed, after a delay, by a prolonged inhibition response. Growth stimulation in rice correlated with a decrease in the levels of rice ETHYLENE INSENSTIVE3-LIKE2 (OsEIL2) and an increase in rice F-BOX DOMAIN AND LRR CONTAINING PROTEIN7 transcripts. The gibberellin (GA) biosynthesis inhibitor paclobutrazol caused millet seedlings to have a prolonged growth inhibition response when ethylene was applied. A transient ethylene growth inhibition response has previously been reported for Arabidopsis ethylene insensitive3-1 (ein3-1) eil1-1 double mutants. Paclobutrazol caused these mutants to have a prolonged response to ethylene, whereas constitutive GA signaling in this background eliminated ethylene responses. Sensitivity to paclobutrazol inversely correlated with the levels of EIN3 in Arabidopsis. Wild-type Arabidopsis seedlings treated with paclobutrazol and mutants deficient in GA levels or signaling had a delayed growth recovery after ethylene removal. It is interesting to note that ethylene caused alterations in gene expression that are predicted to increase GA levels in the ein3-1 eil1-1 seedlings. These results indicate that ethylene affects GA levels leading to modulation of ethylene growth inhibition kinetics.

Ethylglyoxal bis(guanylhydrazone) as an inhibitor of polyamine biosynthesis in L1210 leukemia cells.

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Seppänen, P; Ruohola, H; Jänne, J

1984-04-16

Ethylglyoxal bis(guanylhydrazone), a close derivative of the known anti-cancer drug methylglyoxal bis(guanylhydrazone), is also a powerful inhibitor of S-adenosylmethionine decarboxylase (EC 4.1.1.50), the enzyme needed for the synthesis of spermidine and spermine. There were, however, marked differences between the ethyl and methyl derivatives of glyoxal bis(guanylhydrazone) when tested in cultured L1210 cells. The cellular accumulation of ethylglyoxal bis(guanylhydrazone) represented only a fraction (20-25%) of that of the methyl derivative. Moreover, polyamine depletion, which is known to strikingly stimulate the uptake of methylglyoxal bis(guanylhydrazone), decreased, if anything, the uptake of ethylglyoxal bis(guanylhydrazone) by L1210 cells. The compound produced spermidine and spermine depletion fully comparable to that achieved with methylglyoxal bis(guanylhydrazone) at micromolar concentrations. Ethylglyoxal bis(guanylhydrazone) was growth-inhibitory to L1210 cells and produced an additive antiproliferative action when used together with 2-difluoromethylornithine. Ethylglyoxal bis(guanylhydrazone) was distinctly less effective than methylglyoxal bis(guanylhydrazone) in releasing bound polyamines from isolated cell organelles in vitro. Ethylglyoxal bis(guanylhydrazone) was also devoid of the early and profound mitochondrial toxicity typical to methylglyoxal bis(guanylhydrazone). These findings may indicate that this compound is a more specific inhibitor of polyamine biosynthesis with less intracellular polyamine 'receptor-site' activity than methylglyoxal bis(guanylhydrazone).

Mechanism of fluridone-induced seed germination of cistanche tubulosa

International Nuclear Information System (INIS)

Chen, Q.L.; Tu, P.

2016-01-01

Our previous study disclosed that fluridone, a synthesis inhibitor of abscisic acid (ABA), could stimulate seed germination in the holoparasitic plant Cistanche tubulosa . Nonetheless, the underlying mechanisms have not been thoroughly elucidated. In the present study, an attempt was made to reveal the mechanism of fluridone breaking seed dormancy in C. tubulosa and to determine the contribution of hormones in this process. The ABA level in seeds initially decreased following fluridone treatment and was subsequently maintained at a concentration of 31 ng g/sup -1/ DW (dry weight) three days later. The contents of gibberellins (GAs) initially in creased and subsequently were maintained at a level of 40 ng g-1 DW after ten days. However, the increment of seed germination induced by fluridone was inhibited after the introduction of exogenous ABA or paclobutrazol (a synthesis inhibitor of GAs). Furthermore, inhibition from paclobutrazol was reversed by an additional treatment with exogenous GA3. When the ratio of endogenous GAs to ABA reached 4:3, C. tubulosa seeds initiated germination. By contrast, although the ratio of endogenous GAs to ABA content reached 2:1 by cold stratification, C. tubulosa seeds could not germinate unless exogenous GA3 was added. In summary, our current study revealed that (i) GAs and ABA play key roles for the seed germination of C. tubulosa , (ii) fluridone inhibited ABA biosynthesis but increased the concentration of GAs in seeds, and (iii) fluridone might initiate other processes associated with germination. (author)

Enhancement of misonidazole radiosensitization by an inhibitor of glutathione biosynthesis

International Nuclear Information System (INIS)

Hodgkiss, R.J.; Middleton, R.W.

1983-01-01

A well known inhibitor of glutathione biosynthesis, buthione sulphoximine (S-n-butyl homocysteine sulphoximine, BSO) depletes non-protein sulphydryls (NPSH) in Chinese hamster cells in vitro, resulting in a marked increase in the radiosensitization efficiency of misonidazole. V79 379A Chinese hamster cells were maintained in suspension cultures and irradiated in monolayers using 250 kVp X-rays at a dose rate of 3.93 Gy/min. Radiosensitization by misonidazole alone gave results within 0.1 sensitizer enhancement ratio (s.e.r.) of the curve reported by Watts et al. (1980). GSH (2 mmol dm - 3 ) added to the extracellular medium resulted in a marked decrease in the radiosensitization efficiency of misonidazole, eliminating the effect at 0.1 mmol dm - 3 misonidazole (s.e.r. = 1.0 relative to nitrogen control). A marked enhancement of the radiosensitization by misonidazole was observed when the cells had been incubated with BSO (0.1 mmol dm - 3 ). BSO alone at this concentration gave s.e.r. = 1.17; misonidazole alone (0.1 mmol dm - 3 ) gave s.e.r. = 1.18 and misonidazole with BSO (both 0.1. mmol dm - 3 ) gave s.e.r. = 1.9. The BSO treatment gave little effect in aerated cells. The concentration of BSO needed to produce these effects in vitro is ca. 40-fold lower than doses tolerated by mice in repeated administrations. (U.K.)

Residues of 14C-paclobutrazol in mangoes

International Nuclear Information System (INIS)

Costa, Maria A.; Tornisielo, Valdemar L.; Castanho, Giuliane M.

2009-01-01

Paclobutrazol (PBZ) is a growth regulator used in agricultural systems whose purpose is the control of vegetative growth, stimulating the reproductive capacity of plants. This growth regulator remains active in soil for a long time and its half-life varies with the type of soil and climatic conditions, can severely affect the development of crops. This work aimed to study the residues / metabolites of 14 C-PBZ in mango pulp Tommy Atkins. The tests were performed with mangoes grown in pots stainless steel and application of 14 C-PBZ was performed by the soil projection of the crown, and the mangoes tested in two periods, one year and two years after application. To evaluate the levels of residues of 14 C-PBZ was realize the burning of 200 mg of pulp on biological oxidized and detached 14 CO 2 was detected by liquid scintillation spectrophotometer. The results were 1.65 % of residue of PBZ on fruit collected after two years of application and 4.30 % of residue of PBZ collected on fruit after a year of application and also can see that the product remained in the soil for more than one year, is translocated to the plant and reach the edible part, the pulp fruit. The identification of residual 14 C- PBZ/metabolites by thin-layer chromatography did not reveal any pattern of PBZ / metabolites due to the low activity detected in the samples. Therefore, another procedure was performed for extraction and then analyzed by high performance liquid chromatography (HPLC) for detection of metabolites in the PBZ of mango pulp. (author)

Exogenous GA3 Application Enhances Xylem Development and Induces the Expression of Secondary Wall Biosynthesis Related Genes in Betula platyphylla

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Huiyan Guo

2015-09-01

Full Text Available Gibberellin (GA is a key signal molecule inducing differentiation of tracheary elements, fibers, and xylogenesis. However the molecular mechanisms underlying the effect of GA on xylem elongation and secondary wall development in tree species remain to be determined. In this study, Betula platyphylla (birch seeds were treated with 300 ppm GA3 and/or 300 ppm paclobutrazol (PAC, seed germination was recorded, and transverse sections of hypocotyls were stained with toluidine blue; the two-month-old seedlings were treated with 50 μM GA3 and/or 50 μM PAC, transverse sections of seedling stems were stained using phloroglucinol–HCl, and secondary wall biosynthesis related genes expression was analyzed by real-time quantitative PCR. Results indicated that germination percentage, energy and time of seeds, hypocotyl height and seedling fresh weight were enhanced by GA3, and reduced by PAC; the xylem development was wider in GA3-treated plants than in the control; the expression of NAC and MYB transcription factors, CESA, PAL, and GA oxidase was up-regulated during GA3 treatment, suggesting their role in GA3-induced xylem development in the birch. Our results suggest that GA3 induces the expression of secondary wall biosynthesis related genes to trigger xylogenesis in the birch plants.

Stable Analogues of OSB-AMP: Potent Inhibitors of MenE the o-succinylbenzoate-CoA Synthetase from Bacterial Menaquinone Biosynthesis

Energy Technology Data Exchange (ETDEWEB)

Lu X.; Swaminathan S.; Zhou R.; Sharma I.; Li X.; Kumar G.; Tonge P. J.; Tan D. S.

2012-01-02

MenE, the o-succinylbenzoate (OSB)-CoA synthetase from bacterial menaquinone biosynthesis, is a promising new antibacterial target. Sulfonyladenosine analogues of the cognate reaction intermediate, OSB-AMP, have been developed as inhibitors of the MenE enzymes from Mycobacterium tuberculosis (mtMenE), Staphylococcus aureus (saMenE) and Escherichia coli (ecMenE). Both a free carboxylate and a ketone moiety on the OSB side chain are required for potent inhibitory activity. OSB-AMS (4) is a competitive inhibitor of mtMenE with respect to ATP (K{sub i} = 5.4 {+-} 0.1 nM) and a noncompetitive inhibitor with respect to OSB (K{sub i} = 11.2 {+-} 0.9 nM). These data are consistent with a Bi Uni Uni Bi Ping-Pong kinetic mechanism for these enzymes. In addition, OSB-AMS inhibits saMenE with K{sub i}{sup app} = 22 {+-} 8 nM and ecMenE with K{sub i}{sup OSB} = 128 {+-} 5 nM. Putative active-site residues, Arg222, which may interact with the OSB aromatic carboxylate, and Ser302, which may bind the OSB ketone oxygen, have been identified through computational docking of OSB-AMP with the unliganded crystal structure of saMenE. A pH-dependent interconversion of the free keto acid and lactol forms of the inhibitors is also described, along with implications for inhibitor design.

Synergism between demethylation inhibitor fungicides or gibberellin inhibitor plant growth regulators and bifenthrin in a pyrethroid-resistant population of Listronotus maculicollis (Coleoptera: Curculionidae).

Science.gov (United States)

Ramoutar, D; Cowles, R S; Requintina, E; Alm, S R

2010-10-01

In 2007-2008, the "annual bluegrass weevil," Listronotus maculicollis Kirby (Coleoptera: Curculionidae), a serious pest of Poa annua L. (Poales: Poaceae) on U.S. golf courses, was shown to be resistant to two pyrethroids, bifenthrin and lambda-cyhalothrin. In 2008, we showed that bifenthrin resistance was principally mediated by oxidase detoxification (cytochrome P450 [P450]). P450s can be inhibited by demethylation inhibitor fungicides and gibberellin inhibitor plant growth regulators, both of which are commonly used on golf courses. We tested these compounds for synergistic activity with bifenthin against a pyrethroid-resistant population of L. maculicollis. The LD50 value for bifenthrin was significantly reduced from 87 ng per insect (without synergists) to 9.6-40 ng per insect after exposure to the fungicides fenarimol, fenpropimorph, prochloraz, propiconazole, and pyrifenox and the plant growth regulators flurprimidol, paclobutrazol, and trinexapac-ethyl. Simulated field exposure with formulated products registered for use on turf revealed enhanced mortality when adult weevils were exposed to bifenthrin (25% mortality, presented alone) combined with field dosages of propiconizole, fenarimol, flurprimidol, or trinexapac-ethyl (range, 49-70% mortality).

Phage display-derived inhibitor of the essential cell wall biosynthesis enzyme MurF

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Blewett Ann

2008-12-01

Full Text Available Abstract Background To develop antibacterial agents having novel modes of action against bacterial cell wall biosynthesis, we targeted the essential MurF enzyme of the antibiotic resistant pathogen Pseudomonas aeruginosa. MurF catalyzes the formation of a peptide bond between D-Alanyl-D-Alanine (D-Ala-D-Ala and the cell wall precursor uridine 5'-diphosphoryl N-acetylmuramoyl-L-alanyl-D-glutamyl-meso-diaminopimelic acid (UDP-MurNAc-Ala-Glu-meso-A2pm with the concomitant hydrolysis of ATP to ADP and inorganic phosphate, yielding UDP-N-acetylmuramyl-pentapeptide. As MurF acts on a dipeptide, we exploited a phage display approach to identify peptide ligands having high binding affinities for the enzyme. Results Screening of a phage display 12-mer library using purified P. aeruginosa MurF yielded to the identification of the MurFp1 peptide. The MurF substrate UDP-MurNAc-Ala-Glumeso-A2pm was synthesized and used to develop a sensitive spectrophotometric assay to quantify MurF kinetics and inhibition. MurFp1 acted as a weak, time-dependent inhibitor of MurF activity but was a potent inhibitor when MurF was pre-incubated with UDP-MurNAc-Ala-Glu-meso-A2pm or ATP. In contrast, adding the substrate D-Ala-D-Ala during the pre-incubation nullified the inhibition. The IC50 value of MurFp1 was evaluated at 250 μM, and the Ki was established at 420 μM with respect to the mixed type of inhibition against D-Ala-D-Ala. Conclusion MurFp1 exerts its inhibitory action by interfering with the utilization of D-Ala-D-Ala by the MurF amide ligase enzyme. We propose that MurFp1 exploits UDP-MurNAc-Ala-Glu-meso-A2pm-induced structural changes for better interaction with the enzyme. We present the first peptide inhibitor of MurF, an enzyme that should be exploited as a target for antimicrobial drug development.

Stable analogues of OSB-AMP: potent inhibitors of MenE, the o-succinylbenzoate-CoA synthetase from bacterial menaquinone biosynthesis.

Science.gov (United States)

Lu, Xuequan; Zhou, Rong; Sharma, Indrajeet; Li, Xiaokai; Kumar, Gyanendra; Swaminathan, Subramanyam; Tonge, Peter J; Tan, Derek S

2012-01-02

MenE, the o-succinylbenzoate (OSB)-CoA synthetase from bacterial menaquinone biosynthesis, is a promising new antibacterial target. Sulfonyladenosine analogues of the cognate reaction intermediate, OSB-AMP, have been developed as inhibitors of the MenE enzymes from Mycobacterium tuberculosis (mtMenE), Staphylococcus aureus (saMenE) and Escherichia coli (ecMenE). Both a free carboxylate and a ketone moiety on the OSB side chain are required for potent inhibitory activity. OSB-AMS (4) is a competitive inhibitor of mtMenE with respect to ATP (K(i) =5.4±0.1 nM) and a noncompetitive inhibitor with respect to OSB (K(i) =11.2±0.9 nM). These data are consistent with a Bi Uni Uni Bi Ping-Pong kinetic mechanism for these enzymes. In addition, OSB-AMS inhibits saMenE with K(i)(app) =22±8 nM and ecMenE with K(i)(OSB) =128±5 nM. Putative active-site residues, Arg222, which may interact with the OSB aromatic carboxylate, and Ser302, which may bind the OSB ketone oxygen, have been identified through computational docking of OSB-AMP with the unliganded crystal structure of saMenE. A pH-dependent interconversion of the free keto acid and lactol forms of the inhibitors is also described, along with implications for inhibitor design. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structure-activity relationship of prenyl-substituted polyphenols from Artocarpus heterophyllus as inhibitors of melanin biosynthesis in cultured melanoma cells.

Science.gov (United States)

Arung, Enos Tangke; Shimizu, Kuniyoshi; Kondo, Ryuichiro

2007-09-01

A series of prenylated, flavone-based polyphenols, compounds 1-8, were isolated from the wood of Artocarpus heterophyllus. These compounds, which have previously been shown not to inhibit tyrosinase activity, were found to be active inhibitors of the in vivo melanin biosynthesis in B16 melanoma cells, with little or no cytotoxicity. To clarify the structural requirement for inhibition, some structure-activity relationships were studied, in comparison with related compounds lacking prenyl side chains. Our experiments indicate that both prenyl and OH groups, as well as the type of substitution pattern, are crucial for the inhibition of melanin production in B16 melanoma cells.

Hinokitiol Enhanced Vegetative Growth Parameters of Tomato cv. �Falkato� Compared with Salicylic Acid and Paclobutrazol under In Vitro Salinity Condition

Directory of Open Access Journals (Sweden)

Behrooz ESMAEILPOUR

2012-02-01

Full Text Available The aim of this study was to assess the potential in vitro effect of hinokitiol on improvement of tomato seedling resistance to salinity stress. Effect of hinokitiol was compared with two anti-stress compounds, salicylic acid and paclobutrazol. Leaf numbers, shoot and root fresh weight and root fresh weight were recorded after about 8 weeks. Salt stress was accomplished by application of two levels of pure NaCl (50 and 100 mM on MS basal medium. The treatments consisted of different concentrations of hinokitiol (0, 1, 5 and 10 ppm, paclobutrazol (0, 1, 2 and 4 ?M and salicylic acid (0, 0.01, 0.1 and 1 mM. Results revealed that salinity blocked seed germination in media containing only 100 mM of pure NaCl without any treatment. In general all three compounds increased tomato seedling growth, indicating these compounds are able to alleviate the negative effect of salinity on tomato plants. However, Hinokitiol was the most efficient compound. Compared with SA, application of hinokitiol significantly increased leaf numbers, shoot length and shoot and root dry weight. Also, media containing different concentrations of hinokitiol produced higher root and shoot fresh weight than control and other treatments. Future physiological studies are needed to clarify the mechanism of induction of salt tolerance activity by hinokitiol.

Paclobutrazol no florescimento e na produção da mangueira 'Tommy Atkins' Paclobutrazol on flowering and production of 'Tommy Atkins' mango

Directory of Open Access Journals (Sweden)

Nelson Fonseca

2004-08-01

Full Text Available Técnicas eficientes de controle da época de produção são importantes para o sucesso econômico no cultivo de mangueira (Mangifera indica L.. Neste estudo, avaliaram-se o florescimento e a produção da mangueira 'Tommy Atkins' em resposta à aplicação de paclobutrazol (PBZ no solo (2 mL do i.a./planta, e foliar em diferentes doses (0,5, 1, 1,5 e 2 mL do i.a. em aplicação única e em duas aplicações sob condições de cultivo irrigado, na região semi-árida do Nordeste do Brasil. As variáveis estudadas foram percentual de florescimento, número de frutos e produção por planta, sendo avaliadas em três épocas após a aplicação do regulador de crescimento (68 a 110, 131 a 179 e 190 a 216 dias após para o florescimento da planta; e 162 a 203, 256 a 287 e 327 a 337 para o número e produção de frutos por planta. O PBZ aplicado no solo determinou percentuais de florescimento, número de frutos e produção por planta superiores à testemunha (água foliar e aos tratamentos com PBZ foliar, além de antecipar o florescimento das plantas. O PBZ foliar, independente do número de aplicação e doses usadas, não superou significativamente a testemunha. O PBZ aplicado no solo antecipou o florescimento em 23 dias, em relação à testemunha e aos tratamentos com PBZ foliar, na segunda época de avaliação.Efficient techniques for controlling the production time are important for the economical success in the mango cultivation. In this study, it was evaluated the flowering and production of mango fruit, variety Tommy Atkins, in response to the paclobutrazol application (PBZ, either in the soil (2 mL of the active ingredient per plant, or on foliage at different doses (0.5, 1, 1.5 and 2 mL of a.i. per plant in single or two applications, under conditions of irrigated cultivation, in the semi-arid area of Northeastern of Brazil. The studied variables were percentile flowering, number of fruits, and production per plant, being appraised in

Residues of {sup 14}C-paclobutrazol in mangoes

Energy Technology Data Exchange (ETDEWEB)

Costa, Maria A.; Tornisielo, Valdemar L.; Castanho, Giuliane M., E-mail: macosta@cena.usp.b [Centro de Energia Nuclear na Agricultura (CENA/USP), Piracicaba, SP (Brazil). Lab. de Ecotoxicologia

2009-07-01

Paclobutrazol (PBZ) is a growth regulator used in agricultural systems whose purpose is the control of vegetative growth, stimulating the reproductive capacity of plants. This growth regulator remains active in soil for a long time and its half-life varies with the type of soil and climatic conditions, can severely affect the development of crops. This work aimed to study the residues / metabolites of {sup 14}C-PBZ in mango pulp Tommy Atkins. The tests were performed with mangoes grown in pots stainless steel and application of {sup 14}C-PBZ was performed by the soil projection of the crown, and the mangoes tested in two periods, one year and two years after application. To evaluate the levels of residues of {sup 14}C-PBZ was realize the burning of 200 mg of pulp on biological oxidized and detached {sup 14}CO{sub 2} was detected by liquid scintillation spectrophotometer. The results were 1.65 % of residue of PBZ on fruit collected after two years of application and 4.30 % of residue of PBZ collected on fruit after a year of application and also can see that the product remained in the soil for more than one year, is translocated to the plant and reach the edible part, the pulp fruit. The identification of residual {sup 14}C- PBZ/metabolites by thin-layer chromatography did not reveal any pattern of PBZ / metabolites due to the low activity detected in the samples. Therefore, another procedure was performed for extraction and then analyzed by high performance liquid chromatography (HPLC) for detection of metabolites in the PBZ of mango pulp. (author)

Biosynthesis of intestinal microvillar proteins. Processing of N-linked carbohydrate is not required for surface expression

DEFF Research Database (Denmark)

Danielsen, Erik Michael; Cowell, G M

1986-01-01

Castanospermine, an inhibitor of glucosidase I, the initial enzyme in the trimming of N-linked carbohydrate, was used to study the importance of carbohydrate processing in the biosynthesis of microvillar enzymes in organ-cultured pig intestinal explants. For aminopeptidase N (EC 3.4.11.2), aminop......Castanospermine, an inhibitor of glucosidase I, the initial enzyme in the trimming of N-linked carbohydrate, was used to study the importance of carbohydrate processing in the biosynthesis of microvillar enzymes in organ-cultured pig intestinal explants. For aminopeptidase N (EC 3...

Chemogenomics profiling of drug targets of peptidoglycan biosynthesis pathway in Leptospira interrogans by virtual screening approaches.

Science.gov (United States)

Bhattacharjee, Biplab; Simon, Rose Mary; Gangadharaiah, Chaithra; Karunakar, Prashantha

2013-06-28

Leptospirosis is a worldwide zoonosis of global concern caused by Leptospira interrogans. The availability of ligand libraries has facilitated the search for novel drug targets using chemogenomics approaches, compared with the traditional method of drug discovery, which is time consuming and yields few leads with little intracellular information for guiding target selection. Recent subtractive genomics studies have revealed the putative drug targets in peptidoglycan biosynthesis pathways in Leptospira interrogans. Aligand library for the murD ligase enzyme in the peptidoglycan pathway has also been identified. Our approach in this research involves screening of the pre-existing ligand library of murD with related protein family members in the putative drug target assembly in the peptidoglycan biosynthesis pathway. A chemogenomics approach has been implemented here, which involves screening of known ligands of a protein family having analogous domain architecture for identification of leads for existing druggable protein family members. By means of this approach, one murC and one murF inhibitor were identified, providing a platform for developing an antileptospirosis drug targeting the peptidoglycan biosynthesis pathway. Given that the peptidoglycan biosynthesis pathway is exclusive to bacteria, the in silico identified mur ligase inhibitors are expected to be broad-spectrum Gram-negative inhibitors if synthesized and tested in in vitro and in vivo assays.

Almacenamiento en frío de los bulbos y uso de paclobutrazol para producir tulipán en macetas Bulb cold storage and use of paclobutrazol for pot tulip production

Directory of Open Access Journals (Sweden)

N. Francescangeli

2007-06-01

Full Text Available Se compararon los efectos del paclobutrazol (PBZ y del tiempo de almacenamiento en frío de los bulbos (TA sobre características comerciales de tulipán Tulipa gesneriana L. (Leen van der Mark en maceta. Se evaluaron las concentraciones de PBZ: 0 y 5 ppm aplicadas por inmersión de bulbos durante 24 horas, previo al transplante, y los TA a 5 °C: 5, 6, 7, 8, 9, 10, 11 y 12 semanas. La temperatura promedio del aire de los ocho ciclos fue: 15 a 15,7 °C. El PBZ no afectó la longitud del ciclo, y los TA explicaron el 83% de la variabilidad en la duración del período transplante- momento de la venta, con una relación inversa. El PBZ no afectó el tamaño de la flor. La altura en el momento de la venta y a fin de floración mostró interacción de los tratamientos: a mayor TA aumentó la altura en todos los casos, pero este incremento fue menor en las plantas tratadas con PBZ. Sin utilizar PBZ y con bulbos de 5 a 8 semanas de almacenamiento fue posible producir plantas comerciales de altura equivalente o menor a las obtenidas con bulbos de 12 semanas de almacenamiento tratados con PBZ a 5 ppm por inmersión durante 24 horas.The effect of bulb cold storage time (ST and paclobutrazol (PBZ on commercial characteristics of pot tulips Tulipa gesneriana L. (Leen van der Mark was tested. PBZ concentrations (0 and 5 ppm applied by dipping bulbs for 24 hours before transplanting and 5 °C ST (5, 6, 7, 8, 9, 10, 11, and 12 weeks were evaluated. Mean air temperature throughout the experiment remained between 15 and 15.7 °C. PBZ did not affect the duration of the cycle. ST explained 83% of the variation in the duration of the period transplant-sale time, and the relationship was inverse. PBZ did not affect flower size. There was a significant interaction between treatments for plant height at sale time and at the end of flowering: plant height increased with ST in all cases, but the increase was lower in the PBZ treated plants. Without PBZ and with bulbs

Tomato strigolactones are derived from carotenoids and their biosynthesis is promoted by phosphate starvation

OpenAIRE

López-Ráez, Juan A.; Charnikhova, Tatsiana;; Gómez-Roldán,Victoria;; Matusova, Radoslava;; Kohlen, Wouter;; De Vos, Ric;; Verstappe, Francel;; Puech-Pages, Virginie;; Bécard, Guillaume;; Mulder, Patrick;; Bouwmeester, Harro;

2008-01-01

Strigolactones are rhizosphere signalling compounds that mediate host location in arbuscular mycorrhizal (AM) fungi and parasitic plants. Here, the regulation of the biosynthesis of strigolactones is studied in tomato (Solanum lycopersicum). * Strigolactone production under phosphate starvation, in the presence of the carotenoid biosynthesis inhibitor fluridone and in the abscisic acid (ABA) mutant notabilis were assessed using a germination bioassay with seeds of Orobanche ramosa; a hyphal b...

Involvement of a lipoxygenase-like enzyme in abscisic Acid biosynthesis.

Science.gov (United States)

Creelman, R A; Bell, E; Mullet, J E

1992-07-01

Several lines of evidence indicate that abscisic acid (ABA) is derived from 9'-cis-neoxanthin or 9'-cis-violaxanthin with xanthoxin as an intermediate. (18)O-labeling experiments show incorporation primarily into the side chain carboxyl group of ABA, suggesting that oxidative cleavage occurs at the 11, 12 (11', 12') double bond of xanthophylls. Carbon monoxide, a strong inhibitor of heme-containing P-450 monooxygenases, did not inhibit ABA accumulation, suggesting that the oxygenase catalyzing the carotenoid cleavage step did not contain heme. This observation, plus the ability of lipoxygenase to make xanthoxin from violaxanthin, suggested that a lipoxygenase-like enzyme is involved in ABA biosynthesis. To test this idea, the ability of several soybean (Glycine max L.) lipoxygenase inhibitors (5,8,11-eicosatriynoic acid, 5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid, and naproxen) to inhibit stress-induced ABA accumulation in soybean cell culture and soybean seedlings was determined. All lipoxygenase inhibitors significantly inhibited ABA accumulation in response to stress. These results suggest that the in vivo oxidative cleavage reaction involved in ABA biosynthesis requires activity of a nonheme oxygenase having lipoxygenase-like properties.

Triterpenoid biosynthesis in Euphorbia lathyris latex

International Nuclear Information System (INIS)

Hawkins, D.R.

1987-11-01

The structures of triterpenols, not previously been known, from Euphorbia lathyris latex are reported. A method for quantifying very small amounts of these compounds was developed. Concerning the biochemistry of the latex, no exogenous cofactors were required for the biosynthesis and the addition of compounds such as NADPAH and ATP do not stimulate the biosynthesis. The addition of DTE or a similar anti-oxidant was found to help reduce the oxidation of the latex, thus increasing the length of time that the latex remains active. The requirement of a divalent cation and the preference for Mn in the pellet was observed. The effect of several inhibitors on the biosynthesis of the triterpenoids was examined. Mevinolin was found to inhibit the biosynthesis of the triterpenoids from acetate, but not mevalonate. A dixon plot of the inhibition of acetate incorporation showed an I 50 concentration of 3.2 μM. Fenpropimorph was found to have little or no effect on the biosynthesis. Tridemorph was found to inhibit the biosynthesis of all of the triterpenoids with an I 50 of 4 μM. It was also observed that the cyclopropyl containing triterpenols, cycloartenol and 24-methylenecycloartenol were inhibited much more strongly than those containing an 8-9 double bond, lanosterol and 24-methylenelanosterol. The evidence indicates, but does not definetely prove, that lanosterol and 24-methylenelanosterol are not made from cycloartenol and 24-methylenecycloartenol via a ring-opening enzyme such as cycloeucalenol-obtusifoliol isomerase. The possibilty that cycloartenol is made via lanosterol was investigated by synthesizing 4-R-4- 3 H-mevalonic acid and incubating latex with a mixture of this and 14 C-mevalonic acid. From the 3 H/ 14 C ratio it was shown that cycloartenol and 24-methylenecycloartenol are not made via an intermediate containing as 8-9 double bond. 88 refs., 15 figs., 30 tabs

Peroxidase enzymes regulate collagen extracellular matrix biosynthesis.

Science.gov (United States)

DeNichilo, Mark O; Panagopoulos, Vasilios; Rayner, Timothy E; Borowicz, Romana A; Greenwood, John E; Evdokiou, Andreas

2015-05-01

Myeloperoxidase and eosinophil peroxidase are heme-containing enzymes often physically associated with fibrotic tissue and cancer in various organs, without any direct involvement in promoting fibroblast recruitment and extracellular matrix (ECM) biosynthesis at these sites. We report herein novel findings that show peroxidase enzymes possess a well-conserved profibrogenic capacity to stimulate the migration of fibroblastic cells and promote their ability to secrete collagenous proteins to generate a functional ECM both in vitro and in vivo. Mechanistic studies conducted using cultured fibroblasts show that these cells are capable of rapidly binding and internalizing both myeloperoxidase and eosinophil peroxidase. Peroxidase enzymes stimulate collagen biosynthesis at a post-translational level in a prolyl 4-hydroxylase-dependent manner that does not require ascorbic acid. This response was blocked by the irreversible myeloperoxidase inhibitor 4-amino-benzoic acid hydrazide, indicating peroxidase catalytic activity is essential for collagen biosynthesis. These results suggest that peroxidase enzymes, such as myeloperoxidase and eosinophil peroxidase, may play a fundamental role in regulating the recruitment of fibroblast and the biosynthesis of collagen ECM at sites of normal tissue repair and fibrosis, with enormous implications for many disease states where infiltrating inflammatory cells deposit peroxidases. Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Biosynthesis of collagen by fibroblasts kept in culture

International Nuclear Information System (INIS)

Machado-Santelli, G.M.

1978-01-01

The sinthesis of collagen is studied in fibroblasts of different origins with the purpose of obtaining an appropriate system for the study of its biosynthesis and processing. The percentage of collagen synthesis vary according to the fibroblast origin. Experiences are performed with fibroblasts kept in culture from: chicken - and guinea pig embryos, carragheenin - induced granulomas in adult guinea pig and from human skin. The collagen pattern synthesized after acetic acid - or saline extractions in the presence of inhibitors is also determined. This pattern is then assayed by poliacrilamide - 5% - SDS gel electrophoresis accompanied by fluorography. The importance of the cell culture system in the elucidation of collagen biosynthesis is pointed out. (M.A.) [pt

Analysis of the Staphylococcus aureus capsule biosynthesis pathway in vitro: characterization of the UDP-GlcNAc C6 dehydratases CapD and CapE and identification of enzyme inhibitors.

Science.gov (United States)

Li, Wenjin; Ulm, Hannah; Rausch, Marvin; Li, Xue; O'Riordan, Katie; Lee, Jean C; Schneider, Tanja; Müller, Christa E

2014-11-01

Polysaccharide capsules significantly contribute to virulence of invasive pathogens, and inhibition of capsule biosynthesis may offer a valuable strategy for novel anti-infective treatment. We purified and characterized the enzymes CapD and CapE of the Staphylococcus aureus serotype 5 biosynthesis cluster, which catalyze the first steps in the synthesis of the soluble capsule precursors UDP-D-FucNAc and UDP-L-FucNAc, respectively. CapD is an integral membrane protein and was obtained for the first time in a purified, active form. A capillary electrophoresis (CE)-based method applying micellar electrokinetic chromatography (MEKC) coupled with UV detection at 260 nm was developed for functional characterization of the enzymes using a fused-silica capillary, electrokinetic injection, and dynamic coating with polybrene at pH 12.4. The limits of detection for the CapD and CapE products UDP-2-acetamido-2,6-dideoxy-α-D-xylo-hex-4-ulose and UDP-2-acetamido-2,6-dideoxy-β-L-arabino-hex-4-ulose, respectively, were below 1 μM. Using this new, robust and sensitive method we performed kinetic studies for CapD and CapE and screened a compound library in search for enzyme inhibitors. Several active compounds were identified and characterized, including suramin (IC50 at CapE 1.82 μM) and ampicillin (IC50 at CapD 40.1 μM). Furthermore, the cell wall precursors UDP-D-MurNAc-pentapeptide and lipid II appear to function as inhibitors of CapD enzymatic activity, suggesting an integrated mechanism of regulation for cell envelope biosynthesis pathways in S. aureus. Corroborating the in vitro findings, staphylococcal cells grown in the presence of subinhibitory concentrations of ampicillin displayed drastically reduced CP production. Our studies contribute to a profound understanding of the capsule biosynthesis in pathogenic bacteria. This approach may lead to the identification of novel anti-virulence and antibiotic drugs. Copyright © 2014 Elsevier GmbH. All rights reserved.

Novel guanidine-based inhibitors of inosine monophosphate dehydrogenase.

Science.gov (United States)

Iwanowicz, Edwin J; Watterson, Scott H; Liu, Chunjian; Gu, Henry H; Mitt, Toomas; Leftheris, Katerina; Barrish, Joel C; Fleener, Catherine A; Rouleau, Katherine; Sherbina, N Z; Hollenbaugh, Diane L

2002-10-21

A series of novel guanidine-based small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH) was explored. IMPDH catalyzes the rate determining step in guanine nucleotide biosynthesis and is a target for anticancer, immunosuppressive and antiviral therapy. The synthesis and the structure-activity relationships (SARs), derived from in vitro studies, for this new series of inhibitors is given.

Involvement of a Lipoxygenase-Like Enzyme in Abscisic Acid Biosynthesis 1

Science.gov (United States)

Creelman, Robert A.; Bell, Erin; Mullet, John E.

1992-01-01

Several lines of evidence indicate that abscisic acid (ABA) is derived from 9′-cis-neoxanthin or 9′-cis-violaxanthin with xanthoxin as an intermediate. 18O-labeling experiments show incorporation primarily into the side chain carboxyl group of ABA, suggesting that oxidative cleavage occurs at the 11, 12 (11′, 12′) double bond of xanthophylls. Carbon monoxide, a strong inhibitor of heme-containing P-450 monooxygenases, did not inhibit ABA accumulation, suggesting that the oxygenase catalyzing the carotenoid cleavage step did not contain heme. This observation, plus the ability of lipoxygenase to make xanthoxin from violaxanthin, suggested that a lipoxygenase-like enzyme is involved in ABA biosynthesis. To test this idea, the ability of several soybean (Glycine max L.) lipoxygenase inhibitors (5,8,11-eicosatriynoic acid, 5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid, and naproxen) to inhibit stress-induced ABA accumulation in soybean cell culture and soybean seedlings was determined. All lipoxygenase inhibitors significantly inhibited ABA accumulation in response to stress. These results suggest that the in vivo oxidative cleavage reaction involved in ABA biosynthesis requires activity of a nonheme oxygenase having lipoxygenase-like properties. PMID:16668998

Inhibition of glycolipid biosynthesis by N-(5-adamantane-1-yl-methoxy-pentyl)-deoxynojirimycin protects against the inflammatory response in hapten-induced colitis

NARCIS (Netherlands)

Shen, Chong; Bullens, Dominique; Kasran, Ahmad; Maerten, Philippe; Boon, Louis; Aerts, Johannes M. F. G.; van Assche, Gert; Geboes, Karel; Rutgeerts, Paul; Ceuppens, Jan L.

2004-01-01

Since glycolipid biosynthesis is potentially involved in immunological and inflammatory responses, we tested the effect of a novel inhibitor of intracellular glycolipid biosynthesis N-(5-adamantane-1-yl-methoxy-pentyl)-deoxynojirimycin (AMP-DNM) in two hapten-induced colitis models: trinitrobenzene

Triterpenoid biosynthesis in Euphorbia lathyris latex

Energy Technology Data Exchange (ETDEWEB)

Hawkins, D.R.

1987-11-01

The structures of triterpenols, not previously been known, from Euphorbia lathyris latex are reported. A method for quantifying very small amounts of these compounds was developed. Concerning the biochemistry of the latex, no exogenous cofactors were required for the biosynthesis and the addition of compounds such as NADPAH and ATP do not stimulate the biosynthesis. The addition of DTE or a similar anti-oxidant was found to help reduce the oxidation of the latex, thus increasing the length of time that the latex remains active. The requirement of a divalent cation and the preference for Mn in the pellet was observed. The effect of several inhibitors on the biosynthesis of the triterpenoids was examined. Mevinolin was found to inhibit the biosynthesis of the triterpenoids from acetate, but not mevalonate. A dixon plot of the inhibition of acetate incorporation showed an I/sub 50/ concentration of 3.2 ..mu..M. Fenpropimorph was found to have little or no effect on the biosynthesis. Tridemorph was found to inhibit the biosynthesis of all of the triterpenoids with an I/sub 50/ of 4 ..mu..M. It was also observed that the cyclopropyl containing triterpenols, cycloartenol and 24-methylenecycloartenol were inhibited much more strongly than those containing an 8-9 double bond, lanosterol and 24-methylenelanosterol. The evidence indicates, but does not definetely prove, that lanosterol and 24-methylenelanosterol are not made from cycloartenol and 24-methylenecycloartenol via a ring-opening enzyme such as cycloeucalenol-obtusifoliol isomerase. The possibilty that cycloartenol is made via lanosterol was investigated by synthesizing 4-R-4-/sup 3/H-mevalonic acid and incubating latex with a mixture of this and /sup 14/C-mevalonic acid. From the /sup 3/H//sup 14/C ratio it was shown that cycloartenol and 24-methylenecycloartenol are not made via an intermediate containing as 8-9 double bond. 88 refs., 15 figs., 30 tabs.

Alpha-Difluoromethylornithine, an Irreversible Inhibitor of Polyamine Biosynthesis, as a Therapeutic Strategy against Hyperproliferative and Infectious Diseases

Directory of Open Access Journals (Sweden)

Nicole LoGiudice

2018-02-01

Full Text Available The fluorinated ornithine analog α-difluoromethylornithine (DFMO, eflornithine, ornidyl is an irreversible suicide inhibitor of ornithine decarboxylase (ODC, the first and rate-limiting enzyme of polyamine biosynthesis. The ubiquitous and essential polyamines have many functions, but are primarily important for rapidly proliferating cells. Thus, ODC is potentially a drug target for any disease state where rapid growth is a key process leading to pathology. The compound was originally discovered as an anticancer drug, but its effectiveness was disappointing. However, DFMO was successfully developed to treat African sleeping sickness and is currently one of few clinically used drugs to combat this neglected tropical disease. The other Food and Drug Administration (FDA approved application for DFMO is as an active ingredient in the hair removal cream Vaniqa. In recent years, renewed interest in DFMO for hyperproliferative diseases has led to increased research and promising preclinical and clinical trials. This review explores the use of DFMO for the treatment of African sleeping sickness and hirsutism, as well as its potential as a chemopreventive and chemotherapeutic agent against colorectal cancer and neuroblastoma.

Purine biosynthesis is the bottleneck in trimethoprim-treated Bacillus subtilis.

Science.gov (United States)

Stepanek, Jennifer Janina; Schäkermann, Sina; Wenzel, Michaela; Prochnow, Pascal; Bandow, Julia Elisabeth

2016-10-01

Trimethoprim is a folate biosynthesis inhibitor. Tetrahydrofolates are essential for the transfer of C 1 units in several biochemical pathways including purine, thymine, methionine, and glycine biosynthesis. This study addressed the effects of folate biosynthesis inhibition on bacterial physiology. Two complementary proteomic approaches were employed to analyze the response of Bacillus subtilis to trimethoprim. Acute changes in protein synthesis rates were monitored by radioactive pulse labeling of newly synthesized proteins and subsequent 2DE analysis. Changes in protein levels were detected using gel-free quantitative MS. Proteins involved in purine and histidine biosynthesis, the σ B -dependent general stress response, and sporulation were upregulated. Most prominently, the PurR-regulon required for de novo purine biosynthesis was derepressed indicating purine depletion. The general stress response was activated energy dependently and in a subpopulation of treated cultures an early onset of sporulation was observed, most likely triggered by low guanosine triphosphate levels. Supplementation of adenosine triphosphate, adenosine, and guanosine to the medium substantially decreased antibacterial activity, showing that purine depletion becomes the bottleneck in trimethoprim-treated B. subtilis. The frequently prescribed antibiotic trimethoprim causes purine depletion in B. subtilis, which can be complemented by supplementing purines to the medium. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Chemical screening and development of novel gibberellin mimics.

Science.gov (United States)

Jiang, Kai; Shimotakahara, Hiroaki; Luo, Ming; Otani, Masato; Nakamura, Hidemitsu; Moselhy, Said Salama; Abualnaja, Khalid Omer; Al-Malki, Abdulrahman Labeed; Kumosani, Taha Abduallah; Kitahata, Nobutaka; Nakano, Takeshi; Nakajima, Masatoshi; Asami, Tadao

2017-08-15

Gibberellin (GA) plays versatile roles in the regulation of plant growth and development and therefore is widely used as a regulator in agriculture. We performed a chemical library screening and identified a chemical, named 67D, as a stimulator of seed germination that was suppressed by paclobutrazol (PAC), a GA biosynthesis inhibitor. In vitro binding assays indicated that 67D binds to the GID1 receptor. Further studies on the structure-activity relationship identified a chemical, named chemical 6, that strongly promoted seed germination suppressed by PAC. Chemical 6 was further confirmed to promote the degradation of RGA (for repressor of ga1-3), a DELLA protein, and suppress the expression levels of GA3ox1 in the same manner as GA does. 67D and its analogs are supposed to be agonists of GID1 and are expected to be utilized in agriculture and basic research as an alternative to GA. Copyright © 2017 Elsevier Ltd. All rights reserved.

Chronic exposure to paclobutrazol causes hepatic steatosis in male rockfish Sebastiscus marmoratus and the mechanism involved

Energy Technology Data Exchange (ETDEWEB)

Sun Lingbin [State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen (China); Li Jinshou [Department of Biological Engineering, Ningde Normal University, Ningde City, Fujian (China); Zuo Zhenghong [State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen (China); Chen Meng [State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen (China); Wang Chonggang, E-mail: cgwang@xmu.edu.cn [State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen (China); State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen (China)

2013-01-15

Paclobutrazol (PBZ) is a triazole-containing fungicide which is widely used in agriculture. Acute toxicity can follow its extensive use but it is generally weaker than traditional pesticides such as organochlorine and organophosphorus. However, its adverse effects on aquatic organisms need to be investigated. This study was conducted to investigate the effect of PBZ exposure on the hepatic lipid metabolism of Sebastiscus marmoratus. After PBZ exposure for 50 days, hepatic lipid droplets were enlarged and the hepatic total lipid, triglyceride, total cholesterol and free fatty acid content had increased in a dose dependent manner compared to the control. The mRNA expression of lipid metabolism associated genes such as peroxisome proliferator-activated receptors (PPARs), androgen receptor, acetyl-CoA carboxylase 1, fatty acid synthesis, fatty acid bing protein 4, liver X receptor {alpha} (LXR{alpha}) and stearoyl-CoA desaturase were up-regulated by PBZ exposure. These results indicated that triazole-containing fungicides might affect the metabolism and health of fish via the multi-signal pathways of nuclear receptors such as PPARs and LXR.

Soilless plant growth media influence the efficacy of phytohormones and phytohormone inhibitors.

Science.gov (United States)

Best, Norman B; Hartwig, Thomas; Budka, Joshua S; Bishop, Brandon J; Brown, Elliot; Potluri, Devi P V; Cooper, Bruce R; Premachandra, Gnanasiri S; Johnston, Cliff T; Schulz, Burkhard

2014-01-01

Plant growth regulators, such as hormones and their respective biosynthesis inhibitors, are effective tools to elucidate the physiological function of phytohormones in plants. A problem of chemical treatments, however, is the potential for interaction of the active compound with the growth media substrate. We studied the interaction and efficacy of propiconazole, a potent and specific inhibitor of brassinosteroid biosynthesis, with common soilless greenhouse growth media for rice, sorghum, and maize. Many of the tested growth media interacted with propiconazole reducing its efficacy up to a hundred fold. To determine the molecular interaction of inhibitors with media substrates, Fourier Transform Infrared Spectroscopy and sorption isotherm analysis was applied. While mica clay substrates absorbed up to 1.3 mg of propiconazole per g substrate, calcined clays bound up to 12 mg of propiconazole per g substrate. The efficacy of the gibberellic acid biosynthesis inhibitor, uniconazole, and the most active brassinosteroid, brassinolide, was impacted similarly by the respective substrates. Conversely, gibberellic acid showed no distinct growth response in different media. Our results suggest that the reduction in efficacy of propiconazole, uniconazole, and brassinolide in bioassays when grown in calcined clay is caused by hydrophobic interactions between the plant growth regulators and the growth media. This was further confirmed by experiments using methanol-water solvent mixes with higher hydrophobicity values, which reduce the interaction of propiconazole and calcined clay.

Paclobutrazol and Cytokinin to Produce Iris (Iris X hollandica Tub. in Pots Paclobutrazol y Citoquinina para la Producción de iris (Iris x hollandica Tub. en Macetas

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Nora Francescangeli

2009-12-01

Full Text Available Because of the increasing and diversified demand for potted plants, there is interest in Argentina in commercial iris production (Iris x hollandica Tub.. Two experiments were performed at the Instituto Nacional de Tecnología Agropecuaria (INTA, San Pedro Agricultural Experimental Station (33º41’ S, 59º41’ W, Buenos Aires Province. The effects of increasing concentrations of paclobutrazol (PBZ and combinations of PBZ and 6-benzylaminopurine (BA were compared. Height, length of the cycle and rate of flowering were registered. Experiment 1: the bulbs were subjected to immersion in nine concentrations of PBZ: 0, 10, 20, 30, 40, 50, 60, 70 or 80 mg L-1 for 24 h. Experiment 2: the bulbs were immersed in three concentrations of PBZ: 0, 10 or 20 mg L-1 for 24 h and then in BA solutions: 0 or 5 mg L-1 for 24 h. In both cases, the cultivars evaluated were `Casablanca´ and `Professor Blaauw’. The bulbs were planted immediately after the treatments in 1 L capacity and 12 cm height pots, on 22 July 2008. PBZ allowed for reducing height in all treated plants. A concentration of 20 mg L-1 would be sufficient to achieve harmonious plants with the containers used: 33 cm tall, average reduction of 41 to 44% compared to the respective control (57 cm. For this concentration, the delay in the duration of the cycle was minimal: 3 to 4 days. There was no effect of BA on the variables. The rate of flowering was not affected by the treatments.En Argentina, ante el aumento y la diversificación de la demanda de plantas en maceta, el iris (Iris x hollandica Tub. se presenta atrayente para la producción comercial. En la Estación Experimental Agropecuaria INTA San Pedro (33º41´ S, 59º41´ O, Provincia de Buenos Aires, se compararon los efectos de concentraciones crecientes de paclobutrazol (PBZ y combinaciones de PBZ y 6-bencilaminopurina (BA, sobre la altura de la planta, la duración de las etapas del ciclo fenológico y la tasa de floración. Para ello se

Induced effect of Ca2+ on dalesconols A and B biosynthesis in the culture of Daldinia eschscholzii via calcium/calmodulin signaling.

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Lu, Yanhua; Pan, Zhenghua; Tao, Jun; An, Faliang

2018-02-01

Dalesconols (dalesconols A and B) were isolated from Daldinia eschscholzii and have remarkable immunosuppressive activity. In this study, the response of fungal growth, intra- and extracellular Ca 2+ , and dalesconols production after CaCl 2 addition were reported for the first time. After supplementation with 5 mM Ca 2+ at 24 h, dalesconols production reached 84.33 mg/L, which resulted in a 1.57-fold enhancement compared to the control. The key role of calcium/calmodulin signaling in dalesconols biosynthesis was confirmed by treatment with Ca 2+ channel and calmodulin inhibitors. The transcriptional levels of dalesconols biosynthetic genes were up-regulated after CaCl 2 addition and down-regulated after inhibitors were added. The results demonstrated that Ca 2+ addition induces dalesconols biosynthesis through up-regulation of dalesconols biosynthesis genes via regulation of calcium/calmodulin signaling. This study provided an efficient strategy for improving dalesconols production and would facilitate further research on the biosynthesis and regulation of dalesconols. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Comparison of the role of gibberellins and ethylene in response to submergence of two lowland rice cultivars, Senia and Bomba.

Science.gov (United States)

Dubois, Vincent; Moritz, Thomas; García-Martínez, José L

2011-02-15

We examined the gibberellin (GA) and ethylene regulation of submergence-induced elongation in seedlings of the submergence-tolerant lowland rice (Oryza sativa L.) cvs Senia and Bomba. Elongation was enhanced after germination to facilitate water escape and reach air. We found that submergence-induced elongation depends on GA because it was counteracted by paclobutrazol (an inhibitor of GA biosynthesis), an effect that was negated by GA(3). Moreover, in the cv Senia, submergence increased the content of active GA(1) and its immediate precursors (GA(53), GA(19) and GA(20)) by enhancing expression of several GA biosynthesis genes (OsGA20ox1 and -2, and OsGA3ox2), but not by decreasing expression of several OsGA2ox (GA inactivating genes). Senia seedlings, in contrast to Bomba seedlings, did not elongate in response to ethylene or 1-aminocyclopropane-1-carboxylic-acid (ACC; an ethylene precursor) application, and submergence-induced elongation was not reduced in the presence of 1-methylcyclopropene (1-MCP; an ethylene perception inhibitor). Ethylene emanation was similar in Senia seedlings grown in air and in submerged-grown seedlings following de-submergence, while it increased in Bomba. The expression of ethylene biosynthesis genes (OsACS1, -2 and -3, and OsACO1) was not affected in Senia, but expression of OsACS5 was rapidly enhanced in Bomba upon submergence. Our results support the conclusion that submergence elongation enhancement of lowland rice is due to alteration of GA metabolism leading to an increase in active GA (GA(1)) content. Interestingly, in the cv Senia, in contrast to cv Bomba, this was triggered through an ethylene-independent mechanism. Copyright © 2010 Elsevier GmbH. All rights reserved.

Inhibition of chitin biosynthesis in cultured imaginal discs: Effects of alpha-amanitin, actinomycin-D, cycloheximide, and puromycin.

Science.gov (United States)

Oberlander, Herbert; Ferkovich, Stephen; Leach, Eddie; Van Essen, Frank

1980-02-01

Wing imaginal discs isolated from last instar larvae of the Indian meal moth,Plodia interpunctella, produced chitin when incubated in vitro with ≧2×10 -7 M 20-hydroxyecdysone. Chitin biosynthesis was initiated 8 h after the conclusion of a 24-h treatment with hormone. Simulataneous incubation of wing discs with 20-hydroxyecdysone and either inhibitors of RNA synthesis (alpha-amanitin, actinomycin-D) or inhibitors of protein systhesis (cycloheximide, puromycin) prevented chitin biosynthesis. We conclude from our results that RNA and protein synthesis must continue undiminished during the hormone-contact period, and that synthesis of protein, but not of new RNA is required during the posthormone culture period. Our findings are consistent with the hypothesis that ecdysteroids stimulate insect metamorphosis by promoting the synthesis of new RNA and protein during a hormone-dependent phase followed by hormone-independent protein synthesis.

Amino Acid Precursor Supply in the Biosynthesis of the RNA Polymerase Inhibitor Streptolydigin by Streptomyces lydicus▿†

OpenAIRE

Gómez, Cristina; Horna, Dina H.; Olano, Carlos; Palomino-Schätzlein, Martina; Pineda-Lucena, Antonio; Carbajo, Rodrigo J.; Braña, Alfredo F.; Méndez, Carmen; Salas, José A.

2011-01-01

Biosynthesis of the hybrid polyketide-nonribosomal peptide antibiotic streptolydigin, 3-methylaspartate, is utilized as precursor of the tetramic acid moiety. The three genes from the Streptomyces lydicus streptolydigin gene cluster slgE1-slgE2-slgE3 are involved in 3-methylaspartate supply. SlgE3, a ferredoxin-dependent glutamate synthase, is responsible for the biosynthesis of glutamate from glutamine and 2-oxoglutarate. In addition to slgE3, housekeeping NADPH- and ferredoxin-dependent glu...

Sterol Biosynthesis Pathway as Target for Anti-trypanosomatid Drugs

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Wanderley de Souza

2009-01-01

Full Text Available Sterols are constituents of the cellular membranes that are essential for their normal structure and function. In mammalian cells, cholesterol is the main sterol found in the various membranes. However, other sterols predominate in eukaryotic microorganisms such as fungi and protozoa. It is now well established that an important metabolic pathway in fungi and in members of the Trypanosomatidae family is one that produces a special class of sterols, including ergosterol, and other 24-methyl sterols, which are required for parasitic growth and viability, but are absent from mammalian host cells. Currently, there are several drugs that interfere with sterol biosynthesis (SB that are in use to treat diseases such as high cholesterol in humans and fungal infections. In this review, we analyze the effects of drugs such as (a statins, which act on the mevalonate pathway by inhibiting HMG-CoA reductase, (b bisphosphonates, which interfere with the isoprenoid pathway in the step catalyzed by farnesyl diphosphate synthase, (c zaragozic acids and quinuclidines, inhibitors of squalene synthase (SQS, which catalyzes the first committed step in sterol biosynthesis, (d allylamines, inhibitors of squalene epoxidase, (e azoles, which inhibit C14α-demethylase, and (f azasterols, which inhibit Δ24(25-sterol methyltransferase (SMT. Inhibition of this last step appears to have high selectivity for fungi and trypanosomatids, since this enzyme is not found in mammalian cells. We review here the IC50 values of these various inhibitors, their effects on the growth of trypanosomatids (both in axenic cultures and in cell cultures, and their effects on protozoan structural organization (as evaluted by light and electron microscopy and lipid composition. The results show that the mitochondrial membrane as well as the membrane lining the protozoan cell body and flagellum are the main targets. Probably as a consequence of these primary effects, other important changes take

Biosynthesis of promatrix metalloproteinase-9/chondroitin sulphate proteoglycan heteromer involves a Rottlerin-sensitive pathway.

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Nabin Malla

Full Text Available BACKGROUND: Previously we have shown that a fraction of the matrix metalloproteinase-9 (MMP-9 synthesized by the macrophage cell line THP-1 was bound to a chondroitin sulphate proteoglycan (CSPG core protein as a reduction sensitive heteromer. Several biochemical properties of the enzyme were changed when it was bound to the CSPG. METHODOLOGY/PRINCIPAL FINDINGS: By use of affinity chromatography, zymography, and radioactive labelling, various macrophage stimulators were tested for their effect on the synthesis of the proMMP-9/CSPG heteromer and its components by THP-1 cells. Of the stimulators, only PMA largely increased the biosynthesis of the heteromer. As PMA is an activator of PKC, we determined which PKC isoenzymes were expressed by performing RT-PCR and Western Blotting. Subsequently specific inhibitors were used to investigate their involvement in the biosynthesis of the heteromer. Of the inhibitors, only Rottlerin repressed the biosynthesis of proMMP-9/CSPG and its two components. Much lower concentrations of Rottlerin were needed to reduce the amount of CSPG than what was needed to repress the synthesis of the heteromer and MMP-9. Furthermore, Rottlerin caused a minor reduction in the activation of the PKC isoenzymes δ, ε, θ and υ (PKD3 in both control and PMA exposed cells. CONCLUSIONS/SIGNIFICANCE: The biosynthesis of the proMMP-9/CSPG heteromer and proMMP-9 in THP-1 cells involves a Rottlerin-sensitive pathway that is different from the Rottlerin sensitive pathway involved in the CSPG biosynthesis. MMP-9 and CSPGs are known to be involved in various physiological and pathological processes. Formation of complexes may influence both the specificity and localization of the enzyme. Therefore, knowledge about biosynthetic pathways and factors involved in the formation of the MMP-9/CSPG heteromer may contribute to insight in the heteromers biological function as well as pointing to future targets for therapeutic agents.

Influence of Paclobutrazol (PP333) and Sridiamin (Human hair-derived aminoacid mixture) on growth and quality of Tomato PKM-1

Science.gov (United States)

Suja, S.; Anusuya, N.

2018-03-01

Tomato is one of the most popular vegetable in subtropics and tropics. Plant growth regulators have potential for manipulating growth of many agricultural crops. Among the plant growth retardants, paclobutrazol (PP333) has been reported to exert profound effects on improving the yield of certain vegetables. Aminoacids are essential prerequisite for plant growth. Sridiamin a natural blend of 17 essential L-aminoacids, fortified with vitamins ensuring better crop growth and higher productivity. Therefore the present study was designed with 5mg and 10 mg concentration of PP333 as soil drench and a foliar spray of sridiamin of 0.5% and 1% concentration as individual and as combined treatment improved the yield and quality of tomato PKM1. Various biometric parameters, along with chlorophyll, starch, aminoacid and protein content were analysed in the leaves. In fruit analysis like titrable acidity, total soluble solids, ascorbic acid, lycopene, total sugars, macronutrients and micronutrients were analysed.

ApoB-100 secretion by HepG2 cells is regulated by the rate of triglyceride biosynthesis but not by intracellular lipid pools.

Science.gov (United States)

Benoist, F; Grand-Perret, T

1996-10-01

Triglycerides (TGs), cholesteryl esters (CEs), cholesterol, and phosphatidylcholine have been independently proposed as playing regulatory roles in apoB-100 secretion; the results depended on the cellular model used. In this study, we reinvestigate the role of lipids in apoB-100 production in HepG2 cells and in particular, we clarify the respective roles of intracellular mass and the biosynthesis of lipids in the regulation of apoB-100 production. In a first set of experiments, the pool size of cholesterol, CEs, and TGs was modulated by a 3-day treatment with either lipid precursors or inhibitors of enzymes involved in lipid synthesis. We used simvastatin (a hydroxymethylglutaryl coenzyme A reductase inhibitor), 58-035 (an acyl coenzyme A cholesterol acyltransferase inhibitor), 5-tetradecyloxy-2-furancarboxylic acid (TOFA, an inhibitor of fatty acid synthesis), and oleic acid. The secretion rate of apoB-100 was not affected by the large modulation of lipid mass induced by these various pre-treatments. In a second set of experiments, the same lipid modulators were added during a 4-hour labeling period. Simvastatin and 58-035 inhibited cholesterol and CE synthesis without affecting apoB-100 secretion. By contrast, treatment of HepG2 cells with TOFA resulted in the inhibition of TG synthesis and apoB-100 secretion. This effect was highly specific for apoB-100 and was reversed by adding oleic acid, which stimulated both TG synthesis and apoB-100 secretion. Moreover, a combination of oleic acid and 58-035 inhibited CE biosynthesis and increased both TG synthesis and apoB-100 secretion. These results show that in HepG2 cells TG biosynthesis regulates apoB-100 secretion, whereas the rate of cholesterol or CE biosynthesis has no effect.

Plastid-to-Nucleus Retrograde Signals Are Essential for the Expression of Nuclear Starch Biosynthesis Genes during Amyloplast Differentiation in Tobacco BY-2 Cultured Cells1[W][OA

Science.gov (United States)

Enami, Kazuhiko; Ozawa, Tomoki; Motohashi, Noriko; Nakamura, Masayuki; Tanaka, Kan; Hanaoka, Mitsumasa

2011-01-01

Amyloplasts, a subtype of plastid, are found in nonphotosynthetic tissues responsible for starch synthesis and storage. When tobacco (Nicotiana tabacum) Bright Yellow-2 cells are cultured in the presence of cytokinin instead of auxin, their plastids differentiate from proplastids to amyloplasts. In this program, it is well known that the expression of nucleus-encoded starch biosynthesis genes, such as ADP-Glucose Pyrophosphorylase (AgpS) and Granule-Bound Starch Synthase (GBSS), is specifically induced. In this study, we investigated the roles of plastid gene expression in amyloplast differentiation. Microarray analysis of plastid genes revealed that no specific transcripts were induced in amyloplasts. Nevertheless, amyloplast development accompanied with starch biosynthesis was drastically inhibited in the presence of plastid transcription/translation inhibitors. Surprisingly, the expression of nuclear AgpS and GBSS was significantly repressed by the addition of these inhibitors, suggesting that a plastid-derived signal(s) that reflects normal plastid gene expression was essential for nuclear gene expression. A series of experiments was performed to examine the effects of intermediates and inhibitors of tetrapyrrole biosynthesis, since some of the intermediates have been characterized as candidates for plastid-to-nucleus retrograde signals. Addition of levulinic acid, an inhibitor of tetrapyrrole biosynthesis, resulted in the up-regulation of nuclear AgpS and GBSS gene expression as well as starch accumulation, while the addition of heme showed opposite effects. Thus, these results indicate that plastid transcription and/or translation are required for normal amyloplast differentiation, regulating the expression of specific nuclear genes by unknown signaling mechanisms that can be partly mediated by tetrapyrrole intermediates. PMID:21771917

2-Fluoro-L-Fucose Is a Metabolically Incorporated Inhibitor of Plant Cell Wall Polysaccharide Fucosylation

Science.gov (United States)

Wallace, Ian S.

2015-01-01

The monosaccharide L-fucose (L-Fuc) is a common component of plant cell wall polysaccharides and other plant glycans, including the hemicellulose xyloglucan, pectic rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II), arabinogalactan proteins, and N-linked glycans. Mutations compromising the biosynthesis of many plant cell wall polysaccharides are lethal, and as a result, small molecule inhibitors of plant cell wall polysaccharide biosynthesis have been developed because these molecules can be applied at defined concentrations and developmental stages. In this study, we characterize novel small molecule inhibitors of plant fucosylation. 2-fluoro-L-fucose (2F-Fuc) analogs caused severe growth phenotypes when applied to Arabidopsis seedlings, including reduced root growth and altered root morphology. These phenotypic defects were dependent upon the L-Fuc salvage pathway enzyme L-Fucose Kinase/ GDP-L-Fucose Pyrophosphorylase (FKGP), suggesting that 2F-Fuc is metabolically converted to the sugar nucleotide GDP-2F-Fuc, which serves as the active inhibitory molecule. The L-Fuc content of cell wall matrix polysaccharides was reduced in plants treated with 2F-Fuc, suggesting that this molecule inhibits the incorporation of L-Fuc into these polysaccharides. Additionally, phenotypic defects induced by 2F-Fuc treatment could be partially relieved by the exogenous application of boric acid, suggesting that 2F-Fuc inhibits RG-II biosynthesis. Overall, the results presented here suggest that 2F-Fuc is a metabolically incorporated inhibitor of plant cellular fucosylation events, and potentially suggest that other 2-fluorinated monosaccharides could serve as useful chemical probes for the inhibition of cell wall polysaccharide biosynthesis. PMID:26414071

3-cyanoindole-based inhibitors of inosine monophosphate dehydrogenase: synthesis and initial structure-activity relationships.

Science.gov (United States)

Dhar, T G Murali; Shen, Zhongqi; Gu, Henry H; Chen, Ping; Norris, Derek; Watterson, Scott H; Ballentine, Shelley K; Fleener, Catherine A; Rouleau, Katherine A; Barrish, Joel C; Townsend, Robert; Hollenbaugh, Diane L; Iwanowicz, Edwin J

2003-10-20

A series of novel small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH), based upon a 3-cyanoindole core, were explored. IMPDH catalyzes the rate determining step in guanine nucleotide biosynthesis and is a target for anticancer, immunosuppressive and antiviral therapy. The synthesis and the structure-activity relationships (SAR), derived from in vitro studies, for this new series of inhibitors is given.

Storage behavior and changes in concentrations of abscisic acid and gibberellins during dormancy break and germination in seeds of Phellodendron amurense var. wilsonii (Rutaceae).

Science.gov (United States)

Chen, Shun-Ying; Chien, Ching-Te; Baskin, Jerry M; Baskin, Carol C

2010-02-01

The medicinal Asian plant genus Phellodendron is known to contain several very important compounds that have biological action. The main purpose of this study was to determine whether seeds of Phellodendron amurense var. wilsonii can be stored and to characterize their dormancy. Seeds of this taxon stored at -20 and -80 degrees C and in liquid nitrogen retained their high germinability, indicating that they have orthodox storage behavior. Intact seeds from freshly collected fruits were dormant and required 12 weeks of cold stratification at 4 degrees C for complete germination. Scarifying the seed coat was partially effective in breaking seed dormancy. Exogenous gibberellins (GA(3), GA(4) and GA(4+7)) promoted germination of scarified seeds, GA(4) and GA(4+7) being more effective than GA(3). Fluridone, an abscisic acid (ABA) biosynthesis inhibitor, was efficient in breaking dormancy, but it was less effective than GA(4) or GA(4+7) alone. Paclobutrazol, a GA biosynthesis inhibitor, inhibited seed germination, and the inhibitory effect was reversed completely by GA(4) and by GA(4+7). ABA content of seeds subjected to cold stratification or to incubation at 35/10 degrees C, which enhanced seed germination, was reduced about four- to sixfold compared to that of fresh seeds. Higher concentrations of GA(3), GA(4) and GA(7) were detected in nondormant seeds and in seeds with an emerged radicle than in fresh seeds. Present results seem to indicate that dormancy in P. amurense var. wilsonii seeds is imposed partially by the seed coat and partially by high ABA content. ABA content decreased and GA(3), GA(4) and GA(7) content increased during germination.

Virtual screening for potential inhibitors of bacterial MurC and MurD ligases.

Science.gov (United States)

Tomašić, Tihomir; Kovač, Andreja; Klebe, Gerhard; Blanot, Didier; Gobec, Stanislav; Kikelj, Danijel; Mašič, Lucija Peterlin

2012-03-01

Mur ligases are bacterial enzymes involved in the cytoplasmic steps of peptidoglycan biosynthesis and are viable targets for antibacterial drug discovery. We have performed virtual screening for potential ATP-competitive inhibitors targeting MurC and MurD ligases, using a protocol of consecutive hierarchical filters. Selected compounds were evaluated for inhibition of MurC and MurD ligases, and weak inhibitors possessing dual inhibitory activity have been identified. These compounds represent new scaffolds for further optimisation towards multiple Mur ligase inhibitors with improved inhibitory potency.

Disruption of Sphingolipid Biosynthesis Blocks Phagocytosis of Candida albicans.

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Fikadu G Tafesse

2015-10-01

Full Text Available The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine-palmitoyltransferase (SPT and ceramide synthase-enzymes involved in sphingolipid biosynthesis- by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9-mediated genome editing to generate Sptlc2-deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2-/- DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2-/- DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2-/- DC2.4 cells express reduced levels of the pattern recognition receptors Dectin-1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans.

Arabidopsis DREB2C modulates ABA biosynthesis during germination.

Science.gov (United States)

Je, Jihyun; Chen, Huan; Song, Chieun; Lim, Chae Oh

2014-09-12

Plant dehydration-responsive element binding factors (DREBs) are transcriptional regulators of the APETELA2/Ethylene Responsive element-binding Factor (AP2/ERF) family that control expression of abiotic stress-related genes. We show here that under conditions of mild heat stress, constitutive overexpression seeds of transgenic DREB2C overexpression Arabidopsis exhibit delayed germination and increased abscisic acid (ABA) content compared to untransformed wild-type (WT). Treatment with fluridone, an inhibitor of the ABA biosynthesis abrogated these effects. Expression of an ABA biosynthesis-related gene, 9-cis-epoxycarotenoid dioxygenase 9 (NCED9) was up-regulated in the DREB2C overexpression lines compared to WT. DREB2C was able to trans-activate expression of NCED9 in Arabidopsis leaf protoplasts in vitro. Direct and specific binding of DREB2C to a complete DRE on the NCED9 promoter was observed in electrophoretic mobility shift assays. Exogenous ABA treatment induced DREB2C expression in germinating seeds of WT. Vegetative growth of transgenic DREB2C overexpression lines was more strongly inhibited by exogenous ABA compared to WT. These results suggest that DREB2C is a stress- and ABA-inducible gene that acts as a positive regulator of ABA biosynthesis in germinating seeds through activating NCED9 expression. Copyright © 2014 Elsevier Inc. All rights reserved.

Marked radiosensitization of cells in culture to x ray by 5-chlorodeoxycytidine coadministered with tetrahydrouridine, and inhibitors of pyrimidine biosynthesis

International Nuclear Information System (INIS)

Perez, L.M.; Mekras, J.A.; Briggle, T.V.; Greer, S.

1984-01-01

The authors approach to overcome the problem of rapid catabolism and general toxicity encountered with 5-halogenated analogues of deoxyuridine (5-bromo, chloro or iododeoxyuridine), which has limited their use as tumor radiosensitizers, is to utilize 5-chlorodeoxycytidine (CldC) with tetrahydrouridine (H 4 U). They propose that CldC, coadministered with H 4 U, is metabolized in the following manner: CldC → CldCMP → CldUMP → → CldUTP → DNA. All the enzymes of this pathway are elevated in many human malignant tumors and in HEp-2 cells. In x irradiation studies with HEp-2 cells, limited to 1 or 2 radiation doses. They obtained 3.0 to 3.8 apparent dose enhancement ratios when cells were preincubated with inhibitors of pyrimidine biosynthesis. Enzymatic studies indicate that this toxicity may be tumor selective. Preliminary toxicity studies indicate that mice will tolerate treatment protocols with marginal weight loss (4%). In this approach the authors seek to obtain preferential conversion of CldC to CldUTP at the tumor site by taking advantage of quantitative differences in enzyme levels between tumors and normal tissues

Evaluation of Paclobutrazol Spraying on Salinity Hardiness of Peach- Almond Hybrid (GF677 Rootstock

Directory of Open Access Journals (Sweden)

azam amiri

2017-02-01

Full Text Available Introduction: Salinity is a common abiotic stress that seriously affects crop production in some parts of the world, particularly in arid and semi-arid regions. The deleterious effects of salinity on plant growth are associated with low osmotic potential of soil solution (water stress, nutritional imbalance, specific ion effect (salt stress, or a combination of these factors. Paclobutrazol (PBZ, a member of the triazole plant growth inhibitor group, is a broad-spectrum gibberellin biosynthesis inhibitor. Triazoles have both fungal toxicity and plant growth regulatory effects. They also increase tolerance of various plant species to biotic and abiotic stresses, including fungal pathogens, drought, air pollutants, and low- and high-temperature stress. According to our knowledge, there are no reports on the effects of exogenous PBZ enhancing vegetative peach- almond hybrid (GF 677 rootstock tolerance to salt stress. Therefore, the objective of this work was the possibility test of this idea that PBZ application would protect GF 677 rootstock from damaging effects of salinity. Materials and Methods: One-year-old rooted cuttings of GF 677 were grown in in plastic pots in the research greenhouse of Agricultural College, Isfahan University of Technology of Iran. The minimum and maximum temperatures during the experiment period were 19 and 32˚C, respectively. After cutting establishment (3 months, the plants were sprayed twice (with a 7 days interval with 0 (control, 20 and 40 mg l-1 PBZ to the point of run-off. One week after the second foliar application of PBZ, each plants was subjected to one of several salt stress treatments. The salt treatments (0, 25 and 50 mM NaCl were applied to the pots intervals in 0.5 l of irrigation water. To avoid osmotic shock, the NaCl concentration was increased gradually. The layout was a 3×3 factorial experiment in a completely randomized design, with four replications. The experimental measurements were carried out

[The inhibitor of free radical processes decrease of protein biosynthesis in gun short wound tissues and weaken development of the general adaptation syndrome].

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Todorov, I N; Bogdanov, G N; Mitrokhin, Iu I; Varfolomeev, V N; Sidorenko, L I; Mishchenko, D V

2006-01-01

The dynamics of total protein biosynthesis and procollagen biosynthesis in skeletal muscle of injury tissues with the antioxidant BHT (dibunol) treatment and with common healing were studied. The obtained date indicate that the AO treatment reduce the rate of biosynthesis both the total proteins and procollagen at the 3th day of healing. Dibunol also considerably reduce the protein biosynthesis in adrenals and brake of corticosteroids biogenesis as measured by ESR-signals intensity of reduced adrenodoxine. AO treatment also reduce the protein biosynthesis in thymus, spleen and bone marrow. The lowering of functional activity of endocrine and immune systems indicate that the AO significantly inhibit the systemic reactions of organism induced by acute wound affect. It was suggested that as "primary mediator" of stress-reaction may be considered lipoperoxide radicals and decay products of lipohydroperoide.

Amino acid precursor supply in the biosynthesis of the RNA polymerase inhibitor streptolydigin by Streptomyces lydicus.

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Gómez, Cristina; Horna, Dina H; Olano, Carlos; Palomino-Schätzlein, Martina; Pineda-Lucena, Antonio; Carbajo, Rodrigo J; Braña, Alfredo F; Méndez, Carmen; Salas, José A

2011-08-01

Biosynthesis of the hybrid polyketide-nonribosomal peptide antibiotic streptolydigin, 3-methylaspartate, is utilized as precursor of the tetramic acid moiety. The three genes from the Streptomyces lydicus streptolydigin gene cluster slgE1-slgE2-slgE3 are involved in 3-methylaspartate supply. SlgE3, a ferredoxin-dependent glutamate synthase, is responsible for the biosynthesis of glutamate from glutamine and 2-oxoglutarate. In addition to slgE3, housekeeping NADPH- and ferredoxin-dependent glutamate synthase genes have been identified in S. lydicus. The expression of slgE3 is increased up to 9-fold at the onset of streptolydigin biosynthesis and later decreases to ∼2-fold over the basal level. In contrast, the expression of housekeeping glutamate synthases decreases when streptolydigin begins to be synthesized. SlgE1 and SlgE2 are the two subunits of a glutamate mutase that would convert glutamate into 3-methylaspartate. Deletion of slgE1-slgE2 led to the production of two compounds containing a lateral side chain derived from glutamate instead of 3-methylaspartate. Expression of this glutamate mutase also reaches a peak increase of up to 5.5-fold coinciding with the onset of antibiotic production. Overexpression of either slgE3 or slgE1-slgE2 in S. lydicus led to an increase in the yield of streptolydigin.

Effect of Plant Growth Regulators and Cold on Improvement of Morphological Characteristics of Cineraria (Pericallis × hybrida

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Kazem Bashiri

2017-12-01

consistent with other studies on iris, lily, tulip and hyacinth. The highest longevity of cineraria was observed in 100 mg/L gibberellin + 250 mg/L paclobutrazol (31.8 days which showed increasing of 24 days compared to control. Gibberellin and cytokinin lengthened the life of flowers, while paclobutrazol reduced the ethylene production and delayed the senescence. The greatest shoot fresh and dry weight was evaluated in 100 mg/L gibberellin + 300 mg/L benzyl adenine, while paclobutrazol decreased the plant growth. Similar results have been reported in zinnia and geranium. Gibberellin and cytokinin increased leaf area, while paclobutrazol decreased it. The highest leaf area was detected using 100 mg/L gibberellin + 300 mg/L benzyl adenine. . Maximum flower number was obtained using 100 mg/L gibberellin + 300 mg/L benzyl adenine, while the lowest number was obtained by 100 mg/L gibberellin + 500 mg/L paclobutrazol application. In addition, observations in orchid and jasmine were showed similar result. Gibberellin enhanced and paclobutrazol decreased plant height. plants Spraying with 200 mg/L gibberellin and cold + 500 mg/L paclobutrazol showed the maximum and minimum height, respectively. Similar observations have been found in cyclamen and tuberose. number. . Also, Paclobutrazol application enlarged flower diameter. The greatest enhancement of flower diameter ( 1 cm was found by cold + 500 mg/L paclobutrazol spray. Flower diameter improvement is related to the severed growth, height and flower number by paclobutrazol. Flower diameter reduction in higher concentrations using gibberellin and cytokinin may be attributed to increasing translocation of carbohydrates to shoots and vegetative growth rather than flowers. Osteospermum and chrysanthemum showed similar and opposite results, respectively with this study. The effect of chemicals inhibitor depends on the number of applications, environmental conditions, plant sensitivity and how it is used. The bestvisual quality was

Study of propagation of Berberis thunbergii L. by cuttings, with using less-known methods of stimulation

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Martin Říha

2007-01-01

Full Text Available The different type of own produce stimulators were tested at Berberis thunbergii L. 'Green Carpet', Berberis thunbergii 'Red Shift' and Berberis thunbergii 'Aureum'. We used the combination of growing inhibitors and quick-dip method, single quick-dip metod in solution of acetone and stimulant in form of gel. Groving inhibitors is including paclobutrazol and CCC in test. We used IBK, NAA, IAA and nicotin acid as auxins in quick-dip method. Medium was aceton solution.

2-Fluoro-L-Fucose Is a Metabolically Incorporated Inhibitor of Plant Cell Wall Polysaccharide Fucosylation.

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Jose A Villalobos

Full Text Available The monosaccharide L-fucose (L-Fuc is a common component of plant cell wall polysaccharides and other plant glycans, including the hemicellulose xyloglucan, pectic rhamnogalacturonan-I (RG-I and rhamnogalacturonan-II (RG-II, arabinogalactan proteins, and N-linked glycans. Mutations compromising the biosynthesis of many plant cell wall polysaccharides are lethal, and as a result, small molecule inhibitors of plant cell wall polysaccharide biosynthesis have been developed because these molecules can be applied at defined concentrations and developmental stages. In this study, we characterize novel small molecule inhibitors of plant fucosylation. 2-fluoro-L-fucose (2F-Fuc analogs caused severe growth phenotypes when applied to Arabidopsis seedlings, including reduced root growth and altered root morphology. These phenotypic defects were dependent upon the L-Fuc salvage pathway enzyme L-Fucose Kinase/ GDP-L-Fucose Pyrophosphorylase (FKGP, suggesting that 2F-Fuc is metabolically converted to the sugar nucleotide GDP-2F-Fuc, which serves as the active inhibitory molecule. The L-Fuc content of cell wall matrix polysaccharides was reduced in plants treated with 2F-Fuc, suggesting that this molecule inhibits the incorporation of L-Fuc into these polysaccharides. Additionally, phenotypic defects induced by 2F-Fuc treatment could be partially relieved by the exogenous application of boric acid, suggesting that 2F-Fuc inhibits RG-II biosynthesis. Overall, the results presented here suggest that 2F-Fuc is a metabolically incorporated inhibitor of plant cellular fucosylation events, and potentially suggest that other 2-fluorinated monosaccharides could serve as useful chemical probes for the inhibition of cell wall polysaccharide biosynthesis.

Elucidation and chemical modulation of sulfolipid-1 biosynthesis in Mycobacterium tuberculosis.

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Seeliger, Jessica C; Holsclaw, Cynthia M; Schelle, Michael W; Botyanszki, Zsofia; Gilmore, Sarah A; Tully, Sarah E; Niederweis, Michael; Cravatt, Benjamin F; Leary, Julie A; Bertozzi, Carolyn R

2012-03-09

Mycobacterium tuberculosis possesses unique cell-surface lipids that have been implicated in virulence. One of the most abundant is sulfolipid-1 (SL-1), a tetraacyl-sulfotrehalose glycolipid. Although the early steps in SL-1 biosynthesis are known, the machinery underlying the final acylation reactions is not understood. We provide genetic and biochemical evidence for the activities of two proteins, Chp1 and Sap (corresponding to gene loci rv3822 and rv3821), that complete this pathway. The membrane-associated acyltransferase Chp1 accepts a synthetic diacyl sulfolipid and transfers an acyl group regioselectively from one donor substrate molecule to a second acceptor molecule in two successive reactions to yield a tetraacylated product. Chp1 is fully active in vitro, but in M. tuberculosis, its function is potentiated by the previously identified sulfolipid transporter MmpL8. We also show that the integral membrane protein Sap and MmpL8 are both essential for sulfolipid transport. Finally, the lipase inhibitor tetrahydrolipstatin disrupts Chp1 activity in M. tuberculosis, suggesting an avenue for perturbing SL-1 biosynthesis in vivo. These data complete the SL-1 biosynthetic pathway and corroborate a model in which lipid biosynthesis and transmembrane transport are coupled at the membrane-cytosol interface through the activity of multiple proteins, possibly as a macromolecular complex.

RhHB1 mediates the antagonism of gibberellins to ABA and ethylene during rose (Rosa hybrida) petal senescence.

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Lü, Peitao; Zhang, Changqing; Liu, Jitao; Liu, Xiaowei; Jiang, Guimei; Jiang, Xinqiang; Khan, Muhammad Ali; Wang, Liangsheng; Hong, Bo; Gao, Junping

2014-05-01

Rose (Rosa hybrida) is one of the most important ornamental plants worldwide; however, senescence of its petals terminates the ornamental value of the flower, resulting in major economic loss. It is known that the hormones abscisic acid (ABA) and ethylene promote petal senescence, while gibberellins (GAs) delay the process. However, the molecular mechanisms underlying the antagonistic effects amongst plant hormones during petal senescence are still unclear. Here we isolated RhHB1, a homeodomain-leucine zipper I transcription factor gene, from rose flowers. Quantitative RT-PCR and GUS reporter analyses showed that RhHB1 was strongly expressed in senescing petals, and its expression was induced by ABA or ethylene in petals. ABA or ethylene treatment clearly accelerated rose petal senescence, while application of the gibberellin GA3 delayed the process. However, silencing of RhHB1 delayed the ABA- or ethylene-mediated senescence, and resulted in higher petal anthocyanin levels and lower expression of RhSAG12. Moreover, treatment with paclobutrazol, an inhibitor of GA biosynthesis, repressed these delays. In addition, silencing of RhHB1 blocked the ABA- or ethylene-induced reduction in expression of the GA20 oxidase encoded by RhGA20ox1, a gene in the GA biosynthetic pathway. Furthermore, RhHB1 directly binds to the RhGA20ox1 promoter, and silencing of RhGA20ox1 promoted petal senescence. Eight senescence-related genes showed substantial differences in expression in petals after treatment with GA3 or paclobutrazol. These results suggest that RhHB1 mediates the antagonistic effect of GAs on ABA and ethylene during rose petal senescence, and that the promotion of petal senescence by ABA or ethylene operates through an RhHB1-RhGA20ox1 regulatory checkpoint. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

How polyamine synthesis inhibitors and cinnamic acid affect tropane alkaloid production.

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Marconi, Patricia L; Alvarez, María A; Pitta-Alvarez, Sandra I

2007-01-01

Hairy roots of Brugmansia candida produce the tropane alkaloids scopolamine and hyoscyamine. In an attempt to divert the carbon flux from competing pathways and thus enhance productivity, the polyamine biosynthesis inhibitors cyclohexylamine (CHA) and methylglyoxal-bis-guanylhydrazone (MGBG) and the phenylalanine-ammonia-lyase inhibitor cinnamic acid were used. CHA decreased the specific productivity of both alkaloids but increased significantly the release of scopolamine (approx 500%) when it was added in the mid-exponential phase. However, when CHA was added for only 48 h during the exponential phase, the specific productivity of both alkaloids increased (approx 200%), favoring scopolamine. Treatment with MGBG was detrimental to growth but promoted release into the medium of both alkaloids. However, when it was added for 48 h during the exponential phase, MGBG increased the specific productivity (approx 200%) and release (250- 1800%) of both alkaloids. Cinnamic acid alone also favored release but not specific productivity. When a combination of CHA or MGBG with cinnamic acid was used, the results obtained were approximately the same as with each polyamine biosynthesis inhibitor alone, although to a lesser extent. Regarding root morphology, CHA inhibited growth of primary roots and ramification. However, it had a positive effect on elongation of lateral roots.

Amino Acid Precursor Supply in the Biosynthesis of the RNA Polymerase Inhibitor Streptolydigin by Streptomyces lydicus▿†

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Gómez, Cristina; Horna, Dina H.; Olano, Carlos; Palomino-Schätzlein, Martina; Pineda-Lucena, Antonio; Carbajo, Rodrigo J.; Braña, Alfredo F.; Méndez, Carmen; Salas, José A.

2011-01-01

Biosynthesis of the hybrid polyketide-nonribosomal peptide antibiotic streptolydigin, 3-methylaspartate, is utilized as precursor of the tetramic acid moiety. The three genes from the Streptomyces lydicus streptolydigin gene cluster slgE1-slgE2-slgE3 are involved in 3-methylaspartate supply. SlgE3, a ferredoxin-dependent glutamate synthase, is responsible for the biosynthesis of glutamate from glutamine and 2-oxoglutarate. In addition to slgE3, housekeeping NADPH- and ferredoxin-dependent glutamate synthase genes have been identified in S. lydicus. The expression of slgE3 is increased up to 9-fold at the onset of streptolydigin biosynthesis and later decreases to ∼2-fold over the basal level. In contrast, the expression of housekeeping glutamate synthases decreases when streptolydigin begins to be synthesized. SlgE1 and SlgE2 are the two subunits of a glutamate mutase that would convert glutamate into 3-methylaspartate. Deletion of slgE1-slgE2 led to the production of two compounds containing a lateral side chain derived from glutamate instead of 3-methylaspartate. Expression of this glutamate mutase also reaches a peak increase of up to 5.5-fold coinciding with the onset of antibiotic production. Overexpression of either slgE3 or slgE1-slgE2 in S. lydicus led to an increase in the yield of streptolydigin. PMID:21665968

Efeito do paclobutrazol sobre o crescimento de plantas e produzção de tomate (Solanum lycopersicumL. em ambiente protegido

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Alexsander Seleguini

2016-01-01

Full Text Available The objective was to study, for hybrid AF 7631, the effect of three concentrations of paclobutrazol (0, 50 and 100 mg ai L-1 and two application methods (soaking seeds and seedlings application on the development of plants, production and fruit quality of tomato in greenhouse in the spring / summer period. The trial was conducted the Finance Teaching, Research and Extension UNESP -Ilha Solteira (SP. Application methods and the increase in PBZ concentrations did not significantly alter the productivity, however, the treatment of seedlings, via irrigation, 15 days after sowing, with increasing concentrations of PBZ induced linear reductions in plant height evaluated at 17, 34, 51 and 65 days after transplanting (DAT in absolute growth rate of height in the range between 17 and 34 DAT at the time of first cluster of insertion and dry weight of leaves and stems. Regardless of PBZ applicationmethod, the concentrations significantly reduced the effect of side shoots and increased production of small fruit. The climatic conditions, especially temperature, prevented the expression of the maximum yield potential of the crop.

Conservation of the 2-keto-3-deoxymanno-octulosonic acid (Kdo) biosynthesis pathway between plants and bacteria.

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Smyth, Kevin M; Marchant, Alan

2013-10-18

The increasing prevalence of multi-drug resistant bacteria is driving efforts in the development of new antibacterial agents. This includes a resurgence of interest in the Gram-negative bacteria lipopolysaccharide (LPS) biosynthesis enzymes as drug targets. The six carbon acidic sugar 2-keto-3-deoxymanno-octulosonic acid (Kdo) is a component of the lipid A moiety of the LPS in Gram-negative bacteria. In most cases the lipid A substituted by Kdo is the minimum requirement for cell growth, thus presenting the possibility of targeting either the synthesis or incorporation of Kdo for the development of antibacterial agents. Indeed, potent in vitro inhibitors of Kdo biosynthesis enzymes have been reported but have so far failed to show sufficient in vivo action against Gram-negative bacteria. As part of an effort to design more potent antibacterial agents targeting Kdo biosynthesis, the crystal structures of the key Kdo biosynthesis enzymes from Escherichia coli have been solved and their structure based mechanisms characterized. In eukaryotes, Kdo is found as a component of the pectic polysaccharide rhamnogalacturonan II in the plant primary cell wall. Interestingly, despite incorporating Kdo into very different macromolecules the Kdo biosynthesis and activation pathway is almost completely conserved between plants and bacteria. This raises the possibility for plant research to exploit the increasingly detailed knowledge and resources being generated by the microbiology community. Likewise, insights into Kdo biosynthesis in plants will be potentially useful in efforts to produce new antimicrobial compounds. Copyright © 2013 Elsevier Ltd. All rights reserved.

Brain modulation of Dufour's gland ester biosynthesis in vitro in the honeybee ( Apis mellifera)

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Katzav-Gozansky, Tamar; Hefetz, Abraham; Soroker, Victoria

2007-05-01

Caste-specific pheromone biosynthesis is a prerequisite for reproductive skew in the honeybee. Nonetheless, this process is not hardwired but plastic, in that egg-laying workers produce a queen-like pheromone. Studies with Dufour’s gland pheromone revealed that, in vivo, workers’ gland biosynthesis matches the social status of the worker, i.e., sterile workers showed a worker-like pattern whereas fertile workers showed a queen-like pattern (production of the queen-specific esters). However, when incubated in vitro, the gland spontaneously exhibits the queen-like pattern, irrespective of its original worker type, prompting the notion that ester production in workers is under inhibitory control that is queen-dependent. We tested this hypothesis by exposing queen or worker Dufour’s glands in vitro to brain extracts of queens, queenright (sterile) workers and males. Unexpectedly, worker brain extracts activated the queen-like esters biosynthesis in workers’ Dufour’s gland. This stimulation was gender-specific; queen or worker brains demonstrated a stimulatory activity, but male brains did not. Queen gland could not be further stimulated. Bioassays with heated and filtered extracts indicate that the stimulatory brain factor is below 3,000 Da. We suggest that pheromone production in Dufour’s gland is under dual, negative positive control. Under queenright conditions, the inhibitor is released and blocks ester biosynthesis, whereas under queenless conditions, the activator is released, activating ester biosynthesis in the gland. This is consistent with the hypothesis that queenright workers are unequivocally recognized as non-fertile, whereas queenless workers try to become “false queens” as part of the reproductive competition.

Growth of Cucurbita maxima L. plants in the presence of the cycloartenol synthase inhibitor U18666A.

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Fenner, G P; Raphiou, I

1995-03-01

Squash, like other Cucurbitaceae, have unique sterol profiles that offer an excellent opportunity to examine the relationship between sterol biosynthesis and plant growth. To determine the effect of sterol biosynthesis inhibition on squash growth, Cucurbita maxima seedlings with and without cotyledons were subjected to increasing concentrations of the cycloarternol synthase (EC 5.4.99.8) inhibitor 3 beta-(2-diethylaminoethoxy)androstenone (U18666A). Inhibition of shoot growth was concentration-dependent (from 0, 2, 5, 10, and 20 microM); plants with intact cotyledons grew to 26.4, 23.7, 21.6, 20.0, and 15.6 cm, respectively, at the above inhibitor concentrations, compared to 25.5, 19.4, 17.0, 12.0, and 11 cm for plants with severed cotyledons. In plants with severed cotyledons, 10 and 20 microM U18666A caused rapid necrosis of the first two, newly emerged, primary leaves, and halted new leaf formation. Secondary root formation was initially affected at all inhibitor concentrations regardless of whether cotyledons were present or not. Vegetative tissue showed a decrease in the accumulation of the major squash sterol, 7,22-stigmastadienol, accompanied by increased accumulation of minor sterol components. Sterol profiles in cotyledons were unaltered. The data show that sterols are crucial for maintaining plant growth and viability, but do not address the cotyledonary effect on growth with respect to sterol biosynthesis.

Effect of Furfural, Vanillin and Syringaldehyde on Candida guilliermondii Growth and Xylitol Biosynthesis

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Kelly, Christine; Jones, Opal; Barnhart, Christopher; Lajoie, Curtis

Xylitol is a five-carbon sugar alcohol with established commercial use as an alternative sweetener and can be produced from hemicellulose hydrolysate. However, there are difficulties with microbiological growth and xylitol biosynthesis on hydrolysate because of the inhibitors formed from hydrolysis of hemicellulose. This research focused on the effect of furfural, vanillin, and syringaldehyde on growth of Candida guilliermondii and xylitol accumulation from xylose in a semi-synthetic medium in microwell plate and bioreactor cultivations. All three compounds reduced specific growth rate, increased lag time, and reduced xylitol production rate. In general, increasing concentration of inhibitor increased the severity of inhibition, except in the case of 0.5 g vanillin per liter, which resulted in a faster late batch phase growth rate and increased biomass yield. At concentrations of 1 g/1 or higher, furfural was the least inhibitory to growth, followed by syringaldehyde. Vanillin most severely reduced specific growth rate. All three inhibitors reduced xylitol production rate approximately to the same degree.

Aryl hydrocarbon receptor 2 mediates the toxicity of Paclobutrazol on the digestive system of zebrafish embryos.

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Wang, Wen-Der; Chen, Guan-Ting; Hsu, Hwei-Jan; Wu, Chang-Yi

2015-02-01

Paclobutrazol (PBZ), a trazole-containing fungicide and plant growth retardant, has been widely used for over 30 years to regulate plant growth and promote early fruit setting. Long-term usage of PBZ in agriculture and natural environments has resulted in residual PBZ in the soil and water. Chronic exposure to waterborne PBZ can cause various physiological effects in fish, including hepatic steatosis, antioxidant activity, and disruption of spermatogenesis. We have previously shown that PBZ also affects the rates of zebrafish embryonic survival and hatching, and causes developmental failure of the head skeleton and eyes; here, we further show that PBZ has embryonic toxic effects on digestive organs of zebrafish, and describe the underlying mechanisms. PBZ treatment of embryos resulted in dose-dependent morphological and functional abnormalities of the digestive organs. Real-time RT-PCR and in situ hybridization were used to show that PBZ strongly induces cyp1a1 expression in the digestive system, and slightly induces ahr2 expression in zebrafish embryos. Knockdown of ahr2 with morpholino oligonucleotides prevents PBZ toxicity. Thus, the toxic effect of PBZ on digestive organs is mediated by AhR2, as was previously reported for retene and TCDD. These findings have implications for understanding the potential toxicity of PBZ during embryogenesis, and thus the potential impact of fungicides on public health and the environment. Copyright © 2014 Elsevier B.V. All rights reserved.

Inhibitors of steroidal cytochrome p450 enzymes as targets for drug development.

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Baston, Eckhard; Leroux, Frédéric R

2007-01-01

Cytochrome P450's are enzymes which catalyze a large number of biological reactions, for example hydroxylation, N-, O-, S- dealkylation, epoxidation or desamination. Their substrates include fatty acids, steroids or prostaglandins. In addition, a high number of various xenobiotics are metabolized by these enzymes. The enzyme 17alpha-hydroxylase-C17,20-lyase (P450(17), CYP 17, androgen synthase), a cytochrome P450 monooxygenase, is the key enzyme for androgen biosynthesis. It catalyzes the last step of the androgen biosynthesis in the testes and adrenal glands and produces androstenedione and dehydroepiandrosterone from progesterone and pregnenolone. The microsomal enzyme aromatase (CYP19) transforms these androgens to estrone and estradiol. Estrogens stimulate tumor growth in hormone dependent breast cancer. In addition, about 80 percent of prostate cancers are androgen dependent. Selective inhibitors of these enzymes are thus important alternatives to treatment options like antiandrogens or antiestrogens. The present article deals with recent patents (focus on publications from 2000 - 2006) concerning P450 inhibitor design where steroidal substrates are involved. In this context a special focus is provided for CYP17 and CYP19. Mechanisms of action will also be discussed. Inhibitors of CYP11B2 (aldosterone synthase) will also be dealt with.

Resistance mutation conserved between insects and mites unravels the benzoylurea insecticide mode of action on chitin biosynthesis.

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Douris, Vassilis; Steinbach, Denise; Panteleri, Rafaela; Livadaras, Ioannis; Pickett, John Anthony; Van Leeuwen, Thomas; Nauen, Ralf; Vontas, John

2016-12-20

Despite the major role of chitin biosynthesis inhibitors such as benzoylureas (BPUs) in the control of pests in agricultural and public health for almost four decades, their molecular mode of action (MoA) has in most cases remained elusive. BPUs interfere with chitin biosynthesis and were thought to interact with sulfonylurea receptors that mediate chitin vesicle transport. Here, we uncover a mutation (I1042M) in the chitin synthase 1 (CHS1) gene of BPU-resistant Plutella xylostella at the same position as the I1017F mutation reported in spider mites that confers etoxazole resistance. Using a genome-editing CRISPR/Cas9 approach coupled with homology-directed repair (HDR) in Drosophila melanogaster, we introduced both substitutions (I1056M/F) in the corresponding fly CHS1 gene (kkv). Homozygous lines bearing either of these mutations were highly resistant to etoxazole and all tested BPUs, as well as buprofezin-an important hemipteran chitin biosynthesis inhibitor. This provides compelling evidence that BPUs, etoxazole, and buprofezin share in fact the same molecular MoA and directly interact with CHS. This finding has immediate effects on resistance management strategies of major agricultural pests but also on mosquito vectors of serious human diseases such as Dengue and Zika, as diflubenzuron, the standard BPU, is one of the few effective larvicides in use. The study elaborates on how genome editing can directly, rapidly, and convincingly elucidate the MoA of bioactive molecules, especially when target sites are complex and hard to reconstitute in vitro.

Paclobutrazol e estresse hídrico no florescimento e produção da mangueira (Mangifera indica 'Tommy Atkins' Paclobutrazol and water stress in the flowering and production of mango (Mangifera indica 'Tommy Atkins'

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Nelson Fonseca

2005-04-01

Full Text Available O uso de reguladores de crescimento e do estresse hídrico são práticas fundamentais para a indução do florescimento e produção da mangueira nas regiões tropicais. Avaliaram-se o florescimento e a produção da mangueira 'Tommy Atkins' na região semi-árida do Nordeste do Brasil, em resposta às aplicações de paclobutrazol (PBZ no solo (2 mL do i.a./planta, com irrigação da planta, e foliares em diferentes doses (0,5 e 1 mL do i.a. em aplicação única e fracionada em duas vezes, sem irrigação da planta, além da testemunha (uma aplicação foliar de água seguida de estresse hídrico. O delineamento experimental foi o inteiramente casualizado, no esquema de parcelas subdivididas no tempo. As variáveis foram avaliadas em três épocas após a aplicação PBZ (68 a 91; 131 a 179 e 190 a 216 dias após para o florescimento da planta; e 180 a 203; 267 a 287 e 299 a 337 para o número e produção de frutos por planta. Os resultados indicaram que o estresse hídrico imposto às plantas, sem uso do PBZ, foi tão eficiente na indução do florescimento e na produção de frutos da mangueira quanto a aplicação de PBZ via solo, usando irrigação, e via foliar, sem irrigação. A segunda época de avaliação foi superior à primeira e à terceira épocas no florescimento e produção por planta. A aplicação do PBZ no solo antecipou em 23 dias o florescimento da planta na segunda época em relação à aplicação do estresse hídrico. O trabalho sugere que o estresse hídrico tem potencial para a indução do florescimento e pode substituir o estresse causado pela aplicação de PBZ em mangueiras, nas condições semi-áridas tropicais.The use of growth regulators and water stress are important practices for the induction of flowering and production of mango in tropical areas. Flowering and fruit production of mango trees cv. Tommy Atkins were evaluated in semi-arid area of Northeast Brazil, in response to paclobutrazol applications

Everybody needs sphingolipids, right! Mining for new drug targets in protozoan sphingolipid biosynthesis.

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Mina, John G M; Denny, P W

2018-02-01

Sphingolipids (SLs) are an integral part of all eukaryotic cellular membranes. In addition, they have indispensable functions as signalling molecules controlling a myriad of cellular events. Disruption of either the de novo synthesis or the degradation pathways has been shown to have detrimental effects. The earlier identification of selective inhibitors of fungal SL biosynthesis promised potent broad-spectrum anti-fungal agents, which later encouraged testing some of those agents against protozoan parasites. In this review we focus on the key enzymes of the SL de novo biosynthetic pathway in protozoan parasites of the Apicomplexa and Kinetoplastidae, outlining the divergence and interconnection between host and pathogen metabolism. The druggability of the SL biosynthesis is considered, alongside recent technology advances that will enable the dissection and analyses of this pathway in the parasitic protozoa. The future impact of these advances for the development of new therapeutics for both globally threatening and neglected infectious diseases is potentially profound.

AmcA - a putative mitochondrial ornithine transporter supporting fungal siderophore biosynthesis

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Lukas eSchafferer

2015-04-01

Full Text Available Iron is an essential nutrient required for a wide range of cellular processes. The opportunistic fungal pathogen Aspergillus fumigatus employs low-molecular mass iron-specific chelators, termed siderophores, for uptake, storage and intracellular iron distribution, which play a crucial role in the pathogenicity of this fungus. Siderophore biosynthesis depends on coordination with the supply of its precursor ornithine, produced mitochondrially from glutamate or cytosolically via hydrolysis of arginine. In this study, we demonstrate a role of the putative mitochondrial transporter AmcA (AFUA_8G02760 in siderophore biosynthesis of A. fumigatus.Consistent with a role in cellular ornithine handling, AmcA-deficiency resulted in decreased cellular ornithine and arginine contents as well as decreased siderophore production on medium containing glutamine as the sole nitrogen source. In support, arginine and ornithine as nitrogen sources did not impact siderophore biosynthesis due to cytosolic ornithine availability. As revealed by Northern blot analysis, transcript levels of siderophore biosynthetic genes were unresponsive to the cellular ornithine level. In contrast to siderophore production, AmcA deficiency did only mildly decrease the cellular polyamine content, demonstrating cellular prioritization of ornithine use. Nevertheless, AmcA-deficiency increased the susceptibility of A. fumigatus to the polyamine biosynthesis inhibitor eflornithine, most likely due to the decreased ornithine pool. AmcA-deficiency decreased the growth rate particularly on ornithine as the sole nitrogen source during iron starvation and sufficiency, indicating an additional role in the metabolism and fitness of A. fumigatus, possibly in mitochondrial ornithine import. In the Galleria mellonella infection model, AmcA-deficiency did not affect virulence of A. fumigatus, most likely due to the residual siderophore production and arginine availability in this host niche.

Assessment of Paclobutrazol’s Time and Concentration of Foliar Application on Production and Germination of Potato (Solanum tuberosum L. Mini-tube

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B Saadatian

2018-02-01

Full Text Available Introduction Health and quality of potato (Solanum tuberosum L. seeds are important in the potato seed production technology. Moreover, the basic seed materials must be free of pathogens. Therefore tissue culture techniques are used at this stage in the laboratory to produce disease free seeds. Mini-tubers can be produced after acclimatization from plantlets as tissue culture which are planted at high densities in the greenhouse in beds or containers using different substrate mixtures, or even in hydroponic culture. Foliar application of growth regulators is a way to increase potato mini-tuber production. Growth regulators influence on potato mini-tuber dormancy. Triazoles, a family which Paclobutrazol belongs to them, have both fungitoxic and plant growth regulatory effects. In addition, they can also protect plants against various stresses. Therefore, the triazoles have been characterized as plant multi-protestants. Paclobutrazol changes the relationship between source and sink and by this way, affects plant production. Also, it can inhibit giberellic acid biosynthesis. Some morphological changes observed in paclobutrazol-treated plants include the inhibition of plant growth, decreased inter-nodal elongation and increased root to shoot ratios. Time of paclobutrazol foliar application is an important factor which changes plant characters (Lim et al., 2004. This study implemented to evaluate foliar application of paclobutrazol concentrations at different time on production and mini-tuber dormancy. Materials and Methods The factorial experiment based on randomized complete block design with four replications was conducted at the faculty of agriculture’s research greenhouse, Ferdowsi University of Mashhad, Iran, in 2013. Plantlets of Agria cv. produced from nodal tissue culture in Murashinge and Skoog (MS medium. After 25 days, plantlets of free disease and uniform exported to plastic pots with 12 cm diameter and 30 cm height. Perlite, cocopite

High glucose suppresses human islet insulin biosynthesis by inducing miR-133a leading to decreased polypyrimidine tract binding protein-expression.

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Rikard G Fred

Full Text Available BACKGROUND: Prolonged periods of high glucose exposure results in human islet dysfunction in vitro. The underlying mechanisms behind this effect of high glucose are, however, unknown. The polypyrimidine tract binding protein (PTB is required for stabilization of insulin mRNA and the PTB mRNA 3'-UTR contains binding sites for the microRNA molecules miR-133a, miR-124a and miR-146. The aim of this study was therefore to investigate whether high glucose increased the levels of these three miRNAs in association with lower PTB levels and lower insulin biosynthesis rates. METHODOLOGY/PRINCIPAL FINDINGS: Human islets were cultured for 24 hours in the presence of low (5.6 mM or high glucose (20 mM. Islets were also exposed to sodium palmitate or the proinflammatory cytokines IL-1beta and IFN-gamma, since saturated free fatty acids and cytokines also cause islet dysfunction. RNA was then isolated for real-time RT-PCR analysis of miR-133a, miR-124a, miR-146, insulin mRNA and PTB mRNA contents. Insulin biosynthesis rates were determined by radioactive labeling and immunoprecipitation. Synthetic miR-133a precursor and inhibitor were delivered to dispersed islet cells by lipofection, and PTB was analyzed by immunoblotting following culture at low or high glucose. Culture in high glucose resulted in increased islet contents of miR-133a and reduced contents of miR-146. Cytokines increased the contents of miR-146. The insulin and PTB mRNA contents were unaffected by high glucose. However, both PTB protein levels and insulin biosynthesis rates were decreased in response to high glucose. The miR-133a inhibitor prevented the high glucose-induced decrease in PTB and insulin biosynthesis, and the miR-133a precursor decreased PTB levels and insulin biosynthesis similarly to high glucose. CONCLUSION: Prolonged high-glucose exposure down-regulates PTB levels and insulin biosynthesis rates in human islets by increasing miR-133a levels. We propose that this mechanism

Crystal structures of Leishmania mexicana arginase complexed with α,α-disubstituted boronic amino-acid inhibitors

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Hai, Yang; Christianson, David W.

2016-03-16

Leishmaniaarginase is a potential drug target for the treatment of leishmaniasis because this binuclear manganese metalloenzyme initiatesde novopolyamine biosynthesis by catalyzing the hydrolysis of L-arginine to generate L-ornithine and urea. The product L-ornithine subsequently undergoes decarboxylation to yield putrescine, which in turn is utilized for spermidine biosynthesis. Polyamines such as spermidine are essential for the growth and survival of the parasite, so inhibition of enzymes in the polyamine-biosynthetic pathway comprises an effective strategy for treating parasitic infections. To this end, two X-ray crystal structures ofL. mexicanaarginase complexed with α,α-disubstituted boronic amino-acid inhibitors based on the molecular scaffold of 2-(S)-amino-6-boronohexanoic acid are now reported. Structural comparisons with human and parasitic arginase complexes reveal interesting differences in the binding modes of the additional α-substituents,i.e.the D side chains, of these inhibitors. Subtle differences in the three-dimensional contours of the outer active-site rims among arginases from different species lead to different conformations of the D side chains and thus different inhibitor-affinity trends. The structures suggest that it is possible to maintain affinity while fine-tuning intermolecular interactions of the D side chain of α,α-disubstituted boronic amino-acid inhibitors in the search for isozyme-specific and species-specific arginase inhibitors.

Investigations on the isoprenoid biosynthesis in the green alga Scenedesmus obliquus by using the 13C-labelling technique

International Nuclear Information System (INIS)

Schwender, J.

1995-01-01

The biosynthesis of several prenyllipids (isoprenoid lipids) of the green alga Scendesmus obliquus was investigated. The aim was to verify, whether the biosynthesis of isopentenyl diphosphate (IPP) in Scenedesmus proceeds according to the classical acetate mevalonate pathway or to an alternative pathway. An alternative pathway for IPP formation has recently been detected in some eubacteria by the group of Prof. M. Rohmer. Some inhibition tests were performed with mevinolin, a specific inhibitor of HMG-CoA reductase which yields mevalonic acid. Mevinolin should block the biosynthesis of such isoprenoids which are formed via the acetate mevalonate pathway. Scenedesmus was grown heterotrophically on 13 C-labelled glucose or acetate. After isolation and purification of 13 C-labelled phytol (side chains of chlorophylls), β-carotene, lutein, plastoquinone-9 and three sterol compounds, the enrichment of 13 C at different carbon-positions of the labelled compounds was determined. This was achieved by the 13 C-NMR technique in cooperation with Miriam Seemann of the group of Prof. M. Rohmer in Mullhouse/France. (orig.) [de

Reaction Intermediate Analogues as Bisubstrate Inhibitors of Pantothenate Synthetase

OpenAIRE

Xu, Zhixiang; Yin, Wei; Martinelli, Leonardo K.; Evans, Joanna; Chen, Jinglei; Yu, Yang; Wilson, Daniel J.; Mizrahi, Valerie; Qiao, Chunhua; Aldrich, Courtney C.

2014-01-01

The biosynthesis of pantothenate, the core of coenzyme A (CoA), has been considered an attractive target for the development of antimicrobial agents since this pathway is essential in prokaryotes, but absent in mammals. Pantothenate synthetase, encoded by the gene panC, catalyzes the final condensation of pantoic acid with β–alanine to afford pantothenate via an intermediate pantoyl adenylate. We describe the synthesis and biochemical characterization of five PanC inhibitors that mimic the in...

SHOEBOX Modulates Root Meristem Size in Rice through Dose-Dependent Effects of Gibberellins on Cell Elongation and Proliferation.

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Li, Jintao; Zhao, Yu; Chu, Huangwei; Wang, Likai; Fu, Yanru; Liu, Ping; Upadhyaya, Narayana; Chen, Chunli; Mou, Tongmin; Feng, Yuqi; Kumar, Prakash; Xu, Jian

2015-08-01

Little is known about how the size of meristem cells is regulated and whether it participates in the control of meristem size in plants. Here, we report our findings on shoebox (shb), a mild gibberellin (GA) deficient rice mutant that has a short root meristem size. Quantitative analysis of cortical cell length and number indicates that shb has shorter, rather than fewer, cells in the root meristem until around the fifth day after sowing, from which the number of cortical cells is also reduced. These defects can be either corrected by exogenous application of bioactive GA or induced in wild-type roots by a dose-dependent inhibitory effect of paclobutrazol on GA biosynthesis, suggesting that GA deficiency is the primary cause of shb mutant phenotypes. SHB encodes an AP2/ERF transcription factor that directly activates transcription of the GA biosynthesis gene KS1. Thus, root meristem size in rice is modulated by SHB-mediated GA biosynthesis that regulates the elongation and proliferation of meristem cells in a developmental stage-specific manner.

SHOEBOX Modulates Root Meristem Size in Rice through Dose-Dependent Effects of Gibberellins on Cell Elongation and Proliferation.

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Jintao Li

2015-08-01

Full Text Available Little is known about how the size of meristem cells is regulated and whether it participates in the control of meristem size in plants. Here, we report our findings on shoebox (shb, a mild gibberellin (GA deficient rice mutant that has a short root meristem size. Quantitative analysis of cortical cell length and number indicates that shb has shorter, rather than fewer, cells in the root meristem until around the fifth day after sowing, from which the number of cortical cells is also reduced. These defects can be either corrected by exogenous application of bioactive GA or induced in wild-type roots by a dose-dependent inhibitory effect of paclobutrazol on GA biosynthesis, suggesting that GA deficiency is the primary cause of shb mutant phenotypes. SHB encodes an AP2/ERF transcription factor that directly activates transcription of the GA biosynthesis gene KS1. Thus, root meristem size in rice is modulated by SHB-mediated GA biosynthesis that regulates the elongation and proliferation of meristem cells in a developmental stage-specific manner.

Serine biosynthesis and transport defects.

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El-Hattab, Ayman W

2016-07-01

l-serine is a non-essential amino acid that is biosynthesized via the enzymes phosphoglycerate dehydrogenase (PGDH), phosphoserine aminotransferase (PSAT), and phosphoserine phosphatase (PSP). Besides its role in protein synthesis, l-serine is a potent neurotrophic factor and a precursor of a number of essential compounds including phosphatidylserine, sphingomyelin, glycine, and d-serine. Serine biosynthesis defects result from impairments of PGDH, PSAT, or PSP leading to systemic serine deficiency. Serine biosynthesis defects present in a broad phenotypic spectrum that includes, at the severe end, Neu-Laxova syndrome, a lethal multiple congenital anomaly disease, intermediately, infantile serine biosynthesis defects with severe neurological manifestations and growth deficiency, and at the mild end, the childhood disease with intellectual disability. A serine transport defect resulting from deficiency of the ASCT1, the main transporter for serine in the central nervous system, has been recently described in children with neurological manifestations that overlap with those observed in serine biosynthesis defects. l-serine therapy may be beneficial in preventing or ameliorating symptoms in serine biosynthesis and transport defects, if started before neurological damage occurs. Herein, we review serine metabolism and transport, the clinical, biochemical, and molecular aspects of serine biosynthesis and transport defects, the mechanisms of these diseases, and the potential role of serine therapy. Copyright © 2016 Elsevier Inc. All rights reserved.

Induction of dopamine biosynthesis by l-DOPA in PC12 cells: implications of L-DOPA influx and cyclic AMP.

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Jin, Chun Mei; Yang, Yoo Jung; Huang, Hai Shan; Lim, Sung Cil; Kai, Masaaki; Lee, Myung Koo

2008-09-04

The effects of 3,4-dihydroxyphenylalanine (l-DOPA) on dopamine biosynthesis and cytotoxicity were investigated in PC12 cells. l-DOPA treatment (20-200 microM) increased the levels of dopamine by 226%-504% after 3-6 h of treatment and enhanced the activities of tyrosine hydroxylase (TH) and aromatic l-amino acid decarboxylase (AADC). l-DOPA (20-200 muM) treatment led to a 562%-937% increase in l-DOPA influx at 1 h, which inhibited the activity of TH, but not AADC, during the same period. The extracellular releases of dopamine were also increased by 231%-570% after treatment with 20 and 200 microM l-DOPA for 0.5-3 h. l-DOPA at a concentration of 100-200 microM, but not 20 microM, exerted apoptotic cytotoxicity towards PC12 cells for 24-48 h. l-DOPA (20-200 microM) increased the intracellular cyclic AMP levels by 318%-557% after 0.5-1 h in a concentration-dependent manner. However, the elevated cyclic AMP levels by l-DOPA could not protect against l-DOPA (100-200 microM)-induced cytotoxicity after 24-48 h. In addition, l-DOPA (20-200 microM)-induced increases in cyclic AMP and dopamine were significantly reduced by treatment with SCH23390 (dopamine D(1) receptor antagonist). The increased levels of dopamine by l-DOPA were also reduced by H89 (protein kinase A, PKA, inhibitor) and GF109203X (protein kinase C inhibitor); however, the reduction by GF109203X was not significant. l-DOPA at 20-200 microM stimulated the phosphorylation of PKA and cyclic AMP-response element binding protein and induced the biosynthesis of the TH protein. These results indicate that 20-200 microM l-DOPA induces dopamine biosynthesis by two pathways. One pathway involves l-DOPA directly entering the cells to convert dopamine through AADC activity (l-DOPA decarboxylation). The other pathway involves l-DOPA and/or released dopamine activating TH to enhance dopamine biosynthesis by the dopamine D(1) receptor-cyclic AMP-PKA signaling system (dopamine biosynthesis by TH).

SEARCH OF NEW SYNTHETIC INHIBITORS OF TYROSINASE

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Yu. Shesterenko

2017-11-01

Full Text Available Melanin pigmentation of skin plays the most important role in the protection of organism against UV-irradiation, but the excessive accumulation of melanin brings to toxic melanodermia, melasma, lentigo and other skin lesions. Tyrosinase is the key enzyme of skin melanin pigment biosynthesis. In spite of certain progress in investigation of natural and synthetic tyrosinase inhibitors, actuality of such studies is of a high level, because the existing inhibitors are in some cases unstable, expensive, toxic, requires complex methods of synthesis or isolation from natural sources. The aim of the work is screening of new tyrosinase inhibitors, using the enzyme, isolated from Agaricus bisporus. Tyrosinase was isolated from Agaricus bisporus mushrooms by a modified method. It was found, that the introduction of polyethylene glycol 4000 in the extraction process promotes 3-fold reduction of polyphenol content, which leads to increase purity of enzyme with an increase in its activity by 25%. A search for new tyrosinase inhibitors among a wide range of compounds, including derivatives of 3-chloro-1,4-naphthoquinone, isatin, 3-hydroxy-2-naphthoic acid, etc was conducted. The studied substances did not displayed inhibitory effect at concentration of 0,1-0,5 mmol/dm3.

Fatty acid biosynthesis is involved in the production of hepatitis B virus particles

International Nuclear Information System (INIS)

Okamura, Hitomi; Nio, Yasunori; Akahori, Yuichi; Kim, Sulyi; Watashi, Koichi; Wakita, Takaji; Hijikata, Makoto

2016-01-01

Hepatitis B virus (HBV) proliferates in hepatocytes after infection, but the host factors that contribute to the HBV lifecycle are poorly understood at the molecular level. We investigated whether fatty acid biosynthesis (FABS), which was recently reported to contribute to the genomic replication of hepatitis C virus, plays a role in HBV proliferation. We examined the effects of inhibitors of the enzymes in the FABS pathway on the HBV lifecycle by using recombinant HBV-producing cultured cells and found that the extracellular HBV DNA level, reflecting HBV particle production, was decreased by treatment with inhibitors suppressed the synthesis of long-chain saturated fatty acids with little cytotoxicity. The reduced HBV DNA level was reversed when palmitic acid, which is the product of fatty acid synthase (FAS) during FABS, was used simultaneously with the inhibitor. We also observed that the amount of intracellular HBV DNA in the cells was increased by FAS inhibitor treatment, suggesting that FABS is associated with HBV particle production but not its genome replication. This suggests that FABS might be a potent target for anti-HBV drug with a mode of action different from current HBV therapy. -- Highlights: •Inhibitors of ACC1 and FAS but not SCD1 decreased production of extracellular HBV DNA. •Products of FABS, long chain fatty acids, increased production of extracellular HBV DNA. •FAS inhibitor increased intracellular levels of HBV DNA and HBcAg. •FABS was suggested to contribute to HBV particle production without significant relation with secretory pathway of the cells.

Fatty acid biosynthesis is involved in the production of hepatitis B virus particles

Energy Technology Data Exchange (ETDEWEB)

Okamura, Hitomi [Laboratory of Human Tumor Viruses, Institute for Virus Research, Kyoto University, 53 Kawaharacho, Shogoin, Sakyoku, Kyoto 606-8507 (Japan); Graduate School of Biostudies, Kyoto University, Yoshida-Konoecho, Sakyoku, Kyoto 606-8501 (Japan); Nio, Yasunori, E-mail: yasunori.nio@takeda.com [Takeda Pharmaceutical Company Limited, Pharmaceutical Research Division, 26-1, Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa 251-8555 (Japan); Akahori, Yuichi [Laboratory of Human Tumor Viruses, Institute for Virus Research, Kyoto University, 53 Kawaharacho, Shogoin, Sakyoku, Kyoto 606-8507 (Japan); Graduate School of Biostudies, Kyoto University, Yoshida-Konoecho, Sakyoku, Kyoto 606-8501 (Japan); Kim, Sulyi [Laboratory of Human Tumor Viruses, Institute for Virus Research, Kyoto University, 53 Kawaharacho, Shogoin, Sakyoku, Kyoto 606-8507 (Japan); Watashi, Koichi [Department of Virology II, National Institute of Infectious Diseases, Tokyo 162-8640 (Japan); Department of Applied Biological Science, Tokyo University of Sciences, Noda 278-8510 (Japan); CREST, Japan Science and Technology Agency (JST), Saitama 332-0012 (Japan); Wakita, Takaji [Department of Virology II, National Institute of Infectious Diseases, Tokyo 162-8640 (Japan); Hijikata, Makoto, E-mail: mhijikat@virus.kyoto-u.ac.jp [Laboratory of Human Tumor Viruses, Institute for Virus Research, Kyoto University, 53 Kawaharacho, Shogoin, Sakyoku, Kyoto 606-8507 (Japan); Graduate School of Biostudies, Kyoto University, Yoshida-Konoecho, Sakyoku, Kyoto 606-8501 (Japan)

2016-06-17

Hepatitis B virus (HBV) proliferates in hepatocytes after infection, but the host factors that contribute to the HBV lifecycle are poorly understood at the molecular level. We investigated whether fatty acid biosynthesis (FABS), which was recently reported to contribute to the genomic replication of hepatitis C virus, plays a role in HBV proliferation. We examined the effects of inhibitors of the enzymes in the FABS pathway on the HBV lifecycle by using recombinant HBV-producing cultured cells and found that the extracellular HBV DNA level, reflecting HBV particle production, was decreased by treatment with inhibitors suppressed the synthesis of long-chain saturated fatty acids with little cytotoxicity. The reduced HBV DNA level was reversed when palmitic acid, which is the product of fatty acid synthase (FAS) during FABS, was used simultaneously with the inhibitor. We also observed that the amount of intracellular HBV DNA in the cells was increased by FAS inhibitor treatment, suggesting that FABS is associated with HBV particle production but not its genome replication. This suggests that FABS might be a potent target for anti-HBV drug with a mode of action different from current HBV therapy. -- Highlights: •Inhibitors of ACC1 and FAS but not SCD1 decreased production of extracellular HBV DNA. •Products of FABS, long chain fatty acids, increased production of extracellular HBV DNA. •FAS inhibitor increased intracellular levels of HBV DNA and HBcAg. •FABS was suggested to contribute to HBV particle production without significant relation with secretory pathway of the cells.

Regulation of cell wall biosynthesis.

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Zhong, Ruiqin; Ye, Zheng-Hua

2007-12-01

Plant cell walls differ in their amount and composition among various cell types and even in different microdomains of the wall of a given cell. Plants must have evolved regulatory mechanisms controlling biosynthesis, targeted secretion, and assembly of wall components to achieve the heterogeneity in cell walls. A number of factors, including hormones, the cytoskeleton, glycosylphosphatidylinositol-anchored proteins, phosphoinositides, and sugar nucleotide supply, have been implicated in the regulation of cell wall biosynthesis or deposition. In the past two years, there have been important discoveries in transcriptional regulation of secondary wall biosynthesis. Several transcription factors in the NAC and MYB families have been shown to be the key switches for activation of secondary wall biosynthesis. These studies suggest a transcriptional network comprised of a hierarchy of transcription factors is involved in regulating secondary wall biosynthesis. Further investigation and integration of the regulatory players participating in the making of cell walls will certainly lead to our understanding of how wall amounts and composition are controlled in a given cell type. This may eventually allow custom design of plant cell walls on the basis of our needs.

Discovery of inhibitors of Leishmania β-1,2-mannosyltransferases using a click-chemistry-derived guanosine monophosphate library.

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Phillip van der Peet

Full Text Available Leishmania spp. are a medically important group of protozoan parasites that synthesize a novel intracellular carbohydrate reserve polymer termed mannogen. Mannogen is a soluble homopolymer of β-1,2-linked mannose residues that accumulates in the major pathogenic stages in the sandfly vector and mammalian host. While several steps in mannogen biosynthesis have been defined, none of the enzymes have been isolated or characterized. We report the development of a simple assay for the GDP-mannose-dependent β-1,2-mannosyltransferases involved in mannogen synthesis. This assay utilizes octyl α-D-mannopyranoside to prime the formation of short mannogen oligomers up to 5 mannose residues. This assay was used to screen a focussed library of 44 GMP-triazole adducts for inhibitors. Several compounds provided effective inhibition of mannogen β-1,2-mannosyltransferases in a cell-free membrane preparation. This assay and inhibitor compounds will be useful for dissecting the role of different mannosyltransferases in regulating de novo biosynthesis and elongation reactions in mannogen metabolism.

The Antibiotic CJ-15,801 is an Antimetabolite which Hijacks and then Inhibits CoA Biosynthesis

Science.gov (United States)

van der Westhuyzen, Renier; Hammons, Justin C.; Meier, Jordan L.; Dahesh, Samira; Moolman, Wessel J. A.; Pelly, Stephen C.; Nizet, Victor; Burkart, Michael D.; Strauss, Erick

2012-01-01

SUMMARY The natural product CJ-15,801 is an inhibitor of Staphylococcus aureus, but not other bacteria. Its close structural resemblance to pantothenic acid, the vitamin precursor of coenzyme A (CoA), and its Michael acceptor moiety suggest that it irreversibly inhibits an enzyme involved in CoA biosynthesis or utilization. However, its mode of action and the basis for its specificity have not been elucidated to date. We demonstrate that CJ-15,801 is transformed by the uniquely selective S. aureus pantothenate kinase, the first CoA biosynthetic enzyme, into a substrate for the next enzyme, phosphopantothenoylcysteine synthetase, which is inhibited through formation of a tight-binding structural mimic of its native reaction intermediate. These findings reveal CJ-15,801 as a vitamin biosynthetic pathway antimetabolite with a mechanism similar to that of the sulfonamide antibiotics, and highlight CoA biosynthesis as a viable antimicrobial drug target. PMID:22633408

Biosynthesis of tylophora alkaloids

International Nuclear Information System (INIS)

Mulchandani, N.B.; Iyer, S.S.; Badheka, L.P.

1974-01-01

Using labelled precursors, biosynthesis of the tylophora alkaloids, tylophorine, tylophorinidine and tylophorinide has been investigated in Tylophora asthmatica plants. The radioactive precursors, phenylalanine-2- 14 C, benzoic acid-1- 14 C, benzoic acid-ring 14 C, acetate-2- 14 C, ornithine-5- 14 C, acetate-2- 14 C, ornithine-5- 14 C and cinnamic acid-2- 14 C were administered to the plants individually by wick technique. Tylophorine was isolated in each case and assayed for its radioactivity to find out the incorporation of the label into it. The results indicate that: (1) phenylalanine via cinnamic acid is an important precursor in the biosynthesis of tylophorine (2) orinithine participates in tylophorine biosynthesis via pyrroline and (3) tylophorinidine may be a direct precursor of tylophorine. (M.G.B.)

Synergistic interactions between a variety of insecticides and an ergosterol biosynthesis inhibitor fungicide in dietary exposures of bumble bees (Bombus terrestris L.).

Science.gov (United States)

Raimets, Risto; Karise, Reet; Mänd, Marika; Kaart, Tanel; Ponting, Sally; Song, Jimao; Cresswell, James E

2018-03-01

In recent years, concern has been raised over honey bee colony losses, and also among wild bees there is evidence for extinctions and range contractions in Europe and North America. Pesticides have been proposed as a potential cause of this decline. Bees are exposed simultaneously to a variety of agrochemicals, which may cause synergistically detrimental impacts, which are incompletely understood. We investigated the toxicity of the fungicide imazalil in mixture with four common insecticides: fipronil (phenylpyrazoid), cypermethrin (pyrethroid), thiamethoxam, and imidacloprid (neonicotinoids). Ergosterol biosynthesis inhibitor (EBI) fungicides like imazalil can inhibit P450 detoxification systems in insects and therefore fungicide - insecticide co-occurrence might produce synergistic toxicity in bees. We assessed the impact of dietary fungicide - insecticide mixtures on the mortality and feeding rates of laboratory bumble bees (Bombus terrestris L.). Regarding mortality, imazalil synergised the toxicity of fipronil, cypermethrin and thiamethoxam, but not imidacloprid. We found no synergistic effects on feeding rates. Our findings suggest that P450-based detoxification processes are differentially important in mitigating the toxicity of certain insecticides, even those of the same chemical class. Our evidence that cocktail effects can arise in bumble bees should extend concern about the potential impacts of agrochemical mixtures to include wild bee species in farmland. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Borrelidin B: isolation, biological activity, and implications for nitrile biosynthesis.

Science.gov (United States)

Schulze, Christopher J; Bray, Walter M; Loganzo, Frank; Lam, My-Hanh; Szal, Teresa; Villalobos, Anabella; Koehn, Frank E; Linington, Roger G

2014-11-26

Borrelidin (1) is a nitrile-containing bacterially derived polyketide that is a potent inhibitor of bacterial and eukaryotic threonyl-tRNA synthetases. We now report the discovery of borrelidin B (2), a tetrahydro-borrelidin derivative containing an aminomethyl group in place of the nitrile functionality in borrelidin. The discovery of this new metabolite has implications for both the biosynthesis of the nitrile group and the bioactivity of the borrelidin compound class. Screening in the SToPS assay for tRNA synthetase inhibition revealed that the nitrile moiety is essential for activity, while profiling using our in-house image-based cytological profiling assay demonstrated that 2 retains biological activity by causing a mitotic stall, even in the absence of the nitrile motif.

Effect of amino acids on tannase biosynthesis by Bacillus licheniformis KBR6.

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Mohapatra, Pradeep K Das; Pati, Bikas R; Mondal, Keshab C

2009-04-01

Microbial tannase (tannin acyl hydrolase, EC 3.1.1.20), a hydrolysable tannin-degrading enzyme, has gained importance in various industrial processes, and is used extensively in the manufacture of instant tea, beer, wine, and gallic acid. Tannase is an inducible enzyme, and hydrolysable tannin, especially tannic acid, is the sole inducer. This study is of the effect of various amino acids and their analogues on tannase biosynthesis by Bacillus licheniformis KBR6 to ascertain the mode of action of these growth factors on tannase biosynthesis from microbial origin. Enzyme production was carried out in enriched tannic acid medium through submerged fermentation for 20 h at 35 degrees C. Different amino acids at a concentration of 0.05 g% (w/v) were added to the culture medium immediately after sterilization. Culture supernatant was used as the source of the enzyme and the quantity of tannase was estimated by the colorimetric assay method. Growth of the organism was estimated according to biomass dry weight. Maximum tannase (2.87-fold that of the control) was synthesized by B. licheniformis KBR6 when alanine was added to the culture medium. Other amino acids, such as DL-serine, L-cystine, glycine, L-ornithine, aspartic acid, L-glutamic acid, DL-valine, L-leucine and L-lysine, also induced tannase synthesis. L-Cysteine monohydrochloride and DL-threonine were the most potent inhibitors. Regulation of tannase biosynthesis by B. licheniformis in the presence of various amino acids is shown. This information will be helpful for formulating an enriched culture medium for industrial-scale tannase production.

GERMINATION STUDIES ON Tabebuia impetiginosa Mart. SEEDS

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Edvaldo Aparecido Amaral da Silva

2004-06-01

Full Text Available Seed germination and seedling production of native forest tree species are an important step in ex situ conservation programs and in the reforestation with ecological purposes. Therefore, understanding seed germination and its regulation is mandatory for the complete success of the conservation programs and revegetation techniques. Thus, morphological studies, temperature requirements for seed germination and its control by gibberellins (GAs were studied in Tabebuia impetiginosa (“ipê-roxo” seeds. The best temperature for germination under constant light was 30oC. The imbibition of T. impetiginosa seeds followed the common triphasic pattern, with most of the seeds attaining phase II at 24 hours and phase III at 72 hours of imbibition. Visible germination, as radicle elongation, started at 30 hours in water-imbibed seeds and at 24 hours in GA-imbibed seeds. Seeds imbibed in Paclobutrazol, an inhibitor of GA biosynthesis, failed to germinate. However, application of exogenous gibberellins overcame inhibition and allowed germination, suggesting that GAs are regulators of Tabebuia impetiginosa seed germination. The results suggested that germination in Tabebuia impetiginosa seeds is controlled by elongation of the radicle and gibberellins may play an important role in regulating it. The possible role of gibberellins is discussed.

Effects of a Squalene Epoxidase Inhibitor, Terbinafine, on Ether Lipid Biosyntheses in a Thermoacidophilic Archaeon, Thermoplasma acidophilum

Science.gov (United States)

Kon, Takahide; Nemoto, Naoki; Oshima, Tairo; Yamagishi, Akihiko

2002-01-01

The archaeal plasma membrane consists mainly of diether lipids and tetraether lipids instead of the usual ester lipids found in other organisms. Although a molecule of tetraether lipid is thought to be synthesized from two molecules of diether lipids, there is no direct information about the biosynthetic pathway(s) or intermediates of tetraether lipid biosynthesis. In this study, we examined the effects of the fungal squalene epoxidase inhibitor terbinafine on the growth and ether lipid biosyntheses in the thermoacidophilic archaeon Thermoplasma acidophilum. Terbinafine was found to inhibit the growth of T. acidophilum in a concentration-dependent manner. When growing T. acidophilum cells were pulse-labeled with [2-14C]mevalonic acid in the presence of terbinafine, incorporation of radioactivity into the tetraether lipid fraction was strongly suppressed, while accumulation of radioactivity was noted at the position corresponding to diether lipids, depending on the concentration of terbinafine. After the cells were washed with fresh medium and incubated further without the radiolabeled substrate and the inhibitor, the accumulated radioactivity in the diether lipid fraction decreased quickly while that in the tetraether lipids increased simultaneously, without significant changes in the total radioactivity of ether lipids. These results strongly suggest that terbinafine inhibits the biosynthesis of tetraether lipids from a diether-type precursor lipid(s). The terbinafine treatment will be a tool for dissecting tetraether lipid biosynthesis in T. acidophilum. PMID:11844769

The Spatial Organization of Glucosinolate Biosynthesis

DEFF Research Database (Denmark)

Nintemann, Sebastian

cells is an open question. Likewise, it is not known how glucosinolate biosynthesis is orchestrated at the subcellular level. These open questions were addressed with several approaches in this project, with the aim of shedding light on the spatial organization of glucosinolate biosynthesis from...... between the individual classes of glucosinolates under constitutive and induced conditions and identified the source tissues of these defense compounds. Protein-protein interaction studies were carried out to investigate the subcellular organization of glucosinolate biosynthesis. We identified a family...

Treatment and Prevention of Breast Cancer Using Multifunctional Inhibitors of Cholesterol Biosynthesis

Science.gov (United States)

2015-08-01

suggesting an allosteric modification of estrogen receptor  RO blocks the production of an estrogen regulated gene ( progesterone receptor) in...alternative targets in the pathway leading to the production of cholesterol, which might be regulated with less toxic inhibitors to control the progression of...to effectively treat and prevent cancers of the breast. Our goal was to identify alternative targets in the pathway leading to the production of

Synthesis and study on biological activity of nitrogen-containing heterocyclic compounds – regulators of enzymes of nucleic acid biosynthesis

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Alexeeva I. V.

2013-07-01

Full Text Available Results of investigations on the development of new regulators of functional activity of nucleic acid biosynthesis enzymes based on polycyclic nitrogen-containing heterosystems are summarized. Computer design and molecular docking in the catalytic site of target enzyme (T7pol allowed to perform the directed optimization of basic structures. Several series of compounds were obtained and efficient inhibitors of herpes family (simple herpes virus type 2, Epstein-Barr virus, influenza A and hepatitis C viruses were identified, as well as compounds with potent antitumor, antibacterial and antifungal activity. It was established that the use of model test systems based on enzymes participating in nucleic acids synthesis is a promising approach to the primary screening of potential inhibitors in vitro.

Gibberellin Deficiency Confers Both Lodging and Drought Tolerance in Small Cereals

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Sonia ePlaza-Wüthrich

2016-05-01

Full Text Available Tef [Eragrostis tef (Zucc. Trotter] and finger millet [Eleusine coracana Gaertn] are staple cereal crops in Africa and Asia with several desirable agronomic and nutritional properties. Tef is becoming a life-style crop as it is gluten-free while finger millet has a low glycemic index which makes it an ideal food for diabetic patients. However, both tef and finger millet have extremely low grain yields mainly due to moisture scarcity and susceptibility of the plants to lodging. In this study, the effects of gibberellic acid (GA inhibitors particularly paclobutrazol (PBZ on diverse physiological and yield-related parameters were investigated and compared to GA mutants in rice (Oryza sativa L.. The application of PBZ to tef and finger millet significantly reduced the plant height and increased lodging tolerance. Remarkably, PBZ also enhanced the tolerance of both tef and finger millet to moisture deficit. Under moisture scarcity, tef plants treated with PBZ did not exhibit drought-related symptoms and their stomatal conductance was unaltered, leading to higher shoot biomass and grain yield. Semi-dwarf rice mutants altered in GA biosynthesis, were also shown to have improved tolerance to dehydration. The combination of traits (drought tolerance, lodging tolerance and increased yield that we found in plants with altered GA pathway is of importance to breeders who would otherwise rely on extensive crossing to introgress each trait individually. The key role played by PBZ in the tolerance to both lodging and drought calls for further studies using mutants in the GA biosynthesis pathway in order to obtain candidate lines which can be incorporated into crop-breeding programs to create lodging tolerant and climate-smart crops.

In silico approach towards identification of potential inhibitors of Helicobacter pylori DapE.

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Mandal, Rahul Shubhra; Das, Santasabuj

2015-01-01

Helicobacter pylori is a gastric mucosal pathogen and is associated with diseases like peptic ulcer and gastric cancer. To combat H. pylori infection, there is an urgent need for new class of antibiotics due to the emergence of drug-resistant strains. Enzymes involved in bacterial lysine biosynthetic pathways may be potential targets for antibacterial drug development, since lysine is an essential component of the bacterial peptidoglycan cell wall. No pathway exists for lysine biosynthesis in humans; hence, the inhibitors targeting bacterial enzymes may have selective toxicity. dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) is a critical enzyme of this pathway and deletion of DapE gene is lethal to H. pylori, since the organism has no alternative pathway for lysine biosynthesis. In this study, we reported a 3D model structure of H. pylorie DapE, which consisted of a catalytic domain and a dimerization domain generated by MODELLER software. We also confirmed the stability of the modeled structure through 10 ns molecular dynamics simulation using GROMACS software. Next, to identify potential small molecule inhibitors of DapE, drug-like small molecule-screening library was generated. This was performed by Tanimoto-based similarity searching in the PubChem Database with DapE substrate L,L-SDAP as a query molecule, followed by fragment-based docking approach using GLIDE XP. This approach identified two potential substrate-competitive small molecule inhibitors of DapE. These new molecules may provide a starting point to search for novel therapeutics.

Propiconazole-enhanced hepatic cell proliferation is associated with dysregulation of the cholesterol biosynthesis pathway leading to activation of Erk1/2 through Ras farnesylation

Energy Technology Data Exchange (ETDEWEB)

Murphy, Lynea A.; Moore, Tanya; Nesnow, Stephen, E-mail: nesnow.stephen@epa.gov

2012-04-15

Propiconazole is a mouse hepatotumorigenic fungicide designed to inhibit CYP51, a key enzyme in the biosynthesis of ergosterol in fungi and is widely used in agriculture to prevent fungal growth. Metabolomic studies in mice revealed that propiconazole increased levels of hepatic cholesterol metabolites and bile acids, and transcriptomic studies revealed that genes within the cholesterol biosynthesis, cholesterol metabolism and bile acid biosyntheses pathways were up-regulated. Hepatic cell proliferation was also increased by propiconazole. AML12 immortalized hepatocytes were used to study propiconazole's effects on cell proliferation focusing on the dysregulation of cholesterol biosynthesis and resulting effects on Ras farnesylation and Erk1/2 activation as a primary pathway. Mevalonate, a key intermediate in the cholesterol biosynthesis pathway, increases cell proliferation in several cancer cell lines and tumors in vivo and serves as the precursor for isoprenoids (e.g. farnesyl pyrophosphate) which are crucial in the farnesylation of the Ras protein by farnesyl transferase. Farnesylation targets Ras to the cell membrane where it is involved in signal transduction, including the mitogen-activated protein kinase (MAPK) pathway. In our studies, mevalonic acid lactone (MVAL), a source of mevalonic acid, increased cell proliferation in AML12 cells which was reduced by farnesyl transferase inhibitors (L-744,832 or manumycin) or simvastatin, an HMG-CoA reductase inhibitor, indicating that this cell system responded to alterations in the cholesterol biosynthesis pathway. Cell proliferation in AML12 cells was increased by propiconazole which was reversed by co-incubation with L-744,832 or simvastatin. Increasing concentrations of exogenous cholesterol muted the proliferative effects of propiconazole and the inhibitory effects of L-733,832, results ascribed to reduced stimulation of the endogenous cholesterol biosynthesis pathway. Western blot analysis of subcellular

Glycopeptide antibiotic biosynthesis.

Science.gov (United States)

Yim, Grace; Thaker, Maulik N; Koteva, Kalinka; Wright, Gerard

2014-01-01

Glycopeptides such as vancomycin, teicoplanin and telavancin are essential for treating infections caused by Gram-positive bacteria. Unfortunately, the dwindled pipeline of new antibiotics into the market and the emergence of glycopeptide-resistant enterococci and other resistant bacteria are increasingly making effective antibiotic treatment difficult. We have now learned a great deal about how bacteria produce antibiotics. This information can be exploited to develop the next generation of antimicrobials. The biosynthesis of glycopeptides via nonribosomal peptide assembly and unusual amino acid synthesis, crosslinking and tailoring enzymes gives rise to intricate chemical structures that target the bacterial cell wall. This review seeks to describe recent advances in our understanding of both biosynthesis and resistance of these important antibiotics.

Dual-Targeting Small-Molecule Inhibitors of the Staphylococcus aureus FMN Riboswitch Disrupt Riboflavin Homeostasis in an Infectious Setting.

Science.gov (United States)

Wang, Hao; Mann, Paul A; Xiao, Li; Gill, Charles; Galgoci, Andrew M; Howe, John A; Villafania, Artjohn; Barbieri, Christopher M; Malinverni, Juliana C; Sher, Xinwei; Mayhood, Todd; McCurry, Megan D; Murgolo, Nicholas; Flattery, Amy; Mack, Matthias; Roemer, Terry

2017-05-18

Riboswitches are bacterial-specific, broadly conserved, non-coding RNA structural elements that control gene expression of numerous metabolic pathways and transport functions essential for cell growth. As such, riboswitch inhibitors represent a new class of potential antibacterial agents. Recently, we identified ribocil-C, a highly selective inhibitor of the flavin mononucleotide (FMN) riboswitch that controls expression of de novo riboflavin (RF, vitamin B2) biosynthesis in Escherichia coli. Here, we provide a mechanistic characterization of the antibacterial effects of ribocil-C as well as of roseoflavin (RoF), an antimetabolite analog of RF, among medically significant Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecalis. We provide genetic, biophysical, computational, biochemical, and pharmacological evidence that ribocil-C and RoF specifically inhibit dual FMN riboswitches, separately controlling RF biosynthesis and uptake processes essential for MRSA growth and pathogenesis. Such a dual-targeting mechanism is specifically required to develop broad-spectrum Gram-positive antibacterial agents targeting RF metabolism. Copyright © 2017 Elsevier Ltd. All rights reserved.

Biosynthesis of silver nanoparticles synthesized by Aspergillus ...

Indian Academy of Sciences (India)

Biotechnology Division, Applied Science Department, University of ... Abstract. In the present study, biosynthesis of silver nanoparticles and its antioxidant, antimicrobial and cytotoxic ... example of the biosynthesis using fungi was that the cell-.

Discovery and quantitative structure-activity relationship study of lepidopteran HMG-CoA reductase inhibitors as selective insecticides.

Science.gov (United States)

Zang, Yang-Yang; Li, Yuan-Mei; Yin, Yue; Chen, Shan-Shan; Kai, Zhen-Peng

2017-09-01

In a previous study we have demonstrated that insect 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) can be a potential selective insecticide target. Three series of inhibitors were designed on the basis of the difference in HMGR structures from Homo sapiens and Manduca sexta, with the aim of discovering potent selective insecticide candidates. An in vitro bioassay showed that gem-difluoromethylenated statin analogues have potent effects on JH biosynthesis of M. sexta and high selectivity between H. sapiens and M. sexta. All series II compounds {1,3,5-trisubstituted [4-tert-butyl 2-(5,5-difluoro-2,2-dimethyl-6-vinyl-4-yl) acetate] pyrazoles} have some effect on JH biosynthesis, whereas most of them are inactive on human HMGR. In particular, the IC 50 value of compound II-12 (37.8 nm) is lower than that of lovastatin (99.5 nm) and similar to that of rosuvastatin (24.2 nm). An in vivo bioassay showed that I-1, I-2, I-3 and II-12 are potential selective insecticides, especially for lepidopteran pest control. A predictable and statistically meaningful CoMFA model of 23 inhibitors (20 as training sets and three as test sets) was obtained with a value of q 2 and r 2 of 0.66 and 0.996 respectively. The final model suggested that a potent insect HMGR inhibitor should contain suitable small and non-electronegative groups in the ring part, and electronegative groups in the side chain. Four analogues were discovered as potent selective lepidopteran HMGR inhibitors, which can specifically be used for lepidopteran pest control. The CoMFA model will be useful for the design of new selective insect HMGR inhibitors that are structurally related to the training set compounds. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

The regulation and biosynthesis of antimycins

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Ryan F. Seipke

2013-11-01

Full Text Available Antimycins (>40 members were discovered nearly 65 years ago but the discovery of the gene cluster encoding antimycin biosynthesis in 2011 has facilitated rapid progress in understanding the unusual biosynthetic pathway. Antimycin A is widely used as a piscicide in the catfish farming industry and also has potent killing activity against insects, nematodes and fungi. The mode of action of antimycins is to inhibit cytochrome c reductase in the electron transport chain and halt respiration. However, more recently, antimycin A has attracted attention as a potent and selective inhibitor of the mitochondrial anti-apoptotic proteins Bcl-2 and Bcl-xL. Remarkably, this inhibition is independent of the main mode of action of antimycins such that an artificial derivative named 2-methoxyantimycin A inhibits Bcl-xL but does not inhibit respiration. The Bcl-2/Bcl-xL family of proteins are over-produced in cancer cells that are resistant to apoptosis-inducing chemotherapy agents, so antimycins have great potential as anticancer drugs used in combination with existing chemotherapeutics. Here we review what is known about antimycins, the regulation of the ant gene cluster and the unusual biosynthetic pathway.

Revealing fosfomycin primary effect on Staphylococcus aureus transcriptome: modulation of cell envelope biosynthesis and phosphoenolpyruvate induced starvation

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Gruden Kristina

2010-06-01

Full Text Available Abstract Background Staphylococcus aureus is a highly adaptable human pathogen and there is a constant search for effective antibiotics. Fosfomycin is a potent irreversible inhibitor of MurA, an enolpyruvyl transferase that uses phosphoenolpyruvate as substrate. The goal of this study was to identify the pathways and processes primarily affected by fosfomycin at the genome-wide transcriptome level to aid development of new drugs. Results S. aureus ATCC 29213 cells were treated with sub-MIC concentrations of fosfomycin and harvested at 10, 20 and 40 minutes after treatment. S. aureus GeneChip statistical data analysis was complemented by gene set enrichment analysis. A visualization tool for mapping gene expression data into biological pathways was developed in order to identify the metabolic processes affected by fosfomycin. We have shown that the number of significantly differentially expressed genes in treated cultures increased with time and with increasing fosfomycin concentration. The target pathway - peptidoglycan biosynthesis - was upregulated following fosfomycin treatment. Modulation of transport processes, cofactor biosynthesis, energy metabolism and nucleic acid biosynthesis was also observed. Conclusions Several pathways and genes downregulated by fosfomycin have been identified, in contrast to previously described cell wall active antibiotics, and was explained by starvation response induced by phosphoenolpyruvate accumulation. Transcriptomic profiling, in combination with meta-analysis, has been shown to be a valuable tool in determining bacterial response to a specific antibiotic.

The guanylhydrazone CNI-1493: an inhibitor with dual activity against malaria-inhibition of host cell pro-inflammatory cytokine release and parasitic deoxyhypusine synthase.

Science.gov (United States)

Specht, Sabine; Sarite, Salem Ramadan; Hauber, Ilona; Hauber, Joachim; Görbig, Ulf F; Meier, Chris; Bevec, Dorian; Hoerauf, Achim; Kaiser, Annette

2008-05-01

Malaria is still a major cause of death in the tropics. There is an urgent need for new anti-malarial drugs because drug-resistant plasmodia frequently occur. Over recent years, we elucidated the biosynthesis of hypusine, a novel amino acid contained in eukaryotic initiation factor 5A (eIF-5A) in Plasmodium. Hypusine biosynthesis involves catalysis of deoxyhypusine synthase (DHS) in the first step of post-translational modification. In a screen for new inhibitors of purified plasmodium DHS, CNI-1493, a novel selective pro-inflammatory cytokine inhibitor used in clinical phase II for the treatment of Crohn's disease, inhibited the enzyme of the parasite 3-fold at a concentration of 2 microM. In vitro experiments with 200 microM CNI-1493 in Plasmodium-infected erythrocytes, which lack nuclei and DHS protein, showed a parasite clearance within 2 days. This can presumably be attributed to an anti-proliferating effect because of the inhibition of DHS by the parasite. The determined IC50 of CNI-1493 was 135.79 microM after 72 h. In vivo application of this substance in Plasmodium berghei ANKA-infected C57BL/6 mice significantly reduced parasitemia after dosage of 1 mg/kg or 4 mg/kg/body weight and prevented death of mice with cerebral malaria. This effect was paralleled by a decrease in serum TNF levels of the mice. We suggest that the new mechanism of CNI-1493 is caused by a decrease in modified eIF-5A biosynthesis with a downstream effect on the TNF synthesis of the host. From the current data, we consider CNI-1493 to be a promising drug for anti-malarial therapy because of its combined action, i.e., the decrease in eIF-5A biosynthesis of the parasite and host cell TNF biosynthesis.

Metabolic disturbance and phytochemical changes in Andrographis paniculata and possible action mode of andrographolide

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Netiya Karaket

2018-01-01

Full Text Available Objective: To explore the effect of gibberellic acid (GA3 and its inhibitor paclobutrazol (PBZ on chemical composition and their pharmacological effects on Andrographis paniculata (Burm. f. Wall. ex Nees, and to clarify action mode of andrographolide. Methods: The chemical composition was extracted by sequential extraction with hexane, dichloromethane, ethyl acetate and methanol, respectively. Andrographolide and its derivatives were evaluated by HPLC. Moreover, the metabolic profiling was analyzed by GC-MS. Inhibitory effect of crude extracts was tested against Staphylococcus aureus using agar well diffusion method. Mode of action was tested against mutant yeast by spotting assay. Andrographolide were tested for their mode of action against eukaryotes. Results: Among different solvents, dichloromethane gave the highest yield of crude (3.58% DW, with the highest andrographolide content (8.3 mg/g DW. The effect of plant hormone (10 mg/L GA3 or PBZ on phytochemical variations and bioactivity of Andrographis paniculata was demonstrated. It was found that PBZ promoted sesquiterpene compounds about 3.5 times over than GA3 treatment. But inhibitory effect of extracts against Staphylococcus aureus was highest in GA3 treated plants; andrographolide and 14-deoxy-11,12-didehydroandrographolide contents were significantly higher than those of water or PBZ. It was found that there were 11 strains involving in ergosterol biosynthesis, V-ATPase activity and homeostasis, and superoxide detoxification process. In this regard, andrographolide might cause the damage on the lipid bilayer of yeast cell and plasma membrane by interfering ergosterol biosynthesis. Conclusions: It is found that GA3 promotes andrographolide and 14-deoxy-11,12-didehydroandrographolide content while PBZ promotes sesquiterpene content. Andrographolide might cause the damage on the lipid bilayer of yeast cell and plasma membrane by interfering ergosterol biosynthesis. It might also affect

Design and Synthesis of New Peptidomimetics as Potential Inhibitors of MurE.

Science.gov (United States)

Zivec, Matej; Turk, Samo; Blanot, Didier; Gobec, Stanislav

2011-03-01

With the continuing emergence and spread of multidrug-resistant bacteria, there is an urgent need for the development of new antimicrobial agents. One possible source of new antibacterial targets is the biosynthesis of the bacterial cell-wall peptidoglycan. The assembly of the peptide stem is carried out by four essential enzymes, known as the Mur ligases (MurC, D, E and F). We have designed and synthesised a focused library of compounds as potential inhibitors of UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:L-lysine ligase (MurE) from Staphylococcus aureus. This was achieved using two approaches: (i) synthesis of transition-state analogues based on the methyleneamino core; and (ii) synthesis of MurE reaction product analogues. Two methyleneamino-based compounds are identified as initial hits for inhibitors of MurE.

FLO11 expression and lipid biosynthesis are required for air-liquid biofilm formation in a Saccharomyces cerevisiae flor strain.

Science.gov (United States)

Zara, Giacomo; Goffrini, Paola; Lodi, Tiziana; Zara, Severino; Mannazzu, Ilaria; Budroni, Marilena

2012-11-01

Air-liquid biofilm formation is largely dependent on Flo11p and seems related to cell lipid content and composition. Here, it is shown that in the presence of cerulenin, a known inhibitor of the fatty acid synthase complex, biofilm formation is inhibited together with FLO11 transcription in a flor strain of Saccharomyces cerevisiae, while the administration of saturated fatty acids to cerulenin-containing medium restores biofilm formation and FLO11 transcription. It is also shown that, in biofilm cells, the FLO11 transcription is accompanied by the transcription of ACC1, ACS1 and INO1 key genes in lipid biosynthesis and that biofilm formation is affected by the lack of inositol in flor medium. These results are compatible with the hypothesis that the air-liquid biofilm formation depends on FLO11 transcription levels as well as on fatty acids biosynthesis. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

A porphodimethene chemical inhibitor of uroporphyrinogen decarboxylase.

Directory of Open Access Journals (Sweden)

Kenneth W Yip

Full Text Available Uroporphyrinogen decarboxylase (UROD catalyzes the conversion of uroporphyrinogen to coproporphyrinogen during heme biosynthesis. This enzyme was recently identified as a potential anticancer target; its inhibition leads to an increase in reactive oxygen species, likely mediated by the Fenton reaction, thereby decreasing cancer cell viability and working in cooperation with radiation and/or cisplatin. Because there is no known chemical UROD inhibitor suitable for use in translational studies, we aimed to design, synthesize, and characterize such a compound. Initial in silico-based design and docking analyses identified a potential porphyrin analogue that was subsequently synthesized. This species, a porphodimethene (named PI-16, was found to inhibit UROD in an enzymatic assay (IC50 = 9.9 µM, but did not affect porphobilinogen deaminase (at 62.5 µM, thereby exhibiting specificity. In cellular assays, PI-16 reduced the viability of FaDu and ME-180 cancer cells with half maximal effective concentrations of 22.7 µM and 26.9 µM, respectively, and only minimally affected normal oral epithelial (NOE cells. PI-16 also combined effectively with radiation and cisplatin, with potent synergy being observed in the case of cisplatin in FaDu cells (Chou-Talalay combination index <1. This work presents the first known synthetic UROD inhibitor, and sets the foundation for the design, synthesis, and characterization of higher affinity and more effective UROD inhibitors.

Inhibitors of polyamine metabolism: review article.

Science.gov (United States)

Wallace, H M; Fraser, A V

2004-07-01

The identification of increased polyamine concentrations in a variety of diseases from cancer and psoriasis to parasitic infections has led to the hypothesis that manipulation of polyamine metabolism is a realistic target for therapeutic or preventative intervention in the treatment of certain diseases. The early development of polyamine biosynthetic single enzyme inhibitors such as alpha-difluoromethylornithine (DFMO) and methylglyoxal bis(guanylhydrazone) showed some interesting early promise as anticancer drugs, but ultimately failed in vivo. Despite this, DFMO is currently in use as an effective anti-parasitic agent and has recently also been shown to have further potential as a chemopreventative agent in colorectal cancer. The initial promise in vitro led to the development and testing of other potential inhibitors of the pathway namely the polyamine analogues. The analogues have met with greater success than the single enzyme inhibitors possibly due to their multiple targets. These include down regulation of polyamine biosynthesis through inhibition of ornithine decarboxylase and S-adenosylmethionine decarboxylase and decreased polyamine uptake. This coupled with increased activity of the catabolic enzymes, polyamine oxidase and spermidine/spermine N1-acetyltransferase, and increased polyamine export has made the analogues more effective in depleting polyamine pools. Recently, the identification of a new oxidase (PAO-h1/SMO) in polyamine catabolism and evidence of induction of both PAO and PAO-h1/SMO in response to polyamine analogue treatment, suggests the analogues may become an important part of future chemotherapeutic and/or chemopreventative regimens.

A comparative modeling and molecular docking study on Mycobacterium tuberculosis targets involved in peptidoglycan biosynthesis.

Science.gov (United States)

Fakhar, Zeynab; Naiker, Suhashni; Alves, Claudio N; Govender, Thavendran; Maguire, Glenn E M; Lameira, Jeronimo; Lamichhane, Gyanu; Kruger, Hendrik G; Honarparvar, Bahareh

2016-11-01

An alarming rise of multidrug-resistant Mycobacterium tuberculosis strains and the continuous high global morbidity of tuberculosis have reinvigorated the need to identify novel targets to combat the disease. The enzymes that catalyze the biosynthesis of peptidoglycan in M. tuberculosis are essential and noteworthy therapeutic targets. In this study, the biochemical function and homology modeling of MurI, MurG, MraY, DapE, DapA, Alr, and Ddl enzymes of the CDC1551 M. tuberculosis strain involved in the biosynthesis of peptidoglycan cell wall are reported. Generation of the 3D structures was achieved with Modeller 9.13. To assess the structural quality of the obtained homology modeled targets, the models were validated using PROCHECK, PDBsum, QMEAN, and ERRAT scores. Molecular dynamics simulations were performed to calculate root mean square deviation (RMSD) and radius of gyration (Rg) of MurI and MurG target proteins and their corresponding templates. For further model validation, RMSD and Rg for selected targets/templates were investigated to compare the close proximity of their dynamic behavior in terms of protein stability and average distances. To identify the potential binding mode required for molecular docking, binding site information of all modeled targets was obtained using two prediction algorithms. A docking study was performed for MurI to determine the potential mode of interaction between the inhibitor and the active site residues. This study presents the first accounts of the 3D structural information for the selected M. tuberculosis targets involved in peptidoglycan biosynthesis.

Jasmonate-induced biosynthesis of andrographolide in Andrographis paniculata.

Science.gov (United States)

Sharma, Shiv Narayan; Jha, Zenu; Sinha, Rakesh Kumar; Geda, Arvind Kumar

2015-02-01

Andrographolide is a prominent secondary metabolite found in Andrographis paniculata that exhibits enormous pharmacological effects. In spite of immense value, the normal biosynthesis of andrographolide results in low amount of the metabolite. To induce the biosynthesis of andrographolide, we attempted elicitor-induced activation of andrographolide biosynthesis in cell cultures of A. paniculata. This was carried out by using methyl jasmonate (MeJA) as an elicitor. Among the various concentrations of MeJA tested at different time periods, 5 µM MeJA yielded 5.25 times more andrographolide content after 24 h of treatment. The accumulation of andrographolide was correlated with the expression level of known regulatory genes (hmgs, hmgr, dxs, dxr, isph and ggps) of mevalonic acid (MVA) and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways. These results established the involvement of MeJA in andrographolide biosynthesis by inducing the transcription of its biosynthetic pathways genes. The coordination of isph, ggps and hmgs expression highly influenced the andrographolide biosynthesis. © 2014 Scandinavian Plant Physiology Society.

QSAR study on the antimalarial activity of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitors.

Science.gov (United States)

Hou, X; Chen, X; Zhang, M; Yan, A

2016-01-01

Plasmodium falciparum, the most fatal parasite that causes malaria, is responsible for over one million deaths per year. P. falciparum dihydroorotate dehydrogenase (PfDHODH) has been validated as a promising drug development target for antimalarial therapy since it catalyzes the rate-limiting step for DNA and RNA biosynthesis. In this study, we investigated the quantitative structure-activity relationships (QSAR) of the antimalarial activity of PfDHODH inhibitors by generating four computational models using a multilinear regression (MLR) and a support vector machine (SVM) based on a dataset of 255 PfDHODH inhibitors. All the models display good prediction quality with a leave-one-out q(2) >0.66, a correlation coefficient (r) >0.85 on both training sets and test sets, and a mean square error (MSE) antimalarial activity. The models are capable of predicting inhibitors' antimalarial activity and the molecular descriptors for building the models could be helpful in the development of new antimalarial drugs.

Comparisons of Prostate Cancer Inhibitors Abiraterone and TOK-001 Binding with CYP17A1 through Molecular Dynamics.

Science.gov (United States)

Xiao, Fei; Yang, Maohua; Xu, Youjun; Vongsangnak, Wanwipa

2015-01-01

Cytochrome P450 17A1 (CYP17A1) is associated in the steroid hormone biosynthesis in human. As cell proliferation of prostate cancer in response to androgen steroid, an inhibition of CYP17A1 becomes an alternative approach to inhibit biosynthesis of androgen and support treatment of prostate cancer. However, biology-driven inhibitor development of prostate cancer is poorly elucidated. The aims of this study are to address structural differences at atomic-level between CYP17A1 and inhibitors i.e., abiraterone and TOK-001, and further investigate the effect of point mutation of CYP17A1 on the active site stability and the local interactions that are hydrophobic interaction and hydrogen bonding throughout molecular dynamics (MD) simulation. After performing multiple comparisons among four different complexes across CYP17A1 and inhibitors, interestingly TOK-001 oriented toward the active pocket and formed larger volume with I-helix of CYP17A1 than abiraterone, whereas abiraterone showed tighter binding and more active site stability. Considering on the effect of hydrophobic interaction and hydrogen bonding between abiraterone and CYP17A1, the key residues of Phe114, Ile371, Val482, and Asn202 were identified. This contributes into tight binding interactions; however abiraterone is effectively weakened along with the global conformation mobility increased in A105L mutation. Surprisingly, overall conformation of the CYP17A1 remained stable when bound to TOK-001. This basic knowledge can guide future experiments on design of efficient inhibitors for CYP17A1, which provides theoretical basis of androgen-dependent disease therapy.

Endogenous sterol biosynthesis is important for mitochondrial function and cell morphology in procyclic forms of Trypanosoma brucei.

Science.gov (United States)

Pérez-Moreno, Guiomar; Sealey-Cardona, Marco; Rodrigues-Poveda, Carlos; Gelb, Michael H; Ruiz-Pérez, Luis Miguel; Castillo-Acosta, Víctor; Urbina, Julio A; González-Pacanowska, Dolores

2012-10-01

Sterol biosynthesis inhibitors are promising entities for the treatment of trypanosomal diseases. Insect forms of Trypanosoma brucei, the causative agent of sleeping sickness, synthesize ergosterol and other 24-alkylated sterols, yet also incorporate cholesterol from the medium. While sterol function has been investigated by pharmacological manipulation of sterol biosynthesis, molecular mechanisms by which endogenous sterols influence cellular processes remain largely unknown in trypanosomes. Here we analyse by RNA interference, the effects of a perturbation of three specific steps of endogenous sterol biosynthesis in order to dissect the role of specific intermediates in proliferation, mitochondrial function and cellular morphology in procyclic cells. A decrease in the levels of squalene synthase and squalene epoxidase resulted in a depletion of cellular sterol intermediates and end products, impaired cell growth and led to aberrant morphologies, DNA fragmentation and a profound modification of mitochondrial structure and function. In contrast, cells deficient in sterol methyl transferase, the enzyme involved in 24-alkylation, exhibited a normal growth phenotype in spite of a complete abolition of the synthesis and content of 24-alkyl sterols. Thus, the data provided indicates that while the depletion of squalene and post-squalene endogenous sterol metabolites results in profound cellular defects, bulk 24-alkyl sterols are not strictly required to support growth in insect forms of T. brucei in vitro. Copyright © 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

The role of peroxisomal fatty acyl-CoA beta-oxidation in bile acid biosynthesis

International Nuclear Information System (INIS)

Hayashi, H.; Miwa, A.

1989-01-01

The physiological role of the peroxisomal fatty acyl-CoA beta-oxidizing system (FAOS) is not yet established. We speculated that there might be a relationship between peroxisomal degradation of long-chain fatty acids in the liver and the biosynthesis of bile acids. This was investigated using [1- 14 C]butyric acid and [1- 14 C]lignoceric acid as substrates of FAOS in mitochondria and peroxisomes, respectively. The incorporation of [ 14 C]lignoceric acid into primary bile acids was approximately four times higher than that of [ 14 C]butyric acid (in terms of C-2 units). The pools of these two fatty acids in the liver were exceedingly small. The incorporations of radioactivity into the primary bile acids were strongly inhibited by administration of aminotriazole, which is a specific inhibitor of peroxisomal FAOS in vivo. Aminotriazole inhibited preferentially the formation of cholate, the major primary bile acid, from both [ 14 C]lignoceric acid and [ 14 C]butyric acid, rather than the formation of chenodeoxycholate. The former inhibition was about 70% and the latter was approximately 40-50%. In view of reports that cholate is biosynthesized from endogenous cholesterol, the above results indicate that peroxisomal FAOS may have an anabolic function, supplying acetyl CoA for bile acid biosynthesis

The role of peroxisomal fatty acyl-CoA beta-oxidation in bile acid biosynthesis

Energy Technology Data Exchange (ETDEWEB)

Hayashi, H.; Miwa, A. (Josai Univ., Saitama (Japan))

1989-11-01

The physiological role of the peroxisomal fatty acyl-CoA beta-oxidizing system (FAOS) is not yet established. We speculated that there might be a relationship between peroxisomal degradation of long-chain fatty acids in the liver and the biosynthesis of bile acids. This was investigated using (1-{sup 14}C)butyric acid and (1-{sup 14}C)lignoceric acid as substrates of FAOS in mitochondria and peroxisomes, respectively. The incorporation of ({sup 14}C)lignoceric acid into primary bile acids was approximately four times higher than that of ({sup 14}C)butyric acid (in terms of C-2 units). The pools of these two fatty acids in the liver were exceedingly small. The incorporations of radioactivity into the primary bile acids were strongly inhibited by administration of aminotriazole, which is a specific inhibitor of peroxisomal FAOS in vivo. Aminotriazole inhibited preferentially the formation of cholate, the major primary bile acid, from both ({sup 14}C)lignoceric acid and ({sup 14}C)butyric acid, rather than the formation of chenodeoxycholate. The former inhibition was about 70% and the latter was approximately 40-50%. In view of reports that cholate is biosynthesized from endogenous cholesterol, the above results indicate that peroxisomal FAOS may have an anabolic function, supplying acetyl CoA for bile acid biosynthesis.

Tomato strigolactones are derived from carotenoids and their biosynthesis is promoted by phosphate starvation.

Science.gov (United States)

López-Ráez, Juan Antonio; Charnikhova, Tatsiana; Gómez-Roldán, Victoria; Matusova, Radoslava; Kohlen, Wouter; De Vos, Ric; Verstappen, Francel; Puech-Pages, Virginie; Bécard, Guillaume; Mulder, Patrick; Bouwmeester, Harro

2008-01-01

* Strigolactones are rhizosphere signalling compounds that mediate host location in arbuscular mycorrhizal (AM) fungi and parasitic plants. Here, the regulation of the biosynthesis of strigolactones is studied in tomato (Solanum lycopersicum). * Strigolactone production under phosphate starvation, in the presence of the carotenoid biosynthesis inhibitor fluridone and in the abscisic acid (ABA) mutant notabilis were assessed using a germination bioassay with seeds of Orobanche ramosa; a hyphal branching assay with Gigaspora spp; and by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis. * The root exudates of tomato cv. MoneyMaker induced O. ramosa seed germination and hyphal branching in AM fungi. Phosphate starvation markedly increased, and fluridone strongly decreased, this activity. Exudates of notabilis induced approx. 40% less germination than the wild-type. The LC-MS/MS analysis confirmed that the biological activity and changes therein were due to the presence of several strigolactones; orobanchol, solanacol and two or three didehydro-orobanchol isomers. * These results show that the AM branching factors and parasitic plant germination stimulants in tomato root exudate are strigolactones and that they are biosynthetically derived from carotenoids. The dual activity of these signalling compounds in attracting beneficial AM fungi and detrimental parasitic plants is further strengthened by environmental conditions such as phosphate availability.

Influence of light, UV-B radiation, and herbicides on wax biosynthesis of cucumber seedlings

International Nuclear Information System (INIS)

Tevini, M.; Steinmüller, D.

1987-01-01

The behavior of cuticular alkane-1-ols and alkanes were studied in different developmental stages of cucumber seedlings grown in the dark or under white light, with or without UV-B radiation or in presence of wax biosynthesis inhibitors, trichloroacetic acid and metolachlor. Accumulation of alkane-1-ols increased light independently with seedling age. Synthesis of alkanes was strictly light and dose dependent. Addition of UV-B radiation did not alter the amounts of alkanes or alcohols, however, the distribution of homologues was shifted towards shorter chain homologues. Treatments with Cl 3 AcOH resulted in strong inhibition of alkane accumulation, whereas the amount of alkane-1-ols was changed neither at low nor at moderate concentrations of Cl 3 AcOH but their homologue distribution shifted towards longer chain lengths. This shifting was depressed in the presence of UV-B. At high concentrations of Cl 3 Ac0H similar homologue distributions as produced by UV-B (shift to shorter homologues) were observed. Metolachlor treatment resulted in an inhibition of alkane-1-ol production connected with rising amounts of alkanes, predominantly of short chain species. A simple model of wax biosynthesis is proposed which describes the interactions with white light, UV-B radiation and herbicides. (author)

Inhibition of the Flavin-Dependent Monooxygenase Siderophore A (SidA) Blocks Siderophore Biosynthesis and Aspergillus fumigatus Growth.

Science.gov (United States)

Martín Del Campo, Julia S; Vogelaar, Nancy; Tolani, Karishma; Kizjakina, Karina; Harich, Kim; Sobrado, Pablo

2016-11-18

Aspergillus fumigatus is an opportunistic fungal pathogen and the most common causative agent of fatal invasive mycoses. The flavin-dependent monooxygenase siderophore A (SidA) catalyzes the oxygen and NADPH dependent hydroxylation of l-ornithine (l-Orn) to N 5 -l-hydroxyornithine in the biosynthetic pathway of hydroxamate-containing siderophores in A. fumigatus. Deletion of the gene that codes for SidA has shown that it is essential in establishing infection in mice models. Here, a fluorescence polarization high-throughput assay was used to screen a 2320 compound library for inhibitors of SidA. Celastrol, a natural quinone methide, was identified as a noncompetitive inhibitor of SidA with a MIC value of 2 μM. Docking experiments suggest that celastrol binds across the NADPH and l-Orn pocket. Celastrol prevents A. fumigatus growth in blood agar. The addition of purified ferric-siderophore abolished the inhibitory effect of celastrol. Thus, celastrol inhibits A. fumigatus growth by blocking siderophore biosynthesis through SidA inhibiton.

Paralytic shellfish toxin biosynthesis in cyanobacteria and dinoflagellates: A molecular overview.

Science.gov (United States)

Wang, Da-Zhi; Zhang, Shu-Fei; Zhang, Yong; Lin, Lin

2016-03-01

Paralytic shellfish toxins (PSTs) are a group of water soluble neurotoxic alkaloids produced by two different kingdoms of life, prokaryotic cyanobacteria and eukaryotic dinoflagellates. Owing to the wide distribution of these organisms, these toxic secondary metabolites account for paralytic shellfish poisonings around the world. On the other hand, their specific binding to voltage-gated sodium channels makes these toxins potentially useful in pharmacological and toxicological applications. Much effort has been devoted to the biosynthetic mechanism of PSTs, and gene clusters encoding 26 proteins involved in PST biosynthesis have been unveiled in several cyanobacterial species. Functional analysis of toxin genes indicates that PST biosynthesis in cyanobacteria is a complex process including biosynthesis, regulation, modification and export. However, less is known about the toxin biosynthesis in dinoflagellates owing to our poor understanding of the massive genome and unique chromosomal characteristics [1]. So far, few genes involved in PST biosynthesis have been identified from dinoflagellates. Moreover, the proteins involved in PST production are far from being totally explored. Thus, the origin and evolution of PST biosynthesis in these two kingdoms are still controversial. In this review, we summarize the recent progress on the characterization of genes and proteins involved in PST biosynthesis in cyanobacteria and dinoflagellates, and discuss the standing evolutionary hypotheses concerning the origin of toxin biosynthesis as well as future perspectives in PST biosynthesis. Paralytic shellfish toxins (PSTs) are a group of potent neurotoxins which specifically block voltage-gated sodium channels in excitable cells and result in paralytic shellfish poisonings (PSPs) around the world. Two different kingdoms of life, cyanobacteria and dinoflagellates are able to produce PSTs. However, in contrast with cyanobacteria, our understanding of PST biosynthesis in

Elucidating the structural basis for differing enzyme inhibitor potency by cryo-EM.

Science.gov (United States)

Rawson, Shaun; Bisson, Claudine; Hurdiss, Daniel L; Fazal, Asif; McPhillie, Martin J; Sedelnikova, Svetlana E; Baker, Patrick J; Rice, David W; Muench, Stephen P

2018-02-20

Histidine biosynthesis is an essential process in plants and microorganisms, making it an attractive target for the development of herbicides and antibacterial agents. Imidazoleglycerol-phosphate dehydratase (IGPD), a key enzyme within this pathway, has been biochemically characterized in both Saccharomyces cerevisiae ( Sc_ IGPD) and Arabidopsis thaliana ( At_ IGPD). The plant enzyme, having been the focus of in-depth structural analysis as part of an inhibitor development program, has revealed details about the reaction mechanism of IGPD, whereas the yeast enzyme has proven intractable to crystallography studies. The structure-activity relationship of potent triazole-phosphonate inhibitors of IGPD has been determined in both homologs, revealing that the lead inhibitor (C348) is an order of magnitude more potent against Sc_ IGPD than At_ IGPD; however, the molecular basis of this difference has not been established. Here we have used single-particle electron microscopy (EM) to study structural differences between the At and Sc_ IGPD homologs, which could influence the difference in inhibitor potency. The resulting EM maps at ∼3 Å are sufficient to de novo build the protein structure and identify the inhibitor binding site, which has been validated against the crystal structure of the At_ IGPD/C348 complex. The structure of Sc _IGPD reveals that a 24-amino acid insertion forms an extended loop region on the enzyme surface that lies adjacent to the active site, forming interactions with the substrate/inhibitor binding loop that may influence inhibitor potency. Overall, this study provides insights into the IGPD family and demonstrates the power of using an EM approach to study inhibitor binding. Copyright © 2018 the Author(s). Published by PNAS.

Elaboration of a fragment library hit produces potent and selective aspartate semialdehyde dehydrogenase inhibitors.

Science.gov (United States)

Thangavelu, Bharani; Bhansali, Pravin; Viola, Ronald E

2015-10-15

Aspartate-β-semialdehyde dehydrogenase (ASADH) lies at the first branch point in the aspartate metabolic pathway which leads to the biosynthesis of several essential amino acids and some important metabolites. This pathway is crucial for many metabolic processes in plants and microbes like bacteria and fungi, but is absent in mammals. Therefore, the key microbial enzymes involved in this pathway are attractive potential targets for development of new antibiotics with novel modes of action. The ASADH enzyme family shares the same substrate binding and active site catalytic groups; however, the enzymes from representative bacterial and fungal species show different inhibition patterns when previously screened against low molecular weight inhibitors identified from fragment library screening. In the present study several approaches, including fragment based drug discovery (FBDD), inhibitor docking, kinetic, and structure-activity relationship (SAR) studies have been used to guide ASADH inhibitor development. Elaboration of a core structure identified by FBDD has led to the synthesis of low micromolar inhibitors of the target enzyme, with high selectivity introduced between the Gram-negative and Gram-positive orthologs of ASADH. This new set of structures open a novel direction for the development of inhibitors against this validated drug-target enzyme. Copyright © 2015 Elsevier Ltd. All rights reserved.

In Vivo Roles of Fatty Acid Biosynthesis Enzymes in Biosynthesis of Biotin and α-Lipoic Acid in Corynebacterium glutamicum.

Science.gov (United States)

Ikeda, Masato; Nagashima, Takashi; Nakamura, Eri; Kato, Ryosuke; Ohshita, Masakazu; Hayashi, Mikiro; Takeno, Seiki

2017-10-01

For fatty acid biosynthesis, Corynebacterium glutamicum uses two type I fatty acid synthases (FAS-I), FasA and FasB, in addition to acetyl-coenzyme A (CoA) carboxylase (ACC) consisting of AccBC, AccD1, and AccE. The in vivo roles of the enzymes in supplying precursors for biotin and α-lipoic acid remain unclear. Here, we report genetic evidence demonstrating that the biosynthesis of these cofactors is linked to fatty acid biosynthesis through the FAS-I pathway. For this study, we used wild-type C. glutamicum and its derived biotin vitamer producer BFI-5, which was engineered to express Escherichia coli bioBF and Bacillus subtilis bioI Disruption of either fasA or fasB in strain BFI-5 led to decreased production of biotin vitamers, whereas its amplification contributed to increased production, with a larger impact of fasA in both cases. Double disruptions of fasA and fasB resulted in no biotin vitamer production. The acc genes showed a positive effect on production when amplified simultaneously. Augmented fatty acid biosynthesis was also reflected in pimelic acid production when carbon flow was blocked at the BioF reaction. These results indicate that carbon flow down the FAS-I pathway is destined for channeling into the biotin biosynthesis pathway, and that FasA in particular has a significant impact on precursor supply. In contrast, fasB disruption resulted in auxotrophy for lipoic acid or its precursor octanoic acid in both wild-type and BFI-5 strains. The phenotypes were fully complemented by plasmid-mediated expression of fasB but not fasA These results reveal that FasB plays a specific physiological role in lipoic acid biosynthesis in C. glutamicum IMPORTANCE For the de novo biosynthesis of fatty acids, C. glutamicum exceptionally uses a eukaryotic multifunctional type I fatty acid synthase (FAS-I) system comprising FasA and FasB, in contrast to most bacteria, such as E. coli and B. subtilis , which use an individual nonaggregating type II fatty acid synthase

Biosynthesis of silver nanoparticles by Aspergillus niger , Fusarium ...

African Journals Online (AJOL)

... scanning electron microscope (SEM). Results indicate the synthesis of silver nanoparticles in the reaction mixture. The synthesis of nanoparticles would be suitable for developing a microbial nanotechnology biosynthesis process for mass scale production. Keywords: Silver nanoparticles, biosynthesis, fungi, Aspergillus.

Increased sesquiterpenoid biosynthesis and an apparent decrease in sterol biosynthesis in elicitor-treated tobacco cell suspension cultures

International Nuclear Information System (INIS)

Voegeli, U.; Bhatt, P.N.; Chappell, J.

1987-01-01

Addition of fungel elicitor prepared from Phytophthora parasitica to tobacco cell suspension cultures leads to an increased production of the phytoalexin capsidiol. Capsidiol is a sesquiterpenoid which is most likely synthesized from farnesylpyrophosphat (FPP) by a bicyclic cyclase reaction. Because FPP is also a substrate for squalene synthetase and therefore a precursor of sterol biosynthesis, the question arises whether or not the accumulation of capsidiol in elicitor-treated cells occurs at the expense of sterol biosynthesis. ( 14 C]-acetate was given to elicitor-treated and control (no treatment) cell cultures and incorporation into sterols and capsidiol determined. No labeled capsidiol was detected in control cells. In elicitor-treated cells about 12-15% of the radioactivity taken up by the cells was incorporated into capsidiol. In contrast, control cells incorporated 4 times more radioactivity into sterols than elicitor-treated cells. Similar results were obtained using ( 3 H)-mevalonate as a precursor of capsidiol and sterol biosynthesis. Likely explanations for the apparently decline in sterol biosynthesis in elicitor-treated cells include: (1) inhibition of squalene synthetase; (2) induction of capsidiol synthesizing enzymes; and (3) metabolic channeling of FPP into capsidiol versus sterols. These possibilities will be discussed further together with other results

High glucose suppresses human islet insulin biosynthesis by inducing miR-133a leading to decreased polypyrimidine tract binding protein-expression

DEFF Research Database (Denmark)

Fred, Rikard G; Bang-Berthelsen, Claus H; Mandrup-Poulsen, Thomas

2010-01-01

BACKGROUND: Prolonged periods of high glucose exposure results in human islet dysfunction in vitro. The underlying mechanisms behind this effect of high glucose are, however, unknown. The polypyrimidine tract binding protein (PTB) is required for stabilization of insulin mRNA and the PTB mRNA 3......'-UTR contains binding sites for the microRNA molecules miR-133a, miR-124a and miR-146. The aim of this study was therefore to investigate whether high glucose increased the levels of these three miRNAs in association with lower PTB levels and lower insulin biosynthesis rates. METHODOLOGY...... for real-time RT-PCR analysis of miR-133a, miR-124a, miR-146, insulin mRNA and PTB mRNA contents. Insulin biosynthesis rates were determined by radioactive labeling and immunoprecipitation. Synthetic miR-133a precursor and inhibitor were delivered to dispersed islet cells by lipofection, and PTB...

Rooting of healthy and CVC-affected 'Valência' sweet orange stem cuttings, through the use of plant regulators

Directory of Open Access Journals (Sweden)

Gustavo Habermann

2006-01-01

Full Text Available Citrus variegated chlorosis (CVC is a disease caused by Xylella fastidiosa. Using different concentrations of plant regulators, such as auxins (indole-3-butyric acid and gibberellic acid biosynthesis-inhibitor (paclobutrazol, physiological rooting capacity of healthy and CVC-affected stem cuttings were evaluated in order to investigate the importance of plant hormone imbalance and xylem occlusion in plants with CVC. The percentages of dead, alive and rooted cuttings, cuttings with callus and mean number of roots per cuttings did not show statistical differences in response to the distinct concentrations of synthetic plant regulators. There were differences only between healthy and CVC-affected cuttings. This showed the importance of xylem occlusion and diffusive disturbances in diseased plants, in relation to root initiation capacity and hormonal translocation in the plant tissue.Clorose variegada dos citros (CVC é uma doença causada por Xylella fastidiosa, podendo determinar oclusão do xilema e desbalanço hormonal, o que por fim está relacionado ao processo de iniciação radicial em estacas. Usando diferentes concentrações de fitorreguladores, como auxinas (ácido 3-indol butírico e inibidores da biossíntese de ácido giberélico (paclobutrazol, que são promotores do enraizamento de estacas, verificou-se a capacidade fisiológica de enraizamento de estacas sadias e com CVC, a fim de investigar a importância do desbalanço hormonal e oclusão do xilema em plantas doentes. As porcentagens de estacas mortas, vivas, enraizadas e com calo e o número médio de raízes por estaca não mostraram diferenças estatísticas em resposta às diferentes concentrações dos reguladores vegetais sintéticos. Houve diferenças apenas entre estacas sadias e doentes. Isto aponta a importância da oclusão do xilema e distúrbios difusivos em plantas doentes, em relação à capacidade de iniciação radicial e à translocação hormonal no tecido

Evidence for a cytoplasmic pathway of oxalate biosynthesis in Aspergillus niger

International Nuclear Information System (INIS)

Kubicek, C.P.; Schreferl-Kunar, G.; Woehrer, W.; Roehr, M.

1988-01-01

Oxalate accumulation of up to 8 g/liter was induced in Aspergillus niger by shifting the pH from 6 to 8. This required the presence of P/sub i/ and a nitrogen source and was inhibited by the protein synthesis inhibitor cycloheximide. Exogenously added 14 CO 2 was not incorporated into oxalate, but was incorporated into acetate and malate, thus indicating the biosynthesis of oxalate by hydrolytic cleavage of oxaloacetate. Inhibition of mitochondrial citrate metabolism by fluorocitrate did not significantly decrease the oxalate yield. The putative enzyme that was responsible for this oxaloacetate hydrolase (EC 3.7.1.1), which was induced de novo during the pH shift. Subcellular fractionation of oxalic acid-forming mycelia of A. niger showed that this enzyme is located in the cytoplasm of A. niger. The results are consistent with a cytoplasmic pathway of oxalate formation which does not involve the tricarboxylic acid cycle

Mycobacterium tuberculosis phosphoribosylpyrophosphate synthetase: biochemical features of a crucial enzyme for mycobacterial cell wall biosynthesis.

Directory of Open Access Journals (Sweden)

Anna P Lucarelli

Full Text Available The selection and soaring spread of Mycobacterium tuberculosis multidrug-resistant (MDR-TB and extensively drug-resistant strains (XDR-TB is a severe public health problem. Currently, there is an urgent need for new drugs for tuberculosis treatment, with novel mechanisms of action and, moreover, the necessity to identify new drug targets. Mycobacterial phosphoribosylpyrophosphate synthetase (MtbPRPPase is a crucial enzyme involved in the biosynthesis of decaprenylphosphoryl-arabinose, an essential precursor for the mycobacterial cell wall biosynthesis. Moreover, phosphoribosylpyrophosphate, which is the product of the PRPPase catalyzed reaction, is the precursor for the biosynthesis of nucleotides and of some amino acids such as histidine and tryptophan. In this context, the elucidation of the molecular and functional features of MtbPRPPase is mandatory. MtbPRPPase was obtained as a recombinant form, purified to homogeneity and characterized. According to its hexameric form, substrate specificity and requirement of phosphate for activity, the enzyme proved to belong to the class I of PRPPases. Although the sulfate mimicked the phosphate, it was less effective and required higher concentrations for the enzyme activation. MtbPRPPase showed hyperbolic response to ribose 5-phosphate, but sigmoidal behaviour towards Mg-ATP. The enzyme resulted to be allosterically activated by Mg(2+ or Mn(2+ and inhibited by Ca(2+ and Cu(2+ but, differently from other characterized PRPPases, it showed a better affinity for the Mn(2+ and Cu(2+ ions, indicating a different cation binding site geometry. Moreover, the enzyme from M. tuberculosis was allosterically inhibited by ADP, but less sensitive to inhibition by GDP. The characterization of M. tuberculosis PRPPase provides the starting point for the development of inhibitors for antitubercular drug design.

Jasmonate mediates salt-induced nicotine biosynthesis in tobacco (Nicotiana tabacum L.

Directory of Open Access Journals (Sweden)

Xiaodong Chen

2016-04-01

Full Text Available Jasmonate (JA, as an important signal, plays a key role in multiple processes of plant growth, development and stress response. Nicotine and related pyridine alkaloids in tobacco (Nicotiana tabacum L. are essential secondary metabolites. Whether environmental factors control nicotine biosynthesis and the underlying mechanism remains previously unreported. Here, we applied physiological and biochemical approaches to investigate how salt stress affects nicotine biosynthesis in tobacco. We found that salt stress induced the biosynthesis of JA, which subsequently triggered the activation of JA-responsive gene expression and, ultimately, nicotine synthesis. Bioinformatics analysis revealed the existence of many NtMYC2a-recognized G-box motifs in the promoter regions of NtLOX, NtAOS, NtAOC and NtOPR genes. Applying exogenous JA increased nicotine content, while suppressing JA biosynthesis reduced nicotine biosynthesis. Salt treatment could not efficiently induce nicotine biosynthesis in transgenic anti-COI1 tobacco plants. These results demonstrate that JA acts as the essential signal which triggers nicotine biosynthesis in tobacco after salt stress.

Suppression of elongation and growth of tomato seedlings by auxin biosynthesis inhibitors and modeling of the growth and environmental response.

Science.gov (United States)

Higashide, Tadahisa; Narukawa, Megumi; Shimada, Yukihisa; Soeno, Kazuo

2014-04-02

To develop a growth inhibitor, the effects of auxin inhibitors were investigated. Application of 30 μM L-α-aminooxy-β-phenylpropionic acid (AOPP) or (S)-methyl 2-((1,3-dioxoisoindolin-2-yl)oxy)-3-phenylpropanoate (KOK1101), decreased the endogenous IAA levels in tomato seedlings at 8 days after sowing. Then, 10-1200 μM AOPP or KOK1101 were sprayed on the leaves and stem of 2-3 leaf stage tomato plants grown under a range of environmental conditions. We predicted plant growth and environmental response using a model based on the observed suppression of leaf enlargement. Spraying AOPP or KOK1101 decreased stem length and leaf area. Concentration-dependent inhibitions and dose response curves were observed. Although the effects of the inhibitors on dry weight varied according to the environmental conditions, the net assimilation rate was not influenced by the inhibitors. Accordingly, the observed decrease in dry weight caused by the inhibitors may result from decreased leaf area. Validation of the model based on observed data independent of the dataset showed good correlations between the observed and predicted values of dry weight and leaf area index.

Biosynthesis and function of chondroitin sulfate.

Science.gov (United States)

Mikami, Tadahisa; Kitagawa, Hiroshi

2013-10-01

Chondroitin sulfate proteoglycans (CSPGs) are principal pericellular and extracellular components that form regulatory milieu involving numerous biological and pathophysiological phenomena. Diverse functions of CSPGs can be mainly attributed to structural variability of their polysaccharide moieties, chondroitin sulfate glycosaminoglycans (CS-GAG). Comprehensive understanding of the regulatory mechanisms for CS biosynthesis and its catabolic processes is required in order to understand those functions. Here, we focus on recent advances in the study of enzymatic regulatory pathways for CS biosynthesis including successive modification/degradation, distinct CS functions, and disease phenotypes that have been revealed by perturbation of the respective enzymes in vitro and in vivo. Fine-tuned machineries for CS production/degradation are crucial for the functional expression of CS chains in developmental and pathophysiological processes. Control of enzymes responsible for CS biosynthesis/catabolism is a potential target for therapeutic intervention for the CS-associated disorders. Copyright © 2013 Elsevier B.V. All rights reserved.

Selective inhibition of the demethylation at C-14 in ergosterol biosynthesis by the fungicide, Denmert (S-1358)

International Nuclear Information System (INIS)

Kato, Toshiro; Kawase, Yasuo

1976-01-01

A direct evidence of the inhibitory effect in a cell-free system of S. cerevisiae was experimentally studied, and the site of action of Denmert (S-n-butyl S'-p-tert-butylbenzyl N-3-pyridyldithiocarbon-imidate) in sterol biosynthesis was examined. 14 C-labeled lanosterol and 14-desmethyl-lanosterol were biosynthetically prepared. DL-mevalonate-2- 14 C was incubated with yeast cell-free homogenates for 3 hr at 28 deg C while being shaked vigorously in atmospheric oxygen. The resultant 14 C-labeled sterol was extracted and chromatographed on a silicic acid-Hyflo Super Cel column. 4,4-dimethyl sterol thus obtained was acetylated with acetic anhydride and pyridine. The separation of lanosteryl acetate and 14-desmethyl lanosteryl acetate was accomplished on alumina thin-layer plates. After the saponification of each steryl acetate, the quantity of the sterol was assessed by gas chromatography with cholesterol as an internal standard. The incubation of the 14 C-labeled sterol was achieved under the same conditions as those for the DL-mevalonate-2- 14 C except the addition of the substrate which was dispersed in 0.1M phosphate buffer. Denmert inhibited the conversion of 14 C-labeled lanosterol to 4-desmethyl sterol, while the conversion of 14 C-labeled 14-desmethyl lanosterol to 4-desmethyl sterol was hardly affected by the fungicide. Therefore, Denmert is a potent selective inhibitor of the demethylation at the C-14 position in ergosterol biosynthesis. The fungicide, triarimol, exhibited the same effect on sterol biosynthesis as that of Denmert. (Iwakiri, K.)

Identification of novel inhibitors of Pseudomonas aeruginosa MurC enzyme derived from phage-displayed peptide libraries.

Science.gov (United States)

El Zoeiby, Ahmed; Sanschagrin, François; Darveau, André; Brisson, Jean-Robert; Levesque, Roger C

2003-03-01

The machinery of peptidoglycan biosynthesis is an ideal site at which to look for novel antimicrobial targets. Phage display was used to develop novel peptide inhibitors for MurC, an essential enzyme involved in the early steps of biosynthesis of peptidoglycan monomer. We cloned and overexpressed the murA, -B and -C genes from Pseudomonas aeruginosa in the pET expression vector, adding a His-tag to their C termini. The three proteins were overproduced in Escherichia coli and purified to homogeneity in milligram quantities. MurA and -B were combinatorially used to synthesize the MurC substrate UDP-N-acetylmuramate, the identity of which was confirmed by mass spectrometry and nuclear magnetic resonance analysis. Two phage-display libraries were screened against MurC in order to identify peptide ligands to the enzyme. Three rounds of biopanning were carried out, successively increasing elution specificity from round 1 to 3. The third round was accomplished with both non-specific elution and competitive elution with each of the three MurC substrates, UDP-N-acetylmuramic acid (UNAM), ATP and L-alanine. The DNA of 10 phage, selected randomly from each group, was extracted and sequenced, and consensus peptide sequences were elucidated. Peptides were synthesized and tested for inhibition of the MurC-catalysed reaction, and two peptides were shown to be inhibitors of MurC activity with IC(50)s of 1.5 and 0.9 mM, respectively. The powerful selection technique of phage display allowed us to identify two peptide inhibitors of the essential bacterial enzyme MurC. The peptide sequences represent the basis for the synthesis of inhibitory peptidomimetic molecules.

Differential feedback regulation of ethylene biosynthesis in pulp and peel tissues of banana fruit.

Science.gov (United States)

Inaba, Akitsugu; Liu, Xuejun; Yokotani, Naoki; Yamane, Miki; Lu, Wang-Jin; Nakano, Ryohei; Kubo, Yasutaka

2007-01-01

The feedback regulation of ethylene biosynthesis in banana [Musa sp. (AAA group, Cavendish subgroup) cv. Grand Nain] fruit was investigated in an attempt to clarify the opposite effect of 1-methylcyclopropene (1-MCP), an ethylene action inhibitor, before and after the onset of ripening. 1-MCP pre-treatment completely prevented the ripening-induced effect of propylene in pre-climacteric banana fruit, whereas treatment after the onset of ripening stimulated ethylene production. In pre-climacteric fruit, higher concentrations of propylene suppressed ethylene production more strongly, despite their earlier ethylene-inducing effect. Exposure of the fruit ripened by propylene to 1-MCP increased ethylene production concomitantly with an increase in 1-aminocyclopropane-1-carboxylate (ACC) synthase activity and ACC content, and prevented a transient decrease in MA-ACS1 transcripts in the pulp tissues. In contrast, in the peel of ripening fruit, 1-MCP prevented the increase in ethylene production and subsequently the ripening process by reduction of the increase in MA-ACS1 and MA-ACO1 transcripts and of ACC synthase and ACC oxidase activities. These results suggest that ethylene biosynthesis in ripening banana fruit may be controlled negatively in the pulp tissue and positively in the peel tissue. This differential regulation by ethylene in pulp and peel tissues was also observed for MA-PL, MA-Exp, and MA-MADS genes.

The influence of abscisic acid on the ethylene biosynthesis pathway in the functioning of the flower abscission zone in Lupinus luteus.

Science.gov (United States)

Wilmowicz, Emilia; Frankowski, Kamil; Kućko, Agata; Świdziński, Michał; de Dios Alché, Juan; Nowakowska, Anna; Kopcewicz, Jan

2016-11-01

Flower abscission is a highly regulated developmental process activated in response to exogenous (e.g. changing environmental conditions) and endogenous stimuli (e.g. phytohormones). Ethylene (ET) and abscisic acid (ABA) are very effective stimulators of flower abortion in Lupinus luteus, which is a widely cultivated species in Poland, Australia and Mediterranean countries. In this paper, we show that artificial activation of abscission by flower removal caused an accumulation of ABA in the abscission zone (AZ). Moreover, the blocking of that phytohormone's biosynthesis by NDGA (nordihydroguaiaretic acid) decreased the number of abscised flowers. However, the application of NBD - an inhibitor of ET action - reversed the stimulatory effect of ABA on flower abscission, indicating that ABA itself is not sufficient to turn on the organ separation. Our analysis revealed that exogenous ABA significantly accelerated the transcriptional activity of the ET biosynthesis genes ACC synthase (LlACS) and oxidase (LlACO), and moreover, strongly increased the level of 1-aminocyclopropane-1-carboxylic acid (ACC) - ET precursor, which was specifically localized within AZ cells. We cannot exclude the possibility that ABA mediates flower abscission processes by enhancing the ET biosynthesis rate. The findings of our study will contribute to the overall basic knowledge on the phytohormone-regulated generative organs abscission in L. luteus. Copyright © 2016 Elsevier GmbH. All rights reserved.

Analysis of ethylene biosynthesis and perception during postharvest cold storage of Marsh and Star Ruby grapefruits.

Science.gov (United States)

Lado, Joanna; Rodrigo, María Jesús; Zacarías, Lorenzo

2015-10-01

Grapefruits are among the citrus species more sensitive to cold and develop chilling injury symptoms during prolonged postharvest storage at temperatures lower than 8 ℃-10 ℃. The plant hormone ethylene has been described either to protect or potentiate chilling injury development in citrus whereas little is known about transcriptional regulation of ethylene biosynthesis, perception and response during cold storage and how the hormone is regulating its own perception and signaling cascade. Then, the objective of the present study was to explore the transcriptional changes in the expression of ethylene biosynthesis, receptors and response genes during cold storage of the white Marsh and the red Star Ruby grapefruits. The effect of the ethylene action inhibitor, 1-MCP, was evaluated to investigate the involvement of ethylene in the regulation of the genes of its own biosynthesis and perception pathway. Ethylene production was very low at the harvest time in fruits of both varieties and experienced only minor changes during storage. By contrast, inhibition of ethylene perception by 1-MCP markedly induced ethylene production, and this increase was highly stimulated during shelf-life at 20 ℃, as well as transcription of ACS and ACO. These results support the auto-inhibitory regulation of ethylene in grapefruits, which acts mainly at the transcriptional level of ACS and ACO genes. Moreover, ethylene receptor1 and ethylene receptor3 were induced by cold while no clear role of ethylene was observed in the induction of ethylene receptors. However, ethylene appears to be implicated in the transcriptional regulation of ERFs both under cold storage and shelf-life. © The Author(s) 2014.

Design and synthesis of novel N-benzylidenesulfonohydrazide inhibitors of MurC and MurD as potential antibacterial agents.

Science.gov (United States)

Frlan, Rok; Kovac, Andreja; Blanot, Didier; Gobec, Stanislav; Pecar, Slavko; Obreza, Ales

2008-01-11

A series of novel N-benzylidenesulfonohydrazide compounds were designed and synthesized as inhibitors of UDP-N-acetylmuramic acid: L-alanine ligase (MurC) and UDP-N-acetylmuramoyl-L-alanine: D-glutamate ligase (MurD) from E. coli, involved in the biosynthesis of bacterial cell-walls. Some compounds possessed inhibitory activity against both enzymes with IC(50) values as low as 30 microM. In addition, a new, one-pot synthesis of amidobenzaldehydes is reported.

Evidence for a cytoplasmic pathway of oxalate biosynthesis in Aspergillus niger

Energy Technology Data Exchange (ETDEWEB)

Kubicek, C.P.; Schreferl-Kunar, G.; Woehrer, W.; Roehr, M.

1988-03-01

Oxalate accumulation of up to 8 g/liter was induced in Aspergillus niger by shifting the pH from 6 to 8. This required the presence of P/sub i/ and a nitrogen source and was inhibited by the protein synthesis inhibitor cycloheximide. Exogenously added /sup 14/CO/sub 2/ was not incorporated into oxalate, but was incorporated into acetate and malate, thus indicating the biosynthesis of oxalate by hydrolytic cleavage of oxaloacetate. Inhibition of mitochondrial citrate metabolism by fluorocitrate did not significantly decrease the oxalate yield. The putative enzyme that was responsible for this oxaloacetate hydrolase (EC 3.7.1.1), which was induced de novo during the pH shift. Subcellular fractionation of oxalic acid-forming mycelia of A. niger showed that this enzyme is located in the cytoplasm of A. niger. The results are consistent with a cytoplasmic pathway of oxalate formation which does not involve the tricarboxylic acid cycle.

Method for determining heterologous biosynthesis pathways

KAUST Repository

Gao, Xin

2017-08-10

The present invention relates to a method and system for dynamically analyzing, determining, predicting and displaying ranked suitable heterologous biosynthesis pathways for a specified host. The present invention addresses the problem of finding suitable pathways for the endogenous metabolism of a host organism because the efficacy of heterologous biosynthesis is affected by competing endogenous pathways. The present invention is called MRE (Metabolic Route Explorer), and it was conceived and developed to systematically and dynamically search for, determine, analyze, and display promising heterologous pathways while considering competing endogenous reactions in a given host organism.

Precursor Amino Acids Inhibit Polymyxin E Biosynthesis in Paenibacillus polymyxa, Probably by Affecting the Expression of Polymyxin E Biosynthesis-Associated Genes

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Zhiliang Yu

2015-01-01

Full Text Available Polymyxin E belongs to cationic polypeptide antibiotic bearing four types of direct precursor amino acids including L-2,4-diaminobutyric acid (L-Dab, L-Leu, D-Leu, and L-Thr. The objective of this study is to evaluate the effect of addition of precursor amino acids during fermentation on polymyxin E biosynthesis in Paenibacillus polymyxa. The results showed that, after 35 h fermentation, addition of direct precursor amino acids to certain concentration significantly inhibited polymyxin E production and affected the expression of genes involved in its biosynthesis. L-Dab repressed the expression of polymyxin synthetase genes pmxA and pmxE, as well as 2,4-diaminobutyrate aminotransferase gene ectB; both L-Leu and D-Leu repressed the pmxA expression. In addition, L-Thr affected the expression of not only pmxA, but also regulatory genes spo0A and abrB. As L-Dab precursor, L-Asp repressed the expression of ectB, pmxA, and pmxE. Moreover, it affected the expression of spo0A and abrB. In contrast, L-Phe, a nonprecursor amino acid, had no obvious effect on polymyxin E biosynthesis and those biosynthesis-related genes expression. Taken together, our data demonstrated that addition of precursor amino acids during fermentation will inhibit polymyxin E production probably by affecting the expression of its biosynthesis-related genes.

Niacin and biosynthesis of PGD₂ by platelet COX-1 in mice and humans

DEFF Research Database (Denmark)

Song, Wen-Liang; Stubbe, Jane; Ricciotti, Emanuela

2012-01-01

during platelet activation in humans and, although vascular expression of DP1 is conserved between humans and mice, platelet DP1 is not present in mice. Despite this, DP1 deletion in mice augmented aneurysm formation and the hypertensive response to Ang II and accelerated atherogenesis and thrombogenesis....... Furthermore, COX inhibitors in humans, as well as platelet depletion, COX-1 knockdown, and COX-2 deletion in mice, revealed that niacin evoked platelet COX-1-derived PGD₂ biosynthesis. Finally, ADP-induced spreading on fibrinogen was augmented by niacin in washed human platelets, coincident with increased...... thromboxane (Tx) formation. However, in platelet-rich plasma, where formation of both Tx and PGD₂ was increased, spreading was not as pronounced and was inhibited by DP1 activation. Thus, PGD₂, like PGI₂, may function as a homeostatic response to thrombogenic and hypertensive stimuli and may have particular...

Protoporphyrinogen oxidase: high affinity tetrahydrophthalimide radioligand for the inhibitor/herbicide-binding site in mouse liver mitochondria.

Science.gov (United States)

Birchfield, N B; Casida, J E

1996-01-01

Protoporphyrinogen oxidase (protox), the last common enzyme in heme and chlorophyll biosynthesis, is the target of several classes of herbicides acting as inhibitors in both plants and mammals. N-(4-Chloro-2-fluoro-5-(propargyloxy)phenyl)-3,4,5,6-tetrahydro phthalimide (a potent protox inhibitor referred to as THP) was synthesized as a candidate radioligand ([3H]-THP) by selective catalytic reduction of 3,6-dihydrophthalic anhydride (DHPA) with tritium gas followed by condensation in 45% yield with 4-chloro-2-fluoro-5-(propargyloxy)aniline. Insertion of tritium at the 3 and 6 carbons of DHPA as well as the expected 4 and 5 carbons resulted in high specific activity [3H]THP (92 Ci/mmol). This radioligand undergoes rapid, specific, saturable, and reversible binding to the inhibitor/herbicide binding site of the protox component of cholate-solubilized mouse liver mitochondria with an apparent Kd of 0.41 nM and Bmax of 0.40 pmol/mg of protein. In the standard assay, mouse preparation (150 micrograms of protein) and [3H]THP (0.5 nM) are incubated in 500 microL of phosphate buffer at pH 7.2 for 15 min at 25 degrees C followed by addition of ammonium sulfate and filtration with glass fiber filters. The potencies of five nitrodiphenyl ethers and two other herbicides as inhibitors of [3H]THP binding correlate well with those for inhibition of protox activity (r2 = 0.97, n = 7), thus validating the binding assay as relevant to enzyme inhibition. It is also suitable to determine in vivo block as illustrated by an approximately 50% decrease in [3H]THP binding in liver mitochondria from mice treated ip with oxyfluorfen at 4 mg/kg. This is the first report of a binding assay for protox in mammals. The high affinity and specific activity of [3H]THP facilitate quantitation of protox and therefore research on a sensitive inhibition site for porphyrin biosynthesis.

Gibberellin biosynthesis and signal transduction is essential for internode elongation in deepwater rice.

Science.gov (United States)

Ayano, Madoka; Kani, Takahiro; Kojima, Mikiko; Sakakibara, Hitoshi; Kitaoka, Takuya; Kuroha, Takeshi; Angeles-Shim, Rosalyn B; Kitano, Hidemi; Nagai, Keisuke; Ashikari, Motoyuki

2014-10-01

Under flooded conditions, the leaves and internodes of deepwater rice can elongate above the water surface to capture oxygen and prevent drowning. Our previous studies showed that three major quantitative trait loci (QTL) regulate deepwater-dependent internode elongation in deepwater rice. In this study, we investigated the age-dependent internode elongation in deepwater rice. We also investigated the relationship between deepwater-dependent internode elongation and the phytohormone gibberellin (GA) by physiological and genetic approach using a QTL pyramiding line (NIL-1 + 3 + 12). Deepwater rice did not show internode elongation before the sixth leaf stage under deepwater condition. Additionally, deepwater-dependent internode elongation occurred on the sixth and seventh internodes during the sixth leaf stage. These results indicate that deepwater rice could not start internode elongation until the sixth leaf stage. Ultra-performance liquid chromatography tandem mass-spectrometry (UPLC-MS/MS) method for the phytohormone contents showed a deepwater-dependent GA1 and GA4 accumulation in deepwater rice. Additionally, a GA inhibitor abolished deepwater-dependent internode elongation in deepwater rice. On the contrary, GA feeding mimicked internode elongation under ordinary growth conditions. However, mutations in GA biosynthesis and signal transduction genes blocked deepwater-dependent internode elongation. These data suggested that GA biosynthesis and signal transduction are essential for deepwater-dependent internode elongation in deepwater rice. © 2014 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.

Niacin and biosynthesis of PGD₂by platelet COX-1 in mice and humans.

Science.gov (United States)

Song, Wen-Liang; Stubbe, Jane; Ricciotti, Emanuela; Alamuddin, Naji; Ibrahim, Salam; Crichton, Irene; Prempeh, Maxwell; Lawson, John A; Wilensky, Robert L; Rasmussen, Lars Melholt; Puré, Ellen; FitzGerald, Garret A

2012-04-01

The clinical use of niacin to treat dyslipidemic conditions is limited by noxious side effects, most commonly facial flushing. In mice, niacin-induced flushing results from COX-1-dependent formation of PGD₂ and PGE₂ followed by COX-2-dependent production of PGE₂. Consistent with this, niacin-induced flushing in humans is attenuated when niacin is combined with an antagonist of the PGD₂ receptor DP1. NSAID-mediated suppression of COX-2-derived PGI₂ has negative cardiovascular consequences, yet little is known about the cardiovascular biology of PGD₂. Here, we show that PGD₂ biosynthesis is augmented during platelet activation in humans and, although vascular expression of DP1 is conserved between humans and mice, platelet DP1 is not present in mice. Despite this, DP1 deletion in mice augmented aneurysm formation and the hypertensive response to Ang II and accelerated atherogenesis and thrombogenesis. Furthermore, COX inhibitors in humans, as well as platelet depletion, COX-1 knockdown, and COX-2 deletion in mice, revealed that niacin evoked platelet COX-1-derived PGD₂ biosynthesis. Finally, ADP-induced spreading on fibrinogen was augmented by niacin in washed human platelets, coincident with increased thromboxane (Tx) formation. However, in platelet-rich plasma, where formation of both Tx and PGD₂ was increased, spreading was not as pronounced and was inhibited by DP1 activation. Thus, PGD₂, like PGI₂, may function as a homeostatic response to thrombogenic and hypertensive stimuli and may have particular relevance as a constraint on platelets during niacin therapy.

Isoprenoid biosynthesis in hereditary periodic fever syndromes and inflammation

NARCIS (Netherlands)

Houten, S. M.; Frenkel, J.; Waterham, H. R.

2003-01-01

Mevalonate kinase (MK) is an essential enzyme in the isoprenoid biosynthesis pathway which produces numerous biomolecules (isoprenoids) involved in a variety of cellular processes. The indispensability of MK and isoprenoid biosynthesis for human health is demonstrated by the identification of its

Regulatory cross-talks and cascades in rice hormone biosynthesis pathways contribute to stress signaling

Directory of Open Access Journals (Sweden)

Arindam Deb

2016-08-01

Full Text Available Crosstalk among different hormone signaling pathways play an important role in modulating plant response to both biotic and abiotic stress. Hormone activity is controlled by its bio-availability, which is again influenced by its biosynthesis. Thus independent hormone biosynthesis pathways must be regulated and co-ordinated to mount an integrated response. One of the possibilities is to use cis-regulatory elements to orchestrate expression of hormone biosynthesis genes. Analysis of CREs, associated with differentially expressed hormone biosynthesis related genes in rice leaf under Magnaporthe oryzae attack and drought stress enabled us to obtain insights about cross-talk among hormone biosynthesis pathways at the transcriptional level. We identified some master transcription regulators that co-ordinate different hormone biosynthesis pathways under stress. We found that Abscisic acid and Brassinosteroid regulate Cytokinin conjugation; conversely Brassinosteroid biosynthesis is affected by both Abscisic acid and Cytokinin. Jasmonic acid and Ethylene biosynthesis may be modulated by Abscisic acid through DREB transcription factors. Jasmonic acid or Salicylic acid biosynthesis pathways are co-regulated but they are unlikely to influence each other’s production directly. Thus multiple hormones may modulate hormone biosynthesis pathways through a complex regulatory network, where biosynthesis of one hormone is affected by several other contributing hormones.

Identification by virtual screening and in vitro testing of human DOPA decarboxylase inhibitors.

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Frederick Daidone

Full Text Available Dopa decarboxylase (DDC, a pyridoxal 5'-phosphate (PLP enzyme responsible for the biosynthesis of dopamine and serotonin, is involved in Parkinson's disease (PD. PD is a neurodegenerative disease mainly due to a progressive loss of dopamine-producing cells in the midbrain. Co-administration of L-Dopa with peripheral DDC inhibitors (carbidopa or benserazide is the most effective symptomatic treatment for PD. Although carbidopa and trihydroxybenzylhydrazine (the in vivo hydrolysis product of benserazide are both powerful irreversible DDC inhibitors, they are not selective because they irreversibly bind to free PLP and PLP-enzymes, thus inducing diverse side effects. Therefore, the main goals of this study were (a to use virtual screening to identify potential human DDC inhibitors and (b to evaluate the reliability of our virtual-screening (VS protocol by experimentally testing the "in vitro" activity of selected molecules. Starting from the crystal structure of the DDC-carbidopa complex, a new VS protocol, integrating pharmacophore searches and molecular docking, was developed. Analysis of 15 selected compounds, obtained by filtering the public ZINC database, yielded two molecules that bind to the active site of human DDC and behave as competitive inhibitors with K(i values ≥10 µM. By performing in silico similarity search on the latter compounds followed by a substructure search using the core of the most active compound we identified several competitive inhibitors of human DDC with K(i values in the low micromolar range, unable to bind free PLP, and predicted to not cross the blood-brain barrier. The most potent inhibitor with a K(i value of 500 nM represents a new lead compound, targeting human DDC, that may be the basis for lead optimization in the development of new DDC inhibitors. To our knowledge, a similar approach has not been reported yet in the field of DDC inhibitors discovery.

Lipoprotein biosynthesis as a target for anti-Wolbachia treatment of filarial nematodes

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Slatko Barton E

2010-10-01

Full Text Available Abstract Background Lymphatic filariasis and onchocerciasis are debilitating diseases caused by filarial nematodes. Disease pathogenesis is induced by inflammatory responses following the death of the parasite. Wolbachia endosymbionts of filariae are potent inducers of innate and adaptive inflammation and bacterial lipoproteins have been identified as the ligands that bind toll-like receptors (TLR 2 and TLR6. Lipoproteins are important structural and functional components of bacteria and therefore enzymes involved in Wolbachia lipoprotein biosynthesis are potential chemotherapeutic targets. Results Globomycin, a signal peptidase II (LspA inhibitor, has activity against Gram-negative bacteria and a putative lspA gene has been identified from the Wolbachia genome of Brugia malayi (wBm. The amino acids required for function are strictly conserved and functionality was verified by complementation tests in a temperature-sensitive Escherichia coli lspA mutant. Also, transformation of wild type E. coli with Wolbachia lspA conferred significant globomycin resistance. A cell-based screen has been developed utilizing a Wolbachia-containing Aedes albopictus cell line to assay novel compounds active against Wolbachia. Globomycin was screened using this assay, which resulted in a dose-dependent reduction in Wolbachia load. Furthermore, globomycin was also effective in reducing the motility and viability of adult B. malayi in vitro. Conclusions These studies validate lipoprotein biosynthesis as a target in an organism for which no genetic tools are available. Further studies to evaluate drugs targeting this pathway are underway as part of the A-WOL drug discovery and development program.

The expanding universe of alkaloid biosynthesis.

Science.gov (United States)

De Luca, V; Laflamme, P

2001-06-01

Characterization of many of the major gene families responsible for the generation of central intermediates and for their decoration, together with the development of large genomics and proteomics databases, has revolutionized our capability to identify exotic and interesting natural-product pathways. Over the next few years, these tools will facilitate dramatic advances in our knowledge of the biosynthesis of alkaloids, which will far surpass that which we have learned in the past 50 years. These tools will also be exploited for the rapid characterization of regulatory genes, which control the development of specialized cell factories for alkaloid biosynthesis.

Mammalian protein secretion without signal peptide removal. Biosynthesis of plasminogen activator inhibitor-2 in U-937 cells

International Nuclear Information System (INIS)

Ye, R.D.; Wun, T.C.; Sadler, J.E.

1988-01-01

Plasminogen activator inhibitor-2 (PAI-2) is a serine protease inhibitor that regulates plasmin generation by inhibiting urokinase and tissue plasminogen activator. The primary structure of PAI-2 suggests that it may be secreted without cleavage of a single peptide. To confirm this hypothesis we have studied the glycosylation and secretion of PAI-2 in human monocytic U-937 cells by metabolic labeling, immunoprecipitation, glycosidase digestion, and protein sequencing. PAI-2 is variably glycosylated on asparagine residues to yield intracellular intermediates with zero, one, two, or three high mannose-type oligosaccharide units. Secretion of the N-glycosylated species began by 1 h of chase and the secreted molecules contained both complex-type N-linked and O-linked oligosaccharides. Enzymatically deglycosylated PAI-2 had an electrophoretic mobility identical to that of the nonglycosylated precursor and also to that of PAI-2 synthesized in vitro in a rabbit reticulocyte lysate from synthetic mRNA derived from full length PAI-2 cDNA. The amino-terminal protein sequence of secreted PAI-2 began with the initiator methionine residue. These results indicate that PAI-2 is glycosylated and secreted efficiently without the cleavage of a signal peptide. PAI-2 shares this property with its nearest homologue in the serine protease inhibitor family, chicken ovalbumin, and appears to be the first well characterized example of this phenomenon among natural mammalian proteins

Hormonal Regulation of Dormancy in Developing Sorghum Seeds.

Science.gov (United States)

Steinbach, H. S.; Benech-Arnold, R. L.; Sanchez, R. A.

1997-01-01

The role of abscisic acid (ABA) and gibberellic acid (GA) in determining the dormancy level of developing sorghum (Sorghum bicolor [L.] Moench.) seeds from varieties presenting contrasting preharvest sprouting behavior (Redland B2, susceptible; IS 9530, resistant) was investigated. Panicles from both varieties were sprayed soon after pollination with fluridone or paclobutrazol to inhibit ABA and GA synthesis, respectively. Fluridone application to the panicles increased germinability of Redland B2 immature caryopses, whereas early treatment with paclobutrazol completely inhibited germination of this variety during most of the developmental period. Incubating caryopses in the presence of 100 [mu]M GA4+7 overcame the inhibitory effect of paclobutrazol, but also stimulated germination of seeds from other treatments. IS 9530 caryopses presented germination indices close to zero until physiological maturity (44 d after pollination) in control and paclobutrazol-treated particles. However, fluridone-treated caryopses were released from dormancy earlier than control and paclobutrazol-treated caryopses. Incubation in the presence of GA4+7 stimulated germination of caryopses from all treatments. Our results support the proposition that a low dormancy level (which is related to a high preharvest sprouting susceptibility) is determined not only by a low embryonic sensitivity to ABA, but also by a high GA content or sensitivity.

Design and Synthesis of Novel N-Benzylidenesulfonohydrazide Inhibitors of MurC and MurD as Potential Antibacterial Agents

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Aleš Obreza

2008-01-01

Full Text Available A series of novel N-benzylidenesulfonohydrazide compounds were designedand synthesized as inhibitors of UDP-N-acetylmuramic acid:L-alanine ligase (MurC andUDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD from E. coli, involved inthe biosynthesis of bacterial cell-walls. Some compounds possessed inhibitory activityagainst both enzymes with IC50 values as low as 30 μM. In addition, a new, one-potsynthesis of amidobenzaldehydes is reported.

Final Report on Regulation of Guaiacyl and Syringyl Monolignol Biosynthesis

Energy Technology Data Exchange (ETDEWEB)

Vincent L. Chiang

2006-03-09

The focus of this research is to understand syringyl monolignol biosynthesis that leads to the formation of syringyl lignin, a type of lignin that can be easily removed during biomass conversion. We have achieved the three originally proposed goals for this project. (1) SAD and CAD genes (enzyme catalytic and kinetic properties) and their functional relevance to CAld5H/AldOMT pathway, (2) spatiotemporal expression patterns of Cald5H, AldOMT, SAD and CAD genes, and (3) functions of CAld5H, AldOMT, and SAD genes in vivo using transgenic aspen. Furthermore, we also found that microRNA might be involved in the upstream regulatory network of lignin biosynthesis and wood formation. The achievements are as below. (1) Based on biochemical and molecular studies, we discovered a novel syringyl-specific alcohol dehydrogenase (SAD) involved in monolignol biosynthesis in angiosperm trees. Through CAld5H/OMT/SAD mediation, syringyl monolignol biosynthesis branches out from guaiacyl pathway at coniferaldehyde; (2) The function of CAld5H gene in this syringyl monolignol biosynthesis pathway also was confirmed in vivo in transgenic Populus; (3) The proposed major monolignol biosynthesis pathways were further supported by the involving biochemical functions of CCR based on a detailed kinetic study; (4) Gene promoter activity analysis also supported the cell-type specific expression of SAD and CAD genes in xylem tissue, consistent with the cell-specific locations of SAD and CAD proteins and with the proposed pathways; (5) We have developed a novel small interfering RNA (siRNA)-mediated stable gene-silencing system in transgenic plants; (6) Using the siRNA and P. trichocarpa transformation/regeneration systems we are currently producing transgenic P. trichocarpa to investigate the interactive functions of CAD and SAD in regulating guaiacyl and syringyl lignin biosynthesis; (7) We have cloned for the first time from a tree species, P. trichocarpa, small regulatory RNAs termed micro

Action of Gibberellins on Growth and Metabolism of Arabidopsis Plants Associated with High Concentration of Carbon Dioxide1[W

Science.gov (United States)

Ribeiro, Dimas M.; Araújo, Wagner L.; Fernie, Alisdair R.; Schippers, Jos H.M.; Mueller-Roeber, Bernd

2012-01-01

Although the positive effect of elevated CO2 concentration [CO2] on plant growth is well known, it remains unclear whether global climate change will positively or negatively affect crop yields. In particular, relatively little is known about the role of hormone pathways in controlling the growth responses to elevated [CO2]. Here, we studied the impact of elevated [CO2] on plant biomass and metabolism in Arabidopsis (Arabidopsis thaliana) in relation to the availability of gibberellins (GAs). Inhibition of growth by the GA biosynthesis inhibitor paclobutrazol (PAC) at ambient [CO2] (350 µmol CO2 mol−1) was reverted by elevated [CO2] (750 µmol CO2 mol−1). Thus, we investigated the metabolic adjustment and modulation of gene expression in response to changes in growth of plants imposed by varying the GA regime in ambient and elevated [CO2]. In the presence of PAC (low-GA regime), the activities of enzymes involved in photosynthesis and inorganic nitrogen assimilation were markedly increased at elevated [CO2], whereas the activities of enzymes of organic acid metabolism were decreased. Under ambient [CO2], nitrate, amino acids, and protein accumulated upon PAC treatment; however, this was not the case when plants were grown at elevated [CO2]. These results suggest that only under ambient [CO2] is GA required for the integration of carbohydrate and nitrogen metabolism underlying optimal biomass determination. Our results have implications concerning the action of the Green Revolution genes in future environmental conditions. PMID:23090585

Gibberellin Promotes Shoot Branching in the Perennial Woody Plant Jatropha curcas.

Science.gov (United States)

Ni, Jun; Gao, Congcong; Chen, Mao-Sheng; Pan, Bang-Zhen; Ye, Kaiqin; Xu, Zeng-Fu

2015-08-01

Strigolactone (SL), auxin and cytokinin (CK) interact to regulate shoot branching. CK has long been considered to be the only key phytohormone to promote lateral bud outgrowth. Here we report that gibberellin also acts as a positive regulator in the control of shoot branching in the woody plant Jatropha curcas. We show that gibberellin and CK synergistically promote lateral bud outgrowth, and that both hormones influence the expression of putative branching regulators, J. curcas BRANCHED1 and BRANCHED2, which are key transcription factors maintaining bud dormancy. Moreover, treatment with paclobutrazol, an inhibitor of de novo gibberellin biosynthesis, significantly reduced the promotion of bud outgrowth by CK, suggesting that gibberellin is required for CK-mediated axillary bud outgrowth. In addition, SL, a plant hormone involved in the repression of shoot branching, acted antagonistically to both gibberellin and CK in the control of lateral bud outgrowth. Consistent with this, the expression of JcMAX2, a J. curcas homolog of Arabidopsis MORE AXILLARY GROWTH 2 encoding an F-box protein in the SL signaling pathway, was repressed by gibberellin and CK treatment. We also provide physiological evidence that gibberellin also induces shoot branching in many other trees, such as papaya, indicating that a more complicated regulatory network occurs in the control of shoot branching in some perennial woody plants. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

The Mitochondrion-Located Protein OsB12D1 Enhances Flooding Tolerance during Seed Germination and Early Seedling Growth in Rice

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Dongli He

2014-07-01

Full Text Available B12D belongs to a function unknown subgroup of the Balem (Barley aleurone and embryo proteins. In our previous work on rice seed germination, we identified a B12D-like protein encoded by LOC_Os7g41350 (named OsB12D1. OsB12D1 pertains to an ancient protein family with an amino acid sequence highly conserved from moss to angiosperms. Among the six OsB12Ds, OsB12D1 is one of the major transcripts and is primarily expressed in germinating seed and root. Bioinformatics analyses indicated that OsB12D1 is an anoxic or submergence resistance-related gene. RT-PCR results showed OsB12D1 is induced remarkably in the coleoptiles or roots by flooding during seed germination and early seedling growth. The OsB12D1-overexpressed rice seeds could protrude radicles in 8 cm deep water, further exhibiting significant flooding tolerance compared to the wild type. Moreover, this tolerance was not affected by the gibberellin biosynthesis inhibitor paclobutrazol. OsB12D1 was identified in the mitochondrion by subcellular localization analysis and possibly enhances electron transport through mediating Fe and oxygen availability under flooded conditions. This work indicated that OsB12D1 is a promising gene that can help to enhance rice seedling establishment in farming practices, especially for direct seeding.

Rapid biosynthesis of cadmium sulfide (CdS) nanoparticles using ...

African Journals Online (AJOL)

Rapid biosynthesis of cadmium sulfide (CdS) nanoparticles using culture supernatants of Escherichia coli ATCC 8739, Bacillus subtilis ATCC 6633 and Lactobacillus ... The process of extracellular and fast biosynthesis may help in the development of an easy and eco-friendly route for the synthesis of CdS nanoparticles.

Natural product inhibitors of fatty acid biosynthesis: synthesis of the marine microbial metabolites pseudopyronines A and B and evaluation of their anti-infective activities

DEFF Research Database (Denmark)

Giddens, Anna C.; Nielsen, Lone; Boshoff, Helena I.

2007-01-01

of pathogenic microorganisms and were found to exhibit good potency (IC50≥0.46 μg/mL) and selectivity towards Leishmania donovani. Several of the compounds inhibited recombinant fatty acid biosynthesis enzymes from both Plasmodium falciparum and Mycobacterium tuberculosis, validating these targets in the search...

Two tomato GDP-D-mannose epimerase isoforms involved in ascorbate biosynthesis play specific roles in cell wall biosynthesis and development.

Science.gov (United States)

Mounet-Gilbert, Louise; Dumont, Marie; Ferrand, Carine; Bournonville, Céline; Monier, Antoine; Jorly, Joana; Lemaire-Chamley, Martine; Mori, Kentaro; Atienza, Isabelle; Hernould, Michel; Stevens, Rebecca; Lehner, Arnaud; Mollet, Jean Claude; Rothan, Christophe; Lerouge, Patrice; Baldet, Pierre

2016-08-01

GDP-D-mannose epimerase (GME, EC 5.1.3.18) converts GDP-D-mannose to GDP-L-galactose, and is considered to be a central enzyme connecting the major ascorbate biosynthesis pathway to primary cell wall metabolism in higher plants. Our previous work demonstrated that GME is crucial for both ascorbate and cell wall biosynthesis in tomato. The aim of the present study was to investigate the respective role in ascorbate and cell wall biosynthesis of the two SlGME genes present in tomato by targeting each of them through an RNAi-silencing approach. Taken individually SlGME1 and SlGME2 allowed normal ascorbate accumulation in the leaf and fruits, thus suggesting the same function regarding ascorbate. However, SlGME1 and SlGME2 were shown to play distinct roles in cell wall biosynthesis, depending on the tissue considered. The RNAi-SlGME1 plants harbored small and poorly seeded fruits resulting from alterations of pollen development and of pollination process. In contrast, the RNAi-SlGME2 plants exhibited vegetative growth delay while fruits remained unaffected. Analysis of SlGME1- and SlGME2-silenced seeds and seedlings further showed that the dimerization state of pectin rhamnogalacturonan-II (RG-II) was altered only in the RNAi-SlGME2 lines. Taken together with the preferential expression of each SlGME gene in different tomato tissues, these results suggest sub-functionalization of SlGME1 and SlGME2 and their specialization for cell wall biosynthesis in specific tomato tissues. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Role of tomato lipoxygenase D in wound-induced jasmonate biosynthesis and plant immunity to insect herbivores.

Science.gov (United States)

Yan, Liuhua; Zhai, Qingzhe; Wei, Jianing; Li, Shuyu; Wang, Bao; Huang, Tingting; Du, Minmin; Sun, Jiaqiang; Kang, Le; Li, Chang-Bao; Li, Chuanyou

2013-01-01

In response to insect attack and mechanical wounding, plants activate the expression of genes involved in various defense-related processes. A fascinating feature of these inducible defenses is their occurrence both locally at the wounding site and systemically in undamaged leaves throughout the plant. Wound-inducible proteinase inhibitors (PIs) in tomato (Solanum lycopersicum) provide an attractive model to understand the signal transduction events leading from localized injury to the systemic expression of defense-related genes. Among the identified intercellular molecules in regulating systemic wound response of tomato are the peptide signal systemin and the oxylipin signal jasmonic acid (JA). The systemin/JA signaling pathway provides a unique opportunity to investigate, in a single experimental system, the mechanism by which peptide and oxylipin signals interact to coordinate plant systemic immunity. Here we describe the characterization of the tomato suppressor of prosystemin-mediated responses8 (spr8) mutant, which was isolated as a suppressor of (pro)systemin-mediated signaling. spr8 plants exhibit a series of JA-dependent immune deficiencies, including the inability to express wound-responsive genes, abnormal development of glandular trichomes, and severely compromised resistance to cotton bollworm (Helicoverpa armigera) and Botrytis cinerea. Map-based cloning studies demonstrate that the spr8 mutant phenotype results from a point mutation in the catalytic domain of TomLoxD, a chloroplast-localized lipoxygenase involved in JA biosynthesis. We present evidence that overexpression of TomLoxD leads to elevated wound-induced JA biosynthesis, increased expression of wound-responsive genes and, therefore, enhanced resistance to insect herbivory attack and necrotrophic pathogen infection. These results indicate that TomLoxD is involved in wound-induced JA biosynthesis and highlight the application potential of this gene for crop protection against insects and

Role of tomato lipoxygenase D in wound-induced jasmonate biosynthesis and plant immunity to insect herbivores.

Directory of Open Access Journals (Sweden)

Liuhua Yan

Full Text Available In response to insect attack and mechanical wounding, plants activate the expression of genes involved in various defense-related processes. A fascinating feature of these inducible defenses is their occurrence both locally at the wounding site and systemically in undamaged leaves throughout the plant. Wound-inducible proteinase inhibitors (PIs in tomato (Solanum lycopersicum provide an attractive model to understand the signal transduction events leading from localized injury to the systemic expression of defense-related genes. Among the identified intercellular molecules in regulating systemic wound response of tomato are the peptide signal systemin and the oxylipin signal jasmonic acid (JA. The systemin/JA signaling pathway provides a unique opportunity to investigate, in a single experimental system, the mechanism by which peptide and oxylipin signals interact to coordinate plant systemic immunity. Here we describe the characterization of the tomato suppressor of prosystemin-mediated responses8 (spr8 mutant, which was isolated as a suppressor of (prosystemin-mediated signaling. spr8 plants exhibit a series of JA-dependent immune deficiencies, including the inability to express wound-responsive genes, abnormal development of glandular trichomes, and severely compromised resistance to cotton bollworm (Helicoverpa armigera and Botrytis cinerea. Map-based cloning studies demonstrate that the spr8 mutant phenotype results from a point mutation in the catalytic domain of TomLoxD, a chloroplast-localized lipoxygenase involved in JA biosynthesis. We present evidence that overexpression of TomLoxD leads to elevated wound-induced JA biosynthesis, increased expression of wound-responsive genes and, therefore, enhanced resistance to insect herbivory attack and necrotrophic pathogen infection. These results indicate that TomLoxD is involved in wound-induced JA biosynthesis and highlight the application potential of this gene for crop protection against

PLANT VOLATILES. Biosynthesis of monoterpene scent compounds in roses.

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Magnard, Jean-Louis; Roccia, Aymeric; Caissard, Jean-Claude; Vergne, Philippe; Sun, Pulu; Hecquet, Romain; Dubois, Annick; Hibrand-Saint Oyant, Laurence; Jullien, Frédéric; Nicolè, Florence; Raymond, Olivier; Huguet, Stéphanie; Baltenweck, Raymonde; Meyer, Sophie; Claudel, Patricia; Jeauffre, Julien; Rohmer, Michel; Foucher, Fabrice; Hugueney, Philippe; Bendahmane, Mohammed; Baudino, Sylvie

2015-07-03

The scent of roses (Rosa x hybrida) is composed of hundreds of volatile molecules. Monoterpenes represent up to 70% percent of the scent content in some cultivars, such as the Papa Meilland rose. Monoterpene biosynthesis in plants relies on plastid-localized terpene synthases. Combining transcriptomic and genetic approaches, we show that the Nudix hydrolase RhNUDX1, localized in the cytoplasm, is part of a pathway for the biosynthesis of free monoterpene alcohols that contribute to fragrance in roses. The RhNUDX1 protein shows geranyl diphosphate diphosphohydrolase activity in vitro and supports geraniol biosynthesis in planta. Copyright © 2015, American Association for the Advancement of Science.

Transcriptional changes associated with resistance to inhibitors of epidermal growth factor receptor revealed using metaanalysis

International Nuclear Information System (INIS)

Younis, Sidra; Javed, Qamar; Blumenberg, Miroslav

2015-01-01

EGFR is important in maintaining metabolic homeostasis in healthy cells, but in tumors it activates downstream signaling pathways, causing proliferation, angiogenesis, invasion and metastasis. Consequently, EGFR is targeted in cancers using reversible, irreversible or antibody inhibitors. Unfortunately, tumors develop inhibitor resistance by mutations or overexpressing EGFR, or its ligand, or activating secondary, EGFR-independent pathways. Here we present a global metaanalysis comparing transcriptional profiles from matched pairs of EGFR inhibitor-sensitive vs. -resistant cell lines, using 15 datasets comprising 274 microarrays. We also analyzed separately pairs of cell lines derived using reversible, irreversible or antibody inhibitors. The metaanalysis identifies commonalities in cell lines resistant to EGFR inhibitors: in sensitive cell lines, the ontological categories involving the ErbB receptors pathways, cell adhesion and lipid metabolism are overexpressed; however, resistance to EGFR inhibitors is associated with overexpression of genes for ErbB receptors-independent oncogenic pathways, regulation of cell motility, energy metabolism, immunity especially inflammatory cytokines biosynthesis, cell cycle and responses to exogenous and endogenous stimuli. Specifically in Gefitinib-resistant cell lines, the immunity-associated genes are overexpressed, whereas in Erlotinib-resistant ones so are the mitochondrial genes and processes. Unexpectedly, lines selected using EGFR-targeting antibodies overexpress different gene ontologies from ones selected using kinase inhibitors. Specifically, they have reduced expression of genes for proliferation, chemotaxis, immunity and angiogenesis. This metaanalysis suggests that ‘combination therapies’ can improve cancer treatment outcomes. Potentially, use of mitochondrial blockers with Erlotinib, immunity blockers with Gefitinib, tyrosine kinase inhibitors with antibody inhibitors, may have better chance of avoiding

Microbial biosynthesis of nontoxic gold nanoparticles

International Nuclear Information System (INIS)

Roy, Swarup; Das, Tapan Kumar; Maiti, Guru Prasad; Basu, Utpal

2016-01-01

Graphical abstract: The manuscript deals with the fungus mediated optimized biologically synthesized GNPs using Aspergillus foetidus and characterization of biosynthesized GNPs using various physico-chemical methods. The fairly stable synthesized nanoparticles have size in the range of 10–40 nm. Cytotoxicity study of biosynthesized GNPs on Human lung cancer cell line A549 showed no significant toxicity of GNPs. - Highlights: • A novel biosynthesis process of GNPs using Aspergillus foetidus. • Biosynthesized GNPs are in the range of 10–40 nm as observed from TEM. • This process of synthesis is an optimized biosynthesis process of GNPs. • Biosynthesized GNPs are noncytotoxic against A549 cell line. - Abstract: We study the extracellular biosynthesis of gold nanoparticles (GNPs) using the fungal species Aspergillus foetidus. The formation of GNPs were initially monitored by visual observation and then characterized with the help of various characterization techniques. X-ray diffraction (XRD) results revealed distinctive formation of face centered cubic crystalline GNPs. From field emission scanning electron microscopy (FESEM) the morphology of the nanoparticles were found to be roughly spherical and within the size range of 30–50 nm. The spherical and polydispersed GNPs in the range of 10–40 nm were observed by transmission electron microscopy (TEM) analysis. It was established that alkaline pH, 1 mM gold salt concentration and 75 °C temperature were the respective optimum parameter for biosynthesis of GNPs. Cell cytotoxicity of GNP was compared with that of normal gold salt solution on A549 cell. The A549 cell growth in presence of GNPs was found to be comparatively less toxic than the gold ion.

Microbial biosynthesis of nontoxic gold nanoparticles

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Roy, Swarup, E-mail: swaruproy@klyuniv.ac.in [Department of Biochemistry and Biophysics, University of Kalyani, Kalyani 741235, West Bengal (India); Das, Tapan Kumar [Department of Biochemistry and Biophysics, University of Kalyani, Kalyani 741235, West Bengal (India); Maiti, Guru Prasad [Department of Molecular Biology and Biotechnology, University of Kalyani, Kalyani 741235, West Bengal (India); Department of Anesthesiology, Texas Tech University Health science Center, 3601 4th Street, Lubbock, TX 79430 (United States); Basu, Utpal [Department of Molecular Biology and Biotechnology, University of Kalyani, Kalyani 741235, West Bengal (India)

2016-01-15

Graphical abstract: The manuscript deals with the fungus mediated optimized biologically synthesized GNPs using Aspergillus foetidus and characterization of biosynthesized GNPs using various physico-chemical methods. The fairly stable synthesized nanoparticles have size in the range of 10–40 nm. Cytotoxicity study of biosynthesized GNPs on Human lung cancer cell line A549 showed no significant toxicity of GNPs. - Highlights: • A novel biosynthesis process of GNPs using Aspergillus foetidus. • Biosynthesized GNPs are in the range of 10–40 nm as observed from TEM. • This process of synthesis is an optimized biosynthesis process of GNPs. • Biosynthesized GNPs are noncytotoxic against A549 cell line. - Abstract: We study the extracellular biosynthesis of gold nanoparticles (GNPs) using the fungal species Aspergillus foetidus. The formation of GNPs were initially monitored by visual observation and then characterized with the help of various characterization techniques. X-ray diffraction (XRD) results revealed distinctive formation of face centered cubic crystalline GNPs. From field emission scanning electron microscopy (FESEM) the morphology of the nanoparticles were found to be roughly spherical and within the size range of 30–50 nm. The spherical and polydispersed GNPs in the range of 10–40 nm were observed by transmission electron microscopy (TEM) analysis. It was established that alkaline pH, 1 mM gold salt concentration and 75 °C temperature were the respective optimum parameter for biosynthesis of GNPs. Cell cytotoxicity of GNP was compared with that of normal gold salt solution on A549 cell. The A549 cell growth in presence of GNPs was found to be comparatively less toxic than the gold ion.

Nucleoside antibiotics: biosynthesis, regulation, and biotechnology.

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Niu, Guoqing; Tan, Huarong

2015-02-01

The alarming rise in antibiotic-resistant pathogens has coincided with a decline in the supply of new antibiotics. It is therefore of great importance to find and create new antibiotics. Nucleoside antibiotics are a large family of natural products with diverse biological functions. Their biosynthesis is a complex process through multistep enzymatic reactions and is subject to hierarchical regulation. Genetic and biochemical studies of the biosynthetic machinery have provided the basis for pathway engineering and combinatorial biosynthesis to create new or hybrid nucleoside antibiotics. Dissection of regulatory mechanisms is leading to strategies to increase the titer of bioactive nucleoside antibiotics. Copyright © 2014. Published by Elsevier Ltd.

Rare cause of post-squalene disorder of cholesterol biosynthesis ...

African Journals Online (AJOL)

Errors of cholesterol biosynthesis represent a heterogeneous group of metabolic disorders. The aim of the authors of this article is to present a case of a patient with typical symptoms of a rare post-squalene disorder of cholesterol biosynthesis, its diagnostics and progress in neonatal period. The differential diagnosis of a ...

Pharmacological Inhibitors of NAD Biosynthesis as Potential An ticancer Agents.

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Lucas, Stephanie; Soave, Claire; Nabil, Ghazal; Ahmed, Zainab Sabry Othman; Chen, Guohua; El-Banna, Hossny Awad; Dou, Q Ping; Wang, Jian

2017-01-01

Alteration of cellular metabolism is a hallmark of cancer, which underlies exciting opportunities to develop effective, anti-cancer therapeutics through inhibition of cancer metabolism. Nicotinamide Adenine Dinucleotide (NAD+), an essential coenzyme of energy metabolism and a signaling molecule linking cellular energy status to a spectrum of molecular regulation, has been shown to be in high demand in a variety of cancer cells. Depletion of NAD+ by inhibition of its key biosynthetic enzymes has become an attractive strategy to target cancer. The main objective of this article is to review the recent patents which develop and implicate the chemical inhibitors of the key NAD+ biosynthetic enzymes for cancer treatment. We first discuss the biological principles of NAD+ metabolism in normal and malignant cells, with a focus on the feasibility of selectively targeting cancer cells by pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT) and indoleamine/tryptophan 2,3-dioxygenases (IDO/TDO), the rate-limiting salvage and de novo NAD+ biosynthetic enzymes, respectively. We then analyze a series of recent patents on development and optimization of chemical scaffolds for inhibiting NAMPT or IDO/TDO enzymes as potential anticancer drugs. Conclusion and Results: We have reviewed 16 relevant patents published since 2015, and summarized the chemical properties, mechanisms of action and proposed applications of the patented compounds. Without a better understanding of the properties of these compounds, their utility for further optimization and clinical use is unknown. For the compounds that have been tested using cell and mouse models of cancer, results look promising and clinical trials are currently ongoing to see if these results translate to improved cancer treatments. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

46_ _267 - 278__Aminu- Biosynthesis

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ISSN 2006 – 6996. BIOSYNTHESIS, CHARACTERIZATION AND ANTIMICROBIAL STUDY OF .... the excitation of surface Plasmon vibration with. AgNPs. ... Thin films of the sample were prepared on a carbon ... The resulting film on the SEM.

A novel cofactor-binding mode in bacterial IMP dehydrogenases explains inhibitor selectivity.

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Makowska-Grzyska, Magdalena; Kim, Youngchang; Maltseva, Natalia; Osipiuk, Jerzy; Gu, Minyi; Zhang, Minjia; Mandapati, Kavitha; Gollapalli, Deviprasad R; Gorla, Suresh Kumar; Hedstrom, Lizbeth; Joachimiak, Andrzej

2015-02-27

The steadily rising frequency of emerging diseases and antibiotic resistance creates an urgent need for new drugs and targets. Inosine 5'-monophosphate dehydrogenase (IMP dehydrogenase or IMPDH) is a promising target for the development of new antimicrobial agents. IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD(+), which is the pivotal step in the biosynthesis of guanine nucleotides. Potent inhibitors of bacterial IMPDHs have been identified that bind in a structurally distinct pocket that is absent in eukaryotic IMPDHs. The physiological role of this pocket was not understood. Here, we report the structures of complexes with different classes of inhibitors of Bacillus anthracis, Campylobacter jejuni, and Clostridium perfringens IMPDHs. These structures in combination with inhibition studies provide important insights into the interactions that modulate selectivity and potency. We also present two structures of the Vibrio cholerae IMPDH in complex with IMP/NAD(+) and XMP/NAD(+). In both structures, the cofactor assumes a dramatically different conformation than reported previously for eukaryotic IMPDHs and other dehydrogenases, with the major change observed for the position of the NAD(+) adenosine moiety. More importantly, this new NAD(+)-binding site involves the same pocket that is utilized by the inhibitors. Thus, the bacterial IMPDH-specific NAD(+)-binding mode helps to rationalize the conformation adopted by several classes of prokaryotic IMPDH inhibitors. These findings offer a potential strategy for further ligand optimization. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A Novel Cofactor-binding Mode in Bacterial IMP Dehydrogenases Explains Inhibitor Selectivity*

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Makowska-Grzyska, Magdalena; Kim, Youngchang; Maltseva, Natalia; Osipiuk, Jerzy; Gu, Minyi; Zhang, Minjia; Mandapati, Kavitha; Gollapalli, Deviprasad R.; Gorla, Suresh Kumar; Hedstrom, Lizbeth; Joachimiak, Andrzej

2015-01-01

The steadily rising frequency of emerging diseases and antibiotic resistance creates an urgent need for new drugs and targets. Inosine 5′-monophosphate dehydrogenase (IMP dehydrogenase or IMPDH) is a promising target for the development of new antimicrobial agents. IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD+, which is the pivotal step in the biosynthesis of guanine nucleotides. Potent inhibitors of bacterial IMPDHs have been identified that bind in a structurally distinct pocket that is absent in eukaryotic IMPDHs. The physiological role of this pocket was not understood. Here, we report the structures of complexes with different classes of inhibitors of Bacillus anthracis, Campylobacter jejuni, and Clostridium perfringens IMPDHs. These structures in combination with inhibition studies provide important insights into the interactions that modulate selectivity and potency. We also present two structures of the Vibrio cholerae IMPDH in complex with IMP/NAD+ and XMP/NAD+. In both structures, the cofactor assumes a dramatically different conformation than reported previously for eukaryotic IMPDHs and other dehydrogenases, with the major change observed for the position of the NAD+ adenosine moiety. More importantly, this new NAD+-binding site involves the same pocket that is utilized by the inhibitors. Thus, the bacterial IMPDH-specific NAD+-binding mode helps to rationalize the conformation adopted by several classes of prokaryotic IMPDH inhibitors. These findings offer a potential strategy for further ligand optimization. PMID:25572472

Reversibility of membrane N-glycome of HeLa cells upon treatment with epigenetic inhibitors.

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Tomislav Horvat

Full Text Available Glycans are essential regulators of protein function and are now in the focus of research in many physiological and pathophysiological processes. There are numerous modes of regulating their biosynthesis, including epigenetic mechanisms implicated in the expression of glyco-genes. Since N-glycans located at the cell membrane define intercellular communication as well as a cellular response to a given environment, we developed a method to preferentially analyze this fraction of glycans. The method is based on incorporation of living cells into polyacrylamide gels, partial denaturation of membrane proteins with 3 M urea and subsequent release of N-glycans with PNGase F followed by HPLC analysis. Using this newly developed method, we revealed multiple effects of epigenetic inhibitors Trichostatin A, sodium butyrate and zebularine on the composition of N-glycans in human cells. The induced changes were found to be reversible after inhibitor removal. Given that many epigenetic inhibitors are currently explored as a therapeutic strategy in treatment of cancer, wherein surface glycans play an important role, the presented work contributes to our understanding of their efficiency in altering the N-glycan profile of cancer cells in culture.

Polyamine biosynthesis is critical for growth and differentiation of the pancreas

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Mastracci, Teresa L.; Robertson, Morgan A.; Mirmira, Raghavendra G.; Anderson, Ryan M.

2015-01-01

The pancreas, in most studied vertebrates, is a compound organ with both exocrine and endocrine functions. The exocrine compartment makes and secretes digestive enzymes, while the endocrine compartment, organized into islets of Langerhans, produces hormones that regulate blood glucose. High concentrations of polyamines, which are aliphatic amines, are reported in exocrine and endocrine cells, with insulin-producing β cells showing the highest concentrations. We utilized zebrafish as a model organism, together with pharmacological inhibition or genetic manipulation, to determine how polyamine biosynthesis functions in pancreatic organogenesis. We identified that inhibition of polyamine biosynthesis reduces exocrine pancreas and β cell mass, and that these reductions are at the level of differentiation. Moreover, we demonstrate that inhibition of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, phenocopies inhibition or knockdown of the enzyme deoxyhypusine synthase (DHS). These data identify that the pancreatic requirement for polyamine biosynthesis is largely mediated through a requirement for spermidine for the downstream posttranslational modification of eIF5A by its enzymatic activator DHS, which in turn impacts mRNA translation. Altogether, we have uncovered a role for polyamine biosynthesis in pancreatic organogenesis and identified that it may be possible to exploit polyamine biosynthesis to manipulate pancreatic cell differentiation. PMID:26299433

Recent advances in combinatorial biosynthesis for drug discovery

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Sun H

2015-02-01

Full Text Available Huihua Sun,1,* Zihe Liu,1,* Huimin Zhao,1,2 Ee Lui Ang1 1Metabolic Engineering Research Laboratory, Institute of Chemical and Engineering Sciences, Agency for Science, Technology and Research, Singapore; 2Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA *These authors contributed equally to this work Abstract: Because of extraordinary structural diversity and broad biological activities, natural products have played a significant role in drug discovery. These therapeutically important secondary metabolites are assembled and modified by dedicated biosynthetic pathways in their host living organisms. Traditionally, chemists have attempted to synthesize natural product analogs that are important sources of new drugs. However, the extraordinary structural complexity of natural products sometimes makes it challenging for traditional chemical synthesis, which usually involves multiple steps, harsh conditions, toxic organic solvents, and byproduct wastes. In contrast, combinatorial biosynthesis exploits substrate promiscuity and employs engineered enzymes and pathways to produce novel “unnatural” natural products, substantially expanding the structural diversity of natural products with potential pharmaceutical value. Thus, combinatorial biosynthesis provides an environmentally friendly way to produce natural product analogs. Efficient expression of the combinatorial biosynthetic pathway in genetically tractable heterologous hosts can increase the titer of the compound, eventually resulting in less expensive drugs. In this review, we will discuss three major strategies for combinatorial biosynthesis: 1 precursor-directed biosynthesis; 2 enzyme-level modification, which includes swapping of the entire domains, modules and subunits, site-specific mutagenesis, and directed evolution; 3 pathway-level recombination. Recent examples of combinatorial biosynthesis employing these

Uridine monophosphate synthetase enables eukaryotic de novo NAD+ biosynthesis from quinolinic acid.

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McReynolds, Melanie R; Wang, Wenqing; Holleran, Lauren M; Hanna-Rose, Wendy

2017-07-07

NAD + biosynthesis is an attractive and promising therapeutic target for influencing health span and obesity-related phenotypes as well as tumor growth. Full and effective use of this target for therapeutic benefit requires a complete understanding of NAD + biosynthetic pathways. Here, we report a previously unrecognized role for a conserved phosphoribosyltransferase in NAD + biosynthesis. Because a required quinolinic acid phosphoribosyltransferase (QPRTase) is not encoded in its genome, Caenorhabditis elegans are reported to lack a de novo NAD + biosynthetic pathway. However, all the genes of the kynurenine pathway required for quinolinic acid (QA) production from tryptophan are present. Thus, we investigated the presence of de novo NAD + biosynthesis in this organism. By combining isotope-tracing and genetic experiments, we have demonstrated the presence of an intact de novo biosynthesis pathway for NAD + from tryptophan via QA, highlighting the functional conservation of this important biosynthetic activity. Supplementation with kynurenine pathway intermediates also boosted NAD + levels and partially reversed NAD + -dependent phenotypes caused by mutation of pnc-1 , which encodes a nicotinamidase required for NAD + salvage biosynthesis, demonstrating contribution of de novo synthesis to NAD + homeostasis. By investigating candidate phosphoribosyltransferase genes in the genome, we determined that the conserved uridine monophosphate phosphoribosyltransferase (UMPS), which acts in pyrimidine biosynthesis, is required for NAD + biosynthesis in place of the missing QPRTase. We suggest that similar underground metabolic activity of UMPS may function in other organisms. This mechanism for NAD + biosynthesis creates novel possibilities for manipulating NAD + biosynthetic pathways, which is key for the future of therapeutics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Simultaneous biosynthesis of putrebactin, avaroferrin and bisucaberin by Shewanella putrefaciens and characterisation of complexes with iron(III), molybdenum(VI) or chromium(V).

Science.gov (United States)

Soe, Cho Zin; Telfer, Thomas J; Levina, Aviva; Lay, Peter A; Codd, Rachel

2016-09-01

Cultures of Shewanella putrefaciens grown in medium containing 10mM 1,4-diamino-2-butanone (DBO) as an inhibitor of ornithine decarboxylase and 10mM 1,5-diaminopentane (cadaverine) showed the simultaneous biosynthesis of the macrocyclic dihydroxamic acids: putrebactin (pbH 2 ), avaroferrin (avH 2 ) and bisucaberin (bsH 2 ). The level of DBO did not completely repress the production of endogenous 1,4-diaminobutane (putrescine) as the native diamine substrate of pbH 2 . The relative concentration of pbH 2 :avH 2 :bsH 2 was 1:2:1, which correlated with the substrate selection of putrescine:cadaverine in a ratio of 1:1. The macrocycles were characterised using LC-MS as free ligands and as 1:1 complexes with Fe(III) of the form [Fe(pb)] + , [Fe(av)] + or [Fe(bs)] + , with labile ancillary ligands in six-coordinate complexes displaced during ESI-MS acquisition; or with Mo(VI) of the form [Mo(O) 2 (pb)], [Mo(O) 2 (av)] or [Mo(O) 2 (bs)]. Chromium(V) complexes of the form [CrO(pb)] + were detected from solutions of Cr(VI) and pbH 2 in DMF using X-band EPR spectroscopy. Supplementation of S. putrefaciens medium with DBO and 1,3-diaminopropane, 1,6-diaminohexane or 1,4-diamino-2(Z)-butene (Z-DBE) resulted only in the biosynthesis of pbH 2 . The work has identified a native system for the simultaneous biosynthesis of a suite of three macrocyclic dihydroxamic acid siderophores and highlights both the utility of precursor-directed biosynthesis for expanding the structural diversity of siderophores, and the breadth of their coordination chemistry. Copyright © 2015 Elsevier Inc. All rights reserved.

Very-long-chain fatty acid biosynthesis is inhibited by cafenstrole, N,N-diethyl-3-mesitylsulfonyl-1H-1,2,4-triazole-1-carboxamide and its analogs

Energy Technology Data Exchange (ETDEWEB)

Takahashi, H.; Ohki, A.; Sato, Y.; Wakabayashi, K. [Tamagawa Univ., Tokyo (Japan). Graduate School of Agricultural Science; Kanzaki, M. [Regulatory Affairs Dept., Chugai Pharmaceutical Co. Ltd., Tokyo (Japan); Tanaka, A. [Showa Univ., Tokyo (Japan). School of Pharmaceutical Sciences; Matthes, B.; Boeger, P. [Konstanz Univ. (Germany). Lehrstuhl fuer Physiologie und Biochemie der Pflanzen

2001-10-01

The rice herbicide cafenstrole and its analogs inhibited the incorporation of [1-{sup 14}C]-oleate and [2-{sup 14}C]-malonate into very-long-chain fatty acids (VLCFAs), using Scenedesmus cells and leek microsomes from Allium porrum. Although the precise mode of interaction of cafenstrole at the molecular level is not completely clarified by the present study, it is concluded that cafenstrole acts as a specific inhibitor of the microsomal elongase enzyme involved in the biosynthesis of fatty acids with alkyl chains longer than C{sub 18}. For a strong VLCFA biosynthesis inhibition an -SO{sub 2}- linkage of the 1,2,4-triazole-1-carboxamides was required. Furthermore, N,N-dialkyl substitution of the carbamoyl nitrogen and electron-donating groups such as methyl at the benzene ring of 1,2,4-triazole-1-carboxamides produced a strong inhibition of VLCFA formation. A correlation was found between the phytotoxic effect against barnyardgrass (Echinochloa oryzicola) and impaired VLCFA formation. (orig.)

Very-long-chain fatty acid biosynthesis is inhibited by cafenstrole, N,N-diethyl-3-mesitylsulfonyl-1H-1,2,4-triazole-1-carboxamide and its analogs

International Nuclear Information System (INIS)

Takahashi, H.; Ohki, A.; Sato, Y.; Wakabayashi, K.; Tanaka, A.; Matthes, B.; Boeger, P.

2001-01-01

The rice herbicide cafenstrole and its analogs inhibited the incorporation of [1- 14 C]-oleate and [2- 14 C]-malonate into very-long-chain fatty acids (VLCFAs), using Scenedesmus cells and leek microsomes from Allium porrum. Although the precise mode of interaction of cafenstrole at the molecular level is not completely clarified by the present study, it is concluded that cafenstrole acts as a specific inhibitor of the microsomal elongase enzyme involved in the biosynthesis of fatty acids with alkyl chains longer than C 18 . For a strong VLCFA biosynthesis inhibition an -SO 2 - linkage of the 1,2,4-triazole-1-carboxamides was required. Furthermore, N,N-dialkyl substitution of the carbamoyl nitrogen and electron-donating groups such as methyl at the benzene ring of 1,2,4-triazole-1-carboxamides produced a strong inhibition of VLCFA formation. A correlation was found between the phytotoxic effect against barnyardgrass (Echinochloa oryzicola) and impaired VLCFA formation. (orig.)

The LH2 complexes are assembled in the cells of purple sulfur bacterium Ectothiorhodospira haloalkaliphila with inhibition of carotenoid biosynthesis.

Science.gov (United States)

Ashikhmin, Aleksandr; Makhneva, Zoya; Moskalenko, Andrey

2014-03-01

The effect of the inhibitor of carotenoid (Car) biosynthesis, diphenylamine (DPA), on the cells of the purple sulfur bacterium Ectothiorhodospira (Ect.) haloalkaliphila has been studied. There occurs an inhibition of the biosynthesis of colored Cars (≥99 %) at 71 μM DPA. Considering "empty" Car pockets (Moskalenko and Makhneva 2012) the content of Cars in the DPA-treated samples is first calculated more correctly. The total content of the colored Cars in the sample at 71 μM DPA does not exceed 1 % of the wild type. In the DPA-treated cells (membranes) a complete set of pigment-protein complexes is retained. The LH2 complex at 71 μM DPA is isolated, which is identical to the LH2 complex of the wild type in near IR absorption spectra. This suggests that the principles for assembling this LH2 complex in vivo in the absence of colored Cars remain the same. These results are in full agreement with the data obtained earlier for Allochromatium (Alc.) minutissimum (Moskalenko and Makhneva 2012). They are as follows: (1) DPA almost entirely inhibits the biosynthesis of the colored Cars in Ect. haloalkaliphila cells. (2) In the DPA-treated samples non-colored Cars are detected at 53.25 μM DPA (as traces) and at 71 μM DPA. (3) DPA may affect both phytoene synthase (at ≤71 μM DPA) and phytoene desaturase (at ≥53.25 μM DPA). (4) The assembly of LH2 complex does occur without any colored Cars.

In Silico Design of Human IMPDH Inhibitors Using Pharmacophore Mapping and Molecular Docking Approaches

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Rui-Juan Li

2015-01-01

Full Text Available Inosine 5′-monophosphate dehydrogenase (IMPDH is one of the crucial enzymes in the de novo biosynthesis of guanosine nucleotides. It has served as an attractive target in immunosuppressive, anticancer, antiviral, and antiparasitic therapeutic strategies. In this study, pharmacophore mapping and molecular docking approaches were employed to discover novel Homo sapiens IMPDH (hIMPDH inhibitors. The Güner-Henry (GH scoring method was used to evaluate the quality of generated pharmacophore hypotheses. One of the generated pharmacophore hypotheses was found to possess a GH score of 0.67. Ten potential compounds were selected from the ZINC database using a pharmacophore mapping approach and docked into the IMPDH active site. We find two hits (i.e., ZINC02090792 and ZINC00048033 that match well the optimal pharmacophore features used in this investigation, and it is found that they form interactions with key residues of IMPDH. We propose that these two hits are lead compounds for the development of novel hIMPDH inhibitors.

Carotenoid inhibitors reduce strigolactone production and Striga hermonthica infection in rice.

Science.gov (United States)

Jamil, Muhammad; Charnikhova, Tatsiana; Verstappen, Francel; Bouwmeester, Harro

2010-12-01

The strigolactones are internal and rhizosphere signalling molecules in plants that are biosynthesised through carotenoid cleavage. They are secreted by host roots into the rhizosphere where they signal host-presence to the symbiotic arbuscular mycrorrhizal (AM) fungi and the parasitic plants of the Orobanche, Phelipanche and Striga genera. The seeds of these parasitic plants germinate after perceiving these signalling molecules. After attachment to the host root, the parasite negatively affects the host plant by withdrawing water, nutrients and assimilates through a direct connection with the host xylem. In many areas of the world these parasites are a threat to agriculture but so far very limited success has been achieved to minimize losses due to these parasitic weeds. Considering the carotenoid origin of the strigolactones, in the present study we investigated the possibilities to reduce strigolactone production in the roots of plants by blocking carotenoid biosynthesis using carotenoid inhibitors. Hereto the carotenoid inhibitors fluridone, norflurazon, clomazone and amitrole were applied to rice either through irrigation or through foliar spray. Irrigation application of all carotenoid inhibitors and spray application of amitrole significantly decreased strigolactone production, Striga hermonthica germination and Striga infection, also in concentrations too low to affect growth and development of the host plant. Hence, we demonstrate that the application of carotenoid inhibitors to plants can affect S. hermonthica germination and attachment indirectly by reducing the strigolactone concentration in the rhizosphere. This finding is useful for further studies on the relevance of the strigolactones in rhizosphere signalling. Since these inhibitors are available and accessible, they may represent an efficient technology for farmers, including poor subsistence farmers in the African continent, to control these harmful parasitic weeds. Copyright © 2010 Elsevier Inc

Structure, Biosynthesis, and Occurrence of Bacterial Pyrrolizidine Alkaloids.

Science.gov (United States)

Schimming, Olivia; Challinor, Victoria L; Tobias, Nicholas J; Adihou, Hélène; Grün, Peter; Pöschel, Laura; Richter, Christian; Schwalbe, Harald; Bode, Helge B

2015-10-19

Pyrrolizidine alkaloids (PAs) are widespread plant natural products with potent toxicity and bioactivity. Herein, the identification of bacterial PAs from entomopathogenic bacteria using differential analysis by 2D NMR spectroscopy (DANS) and mass spectrometry is described. Their biosynthesis was elucidated to involve a non-ribosomal peptide synthetase. The occurrence of these biosynthesis gene clusters in Gram-negative and Gram-positive bacteria indicates an important biological function in bacteria. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Water-Deficit Tolerance in Sweet Potato [Ipomoea batatas (L. Lam.] by Foliar Application of Paclobutrazol: Role of Soluble Sugar and Free Proline

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Suravoot Yooyongwech

2017-08-01

Full Text Available The objective of this study was to elevate water deficit tolerance by improving soluble sugar and free proline accumulation, photosynthetic pigment stabilization, photosynthetic abilities, growth performance and storage root yield in sweet potato cv. ‘Tainung 57’ using a foliar application of paclobutrazol (PBZ. The experiment followed a Completely Randomized Block Design with four concentrations of PBZ: 0 (control, 17, 34, and 51 μM before exposure to 47.5% (well irrigation, 32.3% (mild water deficit or 17.5% (severe water deficit soil water content. A sweet potato cultivar, ‘Japanese Yellow’, with water deficit tolerance attributes was the positive check in this study. Total soluble sugar content (sucrose, glucose, and fructose increased by 3.96-folds in ‘Tainung 57’ plants treated with 34 μM PBZ grown under 32.3% soil water content (SWC compared to the untreated plants, adjusting osmotic potential in the leaves and controlling stomatal closure (represented by stomatal conductance and transpiration rate. In addition, under the same treatment, free proline content (2.15 μmol g-1 FW increased by 3.84-folds when exposed to 17.5% SWC. PBZ had an improved effect on leaf size, vine length, photosynthetic pigment stability, chlorophyll fluorescence, and net photosynthetic rate; hence, delaying wilting symptoms and maintaining storage root yield (26.93 g plant-1 at the harvesting stage. A positive relationship between photon yield of PSII (ΦPSII and net photosynthetic rate was demonstrated (r2 = 0.73. The study concludes that soluble sugar and free proline enrichment in PBZ-pretreated plants may play a critical role as major osmoprotectant to control leaf osmotic potential and stomatal closure when plants were subjected to low soil water content, therefore, maintaining the physiological and morphological characters as well as storage root yield.

Water-Deficit Tolerance in Sweet Potato [Ipomoea batatas (L.) Lam.] by Foliar Application of Paclobutrazol: Role of Soluble Sugar and Free Proline.

Science.gov (United States)

Yooyongwech, Suravoot; Samphumphuang, Thapanee; Tisarum, Rujira; Theerawitaya, Cattarin; Cha-Um, Suriyan

2017-01-01

The objective of this study was to elevate water deficit tolerance by improving soluble sugar and free proline accumulation, photosynthetic pigment stabilization, photosynthetic abilities, growth performance and storage root yield in sweet potato cv. 'Tainung 57' using a foliar application of paclobutrazol (PBZ). The experiment followed a Completely Randomized Block Design with four concentrations of PBZ: 0 (control), 17, 34, and 51 μM before exposure to 47.5% (well irrigation), 32.3% (mild water deficit) or 17.5% (severe water deficit) soil water content. A sweet potato cultivar, 'Japanese Yellow', with water deficit tolerance attributes was the positive check in this study. Total soluble sugar content (sucrose, glucose, and fructose) increased by 3.96-folds in 'Tainung 57' plants treated with 34 μM PBZ grown under 32.3% soil water content (SWC) compared to the untreated plants, adjusting osmotic potential in the leaves and controlling stomatal closure (represented by stomatal conductance and transpiration rate). In addition, under the same treatment, free proline content (2.15 μmol g -1 FW) increased by 3.84-folds when exposed to 17.5% SWC. PBZ had an improved effect on leaf size, vine length, photosynthetic pigment stability, chlorophyll fluorescence, and net photosynthetic rate; hence, delaying wilting symptoms and maintaining storage root yield (26.93 g plant -1 ) at the harvesting stage. A positive relationship between photon yield of PSII (Φ PSII ) and net photosynthetic rate was demonstrated ( r 2 = 0.73). The study concludes that soluble sugar and free proline enrichment in PBZ-pretreated plants may play a critical role as major osmoprotectant to control leaf osmotic potential and stomatal closure when plants were subjected to low soil water content, therefore, maintaining the physiological and morphological characters as well as storage root yield.

Heme biosynthesis and its regulation : Toward understanding and improvement of heme biosynthesis in filamentous fungi.

NARCIS (Netherlands)

S. de Weert; P.J. Punt; Christien Lokman; C.A. van den Hondel; A.C. Franken; A.F. Ram

2011-01-01

Heme biosynthesis in fungal host strains has acquired considerable interest in relation to the production of secreted heme-containing peroxidases. Class II peroxidase enzymes have been suggested as eco-friendly replacements of polluting chemical processes in industry. These peroxidases are naturally

Heme biosynthesis and its regulation: Towards understanding and improvement of heme biosynthesis in filamentous fungi

NARCIS (Netherlands)

Franken, A.C.W.; Lokman, B.C.; Ram, A.F.J.; Punt, P.J.; Hondel, C.A.M.J.J. van den; Weert, S. de

2011-01-01

Heme biosynthesis in fungal host strains has acquired considerable interest in relation to the production of secreted heme-containing peroxidases. Class II peroxidase enzymes have been suggested as eco-friendly replacements of polluting chemical processes in industry. These peroxidases are naturally

Dificuldades en el uso de retardantes de crecimento en Viola wittrockiana.

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Carlos Boschi

2007-06-01

Full Text Available B-Nine es la formulación de daminozide (Uniroyal Chemicals, Middlebury, CT usada durante los últimos 25 años para este fin. Recientemente se han desarrollado nuevos retardantes (paclobutrazol aunque la respuesta a dosis extremadamente bajas ha sido errática en especies como la Viola wittrockiana. El resultado es un excesivo arrosetamieto, un alargamiento del ciclo productivo y una disminución de la calidad comercial. El objetivo de este trabajo ha sido evaluar el efecto de la calidad del sustrato de crecimiento sobre la respuesta de plantas de Viola wittrockiana tratadas con diferentes reguladores durante el período de crecimiento inicial en contenedores pequeños (plug. Se utilizaron plantas de Viola wittrockiana ‘Banner Orange’. A los 25 días de la siembra se asperjaron las plantas con: (1 Agua (Testigo, (2 Paclobutrazol: 0,5 mg L-1, (3 Paclobutrazol: 1,0 mg L-1, (4 Paclobutrazol: 2,5 mg L-1, (5 Paclobutrazol: 5,0 mg L-1, (6 Daminozide: 2500 mg L-1. Se trasplantaron posteriormente a macetas de 800 cm3 (35 días desde la siembra conteniendo los siguientes substratos: (1 Fafard Growing Mix® (100%, (2 Fafard Growing Mix® (50% + Suelo (50%, (3 Suelo (100%, (4 Fafard Growing Mix® + Resaca de río (50%, (5 Resaca de río (100%. Nuestros resultados indican que la respuesta para un sustrato de crecimiento dado pueden variar considerablemente dentro de un rango pequeño para Viola wittrockiana y que la producción de fotoasimilados varía según la dosis utilizada de paclobutrazol así como el sustrato de crecimiento utilizado (figura 1.

Structure and mechanism of a bacterial t6A biosynthesis system

OpenAIRE

Luthra, Amit; Swinehart, William; Bayooz, Susan; Phan, Phuc; Stec, Boguslaw; Iwata-Reuyl, Dirk; Swairjo, Manal A

2018-01-01

Abstract The universal N(6)-threonylcarbamoyladenosine (t6A) modification at position 37 of ANN-decoding tRNAs is central to translational fidelity. In bacteria, t6A biosynthesis is catalyzed by the proteins TsaB, TsaC/TsaC2, TsaD and TsaE. Despite intense research, the molecular mechanisms underlying t6A biosynthesis are poorly understood. Here, we report biochemical and biophysical studies of the t6A biosynthesis system from Thermotoga maritima. Small angle X-ray scattering analysis reveals...

The enzymology of polyether biosynthesis.

Science.gov (United States)

Liu, Tiangang; Cane, David E; Deng, Zixin

2009-01-01

Polyether ionophore antibiotics are a special class of polyketides widely used in veterinary medicine, and as food additives in animal husbandry. In this article, we review current knowledge about the mechanism of polyether biosynthesis, and the genetic and biochemical strategies used for its study. Several clear differences distinguish it from traditional type I modular polyketide biosynthesis: polyether backbones are assembled by modular polyketide synthases but are modified by two key enzymes, epoxidase and epoxide hydrolase, to generate the product. All double bonds involved in the oxidative cyclization in the polyketide backbone are of E geometry. Chain release in the polyether biosynthetic pathway requires a special type II thioesterase which specifically hydrolyzes the polyether thioester. All these discoveries should be very helpful for a deep understanding of the biosynthetic mechanism of this class of important natural compounds, and for the targeted engineering of polyether derivatives.

Purine biosynthesis de novo by lymphocytes in gout

International Nuclear Information System (INIS)

Kamoun, P.; Chanard, J.; Brami, M.; Funck-Brentano, J.L.

1978-01-01

A method of measurement in vitro of purine biosynthesis de novo in human circulating blood lymphocytes is proposed. The rate of early reactions of purine biosynthesis de novo was determined by the incorporation of [ 14 C]formate into N-formyl glycinamide ribonucleotide when the subsequent reactions of the metabolic pathway were completely inhibited by the antibiotic azaserine. Synthesis of 14 C-labelled N-formyl glycinamide ribonucleotide by lymphocytes was measured in healthy control subjects and patients with primary gout or hyperuricaemia secondary to renal failure, with or without allopurinol therapy. The average synthesis was higher in gouty patients without therapy than in control subjects, but the values contained overlap the normal range. In secondary hyperuricaemia the synthesis was at same value as in control subjects. These results are in agreement with the inconstant acceleration of purine biosynthesis de novo in gouty patients as seen by others with measurement of [ 14 C]glycine incorporation into urinary uric acid. (author)

Methoxypyrazines biosynthesis and metabolism in grape: A review.

Science.gov (United States)

Lei, Yujuan; Xie, Sha; Guan, Xueqiang; Song, Changzheng; Zhang, Zhenwen; Meng, Jiangfei

2018-04-15

This review summarizes research on the discovery, biosynthesis, accumulation, transport, and metabolism of 3-alkyl-2-methoxypyrazines (MPs) in grape. The MPs are a family of potent volatile compounds distributed throughout biological kingdoms. These compounds impart herbaceous/green/vegetal sensory attributes to certain varieties of wine. Generally, high levels of MPs in wine are derived mainly from the corresponding grapes. Although two pathways for MPs biosynthesis have been proposed, only the final step and the enzymes that catalyze it has been confirmed in grape, and the metabolic intermediates and key enzymes involved in other steps are still unknown. The limited understanding of MPs metabolism has restricted research on these compounds, and some empirical results cannot be explained by the current knowledge of MPs metabolism. This review provides insights into research on MPs biosynthesis and metabolism, and proposes directions for further research on this important class of flavour/odour compounds. Copyright © 2017 Elsevier Ltd. All rights reserved.

Niacin and biosynthesis of PGD2 by platelet COX-1 in mice and humans

Science.gov (United States)

Song, Wen-Liang; Stubbe, Jane; Ricciotti, Emanuela; Alamuddin, Naji; Ibrahim, Salam; Crichton, Irene; Prempeh, Maxwell; Lawson, John A.; Wilensky, Robert L.; Rasmussen, Lars Melholt; Puré, Ellen; FitzGerald, Garret A.

2012-01-01

The clinical use of niacin to treat dyslipidemic conditions is limited by noxious side effects, most commonly facial flushing. In mice, niacin-induced flushing results from COX-1–dependent formation of PGD2 and PGE2 followed by COX-2–dependent production of PGE2. Consistent with this, niacin-induced flushing in humans is attenuated when niacin is combined with an antagonist of the PGD2 receptor DP1. NSAID-mediated suppression of COX-2–derived PGI2 has negative cardiovascular consequences, yet little is known about the cardiovascular biology of PGD2. Here, we show that PGD2 biosynthesis is augmented during platelet activation in humans and, although vascular expression of DP1 is conserved between humans and mice, platelet DP1 is not present in mice. Despite this, DP1 deletion in mice augmented aneurysm formation and the hypertensive response to Ang II and accelerated atherogenesis and thrombogenesis. Furthermore, COX inhibitors in humans, as well as platelet depletion, COX-1 knockdown, and COX-2 deletion in mice, revealed that niacin evoked platelet COX-1–derived PGD2 biosynthesis. Finally, ADP-induced spreading on fibrinogen was augmented by niacin in washed human platelets, coincident with increased thromboxane (Tx) formation. However, in platelet-rich plasma, where formation of both Tx and PGD2 was increased, spreading was not as pronounced and was inhibited by DP1 activation. Thus, PGD2, like PGI2, may function as a homeostatic response to thrombogenic and hypertensive stimuli and may have particular relevance as a constraint on platelets during niacin therapy. PMID:22406532

Exploring the fungal protein cadre in the biosynthesis of PbSe quantum dots

Energy Technology Data Exchange (ETDEWEB)

Jacob, Jaya Mary; Sharma, Sumit; Balakrishnan, Raj Mohan, E-mail: rajmohanbala@gmail.com

2017-02-15

Highlights: • Pb and Se stress activates specific metal detoxification surge in the fungus. • Fungus releases phytochelatins, metallothioneins, super oxide dismutases etc. • These mechanisms capacitate the fungi as bio-factories for synthesis of PbSe QDs. • A pathway for PbSe QD biosynthesis by marine Aspergillus terreus was elucidated - Abstract: While a large number of microbial sources have recently emerged as potent sources for biosynthesis of chalcogenide quantum dots (QDs), studies regarding their biomimetic strategies that initiate QD biosynthesis are scarce. The present study describes several mechanistic aspects of PbSe QD biosynthesis using marine Aspergillus terreus. Scanning electron microscopic (SEM) studies indicated distinctive morphological features such as abrasion and agglomeration on the fungal biomass after the biosynthesis reaction. Further, the biomass subsequent to the heavy metal/metalloid precursor was characterized with spectral signatures typical to primary and secondary stress factors such as thiol compounds and oxalic acid using Fourier Transform Infra-Red Spectroscopic (FTIR) analysis. An increase in the total protein content in the reaction mixture after biosynthesis was another noteworthy observation. Further, metal-phytochelatins were identified as the prominent metal-ion trafficking components in the reaction mixture using Liquid Chromatography Mass Spectroscopic analysis (LCMS). Subsequent assays confirmed the involvement of metal binding peptides namely metallothioneins and other anti-oxidant enzymes that might have played a prominent role in the microbial metal detoxification system for the biosynthesis of PbSe QDs. Based on these findings a possible mechanism for the biosynthesis of PbSe QDs by marine A. terreus has been elucidated.

Convergent Evolution of Ergothioneine Biosynthesis in Cyanobacteria.

Science.gov (United States)

Liao, Cangsong; Seebeck, Florian P

2017-11-02

Biosynthesis of N-α-trimethyl-2-thiohistidine (ergothioneine) is a frequent trait in cyanobacteria. This sulfur compound may provide essential relief from oxidative stress related to oxygenic photosynthesis. The central steps in ergothioneine biosynthesis are catalyzed by a histidine methyltransferase and an iron-dependent sulfoxide synthase. In this report, we present evidence that some cyanobacteria recruited and adapted a sulfoxide synthase from a different biosynthetic pathway to make ergothioneine. The discovery of a second origin of ergothioneine production underscores the physiological importance of this metabolite and highlights the evolutionary malleability of the thiohistidine biosynthetic machinery. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hit discovery of Mycobacterium tuberculosis inosine 5'-monophosphate dehydrogenase, GuaB2, inhibitors.

Science.gov (United States)

Sahu, Niteshkumar U; Singh, Vinayak; Ferraris, Davide M; Rizzi, Menico; Kharkar, Prashant S

2018-04-18

Tuberculosis remains a global concern. There is an urgent need of newer antitubercular drugs due to the development of resistant forms of Mycobacterium tuberculosis (Mtb). Inosine 5'-monophosphate dehydrogenase (IMPDH), guaB2, of Mtb, required for guanine nucleotide biosynthesis, is an attractive target for drug development. In this study, we screened a focused library of 73 drug-like molecules with desirable calculated/predicted physicochemical properties, for growth inhibitory activity against drug-sensitive MtbH37Rv. The eight hits and mycophenolic acid, a prototype IMPDH inhibitor, were further evaluated for activity on purified Mtb-GuaB2 enzyme, target selectivity using a conditional knockdown mutant of guaB2 in Mtb, followed by cross-resistance to IMPDH inhibitor-resistant SRMV2.6 strain of Mtb, and activity on human IMPDH2 isoform. One of the hits, 13, a 5-amidophthalide derivative, has shown growth inhibitory potential and target specificity against the Mtb-GuaB2 enzyme. The hit, 13, is a promising molecule with potential for further development as an antitubercular agent. Copyright © 2018 Elsevier Ltd. All rights reserved.

Methionine sulfoximine supplementation enhances productivity in GS-CHOK1SV cell lines through glutathione biosynthesis.

Science.gov (United States)

Feary, Marc; Racher, Andrew J; Young, Robert J; Smales, C Mark

2017-01-01

In Lonza Biologics' GS Gene Expression System™, recombinant protein-producing GS-CHOK1SV cell lines are generated by transfection with an expression vector encoding both GS and the protein product genes followed by selection in MSX and glutamine-free medium. MSX is required to inhibit endogenous CHOK1SV GS, and in effect create a glutamine auxotrophy in the host that can be complemented by the expression vector encoded GS in selected cell lines. However, MSX is not a specific inhibitor of GS as it also inhibits the activity of GCL (a key enzyme in the glutathione biosynthesis pathway) to a similar extent. Glutathione species (GSH and GSSG) have been shown to provide both oxidizing and reducing equivalents to ER-resident oxidoreductases, raising the possibility that selection for transfectants with increased GCL expression could result in the isolation of GS-CHOKISV cell lines with improved capacity for recombinant protein production. In this study we have begun to address the relationship between MSX supplementation, the amount of intracellular GCL subunit and mAb production from a panel of GS-CHOK1SV cell lines. We then evaluated the influence of reduced GCL activity on batch culture of an industrially relevant mAb-producing GS-CHOK1SV cell line. To the best of our knowledge, this paper describes for the first time the change in expression of GCL subunits and recombinant mAb production in these cell lines with the degree of MSX supplementation in routine subculture. Our data also shows that partial inhibition of GCL activity in medium containing 75 µM MSX increases mAb productivity, and its more specific inhibitor BSO used at a concentration of 80 µM in medium increases the specific rate of mAb production eight-fold and the concentration in harvest medium by two-fold. These findings support a link between the inhibition of glutathione biosynthesis and recombinant protein production in industrially relevant systems and provide a process-driven method for

FUNCTIONAL SPECIALIZATION OF DUPLICATED FLAVONOID BIOSYNTHESIS GENES IN WHEAT

Directory of Open Access Journals (Sweden)

Khlestkina E.

2012-08-01

Full Text Available Gene duplication followed by subfunctionalization and neofunctionalization is of a great evolutionary importance. In plant genomes, duplicated genes may result from either polyploidization (homoeologous genes or segmental chromosome duplications (paralogous genes. In allohexaploid wheat Triticum aestivum L. (2n=6x=42, genome BBAADD, both homoeologous and paralogous copies were found for the regulatory gene Myc encoding MYC-like transcriptional factor in the biosynthesis of flavonoid pigments, anthocyanins, and for the structural gene F3h encoding one of the key enzymes of flavonoid biosynthesis, flavanone 3-hydroxylase. From the 5 copies (3 homoeologous and 2 paralogous of the Myc gene found in T. aestivum, only one plays a regulatory role in anthocyanin biosynthesis, interacting complementary with another transcriptional factor (MYB-like to confer purple pigmentation of grain pericarp in wheat. The role and functionality of the other 4 copies of the Myc gene remain unknown. From the 4 functional copies of the F3h gene in T. aestivum, three homoeologues have similar function. They are expressed in wheat organs colored with anthocyanins or in the endosperm, participating there in biosynthesis of uncolored flavonoid substances. The fourth copy (the B-genomic paralogue is transcribed neither in wheat organs colored with anthocyanins nor in seeds, however, it’s expression has been noticed in roots of aluminium-stressed plants, where the three homoeologous copies are not active. Functional diversification of the duplicated flavonoid biosynthesis genes in wheat may be a reason for maintenance of the duplicated copies and preventing them from pseudogenization.The study was supported by RFBR (11-04-92707. We also thank Ms. Galina Generalova for technical assistance.

Biosynthesis and Characterization of Silver Nanoparticles by Aspergillus Species

Science.gov (United States)

Pourshahid, Seyedmohammad; Mehryar, Pouyan; Pakshir, Keyvan; Rahimi, Mohammad Javad; Arabi Monfared, Ali

2016-01-01

Currently, researchers turn to natural processes such as using biological microorganisms in order to develop reliable and ecofriendly methods for the synthesis of metallic nanoparticles. In this study, we have investigated extracellular biosynthesis of silver nanoparticles using four Aspergillus species including A. fumigatus, A. clavatus, A. niger, and A. flavus. We have also analyzed nitrate reductase activity in the studied species in order to determine the probable role of this enzyme in the biosynthesis of silver nanoparticles. The formation of silver nanoparticles in the cell filtrates was confirmed by the passage of laser light, change in the color of cell filtrates, absorption peak at 430 nm in UV-Vis spectra, and atomic force microscopy (AFM). There was a logical relationship between the efficiencies of studied Aspergillus species in the production of silver nanoparticles and their nitrate reductase activity. A. fumigatus as the most efficient species showed the highest nitrate reductase activity among the studied species while A. flavus exhibited the lowest capacity in the biosynthesis of silver nanoparticles which was in accord with its low nitrate reductase activity. The present study showed that Aspergillus species had potential for the biosynthesis of silver nanoparticles depending on their nitrate reductase activity. PMID:27652264

BIOLOGICAL ROLE OF ALDO-KETO REDUCTASES IN RETINOIC ACID BIOSYNTHESIS AND SIGNALING

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F. Xavier eRuiz

2012-04-01

Full Text Available Several aldo-keto reductase (AKR enzymes from subfamilies 1B and 1C show retinaldehyde reductase activity, having low Km and kcat values. Only AKR1B10 and 1B12, with all-trans-retinaldehyde, and AKR1C3, with 9-cis-retinaldehyde, display high catalytic efficiency. Major structural determinants for retinaldehyde isomer specificity are located in the external loops (A and C for AKR1B10, and B for AKR1C3, as assessed by site-directed mutagenesis and molecular dynamics. Cellular models have shown that AKR1B and 1C enzymes are well suited to work in vivo as retinaldehyde reductases and to regulate retinoic acid (RA biosynthesis at hormone pre-receptor level. An additional physiological role for the retinaldehyde reductase activity of these enzymes, consistent with their tissue localization, is their participation in β-carotene absorption. Retinaldehyde metabolism may be subjected to subcellular compartmentalization, based on enzyme localization. While retinaldehyde oxidation to RA takes place in the cytosol, reduction to retinol could take place in the cytosol by AKRs or in the membranes of endoplasmic reticulum by microsomal retinaldehyde reductases. Upregulation of some AKR1 enzymes in different cancer types may be linked to their induction by oxidative stress and to their participation in different signaling pathways related to cell proliferation. AKR1B10 and AKR1C3, through their retinaldehyde reductase activity, trigger a decrease in the RA biosynthesis flow, resulting in RA deprivation and consequently lower differentiation, with an increased cancer risk in target tissues. Rational design of selective AKR inhibitors could lead to development of novel drugs for cancer treatment as well as reduction of chemotherapeutic drug resistance.

Zincophorin – biosynthesis in Streptomyces griseus and antibiotic properties

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Walther, Elisabeth

2016-11-01

Full Text Available Zincophorin is a polyketide antibiotic that possesses potent activity against Gram-positive bacteria, including human pathogens. While a number of total syntheses of this highly functionalized natural product were reported since its initial discovery, the genetic basis for the biosynthesis of zincophorin has remained unclear. In this study, the co-linearity inherent to polyketide pathways was used to identify the zincophorin biosynthesis gene cluster in the genome of the natural producer HKI 0741. Interestingly, the same locus is fully conserved in the streptomycin-producing actinomycete IFO 13350, suggesting that the latter bacterium is also capable of zincophorin biosynthesis. Biological profiling of zincophorin revealed a dose-dependent inhibition of the Gram-positive bacterium . The antibacterial effect, however, is accompanied by cytotoxicity. Antibiotic and cytotoxic activities were completely abolished upon esterification of the carboxylic acid group in zincophorin.

Induction of sesquiterpenoid biosynthesis in tobacco cell suspension cultures by fungal elicitor

International Nuclear Information System (INIS)

Chappell, J.; Nable, R.

1987-01-01

Large amounts of the sesquiterpenoid capsidiol accumulated in the media of tobacco (Nicotiana tabacum L. cv KY14) cell suspension cultures upon addition of fungal elicitor. Capsidiol accumulation was proportional to the amount of elicitor added. The accumulation of capsidiol was preceded by a transient increase in the capsidiol de novo synthesis rate as measured by the incorporation of exogenous [ 14 C]acetate. Changes in 3-hydroxy-3-methylglutaryl-CoA reductase activity, an enzyme of general isoprenoid metabolism, paralleled the changes in [ 14 C]acetate incorporation into capsidiol. Incubation of the cell cultures with mevinolin, a potent in vitro inhibitor of the tobacco HMGR enzyme activity, inhibited the elicitor-induced capsidiol accumulation in a concentration dependent manner. [ 14 C]Acetate incorporation into capsidiol was likewise inhibited by mevinolin treatment. Unexpectedly, [ 3 H] mevalonate incorporation into capsidiol was also partially inhibited by mevinolin, suggesting that mevinolin may effect secondary sites of sesquiterpenoid biosynthesis in vivo beyond HMGR. The data indicated the importance of the induced HMGR activity for capsidiol production in elicitor-treated tobacco cell suspension cultures

Type 1 plaminogen activator inhibitor gene: Functional analysis and glucocorticoid regulation of its promoter

International Nuclear Information System (INIS)

Van Zonneveld, A.J.; Curriden, S.A.; Loskutoff, D.J.

1988-01-01

Plasminogen activator inhibitor type 1 is an important component of the fibrinolytic system and its biosynthesis is subject to complex regulation. To study this regulation at the level of transcription, the authors have identified and sequenced the promoter of the human plasminogen activator inhibitor type 1 gene. Nuclease protection experiments were performed by using endothelial cell mRNA and the transcription initiation (cap) site was established. Sequence analysis of the 5' flanking region of the gene revealed a perfect TATA box at position -28 to position -23, the conserved distance from the cap site. Comparative functional studies with the firefly luciferase gene as a reporter gene showed that fragments derived from this 5' flanking region exhibited high promoter activity when transfected into bovine aortic endothelial cells and mouse Ltk - fibroblasts but were inactive when introduced into HeLa cells. These studies indicate that the fragments contain the plasminogen activator inhibitor type 1 promoter and that it is expressed in a tissue-specific manner. Although the fragments were also silent in rat FTO2B hepatoma cells, their promoter activity could be induced up to 40-fold with the synthetic glucocorticoid dexamethasone. Promoter deletion mapping experiments and studies involving the fusion of promoter fragments to a heterologous gene indicated that dexamethasone induction is mediated by a glucocorticoid responsive element with enhancer-like properties located within the region between nucleotides -305 and +75 of the plasminogen activator inhibitor type 1 gene

Method for determining heterologous biosynthesis pathways

KAUST Repository

Gao, Xin; Kuwahara, Hiroyuki; Alazmi, Meshari Saud; Cui, Xuefeng

2017-01-01

suitable pathways for the endogenous metabolism of a host organism because the efficacy of heterologous biosynthesis is affected by competing endogenous pathways. The present invention is called MRE (Metabolic Route Explorer), and it was conceived

[Characteristics of polyamine biosynthesis regulation and tumor growth rate in hormone-dependant grafted breast tumors of mice and rats].

Science.gov (United States)

Orlovskiĭ, A A

2007-01-01

Effect of the inhibitors of polyamines biosynthesis on completely or partially hormone-dependant breast tumors (mouse Ca755 carcinoma and Walker W-256 carcinosarcoma) is essentially special: in contrary to hormone-dependant tumors, this effect may be not only breaking but stimulating as well. Change-over from one to another mode of reaction is conditioned, most probable, by hormonal status, which is determined by one or another estral cycle phase. Biochemical mechanisms of this change-over are closely connected with polyamines metabolism, namely the degree of polyamines (especially spermine) interconvertion and physiological reactivity level of the system controlling expression of ornithin-decarboxilase. At that, the first of these pathways is predominant for completely hormone-dependant Ca755 and the second one -for partially hormone-dependant W-256.

NAD+ biosynthesis, aging, and disease [version 1; referees: 2 approved

Directory of Open Access Journals (Sweden)

Sean Johnson

2018-02-01

Full Text Available Nicotinamide adenine dinucleotide (NAD+ biosynthesis and its regulation have recently been attracting markedly increasing interest. Aging is marked by a systemic decrease in NAD+ across multiple tissues. The dysfunction of NAD+ biosynthesis plays a critical role in the pathophysiologies of multiple diseases, including age-associated metabolic disorders, neurodegenerative diseases, and mental disorders. As downstream effectors, NAD+-dependent enzymes, such as sirtuins, are involved in the progression of such disorders. These recent studies implicate NAD+ biosynthesis as a potential target for preventing and treating age-associated diseases. Indeed, new studies have demonstrated the therapeutic potential of supplementing NAD+ intermediates, such as nicotinamide mononucleotide and nicotinamide riboside, providing a proof of concept for the development of an effective anti-aging intervention.

In vivo trypanocidal activities of new S-adenosylmethionine decarboxylase inhibitors.

Science.gov (United States)

Bacchi, C J; Brun, R; Croft, S L; Alicea, K; Bühler, Y

1996-01-01

A series of novel aromatic derivatives based on the structure of methylglyoxal bis(guanylhydrazone) (MGBG) was examined for trypanocidal activities in human and veterinary trypanosomes of African origin. One agent, CGP 40215A, a bicyclic analog of MGBG which also resembles the diamidines diminazene (Berenil) and pentamidine, was curative of infections by 19 isolates of Trypanosoma brucei subspecies as well as a Trypanosoma congolense isolate. Several of these isolates were resistant to standard trypanocides. Curative doses were < or = 25 mg/kg of body weight/day for 3 days in these acute laboratory model infections. In addition, CGP 40215A also cured a model central nervous system infection in combination with the ornithine decarboxylase inhibitor DL-alpha-difluoromethylornithine (DFMO; Ornidyl, eflornithine). Curative combinations were 14 days of oral 2% DFMO (approximately 5 g/kg/day) plus 5, 10, or 25 mg/kg/day for 3 or 7 days given by intraperitoneal injection or with a miniosmotic pump. Combinations were most effective if CGP 40215A was given in the second half or at the end of the DFMO regimen. MGBG has modest activity as an inhibitor of trypanosome S-adenosylmethionine decarboxylase (50% inhibitory concentration [IC50]. 130 microM), while CGP 40215A was a more active inhibitor (IC50, 20 microM). Preincubation of trypanosomes with CGP 40215A for 1 h caused a reduction in spermidine content (36%) and an increase in putrescine content (20%), indicating that one possible mechanism of its action may be inhibition of polyamine biosynthesis. PMID:8726018

Acinetobacter baumannii FolD ligand complexes --potent inhibitors of folate metabolism and a re-evaluation of the structure of LY374571.

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Eadsforth, Thomas C; Maluf, Fernando V; Hunter, William N

2012-12-01

The bifunctional N(5),N(10)-methylenetetrahydrofolate dehydrogenase/cyclohydrolase (DHCH or FolD), which is widely distributed in prokaryotes and eukaryotes, is involved in the biosynthesis of folate cofactors that are essential for growth and cellular development. The enzyme activities represent a potential antimicrobial drug target. We have characterized the kinetic properties of FolD from the Gram-negative pathogen Acinetobacter baumanni and determined high-resolution crystal structures of complexes with a cofactor and two potent inhibitors. The data reveal new details with respect to the molecular basis of catalysis and potent inhibition. A unexpected finding was that our crystallographic data revealed a different structure for LY374571 (an inhibitor studied as an antifolate) than that previously published. The implications of this observation are discussed. © 2012 The Authors Journal compilation © 2012 FEBS.

The putative glutamate receptor 1.1 (AtGLR1.1) in Arabidopsis thaliana regulates abscisic acid biosynthesis and signaling to control development and water loss.

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Kang, Jiman; Mehta, Sohum; Turano, Frank J

2004-10-01

The involvement of the putative glutamate receptor 1.1 (AtGLR1.1) gene in the regulation of abscisic acid (ABA) biosynthesis and signaling was investigated in Arabidopsis. Seeds from AtGLR1.1-deficient (antiAtGLR1.1) lines had increased sensitivity to exogenous ABA with regard to the effect of the hormone on the inhibition of seed germination and root growth. Seed germination, which was inhibited by an animal ionotropic glutamate receptor antagonist, 6,7-dinitroquinoxaline-2,3-[1H,4H]-dione, was restored by co-incubation with an inhibitor of ABA biosynthesis, fluridone. These results confirm that germination in antiAtGLR1.1 lines was inhibited by increased ABA. When antiAtGLR1.1 and WT seeds were co-incubated in fluridone and exogenous ABA, the antiAtGLR1.1 seeds were more sensitive to ABA. In addition, the antiAtGLR1.1 lines exhibited altered expression of ABA biosynthetic (ABA) and signaling (ABI) genes, when compared with WT. Combining the physiological and molecular results suggest that ABA biosynthesis and signaling in antiAtGLR1.1 lines are altered. ABA levels in leaves of antiAtGLR1.1 lines are higher than those in WT. In addition, the antiAtGLR1.1 lines had reduced stomatal apertures, and exhibited enhanced drought tolerance due to deceased water loss compared with WT lines. The results from these experiments imply that ABA biosynthesis and signaling can be regulated through AtGLR1.1 to trigger pre- and post-germination arrest and changes in whole plant responses to water stress. Combined with our earlier results, these findings suggest that AtGLR1.1 integrates and regulates the different aspects of C, N and water balance that are required for normal plant growth and development.

Evolution of the biosynthesis of the branched-chain amino acids

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Keefe, Anthony D.; Lazcano, Antonio; Miller, Stanley L.

1995-01-01

The origins of the biosynthetic pathways for the branched-chain amino acids cannot be understood in terms of the backwards development of the present acetolactate pathway because it contains unstable intermediates. We propose that the first biosynthesis of the branched-chain amino acids was by the reductive carboxylation of short branched chain fatty acids giving keto acids which were then transaminated. Similar reaction sequences mediated by nonspecific enzymes would produce serine and threomine from the abundant prebiotic compounds glycolic and lactic acids. The aromatic amino acids may also have first been synthesized in this way, e.g. tryptophan from indole acetic acid. The next step would have been the biosynthesis of leucine from alpha-ketoisovalerc acid. The acetolactate pathway developed subsequently. The first version of the Krebs cycle, which was used for amino acid biosynthesis, would have been assembled by making use fo the reductive carboxylation and leucine biosynthesis enzymes, and completed with the development of a single new enzyme, succinate dehydrogenase. This evolutionary scheme suggests that there may be limitations to inferring the origins of metabolism by a simple back extrapolation of current pathways.

The effects of photosensitizing antibiotics and ultraviolet irradiation on the biosynthesis of prostaglandins

International Nuclear Information System (INIS)

Lord, J.T.; Ziboh, V.A.; Blick, G.; Poitier, J.; Kursunoglu, I.; Penneys, N.S.

1978-01-01

Oxygenation of arachidonic acid in vitro by calf skin microsomal acetone powder was enhanced by UV-irradiation at wavelengths of 254 and 360 nm. Further enhancement of the oxygenation reaction was observed in the presence of two photosensitizing cyclic antibiotics, tetracycline and demethylchlortetracycline. To test whether or not the oxygenation of arachidonic acid was related to the biosynthesis of prostaglandins, [I- 14 C]-arachidonic acid was incubated with calf skin acetone powder in the presence of UV-irradiation and the cyclic antibiotics. Prostaglandin biosynthesis from arachidonic acid by the calf skin microsomal acetone powder was enhanced after exposure to UV-irradiation at 254 nm and moderately at 360 nm. Incubation in the presence of demethylchlortetra-cycline (0.2 mM) increased prostaglandin biosynthesis approximately 95% over control by UV-irradiation at 254 nm. No significant stimulation of prostaglandin biosynthesis was observed at 360 nm. Non-photosensitizing antibiotics had no effect either on the oxygenation of arachidonic acid or on the biosynthesis of prostaglandin with or without UV-irradiation. It is suggested that the inflammatory reactions associated with these photo-reactive antibiotics may in part, be via the biosynthesis and release of the prostaglandins which are known to produce cutaneous inflammatory reactions. (author)

Light quality affects flavonoid biosynthesis in young berries of Cabernet Sauvignon grape.

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Koyama, Kazuya; Ikeda, Hiroko; Poudel, Puspa Raj; Goto-Yamamoto, Nami

2012-06-01

Biosynthesis of phenolic compounds is known to be sensitive to light environments, which reflects the possible role of these compounds for photoprotection in plants. Herein, the effects of UV and visible light on biosynthesis of flavonoids was investigated, i.e., proanthocyanidins (PAs) and flavonols, in young berry skins of a red-wine grape, Vitis vinifera cv. Cabernet Sauvignon. Shading with light-proof boxes from the flowering stage until 49 days after treatment (DAT) partially decreased PA concentrations, and completely decreased flavonol concentrations in the berry skins. Shading decreased the transcript abundance of a flavonol-related gene more remarkably than those of PA-related genes. In addition, light exclusion influenced the composition of PAs, such as the decrease in the proportion of trihydroxylated subunits and the mean degree of polymerization (mDP) within PAs. However, solar UV exclusion did not affect the concentration and composition of PAs, whereas this exclusion remarkably decreased the flavonol concentration. Consistently, UV exclusion did not influence the transcript levels of PA-related genes, whereas it dramatically decreased that of flavonol-related genes. These findings indicated a different light regulation of the biosynthesis of these flavonoids in young berry skins of wine grape. Visible light primarily induces biosynthesis of PAs and affects their composition, whereas UV light specifically induces biosynthesis of flavonols. Distinct roles of members of a MYB transcription factor family for light regulation of flavonoid biosynthesis were proposed. Copyright © 2012 Elsevier Ltd. All rights reserved.

Genetic variation for lettuce seed thermoinhibition is associated with temperature-sensitive expression of abscisic Acid, gibberellin, and ethylene biosynthesis, metabolism, and response genes.

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Argyris, Jason; Dahal, Peetambar; Hayashi, Eiji; Still, David W; Bradford, Kent J

2008-10-01

Lettuce (Lactuca sativa 'Salinas') seeds fail to germinate when imbibed at temperatures above 25 degrees C to 30 degrees C (termed thermoinhibition). However, seeds of an accession of Lactuca serriola (UC96US23) do not exhibit thermoinhibition up to 37 degrees C in the light. Comparative genetics, physiology, and gene expression were analyzed in these genotypes to determine the mechanisms governing the regulation of seed germination by temperature. Germination of the two genotypes was differentially sensitive to abscisic acid (ABA) and gibberellin (GA) at elevated temperatures. Quantitative trait loci associated with these phenotypes colocated with a major quantitative trait locus (Htg6.1) from UC96US23 conferring germination thermotolerance. ABA contents were elevated in Salinas seeds that exhibited thermoinhibition, consistent with the ability of fluridone (an ABA biosynthesis inhibitor) to improve germination at high temperatures. Expression of many genes involved in ABA, GA, and ethylene biosynthesis, metabolism, and response was differentially affected by high temperature and light in the two genotypes. In general, ABA-related genes were more highly expressed when germination was inhibited, and GA- and ethylene-related genes were more highly expressed when germination was permitted. In particular, LsNCED4, a gene encoding an enzyme in the ABA biosynthetic pathway, was up-regulated by high temperature only in Salinas seeds and also colocated with Htg6.1. The temperature sensitivity of expression of LsNCED4 may determine the upper temperature limit for lettuce seed germination and may indirectly influence other regulatory pathways via interconnected effects of increased ABA biosynthesis.

Pseudopterosin Biosynthesis: Aromatization of the Diterpene Cyclase Product, Elisabethatriene

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Amber C. Kohl

2003-11-01

Full Text Available Abstract: Putative precursors in pseudopterosin biosynthesis, the hydrocarbons isoelisabethatriene (10 and erogorgiaene (11, have been identified from an extract of Pseudopterogorgia elisabethae collected in the Florida Keys. Biosynthetic experiments designed to test the utilization of these compounds in pseudopterosin production revealed that erogorgiaene is transformed to pseudopterosins A-D. Together with our previous data, it is now apparent that early steps in pseudopterosin biosynthesis involve the cyclization of geranylgeranyl diphosphate to elisabethatriene followed by the dehydrogenation and aromatization to erogorgiaene.

Structural characterization of the Mycobacterium tuberculosis biotin biosynthesis enzymes 7,8-diaminopelargonic acid synthase and dethiobiotin synthetase .

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Dey, Sanghamitra; Lane, James M; Lee, Richard E; Rubin, Eric J; Sacchettini, James C

2010-08-10

Mycobacterium tuberculosis (Mtb) depends on biotin synthesis for survival during infection. In the absence of biotin, disruption of the biotin biosynthesis pathway results in cell death rather than growth arrest, an unusual phenotype for an Mtb auxotroph. Humans lack the enzymes for biotin production, making the proteins of this essential Mtb pathway promising drug targets. To this end, we have determined the crystal structures of the second and third enzymes of the Mtb biotin biosynthetic pathway, 7,8-diaminopelargonic acid synthase (DAPAS) and dethiobiotin synthetase (DTBS), at respective resolutions of 2.2 and 1.85 A. Superimposition of the DAPAS structures bound either to the SAM analogue sinefungin or to 7-keto-8-aminopelargonic acid (KAPA) allowed us to map the putative binding site for the substrates and to propose a mechanism by which the enzyme accommodates their disparate structures. Comparison of the DTBS structures bound to the substrate 7,8-diaminopelargonic acid (DAPA) or to ADP and the product dethiobiotin (DTB) permitted derivation of an enzyme mechanism. There are significant differences between the Mtb enzymes and those of other organisms; the Bacillus subtilis DAPAS, presented here at a high resolution of 2.2 A, has active site variations and the Escherichia coli and Helicobacter pylori DTBS have alterations in their overall folds. We have begun to exploit the unique characteristics of the Mtb structures to design specific inhibitors against the biotin biosynthesis pathway in Mtb.

Deregulation of S-adenosylmethionine biosynthesis and regeneration improves methylation in the E. coli de novo vanillin biosynthesis pathway.

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Kunjapur, Aditya M; Hyun, Jason C; Prather, Kristala L J

2016-04-11

Vanillin is an industrially valuable molecule that can be produced from simple carbon sources in engineered microorganisms such as Saccharomyces cerevisiae and Escherichia coli. In E. coli, de novo production of vanillin was demonstrated previously as a proof of concept. In this study, a series of data-driven experiments were performed in order to better understand limitations associated with biosynthesis of vanillate, which is the immediate precursor to vanillin. Time-course experiments monitoring production of heterologous metabolites in the E. coli de novo vanillin pathway revealed a bottleneck in conversion of protocatechuate to vanillate. Perturbations in central metabolism intended to increase flux into the heterologous pathway increased average vanillate titers from 132 to 205 mg/L, but protocatechuate remained the dominant heterologous product on a molar basis. SDS-PAGE, in vitro activity measurements, and L-methionine supplementation experiments suggested that the decline in conversion rate was influenced more by limited availability of the co-substrate S-adenosyl-L-methionine (AdoMet or SAM) than by loss of activity of the heterologous O-methyltransferase. The combination of metJ deletion and overexpression of feedback-resistant variants of metA and cysE, which encode enzymes involved in SAM biosynthesis, increased average de novo vanillate titers by an additional 33% (from 205 to 272 mg/L). An orthogonal strategy intended to improve SAM regeneration through overexpression of native mtn and luxS genes resulted in a 25% increase in average de novo vanillate titers (from 205 to 256 mg/L). Vanillate production improved further upon supplementation with methionine (as high as 419 ± 58 mg/L), suggesting potential for additional enhancement by increasing SAM availability. Results from this study demonstrate context dependency of engineered pathways and highlight the limited methylation capacity of E. coli. Unlike in previous efforts to improve SAM or

Biosynthesis of Tropolones in Streptomyces spp: Interweaving Biosynthesis and Degradation of Phenylacetic Acid and Hydroxylations on Tropone Ring.

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Chen, Xuefei; Xu, Min; Lü, Jin; Xu, Jianguo; Wang, Yemin; Lin, Shuangjun; Deng, Zixin; Tao, Meifeng

2018-04-13

Tropolonoids are important natural products that contain a unique seven-membered aromatic tropolone core and exhibit remarkable biological activities. 3,7-Dihydroxytropolone (DHT) isolated from Streptomyces species is a multiply hydroxylated tropolone exhibiting antimicrobial, anticancer, and antiviral activities. Herein, we determined the DHT biosynthetic pathway by heterologous expression, gene deletion, and bioconversion. Nine trl genes and some of the aerobic phenylacetic acid degradation pathway genes ( paa ) located outside of the trl biosynthetic gene cluster are required for the heterologous production of DHT. The trlA gene encodes a single-domain protein homologous to the C-terminal enoyl-CoA hydratase domain of PaaZ. TrlA truncates the phenylacetic acid catabolic pathway and redirects it towards the formation of heptacyclic intermediates. TrlB is a 3-deoxy-D-arabino-heptulosonic acid-7-phosphate (DAHP) synthase homolog. TrlH is an unusual bifunctional protein bearing an N-terminal prephenate dehydratase domain and a C-terminal chorismate mutase domain. TrlB and TrlH enhanced de novo biosynthesis of phenylpyruvate, thereby providing abundant precursor for the prolific production of DHT in Streptomyces Six seven-membered carbocyclic compounds were identified from the gene deletion mutants of trlC , trlD , trlE , and trlF Four of these chemicals, including 1,4,6-cycloheptatriene-1-carboxylic acid, tropone, tropolone and 7-hydroxytropolone, were verified as key biosynthetic intermediates. TrlF is required for the conversion of 1,4,6-cycloheptatriene-1-carboxylic acid into tropone. Monooxygenases TrlE and TrlCD catalyze the regioselective hydroxylations of tropone to afford DHT. This study reveals a natural association of anabolism of chorismate and phenylpyruvate, catabolism of phenylacetic acid, and biosynthesis of tropolones in Streptomyces spp. IMPORTANCE Tropolonoids are promising drug lead compounds because of their versatile bioactivities attributed to

Biosynthesis of antimycins with a reconstituted 3-formamidosalicylate pharmacophore in Escherichia coli.

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Liu, Joyce; Zhu, Xuejun; Seipke, Ryan F; Zhang, Wenjun

2015-05-15

Antimycins are a family of natural products generated from a hybrid nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) assembly line. Although they possess an array of useful biological activities, their structural complexity makes chemical synthesis challenging, and their biosynthesis has thus far been dependent on slow-growing source organisms. Here, we reconstituted the biosynthesis of antimycins in Escherichia coli, a versatile host that is robust and easy to manipulate genetically. Along with Streptomyces genetic studies, the heterologous expression of different combinations of ant genes enabled us to systematically confirm the functions of the modification enzymes, AntHIJKL and AntO, in the biosynthesis of the 3-formamidosalicylate pharmacophore of antimycins. Our E. coli-based antimycin production system can not only be used to engineer the increased production of these bioactive compounds, but it also paves the way for the facile generation of novel and diverse antimycin analogues through combinatorial biosynthesis.

Structural basis for phosphatidylinositol-phosphate biosynthesis

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Clarke, Oliver B.; Tomasek, David; Jorge, Carla D.; Dufrisne, Meagan Belcher; Kim, Minah; Banerjee, Surajit; Rajashankar, Kanagalaghatta R.; Shapiro, Lawrence; Hendrickson, Wayne A.; Santos, Helena; Mancia, Filippo

2015-10-01

Phosphatidylinositol is critical for intracellular signalling and anchoring of carbohydrates and proteins to outer cellular membranes. The defining step in phosphatidylinositol biosynthesis is catalysed by CDP-alcohol phosphotransferases, transmembrane enzymes that use CDP-diacylglycerol as donor substrate for this reaction, and either inositol in eukaryotes or inositol phosphate in prokaryotes as the acceptor alcohol. Here we report the structures of a related enzyme, the phosphatidylinositol-phosphate synthase from Renibacterium salmoninarum, with and without bound CDP-diacylglycerol to 3.6 and 2.5 Å resolution, respectively. These structures reveal the location of the acceptor site, and the molecular determinants of substrate specificity and catalysis. Functional characterization of the 40%-identical ortholog from Mycobacterium tuberculosis, a potential target for the development of novel anti-tuberculosis drugs, supports the proposed mechanism of substrate binding and catalysis. This work therefore provides a structural and functional framework to understand the mechanism of phosphatidylinositol-phosphate biosynthesis.

Skin-specific regulation of SREBP processing and lipid biosynthesis by glycerol kinase 5

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Zhang, Duanwu; Tomisato, Wataru; Su, Lijing; Sun, Lei; Choi, Jin Huk; Zhang, Zhao; Wang, Kuan-wen; Zhan, Xiaoming; Choi, Mihwa; Li, Xiaohong; Tang, Miao; Castro-Perez, Jose M.; Hildebrand, Sara; Murray, Anne R.; Moresco, Eva Marie Y.; Beutler, Bruce

2017-01-01

The recessive N-ethyl-N-nitrosourea–induced phenotype toku is characterized by delayed hair growth, progressive hair loss, and excessive accumulation of dermal cholesterol, triglycerides, and ceramides. The toku phenotype was attributed to a null allele of Gk5, encoding glycerol kinase 5 (GK5), a skin-specific kinase expressed predominantly in sebaceous glands. GK5 formed a complex with the sterol regulatory element-binding proteins (SREBPs) through their C-terminal regulatory domains, inhibiting SREBP processing and activation. In Gk5toku/toku mice, transcriptionally active SREBPs accumulated in the skin, but not in the liver; they were localized to the nucleus and led to elevated lipid synthesis and subsequent hair growth defects. Similar defective hair growth was observed in kinase-inactive GK5 mutant mice. Hair growth defects of homozygous toku mice were partially rescued by treatment with the HMG-CoA reductase inhibitor simvastatin. GK5 exists as part of a skin-specific regulatory mechanism for cholesterol biosynthesis, independent of cholesterol regulation elsewhere in the body. PMID:28607088

Stimultion by angiotensin of prostacyclin biosynthesis in rats and dogs.

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Dusting, G J; Mullins, E M

1980-01-01

1. Stimulation of prostanoid release by angiotensins (AI and AII) in rat isolated mesenteric vasculature and in the circulation of anaesthetized dogs has been investigated by bioassay. 2. AI and AII released a PGI2-like substance into rat mesenteric effluent and arterial blood of dogs; PGE2, PGF2 alpha, or TXA2 were not detected. 3. AI stimulated PGI2 release in both systems largely as a result of its conversion to AII, since PGI2 release was much reduced after treatment with captorpril. 3. AI stimulated PGI2 release in both systems largely as a result of its conversion to AII, since PGI2 release was much reduced after treatment with captopril. 4. Intravenous AII (0.02-1.0 microgram kg-1min-1) in dogs released PGI2 mainly from the lungs since right atrial blood contained much less than arterial blood. 5. Indomethacin (1 microgram/ml) abolished AII-induced PGI2 release from the memestery preparation, but intravenous idomethacin (10 mg/kg), meclofenamate (2 mg/kg) or aspirin (100 mg/kg) did not eliminate the pulmonary source of PGI2 in dogs. These findings highlight the dangers of assuming in vivo treatment with cyclo-oxygenase inhibitors abolished biosynthesis of all prostanoids.

In vitro biosynthesis of unnatural enterocin and wailupemycin polyketides.

Science.gov (United States)

Kalaitzis, John A; Cheng, Qian; Thomas, Paul M; Kelleher, Neil L; Moore, Bradley S

2009-03-27

Nature has evolved finely tuned strategies to synthesize rare and complex natural products such as the enterocin family of polyketides from the marine bacterium Streptomyces maritimus. Herein we report the directed ex vivo multienzyme syntheses of 24 unnatural 5-deoxyenterocin and wailupemycin F and G analogues, 18 of which are new. We have generated molecular diversity by priming the enterocin biosynthesis enzymes with unnatural substrates and have illustrated further the uniqueness of this type II polyketide synthase by way of exploiting its unusual starter unit biosynthesis pathways.

Suppression of phospholipid biosynthesis by cerulenin in the condensed Single-Protein-Production (cSPP) system

International Nuclear Information System (INIS)

Mao, Lili; Inoue, Koichi; Tao, Yisong; Montelione, Gaetano T.; McDermott, Ann E.; Inouye, Masayori

2011-01-01

Using the single-protein-production (SPP) system, a protein of interest can be exclusively produced in high yield from its ACA-less gene in Escherichia coli expressing MazF, an ACA-specific mRNA interferase. It is thus feasible to study a membrane protein by solid-state NMR (SSNMR) directly in natural membrane fractions. In developing isotope-enrichment methods, we observed that 13 C was also incorporated into phospholipids, generating spurious signals in SSNMR spectra. Notable, with the SPP system a protein can be produced in total absence of cell growth caused by antibiotics. Here, we demonstrate that cerulenin, an inhibitor of phospholipid biosynthesis, can suppress isotope incorporation in the lipids without affecting membrane protein yield in the SPP system. SSNMR analysis of ATP synthase subunit c, an E. coli inner membrane protein, produced by the SPP method using cerulenin revealed that 13 C resonance signals from phospholipid were markedly reduced, while signals for the isotope-enriched protein were clearly present.

The Arabidopsis Vacuolar Sorting Receptor1 Is Required for Osmotic Stress-Induced Abscisic Acid Biosynthesis

KAUST Repository

Wang, Zhen-Yu; Gehring, Christoph A; Zhu, Jianhua; Li, Feng-Min; Zhu, Jian-Kang; Xiong, Liming

2014-01-01

Osmotic stress activates the biosynthesis of the phytohormone abscisic acid (ABA) through a pathway that is rate limited by the carotenoid cleavage enzyme 9-cis-epoxycarotenoid dioxygenase (NCED). To understand the signal transduction mechanism underlying the activation of ABA biosynthesis, we performed a forward genetic screen to isolate mutants defective in osmotic stress regulation of the NCED3 gene. Here, we identified the Arabidopsis (Arabidopsis thaliana) Vacuolar Sorting Receptor1 (VSR1) as a unique regulator of ABA biosynthesis. The vsr1 mutant not only shows increased sensitivity to osmotic stress, but also is defective in the feedback regulation of ABA biosynthesis by ABA. Further analysis revealed that vacuolar trafficking mediated by VSR1 is required for osmotic stress-responsive ABA biosynthesis and osmotic stress tolerance. Moreover, under osmotic stress conditions, the membrane potential, calcium flux, and vacuolar pH changes in the vsr1 mutant differ from those in the wild type. Given that manipulation of the intracellular pH is sufficient to modulate the expression of ABA biosynthesis genes, including NCED3, and ABA accumulation, we propose that intracellular pH changes caused by osmotic stress may play a signaling role in regulating ABA biosynthesis and that this regulation is dependent on functional VSR1.

The Arabidopsis Vacuolar Sorting Receptor1 Is Required for Osmotic Stress-Induced Abscisic Acid Biosynthesis

KAUST Repository

Wang, Zhen-Yu

2014-11-21

Osmotic stress activates the biosynthesis of the phytohormone abscisic acid (ABA) through a pathway that is rate limited by the carotenoid cleavage enzyme 9-cis-epoxycarotenoid dioxygenase (NCED). To understand the signal transduction mechanism underlying the activation of ABA biosynthesis, we performed a forward genetic screen to isolate mutants defective in osmotic stress regulation of the NCED3 gene. Here, we identified the Arabidopsis (Arabidopsis thaliana) Vacuolar Sorting Receptor1 (VSR1) as a unique regulator of ABA biosynthesis. The vsr1 mutant not only shows increased sensitivity to osmotic stress, but also is defective in the feedback regulation of ABA biosynthesis by ABA. Further analysis revealed that vacuolar trafficking mediated by VSR1 is required for osmotic stress-responsive ABA biosynthesis and osmotic stress tolerance. Moreover, under osmotic stress conditions, the membrane potential, calcium flux, and vacuolar pH changes in the vsr1 mutant differ from those in the wild type. Given that manipulation of the intracellular pH is sufficient to modulate the expression of ABA biosynthesis genes, including NCED3, and ABA accumulation, we propose that intracellular pH changes caused by osmotic stress may play a signaling role in regulating ABA biosynthesis and that this regulation is dependent on functional VSR1.

The effect of MEP pathway and other inhibitors on the intracellular localization of a plasma membrane-targeted, isoprenylable GFP reporter protein in tobacco BY-2 cells

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Bach, Thomas J

2013-01-01

We have established an in vivo visualization system for the geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, based on the expression of a dexamethasone-inducible GFP fused to the carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL). By using pathway-specific inhibitors it was demonstrated that inhibition of the methylerythritol phosphate (MEP) pathway with known inhibitors like oxoclomazone and fosmidomycin, as well as inhibition of the protein geranylgeranyltransferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect the localization. During the present work, this test system has been used to examine the effect of newly designed inhibitors of the MEP pathway and inhibitors of sterol biosynthesis such as squalestatin, terbinafine and Ro48-8071. In addition, we also studied the impact of different post-prenylation inhibitors or those suspected to affect the transport of proteins to the plasma membrane on the localization of the geranylgeranylable fusion protein GFP-BD-CVIL. PMID:24555083

Two natural products, trans-phytol and (22E)-ergosta-6,9,22-triene-3β,5α,8α-triol, inhibit the biosynthesis of estrogen in human ovarian granulosa cells by aromatase (CYP19)

Energy Technology Data Exchange (ETDEWEB)

Guo, Jiajia [Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu (China); Yuan, Yun [Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu (China); School of Life Science and Engineering, Southwest University of Science and Technology, Mianyang (China); Lu, Danfeng; Du, Baowen [Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu (China); Xiong, Liang; Shi, Jiangong [State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing (China); Yang, Lijuan [Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu (China); Liu, Wanli [MOE Key Laboratory of Protein Science, School of Life Sciences, Tsinghua University, Beijing 100084 (China); Yuan, Xiaohong [School of Life Science and Engineering, Southwest University of Science and Technology, Mianyang (China); Zhang, Guolin, E-mail: zhanggl@cib.ac.cn [Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu (China); Chinese Academy of Sciences Sichuan Translational Medicine Research Hospital, Chengdu (China); Wang, Fei, E-mail: wangfei@cib.ac.cn [Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu (China); Chinese Academy of Sciences Sichuan Translational Medicine Research Hospital, Chengdu (China)

2014-08-15

Aromatase is the only enzyme in vertebrates to catalyze the biosynthesis of estrogens. Although inhibitors of aromatase have been developed for the treatment of estrogen-dependent breast cancer, the whole-body inhibition of aromatase causes severe adverse effects. Thus, tissue-selective aromatase inhibitors are important for the treatment of estrogen-dependent cancers. In this study, 63 natural products with diverse structures were examined for their effects on estrogen biosynthesis in human ovarian granulosa-like KGN cells. Two compounds—trans-phytol (SA-20) and (22E)-ergosta-6,9,22-triene-3β,5α,8α-triol (SA-48)—were found to potently inhibit estrogen biosynthesis (IC{sub 50}: 1 μM and 0.5 μM, respectively). Both compounds decreased aromatase mRNA and protein expression levels in KGN cells, but had no effect on the aromatase catalytic activity in aromatase-overexpressing HEK293A cells and recombinant expressed aromatase. The two compounds decreased the expression of aromatase promoter I.3/II. Neither compound affected intracellular cyclic AMP (cAMP) levels, but they inhibited the phosphorylation or protein expression of cAMP response element-binding protein (CREB). The effects of these two compounds on extracellular regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinases (MAPKs), and AKT/phosphoinositide 3-kinase (PI3K) pathway were examined. Inhibition of p38 MAPK could be the mechanism underpinning the actions of these compounds. Our results suggests that natural products structurally similar to SA-20 and SA-48 may be a new source of tissue-selective aromatase modulators, and that p38 MAPK is important in the basal control of aromatase in ovarian granulosa cells. SA-20 and SA-48 warrant further investigation as new pharmaceutical tools for the prevention and treatment of estrogen-dependent cancers. - Highlights: • Two natural products inhibited estrogen biosynthesis in human ovarian granulosa cells. • They

Sequential enzymatic epoxidation involved in polyether lasalocid biosynthesis.

Science.gov (United States)

Minami, Atsushi; Shimaya, Mayu; Suzuki, Gaku; Migita, Akira; Shinde, Sandip S; Sato, Kyohei; Watanabe, Kenji; Tamura, Tomohiro; Oguri, Hiroki; Oikawa, Hideaki

2012-05-02

Enantioselective epoxidation followed by regioselective epoxide opening reaction are the key processes in construction of the polyether skeleton. Recent genetic analysis of ionophore polyether biosynthetic gene clusters suggested that flavin-containing monooxygenases (FMOs) could be involved in the oxidation steps. In vivo and in vitro analyses of Lsd18, an FMO involved in the biosynthesis of polyether lasalocid, using simple olefin or truncated diene of a putative substrate as substrate mimics demonstrated that enantioselective epoxidation affords natural type mono- or bis-epoxide in a stepwise manner. These findings allow us to figure out enzymatic polyether construction in lasalocid biosynthesis. © 2012 American Chemical Society

Biosynthesis of human colonic mucin: Muc2 is the prominent secretory mucin

NARCIS (Netherlands)

Tytgat, K. M.; Büller, H. A.; Opdam, F. J.; Kim, Y. S.; Einerhand, A. W.; Dekker, J.

1994-01-01

Human colonic epithelium produces large amounts of mucin. The aim of this study was to examine mucin biosynthesis in the human colon. Human colonic mucin was isolated using CsCl density gradients, and polyclonal antiserum was raised. Biosynthesis of colonic mucins was studied by labeling colonic

A phase I study of a new polyamine biosynthesis inhibitor, SAM486A, in cancer patients with solid tumours

NARCIS (Netherlands)

Paridaens, R; Uges, DRA; Barbet, N; Choi, L; Seeghers, M; van der Graaf, WTA; Groen, HJM; Dumez, H; Van Buuren, [No Value; Muskiet, F; Capdeville, R; van Oosterom, AT; de Vries, EGE

Because tumour cell proliferation is highly dependent upon up-regulation of de-novo polyamine synthesis, inhibition of the polyamine synthesis pathway represents a potential target for anticancer therapy. SAM486A (CGP 48664) is a new inhibitor of the polyamine biosynthetic enzyme

Synthesis and Evaluation of Bicyclo[3.1.0]hexane-Based UDP-Galf Analogues as Inhibitors of the Mycobacterial Galactofuranosyltransferase GlfT2

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Todd L. Lowary

2016-08-01

Full Text Available UDP-galactofuranose (UDP-Galf is the donor substrate for both bifunctional galactofuranosyltransferases, GlfT1 and GlfT2, which are involved in the biosynthesis of mycobacterial galactan. In this paper, a group of UDP-Galf mimics were synthesized via reductive amination of a bicyclo[3.1.0]hexane-based amine by reacting with aromatic, linear, or uridine-containing aldehydes. These compounds were evaluated against GlfT2 using a coupled spectrophotometric assay, and were shown to be weak inhibitors of the enzyme.

Evolutionary Limitation and Opportunities for Developing tRNA Synthetase Inhibitors with 5-Binding-Mode Classification

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Pengfei Fang

2015-12-01

Full Text Available Aminoacyl-tRNA synthetases (aaRSs are enzymes that catalyze the transfer of amino acids to their cognate tRNAs as building blocks for translation. Each of the aaRS families plays a pivotal role in protein biosynthesis and is indispensable for cell growth and survival. In addition, aaRSs in higher species have evolved important non-translational functions. These translational and non-translational functions of aaRS are attractive for developing antibacterial, antifungal, and antiparasitic agents and for treating other human diseases. The interplay between amino acids, tRNA, ATP, EF-Tu and non-canonical binding partners, had shaped each family with distinct pattern of key sites for regulation, with characters varying among species across the path of evolution. These sporadic variations in the aaRSs offer great opportunity to target these essential enzymes for therapy. Up to this day, growing numbers of aaRS inhibitors have been discovered and developed. Here, we summarize the latest developments and structural studies of aaRS inhibitors, and classify them with distinct binding modes into five categories.

Assessment of Metabolic Changes in Mycobacterium smegmatis Wild-Type and alr Mutant Strains: Evidence of a New Pathway of d-Alanine Biosynthesis.

Science.gov (United States)

Marshall, Darrell D; Halouska, Steven; Zinniel, Denise K; Fenton, Robert J; Kenealy, Katie; Chahal, Harpreet K; Rathnaiah, Govardhan; Barletta, Raúl G; Powers, Robert

2017-03-03

In mycobacteria, d-alanine is an essential precursor for peptidoglycan biosynthesis. The only confirmed enzymatic pathway to form d-alanine is through the racemization of l-alanine by alanine racemase (Alr, EC 5.1.1.1). Nevertheless, the essentiality of Alr in Mycobacterium tuberculosis and Mycobacterium smegmatis for cell survivability in the absence of d-alanine has been a point of controversy with contradictory results reported in the literature. To address this issue, we examined the effects of alr inactivation on the cellular metabolism of M. smegmatis. The M. smegmatis alr insertion mutant TAM23 exhibited essentially identical growth to wild-type mc 2 155 in the absence of d-alanine. NMR metabolomics revealed drastically distinct phenotypes between mc 2 155 and TAM23. A metabolic switch was observed for TAM23 as a function of supplemented d-alanine. In the absence of d-alanine, the metabolic response directed carbon through an unidentified transaminase to provide the essential d-alanine required for survival. The process is reversed when d-alanine is available, in which the d-alanine is directed to peptidoglycan biosynthesis. Our results provide further support for the hypothesis that Alr is not an essential function of M. smegmatis and that specific Alr inhibitors will have no bactericidal action.

Occurrence and biosynthesis of carotenoids in phytoplankton.

Science.gov (United States)

Huang, Jim Junhui; Lin, Shaoling; Xu, Wenwen; Cheung, Peter Chi Keung

2017-09-01

Naturally occurring carotenoids are important sources of antioxidants, anti-cancer compounds and anti-inflammatory agents and there is thus considerable market demand for their pharmaceutical applications. Carotenoids are widely distributed in marine and freshwater organisms including microalgae, phytoplankton, crustaceans and fish, as well as in terrestrial plants and birds. Recently, phytoplankton-derived carotenoids have received much attention due to their abundance, rapid rate of biosynthesis and unique composition. The carotenoids that accumulate in particular phytoplankton phyla are synthesized by specific enzymes and play unique physiological roles. This review focuses on studies related to the occurrence of carotenoids in different phytoplankton phyla and the molecular aspects of their biosynthesis. Recent biotechnological advances in the isolation and characterization of some representative carotenoid synthases in phytoplankton are also discussed. Copyright © 2017 Elsevier Inc. All rights reserved.

Intracellular Biosynthesis and Antibacterial Activity of Silver Nanoparticles Using Edible Mushrooms

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Sankaran MIRUNALINI

2012-11-01

Full Text Available The process of biosynthesis of silver nanoparticles is a simple, cost effective and eco-friendly approach. Biosynthesis of silver nanoparticles using some commonly available edible mushroom extracts and their antimicrobial activity was demonstrated in the current study. The formation of silver nanoparticles was confirmed by UV, FTIR and SEM and antibacterial activity was tested using disc diffusion method. From the results it is confirmed the successful formation of silver nanoparticles using mushroom extracts; they performed their role as a reducing and capping agent and also exhibited a potent antibacterial activity against S. aureus (gram positive bacteria. Thus the biosynthesis of silver nanoparticles using edible mushroom extract will deserve to be a good candidate as an antibacterial agent.

Comparative proteomic analysis reveals proteins putatively involved in toxin biosynthesis in the marine dinoflagellate Alexandrium catenella.

Science.gov (United States)

Wang, Da-Zhi; Gao, Yue; Lin, Lin; Hong, Hua-Sheng

2013-01-22

Alexandrium is a neurotoxin-producing dinoflagellate genus resulting in paralytic shellfish poisonings around the world. However, little is known about the toxin biosynthesis mechanism in Alexandrium. This study compared protein profiles of A. catenella collected at different toxin biosynthesis stages (non-toxin synthesis, initial toxin synthesis and toxin synthesizing) coupled with the cell cycle, and identified differentially expressed proteins using 2-DE and MALDI-TOF-TOF mass spectrometry. The results showed that toxin biosynthesis of A. catenella occurred within a defined time frame in the G1 phase of the cell cycle. Proteomic analysis indicated that 102 protein spots altered significantly in abundance (P translation. Among them, nine proteins with known functions in paralytic shellfish toxin-producing cyanobacteria, i.e., methionine S-adenosyltransferase, chloroplast ferredoxin-NADP+ reductase, S-adenosylhomocysteinase, adenosylhomocysteinase, ornithine carbamoyltransferase, inorganic pyrophosphatase, sulfotransferase (similar to), alcohol dehydrogenase and arginine deiminase, varied significantly at different toxin biosynthesis stages and formed an interaction network, indicating that they might be involved in toxin biosynthesis in A. catenella. This study is the first step in the dissection of the behavior of the A. catenella proteome during different toxin biosynthesis stages and provides new insights into toxin biosynthesis in dinoflagellates.

Genetic control and regulatory mechanisms of succinoglycan and curdlan biosynthesis in genus Agrobacterium.

Science.gov (United States)

Wu, Dan; Li, Ang; Ma, Fang; Yang, Jixian; Xie, Yutong

2016-07-01

Agrobacterium is a genus of gram-negative bacteria that can produce several typical exopolysaccharides with commercial uses in the food and pharmaceutical fields. In particular, succinoglycan and curdlan, due to their good quality in high yield, have been employed on an industrial scale comparatively early. Exopolysaccharide biosynthesis is a multiple-step process controlled by different functional genes, and various environmental factors cause changes in exopolysaccharide biosynthesis through regulatory mechanisms. In this mini-review, we focus on the genetic control and regulatory mechanisms of succinoglycan and curdlan produced by Agrobacterium. Some key functional genes and regulatory mechanisms for exopolysaccharide biosynthesis are described, possessing a high potential for application in metabolic engineering to modify exopolysaccharide production and physicochemical properties. This review may contribute to the understanding of exopolysaccharide biosynthesis and exopolysaccharide modification by metabolic engineering methods in Agrobacterium.

Bioregulation of aflatoxin biosynthesis by unirradiated and irradiated conidia of Aspergillus flavus

International Nuclear Information System (INIS)

Aziz, N.H.; Abu-Shady, M.R.; El-Fouly, M.Z.; Moussa, L.A.

1996-01-01

A sequential technique involving the transfer of mycelia from peptone-based, aflatoxin-non-supporting medium to glucose based, aflatoxin-supporting medium was used to study the effect of γ-irradiation on the regulation of aflatoxin biosynthesis by Aspergillus flavus. Analysis indicated that irradiation at a dose of 1.00 kGy produced enhancement of aflatoxin biosynthesis in peptone-glucose mineral salt cultures with an increase of adenine nucleotide levels and fatty acid patterns of microsomes and mitochondria. The results suggest that aflatoxin synthesis is not regulated by the overall energy status of the fungal cell but that lipoperoxidation by γ-irradiation plays a role in aflatoxin biosynthesis

Cloning and Characterization of the Polyether Salinomycin Biosynthesis Gene Cluster of Streptomyces albus XM211

Science.gov (United States)

Jiang, Chunyan; Wang, Hougen; Kang, Qianjin; Liu, Jing

2012-01-01

Salinomycin is widely used in animal husbandry as a food additive due to its antibacterial and anticoccidial activities. However, its biosynthesis had only been studied by feeding experiments with isotope-labeled precursors. A strategy with degenerate primers based on the polyether-specific epoxidase sequences was successfully developed to clone the salinomycin gene cluster. Using this strategy, a putative epoxidase gene, slnC, was cloned from the salinomycin producer Streptomyces albus XM211. The targeted replacement of slnC and subsequent trans-complementation proved its involvement in salinomycin biosynthesis. A 127-kb DNA region containing slnC was sequenced, including genes for polyketide assembly and release, oxidative cyclization, modification, export, and regulation. In order to gain insight into the salinomycin biosynthesis mechanism, 13 gene replacements and deletions were conducted. Including slnC, 7 genes were identified as essential for salinomycin biosynthesis and putatively responsible for polyketide chain release, oxidative cyclization, modification, and regulation. Moreover, 6 genes were found to be relevant to salinomycin biosynthesis and possibly involved in precursor supply, removal of aberrant extender units, and regulation. Sequence analysis and a series of gene replacements suggest a proposed pathway for the biosynthesis of salinomycin. The information presented here expands the understanding of polyether biosynthesis mechanisms and paves the way for targeted engineering of salinomycin activity and productivity. PMID:22156425

Fungal biosynthesis of gold nanoparticles: mechanism and scale up.

Science.gov (United States)

Kitching, Michael; Ramani, Meghana; Marsili, Enrico

2015-11-01

Gold nanoparticles (AuNPs) are a widespread research tool because of their oxidation resistance, biocompatibility and stability. Chemical methods for AuNP synthesis often produce toxic residues that raise environmental concern. On the other hand, the biological synthesis of AuNPs in viable microorganisms and their cell-free extracts is an environmentally friendly and low-cost process. In general, fungi tolerate higher metal concentrations than bacteria and secrete abundant extracellular redox proteins to reduce soluble metal ions to their insoluble form and eventually to nanocrystals. Fungi harbour untapped biological diversity and may provide novel metal reductases for metal detoxification and bioreduction. A thorough understanding of the biosynthetic mechanism of AuNPs in fungi is needed to reduce the time of biosynthesis and to scale up the AuNP production process. In this review, we describe the known mechanisms for AuNP biosynthesis in viable fungi and fungal protein extracts and discuss the most suitable bioreactors for industrial AuNP biosynthesis. © 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Microbial Biosynthesis of Silver Nanoparticles in Different Culture Media.

Science.gov (United States)

Luo, Ke; Jung, Samuel; Park, Kyu-Hwan; Kim, Young-Rok

2018-01-31

Microbial biosynthesis of metal nanoparticles has been extensively studied for the applications in biomedical sciences and engineering. However, the mechanism for their synthesis through microorganism is not completely understood. In this study, several culture media were investigated for their roles in the microbial biosynthesis of silver nanoparticles (AgNPs). The size and morphology of the synthesized AgNPs were analyzed by UV-vis spectroscopy, Fourier-transform-infrared (FT-IR), transmission electron microscopy (TEM), and dynamic light scattering (DLS). The results demonstrated that nutrient broth (NB) and Mueller-Hinton broth (MHB) among tested media effectively reduced silver ions to form AgNPs with different particle size and shape. Although the involved microorganism enhanced the reduction of silver ions, the size and shape of the particles were shown to mainly depend on the culture media. Our findings suggest that the growth media of bacterial culture play an important role in the synthesis of metallic nanoparticles with regard to their size and shape. We believe our findings would provide useful information for further exploration of microbial biosynthesis of AgNPs and their biomedical applications.

Genes involved in long-chain alkene biosynthesis in Micrococcus luteus

Energy Technology Data Exchange (ETDEWEB)

Beller, Harry R.; Goh, Ee-Been; Keasling, Jay D.

2010-01-07

Aliphatic hydrocarbons are highly appealing targets for advanced cellulosic biofuels, as they are already predominant components of petroleum-based gasoline and diesel fuels. We have studied alkene biosynthesis in Micrococcus luteus ATCC 4698, a close relative of Sarcina lutea (now Kocuria rhizophila), which four decades ago was reported to biosynthesize iso- and anteiso branched, long-chain alkenes. The underlying biochemistry and genetics of alkene biosynthesis were not elucidated in those studies. We show here that heterologous expression of a three-gene cluster from M. luteus (Mlut_13230-13250) in a fatty-acid overproducing E. coli strain resulted in production of long-chain alkenes, predominantly 27:3 and 29:3 (no. carbon atoms: no. C=C bonds). Heterologous expression of Mlut_13230 (oleA) alone produced no long-chain alkenes but unsaturated aliphatic monoketones, predominantly 27:2, and in vitro studies with the purified Mlut_13230 protein and tetradecanoyl-CoA produced the same C27 monoketone. Gas chromatography-time of flight mass spectrometry confirmed the elemental composition of all detected long-chain alkenes and monoketones (putative intermediates of alkene biosynthesis). Negative controls demonstrated that the M. luteus genes were responsible for production of these metabolites. Studies with wild-type M. luteus showed that the transcript copy number of Mlut_13230-13250 and the concentrations of 29:1 alkene isomers (the dominant alkenes produced by this strain) generally corresponded with bacterial population over time. We propose a metabolic pathway for alkene biosynthesis starting with acyl-CoA (or -ACP) thioesters and involving decarboxylative Claisen condensation as a key step, which we believe is catalyzed by OleA. Such activity is consistent with our data and with the homology (including the conserved Cys-His-Asn catalytic triad) of Mlut_13230 (OleA) to FabH (?-ketoacyl-ACP synthase III), which catalyzes decarboxylative Claisen condensation during

Fatty Acid Biosynthesis Inhibition Increases Reduction Potential in Neuronal Cells under Hypoxia

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Stephen A Brose

2016-11-01

Full Text Available Recently, we have reported a novel neuronal specific pathway for adaptation to hypoxia through increased fatty acid (FA biosynthesis (FAS followed by esterification into lipids. However, the biological role of this pathway under hypoxia remains to be elucidated. In the presented study, we have tested our hypothesis that activation of FAS maintains reduction potential and reduces lactoacidosis in neuronal cells under hypoxia. To address this hypothesis, we measured the effect of FAS inhibition on NADH2+/NAD+ and NADPH2+/NADP+ ratios, and lactic acid levels in neuronal SH-SY5Y cells exposed to normoxic and hypoxic conditions. FAS inhibitors, TOFA (inhibits Acetyl-CoA carboxylase and cerulenin (inhibits FA synthase, increased NADH2+/NAD+ and NADPH2+/NADP+ ratios under hypoxia. Further, FAS inhibition increased lactic acid under both normoxic and hypoxic conditions, and caused cytotoxicity under hypoxia but not normoxia. These results indicate that FA may serve as hydrogen acceptors under hypoxia, thus supporting oxidation reactions including anaerobic glycolysis. These findings may help to identify a radically different approach to attenuate hypoxia related pathophysiology in the nervous system including stroke.

Fatty Acid Biosynthesis Inhibition Increases Reduction Potential in Neuronal Cells under Hypoxia.

Science.gov (United States)

Brose, Stephen A; Golovko, Svetlana A; Golovko, Mikhail Y

2016-01-01

Recently, we have reported a novel neuronal specific pathway for adaptation to hypoxia through increased fatty acid (FA) biosynthesis followed by esterification into lipids. However, the biological role of this pathway under hypoxia remains to be elucidated. In the presented study, we have tested our hypothesis that activation of FA synthesis maintains reduction potential and reduces lactoacidosis in neuronal cells under hypoxia. To address this hypothesis, we measured the effect of FA synthesis inhibition on [Formula: see text]/NAD + and [Formula: see text]/NADP + ratios, and lactic acid levels in neuronal SH-SY5Y cells exposed to normoxic and hypoxic conditions. FA synthesis inhibitors, TOFA (inhibits Acetyl-CoA carboxylase) and cerulenin (inhibits FA synthase), increased [Formula: see text]/NAD + and [Formula: see text]/NADP + ratios under hypoxia. Further, FA synthesis inhibition increased lactic acid under both normoxic and hypoxic conditions, and caused cytotoxicity under hypoxia but not normoxia. These results indicate that FA may serve as hydrogen acceptors under hypoxia, thus supporting oxidation reactions including anaerobic glycolysis. These findings may help to identify a radically different approach to attenuate hypoxia related pathophysiology in the nervous system including stroke.

Biosynthesis of membrane lipids of thermophilic archaebacteria and its implication to early evolution of life

International Nuclear Information System (INIS)

Oshima, Tairo

1995-01-01

The unit lipid of cell membranes of archaebacteria is unique ether lipids, O-dialkylated glycerol with a polar head group at sn-1 position. The chirality of glycerol moiety of the lipids is opposite to that of other kingdoms. The hydrophobic potion consists of saturated C 20 isoprenoid hydrocarbon backbone and is connected to glycerol by an ether linkage. In addition, cell membrane of some of thermophilic archaebacteria are monolayer (in stead of bilayer) of tetraether lipids in which both tails of hydrocarbon chains of two diether lipids are covalently connected in a tail-to-tail fashion. Although the host cell from which contemporary eukaryotes have been derived by endosymbiosis, is speculated to be an archaebacterium, the unique ether lipids raised a serious question to the idea of archabacterial origin of eukaryote cells; why the unique ether lipids are not used to construct cytoplasmic membranes of eukaryotes? The author and his colleagues have studied biosynthesis of membrane liquids of two thermo-acidophilic archaebacteria, Thermoplasma and Sulfolobus. It was found that origins of stereospecificity of glycerol moiety of archaebacterial ether lipids differs form species to species. In Sulfolobus sn-glycerol-1-phosphate (the abnormal isomer of glycerol phosphate) seems to be directly synthesized from glycerol, whereas in Halobacterium stereospecificity of glycerol phosphate is inverted during the lipid synthesis. Recently we found that specific inhibitors for eukaryotes squalene epoxidase inhibit the condensation of diether lipids to tetraether lipids in cell-free extracts of these thermophilic archaebacteria. The results suggest evolutionary implication of archaebacterial tetraether condensing enzyme to eukaryote sterol biosynthesis. Relationships between chemical structures of membrane lipids and early evolution of life will be discussed. (author). Abstract only

Regulation of neurosteroid biosynthesis by neurotransmitters and neuropeptides

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Jean-Luc eDo-Rego

2012-01-01

Full Text Available The enzymatic pathways leading to the synthesis of bioactive steroids in the brain are now almost completely elucidated in various groups of vertebrates and, during the last decade, the neuronal mechanisms involved in the regulation of neurosteroid production have received increasing attention. This report reviews the current knowledge concerning the effects of neurotransmitters, peptide hormones and neuropeptides on the biosynthesis of neurosteroids. Anatomical studies have been carried out to visualize the neurotransmitter- or neuropeptide-containing fibers contacting steroid-synthesizing neurons as well as the neurotransmitter, peptide hormones or neuropeptide receptors expressed in these neurons. Biochemical experiments have been conducted to investigate the effects of neurotransmitters, peptide hormones or neuropeptides on neurosteroid biosynthesis, and to characterize the type of receptors involved. Thus, it has been found that glutamate, acting through kainate and/or AMPA receptors, rapidly inactivates P450arom, and that melatonin produced by the pineal gland and eye inhibits the biosynthesis of 7-hydroxypregnenolone (7-OH-5P, while prolactin produced by the adenohypophysis enhances the formation of 7-OH-5P. It has also been demonstrated that the biosynthesis of neurosteroids is inhibited by GABA, acting through GABAA receptors, and neuropeptide Y, acting through Y1 receptors. In contrast, it has been shown that the octadecaneuropetide ODN, acting through central-type benzodiazepine receptors, the triakontatetraneuropeptide TTN, acting though peripheral-type benzodiazepine receptors, and vasotocine, acting through V1a-like receptors, stimulate the production of neurosteroids. Since neurosteroids are implicated in the control of various neurophysiological and behavioral processes, these data suggest that some of the neurophysiological effects exerted by neurotransmitters and neuropeptides may be mediated via the regulation

Biosynthesis of furanochromones in Pimpinella monoica

Indian Academy of Sciences (India)

polyketide origin of their aromatic and pyrone rings while the furan ring originates via an acetate-mevalonate pathway. The plant also utilises glycine and leucine as substrate via acetate. Biotransformation of 3-H-visnagin to (6) but not to (2) was also observed. Keywords. Biosynthesis; furochromones; polyketide origin; ...

Oleic acid biosynthesis in cyanobacteria

International Nuclear Information System (INIS)

VanDusen, W.J.; Jaworski, J.G.

1986-01-01

The biosynthesis of fatty acids in cyanobacteria is very similar to the well characterized system found in green plants. However, the initial desaturation of stearic acid in cyanobacteria appears to represent a significant departure from plant systems in which stearoyl-ACP is the exclusive substrate for desaturation. In Anabaena variabilis, the substrate appears to be monoglucosyldiacylglycerol, a lipid not found in plants. The authors examined five different cyanobacteria to determine if the pathway in A. variabilis was generally present in other cyanobacteria. The cyanobacteria studied were A. variabilis, Chlorogloeopsis sp., Schizothrix calcicola, Anacystis marina, and Anacystis nidulans. Each were grown in liquid culture, harvested, and examined for stearoyl-ACP desaturase activity or incubated with 14 CO 2 . None of the cyanobacteria contained any stearoyl-ACP desaturase activity in whole homogenates or 105,000g supernatants. All were capable of incorporating 14 CO 2 into monoglucosyldiacylglycerol and results from incubations of 20 min, 1 hr, 1 hr + 10 hr chase were consistent with monoglucosyldiacylglycerol serving as precursor for monogalctosyldiacylglycerol. Thus, initial evidence is consistent with oleic acid biosynthesis occurring by desaturation of stearoyl-monoglucosyldiacylglycerol in all cyanobacteria

Arogenate Dehydratase Isoforms Differentially Regulate Anthocyanin Biosynthesis in Arabidopsis thaliana.

Science.gov (United States)

Chen, Qingbo; Man, Cong; Li, Danning; Tan, Huijuan; Xie, Ye; Huang, Jirong

2016-12-05

Anthocyanins, a group of L-phenylalanine (Phe)-derived flavonoids, have been demonstrated to play important roles in plant stress resistance and interactions between plants and insects. Although the anthocyanin biosynthetic pathway and its regulatory mechanisms have been extensively studied, it remains unclear whether the level of Phe supply affects anthocyanin biosynthesis. Here, we investigated the roles of arogenate dehydratases (ADTs), the key enzymes that catalyze the conversion of arogenate into Phe, in sucrose-induced anthocyanin biosynthesis in Arabidopsis. Genetic analysis showed that all six ADT isoforms function redundantly in anthocyanin biosynthesis but have differential contributions. ADT2 contributes the most to anthocyanin accumulation, followed by ADT1 and ADT3, and ADT4-ADT6. We found that anthocyanin content is positively correlated with the levels of Phe and sucrose-induced ADT transcripts in seedlings. Consistently, addition of Phe to the medium could dramatically increase anthocyanin content in the wild-type plants and rescue the phenotype of the adt1 adt3 double mutant regarding the anthocyanin accumulation. Moreover, transgenic plants overexpressing ADT4, which appears to be less sensitive to Phe than overexpression of ADT2, hyperaccumulate Phe and produce elevated level of anthocyanins. Taken together, our results suggest that the level of Phe is an important regulatory factor for sustaining anthocyanin biosynthesis. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

Discovery of new inhibitors of the bacterial peptidoglycan biosynthesis enzymes MurD and MurF by structure-based virtual screening.

Science.gov (United States)

Turk, Samo; Kovac, Andreja; Boniface, Audrey; Bostock, Julieanne M; Chopra, Ian; Blanot, Didier; Gobec, Stanislav

2009-03-01

The ATP-dependent Mur ligases (MurC, MurD, MurE and MurF) successively add L-Ala, D-Glu, meso-A(2)pm or L-Lys, and D-Ala-D-Ala to the nucleotide precursor UDP-MurNAc, and they represent promising targets for antibacterial drug discovery. We have used the molecular docking programme eHiTS for the virtual screening of 1990 compounds from the National Cancer Institute 'Diversity Set' on MurD and MurF. The 50 top-scoring compounds from screening on each enzyme were selected for experimental biochemical evaluation. Our approach of virtual screening and subsequent in vitro biochemical evaluation of the best ranked compounds has provided four novel MurD inhibitors (best IC(50)=10 microM) and one novel MurF inhibitor (IC(50)=63 microM).

Phosphatidylcholine (PC) biosynthesis in pancreatic islets of Langerhans

International Nuclear Information System (INIS)

Hoffman, J.M.; Laychock, S.G.

1986-01-01

Islets of Langerhans isolated from rat pancreata were incubated with [ 14 C]choline to determine the biosynthesis of PC by the CDP choline to determine the biosynthesis of PC by the CDPcholine pathway. Recovery of [ 14 C]PC in islet membranes was time-related, and stimulated by glucose (17mM) during 60 min. The rate of PC synthesis was constant during 60 min with glucose stimulation. In contrast, the sulfonylurea tolbutamide (2 mM) reduced the recovery of [ 14 C]choline in PC, and 8-bromo-cyclic AMP (5 mM) did not significantly affect [ 14 C]PC recovery. Incubation of islets in Ca 2+ -free medium enhanced glucose-stimulated recovery of [ 14 C]choline-labeled PC due to the inhibition of phospholipase and phospholipid hydrolysis. Inhibition of CTP:phosphocholine cytidylyltransferase with 5-deoxy-5'-isobutylthioadenosine (SIBA) reduced [ 14 C]PC levels and insulin release in a concentration dependent manner. Treatment with SIBA also reduced Mg 2+ -dependent Ca 2+ -ATPase activity in islet microsomes. Quantitation of membrane PC showed that glucose stimulation did not alter islet P levels. Thus, islet PC biosynthesis is linked to glucose stimulation and contributes to the maintenance of PC levels in membranes undergoing exocytosis and phospholipid hydrolysis. Adequate PC levels support Ca 2+ pump activity and secretory mechanisms

Comparative Proteomic Analysis Reveals Proteins Putatively Involved in Toxin Biosynthesis in the Marine Dinoflagellate Alexandrium catenella

Directory of Open Access Journals (Sweden)

Da-Zhi Wang

2013-01-01

Full Text Available Alexandrium is a neurotoxin-producing dinoflagellate genus resulting in paralytic shellfish poisonings around the world. However, little is known about the toxin biosynthesis mechanism in Alexandrium. This study compared protein profiles of A. catenella collected at different toxin biosynthesis stages (non-toxin synthesis, initial toxin synthesis and toxin synthesizing coupled with the cell cycle, and identified differentially expressed proteins using 2-DE and MALDI-TOF-TOF mass spectrometry. The results showed that toxin biosynthesis of A. catenella occurred within a defined time frame in the G1 phase of the cell cycle. Proteomic analysis indicated that 102 protein spots altered significantly in abundance (P < 0.05, and 53 proteins were identified using database searching. These proteins were involved in a variety of biological processes, i.e., protein modification and biosynthesis, metabolism, cell division, oxidative stress, transport, signal transduction, and translation. Among them, nine proteins with known functions in paralytic shellfish toxin-producing cyanobacteria, i.e., methionine S-adenosyltransferase, chloroplast ferredoxin-NADP+ reductase, S-adenosylhomocysteinase, adenosylhomocysteinase, ornithine carbamoyltransferase, inorganic pyrophosphatase, sulfotransferase (similar to, alcohol dehydrogenase and arginine deiminase, varied significantly at different toxin biosynthesis stages and formed an interaction network, indicating that they might be involved in toxin biosynthesis in A. catenella. This study is the first step in the dissection of the behavior of the A. catenella proteome during different toxin biosynthesis stages and provides new insights into toxin biosynthesis in dinoflagellates.

Cloning and bioinformatic analysis of lovastatin biosynthesis regulatory gene lovE.

Science.gov (United States)

Huang, Xin; Li, Hao-ming

2009-08-05

Lovastatin is an effective drug for treatment of hyperlipidemia. This study aimed to clone lovastatin biosynthesis regulatory gene lovE and analyze the structure and function of its encoding protein. According to the lovastatin synthase gene sequence from genebank, primers were designed to amplify and clone the lovastatin biosynthesis regulatory gene lovE from Aspergillus terrus genomic DNA. Bioinformatic analysis of lovE and its encoding animo acid sequence was performed through internet resources and software like DNAMAN. Target fragment lovE, almost 1500 bp in length, was amplified from Aspergillus terrus genomic DNA and the secondary and three-dimensional structures of LovE protein were predicted. In the lovastatin biosynthesis process lovE is a regulatory gene and LovE protein is a GAL4-like transcriptional factor.

Recent advances in the elucidation of enzymatic function in natural product biosynthesis [version 2; referees: 2 approved

Directory of Open Access Journals (Sweden)

Gao-Yi Tan

2016-02-01

Full Text Available With the successful production of artemisinic acid in yeast, the promising potential of synthetic biology for natural product biosynthesis is now being realized. The recent total biosynthesis of opioids in microbes is considered to be another landmark in this field. The importance and significance of enzymes in natural product biosynthetic pathways have been re-emphasized by these advancements. Therefore, the characterization and elucidation of enzymatic function in natural product biosynthesis are undoubtedly fundamental for the development of new drugs and the heterologous biosynthesis of active natural products. Here, discoveries regarding enzymatic function in natural product biosynthesis over the past year are briefly reviewed.

Recent advances in the elucidation of enzymatic function in natural product biosynthesis [version 1; referees: 2 approved

Directory of Open Access Journals (Sweden)

Tan Gao-Yi

2015-12-01

Full Text Available With the successful production of artemisinic acid in yeast, the promising potential of synthetic biology for natural product biosynthesis is now being realized. The recent total biosynthesis of opioids in microbes is considered to be another landmark in this field. The importance and significance of enzymes in natural product biosynthetic pathways have been re-emphasized by these advancements. Therefore, the characterization and elucidation of enzymatic function in natural product biosynthesis are undoubtedly fundamental for the development of new drugs and the heterologous biosynthesis of active natural products. Here, discoveries regarding enzymatic function in natural product biosynthesis over the past year are briefly reviewed.

Topical problems in the biosynthesis of red blood pigment

International Nuclear Information System (INIS)

Franck, B.

1982-01-01

Uroporphyrinogen III plays a key role in the biosynthesis of heme, the red pigment of blood. In vivo studies with specifically 14 C- and 3 H-labeled precursors have revealed that the formation of uroporphyrinogen III in the organism follows several primary and subsidiary pathways. Model experiments on the pattern of biosynthesis have led to simple and effective methods of synthesizing uroporphyrin analogs and have shwon that their production is strongly favored thermodynamically, The biologically important porphyrins thus available permit a mechanistic explanantion of the light-induced dermatoses in porphyria diseases and suggest promising medical applications in diagnosis and therapy. (orig.)

Monoterpene biosynthesis potential of plant subcellular compartments

NARCIS (Netherlands)

Dong, L.; Jongedijk, E.J.; Bouwmeester, H.J.; Krol, van der A.R.

2016-01-01

Subcellular monoterpene biosynthesis capacity based on local geranyl diphosphate (GDP) availability or locally boosted GDP production was determined for plastids, cytosol and mitochondria. A geraniol synthase (GES) was targeted to plastids, cytosol, or mitochondria. Transient expression in Nicotiana

Transcriptional Responses and Gentiopicroside Biosynthesis in Methyl Jasmonate-Treated Gentiana macrophylla Seedlings.

Directory of Open Access Journals (Sweden)

Xiaoyan Cao

Full Text Available Gentiana macrophylla, a medicinal plant with significant pharmacological properties, contains the bioactive compound gentiopicroside. Methyl jasmonate (MeJA is an effective elicitor for enhancing the production of such compounds. However, little is known about MeJA-mediated biosynthesis of gentiopicroside. We investigated this phenomenon as well as gene expression profiles to determine the molecular mechanisms for MeJA-mediated gentiopicroside biosynthesis and regulation in G. macrophylla. Our HPLC results showed that Gentiana macrophylla seedlings exposed to MeJA had significantly higher concentrations of gentiopicroside when compared with control plants. We used RNA sequencing to compare transcriptional profiles in seedlings treated for 5 d with either 0 μmol L-1 MeJA (C or 250 μmol L-1 MeJA (M5 and detected differentially expressed genes (DEGs. In total, 77,482 unique sequences were obtained from approximately 34 million reads. Of these, 48,466 (57.46% sequences were annotated based on BLASTs performed against public databases. We identified 5,206 DEGs between the C and M5 samples, including genes related to the α-lenolenic acid degradation pathway, JA signaling pathway, and gentiopicroside biosynthesis. Expression of numerous enzyme genes in the glycolysis pathway was significantly up-regulated. Many genes encoding transcription factors (e.g. ERF, bHLH, MYB, and WRKY also responded to MeJA elicitation. Rapid acceleration of the glycolysis pathway that supplies precursors for IPP biosynthesis and up-regulates the expression of enzyme genes in that IPP pathway are probably most responsible for MeJA stimulation of gentiopicroside synthesis. Our qRT-PCR results showed that the expression profiles of 12 gentiopicroside biosynthesis genes were consistent with the RNA-Seq data. These results increase our understanding about how the gentiopicroside biosynthesis pathway in G. macrophylla responds to MeJA.

Meleagrin, a new FabI inhibitor from Penicillium chryosogenum with at least one additional mode of action.

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Chang Ji Zheng

Full Text Available Bacterial enoyl-acyl carrier protein reductase (FabI is a promising novel antibacterial target. We isolated a new class of FabI inhibitor from Penicillium chrysogenum, which produces various antibiotics, the mechanisms of some of them are unknown. The isolated FabI inhibitor was determined to be meleagrin by mass spectroscopy and nuclear magnetic resonance spectral analyses, and its more active and inactive derivatives were chemically prepared. Consistent with their selective inhibition of Staphylococcus aureus FabI, meleagrin and its more active derivatives directly bound to S. aureus FabI in a fluorescence quenching assay, inhibited intracellular fatty acid biosynthesis and growth of S. aureus, and increased the minimum inhibitory concentration for fabI-overexpressing S. aureus. The compounds that were not effective against the FabK isoform, however, inhibited the growth of Streptococcus pneumoniae that contained only the FabK isoform. Additionally no resistant mutant to the compounds was obtained. Importantly, fabK-overexpressing Escherichia coli was not resistant to these compounds, but was resistant to triclosan. These results demonstrate that the compounds inhibited another target in addition to FabI. Thus, meleagrin is a new class of FabI inhibitor with at least one additional mode of action that could have potential for treating multidrug-resistant bacteria.

Improvement of Folate Biosynthesis by Lactic Acid Bacteria Using Response Surface Methodology

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Norfarina Muhamad Nor

2010-01-01

Full Text Available Lactic acid bacteria (Lactococcus lactis NZ9000, Lactococcus lactis MG1363, Lactobacillus plantarum I-UL4 and Lactobacillus johnsonii DSM 20553 have been screened for their ability to produce folate intracellularly and/or extracellularly. L. plantarum I-UL4 was shown to be superior producer of folate compared to other strains. Statistically based experimental designs were used to optimize the medium formulation for the growth of L. plantarum I-UL4 and folate biosynthesis. The optimal values of important factors were determined by response surface methodology (RSM. The effects of carbon sources, nitrogen sources and para-aminobenzoic acid (PABA concentrations on folate biosynthesis were determined prior to RSM study. The biosynthesis of folate by L. plantarum I-UL4 increased from 36.36 to 60.39 µg/L using the optimized medium formulation compared to the selective Man de Rogosa Sharpe (MRS medium. Conditions for the optimal growth of L. plantarum I-UL4 and folate biosynthesis as suggested by RSM were as follows: lactose 20 g/L, meat extract 16.57 g/L and PABA 10 µM.

Evolution of the Phosphatidylcholine Biosynthesis Pathways in Green Algae: Combinatorial Diversity of Methyltransferases.

Science.gov (United States)

Hirashima, Takashi; Toyoshima, Masakazu; Moriyama, Takashi; Sato, Naoki

2018-01-01

Phosphatidylcholine (PC) is one of the most common phospholipids in eukaryotes, although some green algae such as Chlamydomonas reinhardtii are known to lack PC. Recently, we detected PC in four species in the genus Chlamydomonas: C. applanata NIES-2202, C. asymmetrica NIES-2207, C. debaryana NIES-2212, and C. sphaeroides NIES-2242. To reveal the PC biosynthesis pathways in green algae and the evolutionary scenario involved in their diversity, we analyzed the PC biosynthesis genes in these four algae using draft genome sequences. Homology searches suggested that PC in these species is synthesized by phosphoethanolamine-N-methyltransferase (PEAMT) and/or phosphatidylethanolamine-N-methyltransferase (PEMT), both of which are absent in C. reinhardtii. Recombinant PEAMTs from these algae showed methyltransferase activity for phosphoethanolamine but not for monomethyl phosphoethanolamine in vitro, in contrast to land plant PEAMT, which catalyzes the three methylations from phosphoethanolamine to phosphocholine. This suggested an involvement of other methyltransferases in PC biosynthesis. Here, we characterized the putative phospholipid-N-methyltransferase (PLMT) genes of these species by genetic and phylogenetic analysis. Complementation assays using a PC biosynthesis-deficient yeast suggested that the PLMTs of these algae can synthesize PC from phosphatidylethanolamine. These results indicated that the PC biosynthesis pathways in green algae differ from those of land plants, although the enzymes involved are homologous. Phylogenetic analysis suggested that the PEAMTs and PLMTs in these algae were inherited from the common ancestor of green algae. The absence of PC biosynthesis in many Chlamydomonas species is likely a result of parallel losses of PEAMT and PLMT in this genus.

Genes encoding enzymes of the lignin biosynthesis pathway in Eucalyptus

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Ricardo Harakava

2005-01-01

Full Text Available Eucalyptus ESTs libraries were screened for genes involved in lignin biosynthesis. This search was performed under the perspective of recent revisions on the monolignols biosynthetic pathway. Eucalyptus orthologues of all genes of the phenylpropanoid pathway leading to lignin biosynthesis reported in other plant species were identified. A library made with mRNAs extracted from wood was enriched for genes involved in lignin biosynthesis and allowed to infer the isoforms of each gene family that play a major role in wood lignin formation. Analysis of the wood library suggests that, besides the enzymes of the phenylpropanoids pathway, chitinases, laccases, and dirigent proteins are also important for lignification. Colocalization of several enzymes on the endoplasmic reticulum membrane, as predicted by amino acid sequence analysis, supports the existence of metabolic channeling in the phenylpropanoid pathway. This study establishes a framework for future investigations on gene expression level, protein expression and enzymatic assays, sequence polymorphisms, and genetic engineering.

IspE inhibitors identified by a combination of in silico and in vitro high-throughput screening.

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Naomi Tidten-Luksch

Full Text Available CDP-ME kinase (IspE contributes to the non-mevalonate or deoxy-xylulose phosphate (DOXP pathway for isoprenoid precursor biosynthesis found in many species of bacteria and apicomplexan parasites. IspE has been shown to be essential by genetic methods and since it is absent from humans it constitutes a promising target for antimicrobial drug development. Using in silico screening directed against the substrate binding site and in vitro high-throughput screening directed against both, the substrate and co-factor binding sites, non-substrate-like IspE inhibitors have been discovered and structure-activity relationships were derived. The best inhibitors in each series have high ligand efficiencies and favourable physico-chemical properties rendering them promising starting points for drug discovery. Putative binding modes of the ligands were suggested which are consistent with established structure-activity relationships. The applied screening methods were complementary in discovering hit compounds, and a comparison of both approaches highlights their strengths and weaknesses. It is noteworthy that compounds identified by virtual screening methods provided the controls for the biochemical screens.

Rational Design of Novel Allosteric Dihydrofolate Reductase Inhibitors Showing Antibacterial Effects on Drug-Resistant Escherichia coli Escape Variants.

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Srinivasan, Bharath; Rodrigues, João V; Tonddast-Navaei, Sam; Shakhnovich, Eugene; Skolnick, Jeffrey

2017-07-21

In drug discovery, systematic variations of substituents on a common scaffold and bioisosteric replacements are often used to generate diversity and obtain molecules with better biological effects. However, this could saturate the small-molecule diversity pool resulting in drug resistance. On the other hand, conventional drug discovery relies on targeting known pockets on protein surfaces leading to drug resistance by mutations of critical pocket residues. Here, we present a two-pronged strategy of designing novel drugs that target unique pockets on a protein's surface to overcome the above problems. Dihydrofolate reductase, DHFR, is a critical enzyme involved in thymidine and purine nucleotide biosynthesis. Several classes of compounds that are structural analogues of the substrate dihydrofolate have been explored for their antifolate activity. Here, we describe 10 novel small-molecule inhibitors of Escherichia coli DHFR, EcDHFR, belonging to the stilbenoid, deoxybenzoin, and chalcone family of compounds discovered by a combination of pocket-based virtual ligand screening and systematic scaffold hopping. These inhibitors show a unique uncompetitive or noncompetitive inhibition mechanism, distinct from those reported for all known inhibitors of DHFR, indicative of binding to a unique pocket distinct from either substrate or cofactor-binding pockets. Furthermore, we demonstrate that rescue mutants of EcDHFR, with reduced affinity to all known classes of DHFR inhibitors, are inhibited at the same concentration as the wild-type. These compounds also exhibit antibacterial activity against E. coli harboring the drug-resistant variant of DHFR. This discovery is the first report on a novel class of inhibitors targeting a unique pocket on EcDHFR.

Epoxide hydrolase-lasalocid a structure provides mechanistic insight into polyether natural product biosynthesis.

Science.gov (United States)

Wong, Fong T; Hotta, Kinya; Chen, Xi; Fang, Minyi; Watanabe, Kenji; Kim, Chu-Young

2015-01-14

Biosynthesis of some polyether natural products involves a kinetically disfavored epoxide-opening cyclic ether formation, a reaction termed anti-Baldwin cyclization. One such example is the biosynthesis of lasalocid A, an ionophore antibiotic polyether. During lasalocid A biosynthesis, an epoxide hydrolase, Lsd19, converts the bisepoxy polyketide intermediate into the tetrahydrofuranyl-tetrahydropyran product. We report the crystal structure of Lsd19 in complex with lasalocid A. The structure unambiguously shows that the C-terminal domain of Lsd19 catalyzes the intriguing anti-Baldwin cyclization. We propose a general mechanism for epoxide selection by ionophore polyether epoxide hydrolases.

Identification and biosynthesis of a novel xanthomonadin-dialkylresorcinol-hybrid from Azoarcus sp. BH72.

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Tim A Schöner

Full Text Available A novel xanthomonadin-dialkylresorcinol hybrid named arcuflavin was identified in Azoarcus sp. BH72 by a combination of feeding experiments, HPLC-MS and MALDI-MS and gene clusters encoding the biosynthesis of this non-isoprenoid aryl-polyene containing pigment are reported. A chorismate-utilizing enzyme from the XanB2-type producing 3- and 4-hydroxybenzoic acid and an AMP-ligase encoded by these gene clusters were characterized, that might perform the first two steps of the polyene biosynthesis. Furthermore, a detailed analysis of the already known or novel biosynthesis gene clusters involved in the biosynthesis of polyene containing pigments like arcuflavin, flexirubin and xanthomonadin revealed the presence of similar gene clusters in a wide range of bacterial taxa, suggesting that polyene and polyene-dialkylresorcinol pigments are more widespread than previously realized.

A fluorescence polarization binding assay to identify inhibitors of flavin-dependent monooxygenases.

Science.gov (United States)

Qi, Jun; Kizjakina, Karina; Robinson, Reeder; Tolani, Karishma; Sobrado, Pablo

2012-06-01

N-Hydroxylating monooxygenases (NMOs) are essential for pathogenesis in fungi and bacteria. NMOs catalyze the hydroxylation of sine and ornithine in the biosynthesis of hydroxamate-containing siderophores. Inhibition of kynurenine monooxygenase (KMO), which catalyzes the conversion of kynurenine to 3-hydroxykynurenine, alleviates neurodegenerative disorders such as Huntington's and Alzheimer's diseases and brain infections caused by the parasite Trypanosoma brucei. These enzymes are examples of flavin-dependent monooxygenases, which are validated drug targets. Here, we describe the development and optimization of a fluorescence polarization assay to identify potential inhibitors of flavin-dependent monooxygenases. Fluorescently labeled ADP molecules were synthesized and tested. An ADP-TAMRA chromophore bound to KMO with a K(d) value of 0.60 ± 0.05 μM and to the NMOs from Aspergillus fumigatus and Mycobacterium smegmatis with K(d) values of 2.1 ± 0.2 and 4.0 ± 0.2 μM, respectively. The assay was tested in competitive binding experiments with substrates and products of KMO and an NMO. Furthermore, we show that this assay can be used to identify inhibitors of NMOs. A Z' factor of 0.77 was calculated, and we show that the assay exhibits good tolerance to temperature, incubation time, and dimethyl sulfoxide concentration. Copyright © 2012 Elsevier Inc. All rights reserved.

Pregnenolone biosynthesis in C6-2B glioma cell mitochondria: regulation by a mitochondrial diazepam binding inhibitor receptor.

OpenAIRE

Papadopoulos, V; Guarneri, P; Kreuger, K E; Guidotti, A; Costa, E

1992-01-01

The C6-2B glioma cell line, rich in mitochondrial receptors that bind with high affinity to benzodiazepines, imidazopyridines, and isoquinolinecarboxamides (previously called peripheral-type benzodiazepine receptors), was investigated as a model to study the significance of the polypeptide diazepam binding inhibitor (DBI) and the putative DBI processing products on mitochondrial receptor-regulated steroidogenesis. DBI and its naturally occurring fragments have been found to be present in high...

Lipid, membrane, and mitochondrial characteristics of Ustilago maydis following exposure to ergosterol biosynthesis inhibitors

Energy Technology Data Exchange (ETDEWEB)

Waterfield, W.F. III

1986-01-01

Pencoazole at 0.5 ..mu..g/ml inhibited ergosterol biosynthesis in U. maydis. Polar lipids of sporidia grown with 0.5 ..mu..g/ml penconazole for 7.5 or 22 hr or 1.0 ..mu..g/ml fenarimol for 7.5 hr contained more 18:2 than 18:1 fatty acids. There was usually more 18:1 than 18:2 fatty acids in polar lipids of untreated sporidia but this ratio was influenced by culture cell density. The high 18:2 to 18:1 ratio in the polar lipids from penconazole grown cells was unaffected by cell density. There was an increase in free fatty acids and these were enriched with 18:2 members in cells grown with 0.5 ..mu..g/ml penconazole for 22 hr. Unsaturation of triglycerides fatty acids did not differ appreciably from that of untreated sporidia. Untreated WT U. maydis protoplasts lysed more slowly in 0.3 M sorbitol than those prepared from WT sporidia grown for 16 hr with 1.0 ..mu..g/ml penconazole or 2.0 ..mu..g/ml fenarimol or from untreated erg-40 sporidia. Protoplasts were more permeable to crystal violet than were those from untreated WT sporidia. Mitochondria from untreated WT sporidia oxidizing pyruvate plus malate or succinate yielded higher ADP/O rations than mitochondria from erg-40 or penconazole grown WT sporidia. The mitochondrial ATPase of control cells had a Km of 0.8 mM ATP whereas the mitochondrial ATPase of penconazole grown WT and erg-40 had a Km value of 3.7 and 3.2 mM ATP, respectively. When the mitochondrial catalytic subunit of the ATPase from these mitochondria were solubilized, the Km did not differ. These studies suggest that changes in sterols and membrane fatty acids resulting from treatments with EBI fungicides cause increased membrane fluidity which affects membrane stability, permeability and activity of the mitochondrial ATPase.

Biosynthesis of anatoxin-a and analogues (anatoxins) in cyanobacteria.

Science.gov (United States)

Méjean, Annick; Paci, Guillaume; Gautier, Valérie; Ploux, Olivier

2014-12-01

Freshwater cyanobacteria produce secondary metabolites that are toxic to humans and animals, the so-called cyanotoxins. Among them, anatoxin-a and homoanatoxin-a are potent neurotoxins that are agonists of the nicotinic acetylcholine receptor. These alkaloids provoke a rapid death if ingested at low doses. Recently, the cluster of genes responsible for the biosynthesis of these toxins, the ana cluster, has been identified in Oscillatoria sp. PCC 6506, and a biosynthetic pathway was proposed. This biosynthesis was reconstituted in vitro using purified enzymes confirming the predicted pathway. One of the enzymes, AnaB a prolyl-acyl carrier protein oxidase, was crystallized and its three dimensional structure solved confirming its reaction mechanism. Three other ana clusters have now been identified and sequenced in other cyanobacteria. These clusters show similarities and some differences suggesting a common evolutionary origin. In particular, the cluster from Cylindrospermum stagnale PCC 7417, possesses an extra gene coding for an F420-dependent oxidoreductase that is likely involved in the biosynthesis of dihydroanatoxin-a. This review summarizes all these new data and discusses them in relation to the production of anatoxins in the environment. Copyright © 2014 Elsevier Ltd. All rights reserved.

Biosynthesis of silver nanoparticles synthesized by Aspergillus

Indian Academy of Sciences (India)

In the present study, biosynthesis of silver nanoparticles and its antioxidant, antimicrobial and cytotoxic activities were investigated. Silver nanoparticles were extracellularly synthesized using Aspergillus flavus and the formation of nanoparticles was observed after 72 h of incubation. The results recorded from colour ...

Cholesterol biosynthesis in polychlorinated biphenyl-treated rats

International Nuclear Information System (INIS)

Kling, D.; Gamble, W.

1982-01-01

After administration of polychlorinated biphenly (PCB) at 0.055 (w/w) of the diet to Wistar rats for 30 days, followed by intraperitioneal injection of tritiated water, [ 14 C]mevalonate, and [ 14 C]acetate, there was a decrease in cholesterol biosynthesis in rat liver. No significant change in cholesterol formation was observed when PCB was administered at 0.01% (w/w) of the diet. In vitro inhibition of cholesterol synthesis by rat liver microsomes was observed with PCB. Squalene 2,3-oxidocyclase activity of rat liver microsomes was not significantly altered. Desmosterol delta 24 reductase activity was inhibited only at relatively high concentrations of PCB. There was increased incorporation of radioactivity into squalene and lanosterol, in vitro, in the presence of PCB. The primary inhibition of cholesterol biosynthesis appears to be at the demethylation and rearrangement reactions between lanosterol and cholesterol in the biosynthetic pathway

In Vitro Biosynthesis of Unnatural Enterocin and Wailupemycin Polyketides¥

Science.gov (United States)

Kalaitzis, John A.; Cheng, Qian; Thomas, Paul M.; Kelleher, Neil L.; Moore, Bradley S.

2009-01-01

Nature has evolved finely tuned strategies to synthesize rare and complex natural products such as the enterocin family of polyketides from the marine bacterium Streptomyces maritimus. Herein we report the directed ex vivo multienzyme syntheses of 24 unnatural 5-deoxyenterocin and wailupemycin F and G analogues, 18 of which are new. We have generated molecular diversity by priming the enterocin biosynthesis enzymes with unnatural substrates and have illustrated further the uniqueness of this type II polyketide synthase by way of exploiting its unusual starter unit biosynthesis pathways. PMID:19215142

Structure and Biosynthesis of Branched Wax Compounds on Wild Type and Wax Biosynthesis Mutants of Arabidopsis thaliana.

Science.gov (United States)

Busta, Lucas; Jetter, Reinhard

2017-06-01

The cuticle is a waxy composite that protects the aerial organs of land plans from non-stomatal water loss. The chemical make-up of the cuticular wax mixture plays a central role in defining the water barrier, but structure-function relationships have not been established so far, in part due to gaps in our understanding of wax structures and biosynthesis. While wax compounds with saturated, linear hydrocarbon tails have been investigated in detail, very little is known about compounds with modified aliphatic tails, which comprise substantial portions of some plant wax mixtures. This study aimed to investigate the structures, abundances and biosynthesis of branched compounds on the species for which wax biosynthesis is best understood: Arabidopsis thaliana. Microscale derivatization, mass spectral interpretation and organic synthesis identified homologous series of iso-alkanes and iso-alcohols on flowers and leaves, respectively. These comprised approximately 10-15% of wild type wax mixtures. The abundances of both branched wax constituents and accompanying unbranched compounds were reduced on the cer6, cer3 and cer1 mutants but not cer4, indicating that branched compounds are in part synthesized by the same machinery as unbranched compounds. In contrast, the abundances of unbranched, but not branched, wax constituents were reduced on the cer2 and cer26 mutants, suggesting that the pathways to both types of compounds deviate in later steps of chain elongation. Finally, the abundances of branched, but not unbranched, wax compounds were reduced on the cer16 mutant, and the (uncharacterized) CER16 protein may therefore be controlling the relative abundances of iso-alkanes and iso-alcohols on Arabidopsis surfaces. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Novel bioassay for the discovery of inhibitors of the 2-C-methyl-D-erythritol 4-phosphate (MEP and terpenoid pathways leading to carotenoid biosynthesis.

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Natália Corniani

Full Text Available The 2-C-methyl-D-erythritol 4-phosphate (MEP pathway leads to the synthesis of isopentenyl diphosphate in plastids. It is a major branch point providing precursors for the synthesis of carotenoids, tocopherols, plastoquinone and the phytyl chain of chlorophylls, as well as the hormones abscisic acid and gibberellins. Consequently, disruption of this pathway is harmful to plants. We developed an in vivo bioassay that can measure the carbon flow through the carotenoid pathway. Leaf cuttings are incubated in the presence of a phytoene desaturase inhibitor to induce phytoene accumulation. Any compound reducing the level of phytoene accumulation is likely to interfere with either one of the steps in the MEP pathway or the synthesis of geranylgeranyl diphosphate. This concept was tested with known inhibitors of steps of the MEP pathway. The specificity of this in vivo bioassay was also verified by testing representative herbicides known to target processes outside of the MEP and carotenoid pathways. This assay enables the rapid screen of new inhibitors of enzymes preceding the synthesis of phytoene, though there are some limitations related to the non-specific effect of some inhibitors on this assay.

Biosynthesis and metabolic fate of phenylalanine in conifers

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María Belén Pascual

2016-07-01

Full Text Available The amino acid phenylalanine (Phe is a critical metabolic node that plays an essential role in the interconnection between primary and secondary metabolism in plants. Phe is used as a protein building block but it is also as a precursor for numerous plant compounds that are crucial for plant reproduction, growth, development and defense against different types of stresses. The metabolism of Phe plays a central role in the channeling of carbon from photosynthesis to the biosynthesis of phenylpropanoids. The study of this metabolic pathway is particularly relevant in trees, which divert large amounts of carbon into the biosynthesis of Phe-derived compounds, particularly lignin, an important constituent of wood. The trunks of trees are metabolic sinks that consume a considerable percentage of carbon and energy from photosynthesis, and carbon is finally immobilized in wood. This paper reviews recent advances in the biosynthesis and metabolic utilization of Phe in conifer trees. Two alternative routes have been identified: the ancient phenylpyruvate pathway that is present in microorganisms, and the arogenate pathway that possibly evolved later during plant evolution. Additionally, an efficient nitrogen recycling mechanism is required to maintain sustained growth during xylem formation. The relevance of phenylalanine metabolic pathways in wood formation, the biotic interactions and ultraviolet protection is discussed. The genetic manipulation and transcriptional regulation of the pathways are also outlined.

Biosynthesis and Metabolic Fate of Phenylalanine in Conifers.

Science.gov (United States)

Pascual, María B; El-Azaz, Jorge; de la Torre, Fernando N; Cañas, Rafael A; Avila, Concepción; Cánovas, Francisco M

2016-01-01

The amino acid phenylalanine (Phe) is a critical metabolic node that plays an essential role in the interconnection between primary and secondary metabolism in plants. Phe is used as a protein building block but it is also as a precursor for numerous plant compounds that are crucial for plant reproduction, growth, development, and defense against different types of stresses. The metabolism of Phe plays a central role in the channeling of carbon from photosynthesis to the biosynthesis of phenylpropanoids. The study of this metabolic pathway is particularly relevant in trees, which divert large amounts of carbon into the biosynthesis of Phe-derived compounds, particularly lignin, an important constituent of wood. The trunks of trees are metabolic sinks that consume a considerable percentage of carbon and energy from photosynthesis, and carbon is finally immobilized in wood. This paper reviews recent advances in the biosynthesis and metabolic utilization of Phe in conifer trees. Two alternative routes have been identified: the ancient phenylpyruvate pathway that is present in microorganisms, and the arogenate pathway that possibly evolved later during plant evolution. Additionally, an efficient nitrogen recycling mechanism is required to maintain sustained growth during xylem formation. The relevance of phenylalanine metabolic pathways in wood formation, the biotic interactions, and ultraviolet protection is discussed. The genetic manipulation and transcriptional regulation of the pathways are also outlined.

Genes involved in long-chain alkene biosynthesis in Micrococcus luteus.

Science.gov (United States)

Beller, Harry R; Goh, Ee-Been; Keasling, Jay D

2010-02-01

Aliphatic hydrocarbons are highly appealing targets for advanced cellulosic biofuels, as they are already predominant components of petroleum-based gasoline and diesel fuels. We have studied alkene biosynthesis in Micrococcus luteus ATCC 4698, a close relative of Sarcina lutea (now Kocuria rhizophila), which 4 decades ago was reported to biosynthesize iso- and anteiso-branched, long-chain alkenes. The underlying biochemistry and genetics of alkene biosynthesis were not elucidated in those studies. We show here that heterologous expression of a three-gene cluster from M. luteus (Mlut_13230-13250) in a fatty acid-overproducing Escherichia coli strain resulted in production of long-chain alkenes, predominantly 27:3 and 29:3 (no. carbon atoms: no. C=C bonds). Heterologous expression of Mlut_13230 (oleA) alone produced no long-chain alkenes but unsaturated aliphatic monoketones, predominantly 27:2, and in vitro studies with the purified Mlut_13230 protein and tetradecanoyl-coenzyme A (CoA) produced the same C(27) monoketone. Gas chromatography-time of flight mass spectrometry confirmed the elemental composition of all detected long-chain alkenes and monoketones (putative intermediates of alkene biosynthesis). Negative controls demonstrated that the M. luteus genes were responsible for production of these metabolites. Studies with wild-type M. luteus showed that the transcript copy number of Mlut_13230-13250 and the concentrations of 29:1 alkene isomers (the dominant alkenes produced by this strain) generally corresponded with bacterial population over time. We propose a metabolic pathway for alkene biosynthesis starting with acyl-CoA (or-ACP [acyl carrier protein]) thioesters and involving decarboxylative Claisen condensation as a key step, which we believe is catalyzed by OleA. Such activity is consistent with our data and with the homology (including the conserved Cys-His-Asn catalytic triad) of Mlut_13230 (OleA) to FabH (beta-ketoacyl-ACP synthase III), which

Metabolic approaches to enhance transdermal drug delivery. 1. Effect of lipid synthesis inhibitors.

Science.gov (United States)

Tsai, J C; Guy, R H; Thornfeldt, C R; Gao, W N; Feingold, K R; Elias, P M

1996-06-01

The intercellular domains of the stratum corneum, which contain a mixture of cholesterol, free fatty acids, and ceramides, mediate both the epidermal permeability barrier and the transdermal delivery of both lipophilic and hydrophilic molecules. Prior studies have shown that each of the three key lipid classes is required for normal barrier function. For example, selective inhibition of either cholesterol, fatty acid, or ceramide synthesis in the epidermis delays barrier recovery rates after barrier perturbation of hairless mouse skin in vivo. In this study, we investigated the potential of certain inhibitors of lipid synthesis to enhance the transdermal delivery of lidocaine or caffeine as a result of their capacity to perturb barrier homeostasis. After acetone disruption of the barrier, the extent of lidocaine delivery and the degree of altered barrier function paralleled each other. Moreover, the further alteration in barrier function produced by either the fatty acid synthesis inhibitor 5-(tetradecyloxy)-2-furancarboxylic acid (TOFA), the cholesterol synthesis inhibitor fluvastatin (FLU), or cholesterol sulfate (CS) resulted in a further increase in lidocaine absorption. Furthermore, coapplications of TOFA and CS together caused an additive increase in lidocaine uptake. Finally, a comparable increase in drug delivery occurred when the barrier was disrupted initially with DMSO instead of acetone; coapplications of TOFA and FLU together again delayed barrier recovery and increased drug delivery by about 8-fold vs delivery from a standard enhancing vehicle. Whereas these metabolic inhibitors also variably increased the octanol/water partitioning of the drugs studied (perhaps via complexion or pH alterations), physicochemical effects of the inhibitors alone did not alter drug uptake in intact skin; i.e., passive mechanisms alone cannot account for the net increase in drug delivery. Our results show that modulations of epidermal lipid biosynthesis, following

Self-enhancement of hepatitis C virus replication by promotion of specific sphingolipid biosynthesis.

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Yuichi Hirata

Full Text Available Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (SGMS1 and 2 encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.

Self-enhancement of hepatitis C virus replication by promotion of specific sphingolipid biosynthesis.

Science.gov (United States)

Hirata, Yuichi; Ikeda, Kazutaka; Sudoh, Masayuki; Tokunaga, Yuko; Suzuki, Akemi; Weng, Leiyun; Ohta, Masatoshi; Tobita, Yoshimi; Okano, Ken; Ozeki, Kazuhisa; Kawasaki, Kenichi; Tsukuda, Takuo; Katsume, Asao; Aoki, Yuko; Umehara, Takuya; Sekiguchi, Satoshi; Toyoda, Tetsuya; Shimotohno, Kunitada; Soga, Tomoyoshi; Nishijima, Masahiro; Taguchi, Ryo; Kohara, Michinori

2012-01-01

Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (SGMS1 and 2) encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM) fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT) inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.

Acetylglutamate synthase in Neurospora crassa: characterization, localization, and genetic behavior of a regulatory enzyme of arginine biosynthesis

International Nuclear Information System (INIS)

Jacobson, J.A.

1988-01-01

This study describes the characterization and localization of the first enzyme of arginine biosynthesis in Neurospora crassa. A radioactive assay was developed to detect this enzyme whereby radioactive substrate and product molecules could be separated by ion-exchange chromatography. The enzyme was found to have a pH optimum of 9.0 and K/sub m/ values for glutamate and acetyl-CoA of approximately 4.7 and 0.45 mM, respectively. The enzyme was shown to be feedback inhibited by arginine. Half-maximal inhibition was observed at 0.13 mM arginine, a concentration which is similar to be in vivo cytosolic concentration of 0.2 mM. Arginine was found to act as a competitive inhibitor with respect to acetyl-CoA. Acetylglutamate synthase was localized to the mitochondrion. However, in contrast to the mitochondrial matrix location of the other ornithine biosynthetic enzymes, this enzyme was found to reside on the mitochondrial inner membrane

Combinatorial biosynthesis of medicinal plant secondary metabolites

NARCIS (Netherlands)

Julsing, Mattijs K.; Koulman, Albert; Woerdenbag, Herman J.; Quax, Wim J.; Kayser, Oliver

2006-01-01

Combinatorial biosynthesis is a new tool in the generation of novel natural products and for the production of rare and expensive natural products. The basic concept is combining metabolic pathways in different organisms on a genetic level. As a consequence heterologous organisms provide precursors

DGAT enzymes and triacylglycerol biosynthesis

OpenAIRE

Yen, Chi-Liang Eric; Stone, Scot J.; Koliwad, Suneil; Harris, Charles; Farese, Robert V.

2008-01-01

Triacylglycerols (triglycerides) (TGs) are the major storage molecules of metabolic energy and FAs in most living organisms. Excessive accumulation of TGs, however, is associated with human diseases, such as obesity, diabetes mellitus, and steatohepatitis. The final and the only committed step in the biosynthesis of TGs is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. The genes encoding two DGAT enzymes, DGAT1 and DGAT2, were identified in the past decade, ...

Deep sequencing of the Camellia chekiangoleosa transcriptome revealed candidate genes for anthocyanin biosynthesis.

Science.gov (United States)

Wang, Zhong-Wei; Jiang, Cong; Wen, Qiang; Wang, Na; Tao, Yuan-Yuan; Xu, Li-An

2014-03-15

Camellia chekiangoleosa is an important species of genus Camellia. It provides high-quality edible oil and has great ornamental value. The flowers are big and red which bloom between February and March. Flower pigmentation is closely related to the accumulation of anthocyanin. Although anthocyanin biosynthesis has been studied extensively in herbaceous plants, little molecular information on the anthocyanin biosynthesis pathway of C. chekiangoleosa is yet known. In the present study, a cDNA library was constructed to obtain detailed and general data from the flowers of C. chekiangoleosa. To explore the transcriptome of C. chekiangoleosa and investigate genes involved in anthocyanin biosynthesis, a 454 GS FLX Titanium platform was used to generate an EST dataset. About 46,279 sequences were obtained, and 24,593 (53.1%) were annotated. Using Blast search against the AGRIS, 1740 unigenes were found homologous to 599 Arabidopsis transcription factor genes. Based on the transcriptome dataset, nine anthocyanin biosynthesis pathway genes (PAL, CHS1, CHS2, CHS3, CHI, F3H, DFR, ANS, and UFGT) were identified and cloned. The spatio-temporal expression patterns of these genes were also analyzed using quantitative real-time polymerase chain reaction. The study results not only enrich the gene resource but also provide valuable information for further studies concerning anthocyanin biosynthesis. Copyright © 2014 Elsevier B.V. All rights reserved.

Fatty Acid Biosynthesis Pathways in Methylomicrobium buryatense 5G(B1).

Science.gov (United States)

Demidenko, Aleksandr; Akberdin, Ilya R; Allemann, Marco; Allen, Eric E; Kalyuzhnaya, Marina G

2016-01-01

Methane utilization by methanotrophic bacteria is an attractive application for biotechnological conversion of natural or biogas into high-added-value products. Haloalcaliphilic methanotrophic bacteria belonging to the genus Methylomicrobium are among the most promising strains for methane-based biotechnology, providing easy and inexpensive cultivation, rapid growth, and the availability of established genetic tools. A number of methane bioconversions using these microbial cultures have been discussed, including the derivation of biodiesel, alkanes, and OMEGA-3 supplements. These compounds are derived from bacterial fatty acid pools. Here, we investigate fatty acid biosynthesis in Methylomicrobium buryatense 5G(B1) . Most of the genes homologous to typical Type II fatty acid biosynthesis pathways could be annotated by bioinformatics analyses, with the exception of fatty acid transport and regulatory elements. Different approaches for improving fatty acid accumulation were investigated. These studies indicated that both fatty acid degradation and acetyl- and malonyl-CoA levels are bottlenecks for higher level fatty acid production. The best strain generated in this study synthesizes 111 ± 2 mg/gDCW of extractable fatty acids, which is ~20% more than the original strain. A candidate gene for fatty acid biosynthesis regulation, farE , was identified and studied. Its deletion resulted in drastic changes to the fatty acid profile, leading to an increased pool of C18-fatty acid methyl ester. The FarE-regulon was further investigated by RNA-seq analysis of gene expression in farE -knockout mutants and farE -overexpressing strains. These gene profiles highlighted a novel set of enzymes and regulators involved in fatty acid biosynthesis. The gene expression and fatty acid profiles of the different farE -strains support the hypothesis that metabolic fluxes upstream of fatty acid biosynthesis restrict fatty acid production in the methanotroph.

Genetic Variation for Lettuce Seed Thermoinhibition Is Associated with Temperature-Sensitive Expression of Abscisic Acid, Gibberellin, and Ethylene Biosynthesis, Metabolism, and Response Genes1[C][W][OA

Science.gov (United States)

Argyris, Jason; Dahal, Peetambar; Hayashi, Eiji; Still, David W.; Bradford, Kent J.

2008-01-01

Lettuce (Lactuca sativa ‘Salinas’) seeds fail to germinate when imbibed at temperatures above 25°C to 30°C (termed thermoinhibition). However, seeds of an accession of Lactuca serriola (UC96US23) do not exhibit thermoinhibition up to 37°C in the light. Comparative genetics, physiology, and gene expression were analyzed in these genotypes to determine the mechanisms governing the regulation of seed germination by temperature. Germination of the two genotypes was differentially sensitive to abscisic acid (ABA) and gibberellin (GA) at elevated temperatures. Quantitative trait loci associated with these phenotypes colocated with a major quantitative trait locus (Htg6.1) from UC96US23 conferring germination thermotolerance. ABA contents were elevated in Salinas seeds that exhibited thermoinhibition, consistent with the ability of fluridone (an ABA biosynthesis inhibitor) to improve germination at high temperatures. Expression of many genes involved in ABA, GA, and ethylene biosynthesis, metabolism, and response was differentially affected by high temperature and light in the two genotypes. In general, ABA-related genes were more highly expressed when germination was inhibited, and GA- and ethylene-related genes were more highly expressed when germination was permitted. In particular, LsNCED4, a gene encoding an enzyme in the ABA biosynthetic pathway, was up-regulated by high temperature only in Salinas seeds and also colocated with Htg6.1. The temperature sensitivity of expression of LsNCED4 may determine the upper temperature limit for lettuce seed germination and may indirectly influence other regulatory pathways via interconnected effects of increased ABA biosynthesis. PMID:18753282

Kunitz Proteinase Inhibitors Limit Water Stress Responses in White Clover (Trifolium repens L.) Plants.

Science.gov (United States)

Islam, Afsana; Leung, Susanna; Nikmatullah, Aluh; Dijkwel, Paul P; McManus, Michael T

2017-01-01

The response of plants to water deficiency or drought is a complex process, the perception of which is triggered at the molecular level before any visible morphological responses are detected. It was found that different groups of plant proteinase inhibitors (PIs) are induced and play an active role during abiotic stress conditions such as drought. Our previous work with the white clover ( Trifolium repens L.) Kunitz Proteinase Inhibitor ( Tr-KPI ) gene family showed that Tr-KPIs are differentially regulated to ontogenetic and biotic stress associated cues and that, at least some members of this gene family may be required to maintain cellular homeostasis. Altered cellular homeostasis may also affect abiotic stress responses and therefore, we aimed to understand if distinct Tr-PKI members function during drought stress. First, the expression level of three Tr-KPI genes, Tr-KPI1 , Tr-KPI2 , and Tr-KPI5 , was measured in two cultivars and one white clover ecotype with differing capacity to tolerate drought. The expression of Tr-KPI1 and Tr-KPI5 increased in response to water deficiency and this was exaggerated when the plants were treated with a previous period of water deficiency. In contrast, proline accumulation and increased expression of Tr-NCED1 , a gene encoding a protein involved in ABA biosynthesis, was delayed in plants that experienced a previous drought period. RNAi knock-down of Tr-KPI1 and Tr-KPI5 resulted in increased proline accumulation in leaf tissue of plants grown under both well-watered and water-deficit conditions. In addition, increased expression of genes involved in ethylene biosynthesis was found. The data suggests that Tr-KPIs , particularly Tr-KPI5 , have an explicit function during water limitation. The results also imply that the Tr-KPI family has different in planta proteinase targets and that the functions of this protein family are not solely restricted to one of storage proteins or in response to biotic stress.

Kunitz Proteinase Inhibitors Limit Water Stress Responses in White Clover (Trifolium repens L. Plants

Directory of Open Access Journals (Sweden)

Afsana Islam

2017-10-01

Full Text Available The response of plants to water deficiency or drought is a complex process, the perception of which is triggered at the molecular level before any visible morphological responses are detected. It was found that different groups of plant proteinase inhibitors (PIs are induced and play an active role during abiotic stress conditions such as drought. Our previous work with the white clover (Trifolium repens L. Kunitz Proteinase Inhibitor (Tr-KPI gene family showed that Tr-KPIs are differentially regulated to ontogenetic and biotic stress associated cues and that, at least some members of this gene family may be required to maintain cellular homeostasis. Altered cellular homeostasis may also affect abiotic stress responses and therefore, we aimed to understand if distinct Tr-PKI members function during drought stress. First, the expression level of three Tr-KPI genes, Tr-KPI1, Tr-KPI2, and Tr-KPI5, was measured in two cultivars and one white clover ecotype with differing capacity to tolerate drought. The expression of Tr-KPI1 and Tr-KPI5 increased in response to water deficiency and this was exaggerated when the plants were treated with a previous period of water deficiency. In contrast, proline accumulation and increased expression of Tr-NCED1, a gene encoding a protein involved in ABA biosynthesis, was delayed in plants that experienced a previous drought period. RNAi knock-down of Tr-KPI1 and Tr-KPI5 resulted in increased proline accumulation in leaf tissue of plants grown under both well-watered and water-deficit conditions. In addition, increased expression of genes involved in ethylene biosynthesis was found. The data suggests that Tr-KPIs, particularly Tr-KPI5, have an explicit function during water limitation. The results also imply that the Tr-KPI family has different in planta proteinase targets and that the functions of this protein family are not solely restricted to one of storage proteins or in response to biotic stress.

Ribosomal protein S6 kinase1 coordinates with TOR-Raptor2 to regulate thylakoid membrane biosynthesis in rice.

Science.gov (United States)

Sun, Linxiao; Yu, Yonghua; Hu, Weiqin; Min, Qiming; Kang, Huiling; Li, Yilu; Hong, Yue; Wang, Xuemin; Hong, Yueyun

2016-07-01

Ribosomal protein S6 kinase (S6K) functions as a key component in the target of rapamycin (TOR) pathway involved in multiple processes in eukaryotes. The role and regulation of TOR-S6K in lipid metabolism remained unknown in plants. Here we provide genetic and pharmacological evidence that TOR-Raptor2-S6K1 is important for thylakoid galactolipid biosynthesis and thylakoid grana modeling in rice (Oryza sativa L.). Genetic suppression of S6K1 caused pale yellow-green leaves, defective thylakoid grana architecture. S6K1 directly interacts with Raptor2, a core component in TOR signaling, and S6K1 activity is regulated by Raptor2 and TOR. Plants with suppressed Raptor2 expression or reduced TOR activity by inhibitors mimicked the S6K1-deficient phenotype. A significant reduction in galactolipid content was found in the s6k1, raptor2 mutant or TOR-inhibited plants, which was accompanied by decreased transcript levels of the set of genes such as lipid phosphate phosphatase α5 (LPPα5), MGDG synthase 1 (MGD1), and DGDG synthase 1 (DGD1) involved in galactolipid synthesis, compared to the control plants. Moreover, loss of LPPα5 exhibited a similar phenotype with pale yellow-green leaves. These results suggest that TOR-Raptor2-S6K1 is important for modulating thylakoid membrane lipid biosynthesis, homeostasis, thus enhancing thylakoid grana architecture and normal photosynthesis ability in rice. Copyright © 2016 Elsevier B.V. All rights reserved.

Metabolic engineering for improved heterologous terpenoid biosynthesis

NARCIS (Netherlands)

Ryden, A.; Melillo, E.; Czepnik, M.; Kayser, O.

Terpenoids belong to the largest class of natural compounds and are produced in all living organisms. The isoprenoid skeleton is based on assembling of C5 building blocks, but the biosynthesis of a great variety of terpenoids ranging from monoterpenoids to polyterpenoids is not fully understood

Ergothioneine Biosynthesis and Functionality in the Opportunistic Fungal Pathogen, Aspergillus fumigatus.

Science.gov (United States)

Sheridan, Kevin J; Lechner, Beatrix Elisabeth; Keeffe, Grainne O'; Keller, Markus A; Werner, Ernst R; Lindner, Herbert; Jones, Gary W; Haas, Hubertus; Doyle, Sean

2016-10-17

Ergothioneine (EGT; 2-mercaptohistidine trimethylbetaine) is a trimethylated and sulphurised histidine derivative which exhibits antioxidant properties. Here we report that deletion of Aspergillus fumigatus egtA (AFUA_2G15650), which encodes a trimodular enzyme, abrogated EGT biosynthesis in this opportunistic pathogen. EGT biosynthetic deficiency in A. fumigatus significantly reduced resistance to elevated H 2 O 2 and menadione, respectively, impaired gliotoxin production and resulted in attenuated conidiation. Quantitative proteomic analysis revealed substantial proteomic remodelling in ΔegtA compared to wild-type under both basal and ROS conditions, whereby the abundance of 290 proteins was altered. Specifically, the reciprocal differential abundance of cystathionine γ-synthase and β-lyase, respectively, influenced cystathionine availability to effect EGT biosynthesis. A combined deficiency in EGT biosynthesis and the oxidative stress response regulator Yap1, which led to extreme oxidative stress susceptibility, decreased resistance to heavy metals and production of the extracellular siderophore triacetylfusarinine C and increased accumulation of the intracellular siderophore ferricrocin. EGT dissipated H 2 O 2 in vitro, and elevated intracellular GSH levels accompanied abrogation of EGT biosynthesis. EGT deficiency only decreased resistance to high H 2 O 2 levels which suggests functionality as an auxiliary antioxidant, required for growth at elevated oxidative stress conditions. Combined, these data reveal new interactions between cellular redox homeostasis, secondary metabolism and metal ion homeostasis.

Agrobacterium mediated transient gene silencing (AMTS in Stevia rebaudiana: insights into steviol glycoside biosynthesis pathway.

Directory of Open Access Journals (Sweden)

Praveen Guleria

Full Text Available Steviol glycoside biosynthesis pathway has emerged as bifurcation from ent-kaurenoic acid, substrate of methyl erythritol phosphate pathway that also leads to gibberellin biosynthesis. However, the genetic regulation of steviol glycoside biosynthesis has not been studied. So, in present study RNA interference (RNAi based Agrobacterium mediated transient gene silencing (AMTS approach was followed. SrKA13H and three SrUGTs (SrUGT85C2, SrUGT74G1 and SrUGT76G1 genes encoding ent-kaurenoic acid-13 hydroxylase and three UDP glycosyltransferases of steviol glycoside biosynthesis pathway were silenced in Stevia rebaudiana to understand its molecular mechanism and association with gibberellins.RNAi mediated AMTS of SrKA13H and three SrUGTs has significantly reduced the expression of targeted endogenous genes as well as total steviol glycoside accumulation. While gibberellins (GA3 content was significantly enhanced on AMTS of SrUGT85C2 and SrKA13H. Silencing of SrKA13H and SrUGT85C2 was found to block the metabolite flux of steviol glycoside pathway and shifted it towards GA3 biosynthesis. Further, molecular docking of three SrUGT proteins has documented highest affinity of SrUGT76G1 for the substrates of alternate pathways synthesizing steviol glycosides. This could be a plausible reason for maximum reduction in steviol glycoside content on silencing of SrUGT76G1 than other genes.SrKA13H and SrUGT85C2 were identified as regulatory genes influencing carbon flux between steviol glycoside and gibberellin biosynthesis. This study has also documented the existence of alternate steviol glycoside biosynthesis route.

Cellular oxido-reductive proteins of Chlamydomonas reinhardtii control the biosynthesis of silver nanoparticles

Directory of Open Access Journals (Sweden)

Barwal Indu

2011-12-01

Full Text Available Abstract Background Elucidation of molecular mechanism of silver nanoparticles (SNPs biosynthesis is important to control its size, shape and monodispersity. The evaluation of molecular mechanism of biosynthesis of SNPs is of prime importance for the commercialization and methodology development for controlling the shape and size (uniform distribution of SNPs. The unicellular algae Chlamydomonas reinhardtii was exploited as a model system to elucidate the role of cellular proteins in SNPs biosynthesis. Results The C. reinhardtii cell free extract (in vitro and in vivo cells mediated synthesis of silver nanoparticles reveals SNPs of size range 5 ± 1 to 15 ± 2 nm and 5 ± 1 to 35 ± 5 nm respectively. In vivo biosynthesized SNPs were localized in the peripheral cytoplasm and at one side of flagella root, the site of pathway of ATP transport and its synthesis related enzymes. This provides an evidence for the involvement of oxidoreductive proteins in biosynthesis and stabilization of SNPs. Alteration in size distribution and decrease of synthesis rate of SNPs in protein-depleted fractions confirmed the involvement of cellular proteins in SNPs biosynthesis. Spectroscopic and SDS-PAGE analysis indicate the association of various proteins on C. reinhardtii mediated in vivo and in vitro biosynthesized SNPs. We have identified various cellular proteins associated with biosynthesized (in vivo and in vitro SNPs by using MALDI-MS-MS, like ATP synthase, superoxide dismutase, carbonic anhydrase, ferredoxin-NADP+ reductase, histone etc. However, these proteins were not associated on the incubation of pre-synthesized silver nanoparticles in vitro. Conclusion Present study provides the indication of involvement of molecular machinery and various cellular proteins in the biosynthesis of silver nanoparticles. In this report, the study is mainly focused towards understanding the role of diverse cellular protein in the synthesis and capping of silver

Abscisic acid regulates seed germination of Vellozia species in response to temperature.

Science.gov (United States)

Vieira, B C; Bicalho, E M; Munné-Bosch, S; Garcia, Q S

2017-03-01

The relationship between the phytohormones, gibberellin (GA) and abscisic acid (ABA) and light and temperature on seed germination is still not well understood. We aimed to investigate the role of the ABA and GA on seed germination of Vellozia caruncularis, V. intermedia and V. alutacea in response to light/dark conditions on different temperature. Seeds were incubated in GA (GA 3 or GA 4 ) or ABA and their respective biosynthesis inhibitors (paclobutrazol - PAC, and fluridone - FLU) solutions at two contrasting temperatures (25 and 40 °C). Furthermore, endogenous concentrations of active GAs and those of ABA were measured in seeds of V. intermedia and V. alutacea during imbibition/germination. Exogenous ABA inhibited the germination of Vellozia species under all conditions tested. GA, FLU and FLU + GA 3 stimulated germination in the dark at 25 °C (GA 4 being more effective than GA 3 ). PAC reduced seed germination in V. caruncularis and V. alutacea, but did not affect germination of V. intermedia at 40 °C either under light or dark conditions. During imbibition in the dark, levels of active GAs decreased in the seeds of V. intermedia, but were not altered in those of V. alutacea. Incubation at 40 °C decreased ABA levels during imbibition in both V. caruncularis and V. alutacea. We conclude that the seeds of Vellozia species studied here require light or high temperature to germinate and ABA has a major role in the regulation of Vellozia seed germination in response to light and temperature. © 2016 German Botanical Society and The Royal Botanical Society of the Netherlands.

Homology modeling, molecular dynamics and inhibitor binding study on MurD ligase of Mycobacterium tuberculosis.

Science.gov (United States)

Arvind, Akanksha; Kumar, Vivek; Saravanan, Parameswaran; Mohan, C Gopi

2012-09-01

The cell wall of mycobacterium offers well validated targets which can be exploited for discovery of new lead compounds. MurC-MurF ligases catalyze a series of irreversible steps in the biosynthesis of peptidoglycan precursor, i.e. MurD catalyzes the ligation of D-glutamate to the nucleotide precursor UMA. The three dimensional structure of Mtb-MurD is not known and was predicted by us for the first time using comparative homology modeling technique. The accuracy and stability of the predicted Mtb-MurD structure was validated using Procheck and molecular dynamics simulation. Key interactions in Mtb-MurD were studied using docking analysis of available transition state inhibitors of E.coli-MurD. The docking analysis revealed that analogues of both L and D forms of glutamic acid have similar interaction profiles with Mtb-MurD. Further, residues His192, Arg382, Ser463, and Tyr470 are proposed to be important for inhibitor-(Mtb-MurD) interactions. We also identified few pharmacophoric features essential for Mtb-MurD ligase inhibitory activity and which can further been utilized for the discovery of putative antitubercular chemotherapy.

Biosynthesis of two dihydropyrrole-polyketides from a marine-derived Penicillium citrinum

Energy Technology Data Exchange (ETDEWEB)

Romminger, Stelamar; Pimenta, Eli F.; Berlinck, Roberto G.S., E-mail: rgsberlinck@iqsc.usp.br [Universidade de Sao Paulo (USP), Sao Carlos, SP (Brazil). Inst. de Quimica; Nascimento, Eduardo S.; Ferreira, Antonio G. [Universidade Federal de Sao Carlos (UFSCAR), SP (Brazil). Dept. de Quimica

2012-10-15

Feeding experiments with {sup 13}C-labeled precursors were performed in order to establish the biosynthesis of two N-acylated dihydro pyrroles, (8E)-1-(2,3-dihydro-1H-pyrrol-1-yl)-2-methyldec- 8-ene-1,3-dione (1) and 1-(2,3-dihydro-1H-pyrrol-1-yl)-2-methyldecane-1,3-dione (2), isolated from the cultures of a marine-derived Penicillium citrinum. The biosynthesis of both, 1 and 2, involves the incorporation of acetate, methionine and ornithine. (author)

Biosynthesis of two dihydropyrrole-polyketides from a marine-derived Penicillium citrinum

International Nuclear Information System (INIS)

Romminger, Stelamar; Pimenta, Eli F.; Berlinck, Roberto G.S.; Nascimento, Eduardo S.; Ferreira, Antonio G.

2012-01-01

Feeding experiments with 13 C-labeled precursors were performed in order to establish the biosynthesis of two N-acylated dihydro pyrroles, (8E)-1-(2,3-dihydro-1H-pyrrol-1-yl)-2-methyldec- 8-ene-1,3-dione (1) and 1-(2,3-dihydro-1H-pyrrol-1-yl)-2-methyldecane-1,3-dione (2), isolated from the cultures of a marine-derived Penicillium citrinum. The biosynthesis of both, 1 and 2, involves the incorporation of acetate, methionine and ornithine. (author)

epsilon-N-trimethyllysine availability regulates the rate of carnitine biosynthesis in the growing rat

International Nuclear Information System (INIS)

Rebouche, C.J.; Lehman, L.J.; Olson, L.

1986-01-01

Rates of carnitine biosynthesis in mammals depend on the availability of substrates and the activity of enzymes subserving the pathway. This study was undertaken to test the hypothesis that the availability of epsilon-N-trimethyllysine is rate-limiting for synthesis of carnitine in the growing rat and to evaluate diet as a source of this precursor for carnitine biosynthesis. Rats apparently absorbed greater than 90% of a tracer dose of [methyl- 3 H]epsilon-N-trimethyllysine, and approximately 30% of that was incorporated into tissues as [ 3 H]carnitine. Rats given oral supplements of epsilon-N-trimethyllysine (0.5-20 mg/d), but no dietary carnitine, excreted more carnitine than control animals receiving no dietary epsilon-N-trimethyllysine or carnitine. Rates of carnitine excretion increased in a dose-dependent manner. Tissue and serum levels of carnitine also increased with dietary epsilon-N-trimethyllysine supplementation. There was no evidence that the capacity for carnitine biosynthesis was saturated even at the highest level of oral epsilon-N-trimethyllysine supplementation. Common dietary proteins (casein, soy protein and wheat gluten) were found to be poor sources of epsilon-N-trimethyllysine for carnitine biosynthesis. The results of this study indicate that the availability of epsilon-N-trimethyllysine limits the rate of carnitine biosynthesis in the growing rat

Biosynthesis of polyhydroxyalkanotes in wildtype yeasts | Desuoky ...

African Journals Online (AJOL)

Biosynthesis of the biodegradable polymers polyhydroxyalkanotes (PHAs) are studied extensively in wild type and genetically modified prokaryotic cells, however the content and structure of PHA in wild type yeasts are not well documented. The purpose of this study was to screen forty yeast isolates collected from different ...

Apicobasal domain identities of expanding tubular membranes depend on glycosphingolipid biosynthesis.

Science.gov (United States)

Zhang, Hongjie; Abraham, Nessy; Khan, Liakot A; Hall, David H; Fleming, John T; Göbel, Verena

2011-09-18

Metazoan internal organs are assembled from polarized tubular epithelia that must set aside an apical membrane domain as a lumenal surface. In a global Caenorhabditis elegans tubulogenesis screen, interference with several distinct fatty-acid-biosynthetic enzymes transformed a contiguous central intestinal lumen into multiple ectopic lumens. We show that multiple-lumen formation is caused by apicobasal polarity conversion, and demonstrate that in situ modulation of lipid biosynthesis is sufficient to reversibly switch apical domain identities on growing membranes of single post-mitotic cells, shifting lumen positions. Follow-on targeted lipid-biosynthesis pathway screens and functional genetic assays were designed to identify a putative single causative lipid species. They demonstrate that fatty-acid biosynthesis affects polarity through sphingolipid synthesis, and reveal ceramide glucosyltransferases (CGTs) as end-point biosynthetic enzymes in this pathway. Our findings identify glycosphingolipids, CGT products and obligate membrane lipids, as critical determinants of in vivo polarity and indicate that they sort new components to the expanding apical membrane.

Biosynthesis of steroidal alkaloids in Solanaceae plants: involvement of an aldehyde intermediate during C-26 amination.

Science.gov (United States)

Ohyama, Kiyoshi; Okawa, Akiko; Moriuchi, Yuka; Fujimoto, Yoshinori

2013-05-01

The C-26 amino group of steroidal alkaloids, such as tomatine, is introduced during an early step of their biosynthesis from cholesterol. In the present study, the mechanism of C-26 amination was reinvestigated by administering stable isotope labeled compounds, such as (26,26,26,27,27,27-(2)H6)cholesterol during biosynthesis of tomatine, solanine and solasonine. The chemical compositions of tomatine and solanine so obtained were analyzed by LC-MS after administering the d6-cholesterol to a tomato seedling and a potato shoot, respectively. The resulting spectra indicated that two deuterium atoms were eliminated from C-26 of cholesterol during biosynthesis. Furthermore, administration of (6-(13)C(2)H3)mevalonate in combination with lovastatin to an eggplant seedling, followed by GC-MS analysis of solasodine after TMS derivatization established that two deuterium atoms were eliminated from C-26 of cholesterol during solasonine biosynthesis. These findings are in contrast to an earlier observation that one hydrogen atom was lost from C-26 during tomatidine biosynthesis, and suggest that C-26 nitrogen atom addition involves an aldehyde intermediate. Thus, it is proposed that the C-26 amination reaction that occurs during steroidal alkaloid biosynthesis proceeds by way of a transamination mechanism. Copyright © 2013 Elsevier Ltd. All rights reserved.

Benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole derivatives as multiple inhibitors of bacterial Mur ligases (MurC-MurF).

Science.gov (United States)

Perdih, Andrej; Hrast, Martina; Barreteau, Hélène; Gobec, Stanislav; Wolber, Gerhard; Solmajer, Tom

2014-08-01

Enzymes catalyzing the biosynthesis of bacterial peptidoglycan represent traditionally a collection of highly selective targets for novel antibacterial drug design. Four members of the bacterial Mur ligase family-MurC, MurD, MurE and MurF-are involved in the intracellular steps of peptidoglycan biosynthesis, catalyzing the synthesis of the peptide moiety of the Park's nucleotide. In our previous virtual screening campaign, a chemical class of benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole derivatives exhibiting dual MurD/MurE inhibition properties was discovered. In the present study we further investigated this class of compounds by performing inhibition assays on all four Mur ligases (MurC-MurF). Furthermore, molecular dynamics (MD) simulation studies of one of the initially discovered compound 1 were performed to explore its geometry as well as its energetic behavior based on the Linear Interaction Energy (LIE) method. Further in silico virtual screening (VS) experiments based on the parent active compound 1 were conducted to optimize the discovered series. Selected hits were assayed against all Escherichia coli MurC-MurF enzymes in biochemical inhibition assays and molecules 10-14 containing benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole coupled with five member-ring rhodanine moiety were found to be multiple inhibitors of the whole MurC-MurF cascade of bacterial enzymes in the micromolar range. Steady-state kinetics studies suggested this class to act as competitive inhibitors of the MurD enzyme towards d-Glu. These compounds represent novel valuable starting point in the development of novel antibacterial agents. Copyright © 2014 Elsevier Ltd. All rights reserved.

Benzylisoquinoline alkaloid biosynthesis in opium poppy.

Science.gov (United States)

Beaudoin, Guillaume A W; Facchini, Peter J

2014-07-01

Opium poppy (Papaver somniferum) is one of the world's oldest medicinal plants and remains the only commercial source for the narcotic analgesics morphine, codeine and semi-synthetic derivatives such as oxycodone and naltrexone. The plant also produces several other benzylisoquinoline alkaloids with potent pharmacological properties including the vasodilator papaverine, the cough suppressant and potential anticancer drug noscapine and the antimicrobial agent sanguinarine. Opium poppy has served as a model system to investigate the biosynthesis of benzylisoquinoline alkaloids in plants. The application of biochemical and functional genomics has resulted in a recent surge in the discovery of biosynthetic genes involved in the formation of major benzylisoquinoline alkaloids in opium poppy. The availability of extensive biochemical genetic tools and information pertaining to benzylisoquinoline alkaloid metabolism is facilitating the study of a wide range of phenomena including the structural biology of novel catalysts, the genomic organization of biosynthetic genes, the cellular and sub-cellular localization of biosynthetic enzymes and a variety of biotechnological applications. In this review, we highlight recent developments and summarize the frontiers of knowledge regarding the biochemistry, cellular biology and biotechnology of benzylisoquinoline alkaloid biosynthesis in opium poppy.

Agrobacterium Mediated Transient Gene Silencing (AMTS) in Stevia rebaudiana: Insights into Steviol Glycoside Biosynthesis Pathway

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Guleria, Praveen; Yadav, Sudesh Kumar

2013-01-01

Background Steviol glycoside biosynthesis pathway has emerged as bifurcation from ent-kaurenoic acid, substrate of methyl erythritol phosphate pathway that also leads to gibberellin biosynthesis. However, the genetic regulation of steviol glycoside biosynthesis has not been studied. So, in present study RNA interference (RNAi) based Agrobacterium mediated transient gene silencing (AMTS) approach was followed. SrKA13H and three SrUGTs (SrUGT85C2, SrUGT74G1 and SrUGT76G1) genes encoding ent-kaurenoic acid-13 hydroxylase and three UDP glycosyltransferases of steviol glycoside biosynthesis pathway were silenced in Stevia rebaudiana to understand its molecular mechanism and association with gibberellins. Methodology/Principal Findings RNAi mediated AMTS of SrKA13H and three SrUGTs has significantly reduced the expression of targeted endogenous genes as well as total steviol glycoside accumulation. While gibberellins (GA3) content was significantly enhanced on AMTS of SrUGT85C2 and SrKA13H. Silencing of SrKA13H and SrUGT85C2 was found to block the metabolite flux of steviol glycoside pathway and shifted it towards GA3 biosynthesis. Further, molecular docking of three SrUGT proteins has documented highest affinity of SrUGT76G1 for the substrates of alternate pathways synthesizing steviol glycosides. This could be a plausible reason for maximum reduction in steviol glycoside content on silencing of SrUGT76G1 than other genes. Conclusions SrKA13H and SrUGT85C2 were identified as regulatory genes influencing carbon flux between steviol glycoside and gibberellin biosynthesis. This study has also documented the existence of alternate steviol glycoside biosynthesis route. PMID:24023961

Direct Ionic Regulation of the Activity of Myo-Inositol Biosynthesis Enzymes in Mozambique Tilapia.

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Fernando D Villarreal

Full Text Available Myo-inositol (Ins is a major compatible osmolyte in many cells, including those of Mozambique tilapia (Oreochromis mossambicus. Ins biosynthesis is highly up-regulated in tilapia and other euryhaline fish exposed to hyperosmotic stress. In this study, enzymatic regulation of two enzymes of Ins biosynthesis, Ins phosphate synthase (MIPS and inositol monophosphatase (IMPase, by direct ionic effects is analyzed. Specific MIPS and IMPase isoforms from Mozambique tilapia (MIPS-160 and IMPase 1 were selected based on experimental, phylogenetic, and structural evidence supporting their role for Ins biosynthesis during hyperosmotic stress. Recombinant tilapia IMPase 1 and MIPS-160 activity was assayed in vitro at ionic conditions that mimic changes in the intracellular milieu during hyperosmotic stress. The in vitro activities of MIPS-160 and IMPase 1 are highest at alkaline pH of 8.8. IMPase 1 catalytic efficiency is strongly increased during hyperosmolality (particularly for the substrate D-Ins-3-phosphate, Ins-3P, mainly as a result of [Na+] elevation. Furthermore, the substrate-specificity of IMPase 1 towards D-Ins-1-phosphate (Ins-1P is lower than towards Ins-3P. Because MIPS catalysis results in Ins-3P this results represents additional evidence for IMPase 1 being the isoform that mediates Ins biosynthesis in tilapia. Our data collectively demonstrate that the Ins biosynthesis enzymes are activated under ionic conditions that cells are exposed to during hypertonicity, resulting in Ins accumulation, which, in turn, results in restoration of intracellular ion homeostasis. We propose that the unique and direct ionic regulation of the activities of Ins biosynthesis enzymes represents an efficient biochemical feedback loop for regulation of intracellular physiological ion homeostasis during hyperosmotic stress.

Identification of Putative Genes Involved in Limonoids Biosynthesis in Citrus by Comparative Transcriptomic Analysis

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Fusheng Wang

2017-05-01

Full Text Available Limonoids produced by citrus are a group of highly bioactive secondary metabolites which provide health benefits for humans. Currently there is a lack of information derived from research on the genetic mechanisms controlling the biosynthesis of limonoids, which has limited the improvement of citrus for high production of limonoids. In this study, the transcriptome sequences of leaves, phloems and seeds of pummelo (Citrus grandis (L. Osbeck at different development stages with variances in limonoids contents were used for digital gene expression profiling analysis in order to identify the genes corresponding to the biosynthesis of limonoids. Pair-wise comparison of transcriptional profiles between different tissues identified 924 differentially expressed genes commonly shared between them. Expression pattern analysis suggested that 382 genes from three conjunctive groups of K-means clustering could be possibly related to the biosynthesis of limonoids. Correlation analysis with the samples from different genotypes, and different developing tissues of the citrus revealed that the expression of 15 candidate genes were highly correlated with the contents of limonoids. Among them, the cytochrome P450s (CYP450s and transcriptional factor MYB demonstrated significantly high correlation coefficients, which indicated the importance of those genes on the biosynthesis of limonoids. CiOSC gene encoding the critical enzyme oxidosqualene cyclase (OSC for biosynthesis of the precursor of triterpene scaffolds was found positively corresponding to the accumulation of limonoids during the development of seeds. Suppressing the expression of CiOSC with VIGS (Virus-induced gene silencing demonstrated that the level of gene silencing was significantly correlated to the reduction of limonoids contents. The results indicated that the CiOSC gene plays a pivotal role in biosynthesis of limonoids.

Direct Ionic Regulation of the Activity of Myo-Inositol Biosynthesis Enzymes in Mozambique Tilapia.

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Villarreal, Fernando D; Kültz, Dietmar

2015-01-01

Myo-inositol (Ins) is a major compatible osmolyte in many cells, including those of Mozambique tilapia (Oreochromis mossambicus). Ins biosynthesis is highly up-regulated in tilapia and other euryhaline fish exposed to hyperosmotic stress. In this study, enzymatic regulation of two enzymes of Ins biosynthesis, Ins phosphate synthase (MIPS) and inositol monophosphatase (IMPase), by direct ionic effects is analyzed. Specific MIPS and IMPase isoforms from Mozambique tilapia (MIPS-160 and IMPase 1) were selected based on experimental, phylogenetic, and structural evidence supporting their role for Ins biosynthesis during hyperosmotic stress. Recombinant tilapia IMPase 1 and MIPS-160 activity was assayed in vitro at ionic conditions that mimic changes in the intracellular milieu during hyperosmotic stress. The in vitro activities of MIPS-160 and IMPase 1 are highest at alkaline pH of 8.8. IMPase 1 catalytic efficiency is strongly increased during hyperosmolality (particularly for the substrate D-Ins-3-phosphate, Ins-3P), mainly as a result of [Na+] elevation. Furthermore, the substrate-specificity of IMPase 1 towards D-Ins-1-phosphate (Ins-1P) is lower than towards Ins-3P. Because MIPS catalysis results in Ins-3P this results represents additional evidence for IMPase 1 being the isoform that mediates Ins biosynthesis in tilapia. Our data collectively demonstrate that the Ins biosynthesis enzymes are activated under ionic conditions that cells are exposed to during hypertonicity, resulting in Ins accumulation, which, in turn, results in restoration of intracellular ion homeostasis. We propose that the unique and direct ionic regulation of the activities of Ins biosynthesis enzymes represents an efficient biochemical feedback loop for regulation of intracellular physiological ion homeostasis during hyperosmotic stress.

De novo fatty acid biosynthesis contributes significantly to establishment of a bioenergetically favorable environment for vaccinia virus infection.

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Greseth, Matthew D; Traktman, Paula

2014-03-01

The poxvirus life cycle, although physically autonomous from the host nucleus, is nevertheless dependent upon cellular functions. A requirement for de novo fatty acid biosynthesis was implied by our previous demonstration that cerulenin, a fatty acid synthase inhibitor, impaired vaccinia virus production. Here we show that additional inhibitors of this pathway, TOFA and C75, reduce viral yield significantly, with partial rescue provided by exogenous palmitate, the pathway's end-product. Palmitate's major role during infection is not for phospholipid synthesis or protein palmitoylation. Instead, the mitochondrial import and β-oxidation of palmitate are essential, as shown by the impact of etomoxir and trimetazidine, which target these two processes respectively. Moreover, the impact of these inhibitors is exacerbated in the absence of exogenous glucose, which is otherwise dispensable for infection. In contrast to glucose, glutamine is essential for productive viral infection, providing intermediates that sustain the TCA cycle (anaplerosis). Cumulatively, these data suggest that productive infection requires the mitochondrial β-oxidation of palmitate which drives the TCA cycle and energy production. Additionally, infection causes a significant rise in the cellular oxygen consumption rate (ATP synthesis) that is ablated by etomoxir. The biochemical progression of the vaccinia life cycle is not impaired in the presence of TOFA, C75, or etomoxir, although the levels of viral DNA and proteins synthesized are somewhat diminished. However, by reversibly arresting infections at the onset of morphogenesis, and then monitoring virus production after release of the block, we determined that virion assembly is highly sensitive to TOFA and C75. Electron microscopic analysis of cells released into C75 revealed fragmented aggregates of viroplasm which failed to be enclosed by developing virion membranes. Taken together, these data indicate that vaccinia infection, and in

De novo fatty acid biosynthesis contributes significantly to establishment of a bioenergetically favorable environment for vaccinia virus infection.

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Matthew D Greseth

2014-03-01

Full Text Available The poxvirus life cycle, although physically autonomous from the host nucleus, is nevertheless dependent upon cellular functions. A requirement for de novo fatty acid biosynthesis was implied by our previous demonstration that cerulenin, a fatty acid synthase inhibitor, impaired vaccinia virus production. Here we show that additional inhibitors of this pathway, TOFA and C75, reduce viral yield significantly, with partial rescue provided by exogenous palmitate, the pathway's end-product. Palmitate's major role during infection is not for phospholipid synthesis or protein palmitoylation. Instead, the mitochondrial import and β-oxidation of palmitate are essential, as shown by the impact of etomoxir and trimetazidine, which target these two processes respectively. Moreover, the impact of these inhibitors is exacerbated in the absence of exogenous glucose, which is otherwise dispensable for infection. In contrast to glucose, glutamine is essential for productive viral infection, providing intermediates that sustain the TCA cycle (anaplerosis. Cumulatively, these data suggest that productive infection requires the mitochondrial β-oxidation of palmitate which drives the TCA cycle and energy production. Additionally, infection causes a significant rise in the cellular oxygen consumption rate (ATP synthesis that is ablated by etomoxir. The biochemical progression of the vaccinia life cycle is not impaired in the presence of TOFA, C75, or etomoxir, although the levels of viral DNA and proteins synthesized are somewhat diminished. However, by reversibly arresting infections at the onset of morphogenesis, and then monitoring virus production after release of the block, we determined that virion assembly is highly sensitive to TOFA and C75. Electron microscopic analysis of cells released into C75 revealed fragmented aggregates of viroplasm which failed to be enclosed by developing virion membranes. Taken together, these data indicate that vaccinia

CD73 Protein as a Source of Extracellular Precursors for Sustained NAD+ Biosynthesis in FK866-treated Tumor Cells*

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Grozio, Alessia; Sociali, Giovanna; Sturla, Laura; Caffa, Irene; Soncini, Debora; Salis, Annalisa; Raffaelli, Nadia; De Flora, Antonio; Nencioni, Alessio; Bruzzone, Santina

2013-01-01

NAD+ is mainly synthesized in human cells via the “salvage” pathways starting from nicotinamide, nicotinic acid, or nicotinamide riboside (NR). The inhibition with FK866 of the enzyme nicotinamide phosphoribosyltransferase (NAMPT), catalyzing the first reaction in the “salvage” pathway from nicotinamide, showed potent antitumor activity in several preclinical models of solid and hematologic cancers. In the clinical studies performed with FK866, however, no tumor remission was observed. Here we demonstrate that low micromolar concentrations of extracellular NAD+ or NAD+ precursors, nicotinamide mononucleotide (NMN) and NR, can reverse the FK866-induced cell death, this representing a plausible explanation for the failure of NAMPT inhibition as an anti-cancer therapy. NMN is a substrate of both ectoenzymes CD38 and CD73, with generation of NAM and NR, respectively. In this study, we investigated the roles of CD38 and CD73 in providing ectocellular NAD+ precursors for NAD+ biosynthesis and in modulating cell susceptibility to FK866. By specifically silencing or overexpressing CD38 and CD73, we demonstrated that endogenous CD73 enables, whereas CD38 impairs, the conversion of extracellular NMN to NR as a precursor for intracellular NAD+ biosynthesis in human cells. Moreover, cell viability in FK866-treated cells supplemented with extracellular NMN was strongly reduced in tumor cells, upon pharmacological inhibition or specific down-regulation of CD73. Thus, our study suggests that genetic or pharmacologic interventions interfering with CD73 activity may prove useful to increase cancer cell sensitivity to NAMPT inhibitors. PMID:23880765

Absence of functional peroxisomes does not lead to deficiency of enzymes involved in cholesterol biosynthesis

NARCIS (Netherlands)

Hogenboom, Sietske; Romeijn, Gerrit Jan; Houten, Sander M.; Baes, Myriam; Wanders, Ronald J. A.; Waterham, Hans R.

2002-01-01

To unravel the conflicting data concerning the dependence of human cholesterol biosynthesis on functional peroxisomes, we determined activities and levels of selected enzymes involved in cholesterol biosynthesis in livers of PEX5 knockout mice, a well-characterized model for human Zellweger

Metformin Antagonizes Cancer Cell Proliferation by Suppressing Mitochondrial-Dependent Biosynthesis.

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Takla Griss

2015-12-01

Full Text Available Metformin is a biguanide widely prescribed to treat Type II diabetes that has gained interest as an antineoplastic agent. Recent work suggests that metformin directly antagonizes cancer cell growth through its actions on complex I of the mitochondrial electron transport chain (ETC. However, the mechanisms by which metformin arrests cancer cell proliferation remain poorly defined. Here we demonstrate that the metabolic checkpoint kinases AMP-activated protein kinase (AMPK and LKB1 are not required for the antiproliferative effects of metformin. Rather, metformin inhibits cancer cell proliferation by suppressing mitochondrial-dependent biosynthetic activity. We show that in vitro metformin decreases the flow of glucose- and glutamine-derived metabolic intermediates into the Tricarboxylic Acid (TCA cycle, leading to reduced citrate production and de novo lipid biosynthesis. Tumor cells lacking functional mitochondria maintain lipid biosynthesis in the presence of metformin via glutamine-dependent reductive carboxylation, and display reduced sensitivity to metformin-induced proliferative arrest. Our data indicate that metformin inhibits cancer cell proliferation by suppressing the production of mitochondrial-dependent metabolic intermediates required for cell growth, and that metabolic adaptations that bypass mitochondrial-dependent biosynthesis may provide a mechanism of tumor cell resistance to biguanide activity.

Comparative Transcriptome Analysis Identifies Putative Genes Involved in Steroid Biosynthesis in Euphorbia tirucalli

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Weibo Qiao

2018-01-01

Full Text Available Phytochemical analysis of different Euphorbia tirucalli tissues revealed a contrasting tissue-specificity for the biosynthesis of euphol and β-sitosterol, which represent the two pharmaceutically active steroids in E. tirucalli. To uncover the molecular mechanism underlying this tissue-specificity for phytochemicals, a comprehensive E. tirucalli transcriptome derived from its root, stem, leaf and latex was constructed, and a total of 91,619 unigenes were generated with 51.08% being successfully annotated against the non-redundant (Nr protein database. A comparison of the transcriptome from different tissues discovered members of unigenes in the upstream steps of sterol backbone biosynthesis leading to this tissue-specific sterol biosynthesis. Among them, the putative oxidosqualene cyclase (OSC encoding genes involved in euphol synthesis were notably identified, and their expressions were significantly up-regulated in the latex. In addition, genome-wide differentially expressed genes (DEGs in the different E. tirucalli tissues were identified. The cluster analysis of those DEGs showed a unique expression pattern in the latex compared with other tissues. The DEGs identified in this study would enrich the insights of sterol biosynthesis and the regulation mechanism of this latex-specificity.

Comparative Analysis of Tocopherol Biosynthesis Genes and Its Transcriptional Regulation in Soybean Seeds.

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T, Vinutha; Bansal, Navita; Kumari, Khushboo; Prashat G, Rama; Sreevathsa, Rohini; Krishnan, Veda; Kumari, Sweta; Dahuja, Anil; Lal, S K; Sachdev, Archana; Praveen, Shelly

2017-12-20

Tocopherols composed of four isoforms (α, β, γ, and δ) and its biosynthesis comprises of three pathways: methylerythritol 4-phosphate (MEP), shikimate (SK) and tocopherol-core pathways regulated by 25 enzymes. To understand pathway regulatory mechanism at transcriptional level, gene expression profile of tocopherol-biosynthesis genes in two soybean genotypes was carried out, the results showed significantly differential expression of 5 genes: 1-deoxy-d-xylulose-5-P-reductoisomerase (DXR), geranyl geranyl reductase (GGDR) from MEP, arogenate dehydrogenase (TyrA), tyrosine aminotransferase (TAT) from SK and γ-tocopherol methyl transferase 3 (γ-TMT3) from tocopherol-core pathways. Expression data were further analyzed for total tocopherol (T-toc) and α-tocopherol (α-toc) content by coregulation network and gene clustering approaches, the results showed least and strong association of γ-TMT3/tocopherol cyclase (TC) and DXR/DXS, respectively, with gene clusters of tocopherol biosynthesis suggested the specific role of γ-TMT3/TC in determining tocopherol accumulation and intricacy of DXR/DXS genes in coordinating precursor pathways toward tocopherol biosynthesis in soybean seeds. Thus, the present study provides insight into the major role of these genes regulating the tocopherol synthesis in soybean seeds.

Blockade of oestrogen biosynthesis in peripubertal boys: effects on lipid metabolism, insulin sensitivity, and body composition.

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Hero, Matti; Ankarberg-Lindgren, Carina; Taskinen, Marja-Riitta; Dunkel, Leo

2006-09-01

In males, the pubertal increase in sex hormone production has been associated with proatherogenic changes in lipid and carbohydrate metabolism. Aromatase inhibitors, a novel treatment modality for some growth disorders, may significantly influence these risk factors for cardiovascular disease by suppressing oestrogen biosynthesis and stimulating gonadal androgen production. In the current study, we explored the effects of aromatase inhibition on lipid metabolism, insulin sensitivity, body composition and serum adiponectin in peripubertal boys. Prospective, double-blind, randomised, placebo-controlled clinical study. Thirty-one boys, aged 9.0-14.5 years, with idiopathic short stature were treated with the aromatase inhibitor letrozole (2.5 mg/day) or placebo for 2 years. During the treatment, the concentrations of sex hormones, IGF-I, lipids, lipoproteins and adiponectin were followed-up. The percentage of fat mass (FM) was assessed by skinfold measurements and insulin resistance by homeostasis model assessment (HOMA) index. In pubertal boys, who received letrozole, high-density lipoprotein cholesterol (HDL-C) decreased by 0.47 mmol/l (P<0.01) during the study. Simultaneously, their percentage of FM decreased from 17.0 to 10.5 (P<0.001), in an inverse relationship with serum testosterone. The concentrations of low-density lipoprotein cholesterol, triglycerides and HOMA index remained at pretreatment level in both groups. Serum adiponectin decreased similarly in letrozole- and placebo-treated pubertal boys (2.9 and 3.3 mg/l respectively). In males, aromatase inhibition reduces HDL-C and decreases relative FM after the start of puberty. The treatment does not adversely affect insulin sensitivity in lean subjects.

Effects of nitrogen availability on polymalic acid biosynthesis in the yeast-like fungus Aureobasidium pullulans.

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Wang, Yongkang; Song, Xiaodan; Zhang, Yongjun; Wang, Bochu; Zou, Xiang

2016-08-22

Polymalic acid (PMA) is a novel polyester polymer that has been broadly used in the medical and food industries. Its monomer, L-malic acid, is also a potential C4 platform chemical. However, little is known about the mechanism of PMA biosynthesis in the yeast-like fungus, Aureobasidium pullulans. In this study, the effects of different nitrogen concentration on cell growth and PMA biosynthesis were investigated via comparative transcriptomics and proteomics analyses, and a related signaling pathway was also evaluated. A high final PMA titer of 44.00 ± 3.65 g/L (49.9 ± 4.14 g/L of malic acid after hydrolysis) was achieved in a 5-L fermentor under low nitrogen concentration (2 g/L of NH4NO3), which was 18.3 % higher yield than that obtained under high nitrogen concentration (10 g/L of NH4NO3). Comparative transcriptomics profiling revealed that a set of genes, related to the ribosome, ribosome biogenesis, proteasome, and nitrogen metabolism, were significantly up- or down-regulated under nitrogen sufficient conditions, which could be regulated by the TOR signaling pathway. Fourteen protein spots were identified via proteomics analysis, and were found to be associated with cell division and growth, energy metabolism, and the glycolytic pathway. qRT-PCR further confirmed that the expression levels of key genes involved in the PMA biosynthetic pathway (GLK, CS, FUM, DAT, and MCL) and the TOR signaling pathway (GS, TOR1, Tap42, and Gat1) were upregulated due to nitrogen limitation. Under rapamycin stress, PMA biosynthesis was obviously inhibited in a dose-dependent manner, and the transcription levels of TOR1, MCL, and DAT were also downregulated. The level of nitrogen could regulate cell growth and PMA biosynthesis. Low concentration of nitrogen was beneficial for PMA biosynthesis, which could upregulate the expression of key genes involved in the PMA biosynthesis pathway. Cell growth and PMA biosynthesis might be mediated by the TOR signaling pathway in

Direct observation of the effects of cellulose synthesis inhibitors using live cell imaging of Cellulose Synthase (CESA) in Physcomitrella patens.

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Tran, Mai L; McCarthy, Thomas W; Sun, Hao; Wu, Shu-Zon; Norris, Joanna H; Bezanilla, Magdalena; Vidali, Luis; Anderson, Charles T; Roberts, Alison W

2018-01-15

Results from live cell imaging of fluorescently tagged Cellulose Synthase (CESA) proteins in Cellulose Synthesis Complexes (CSCs) have enhanced our understanding of cellulose biosynthesis, including the mechanisms of action of cellulose synthesis inhibitors. However, this method has been applied only in Arabidopsis thaliana and Brachypodium distachyon thus far. Results from freeze fracture electron microscopy of protonemal filaments of the moss Funaria hygrometrica indicate that a cellulose synthesis inhibitor, 2,6-dichlorobenzonitrile (DCB), fragments CSCs and clears them from the plasma membrane. This differs from Arabidopsis, in which DCB causes CSC accumulation in the plasma membrane and a different cellulose synthesis inhibitor, isoxaben, clears CSCs from the plasma membrane. In this study, live cell imaging of the moss Physcomitrella patens indicated that DCB and isoxaben have little effect on protonemal growth rates, and that only DCB causes tip rupture. Live cell imaging of mEGFP-PpCESA5 and mEGFP-PpCESA8 showed that DCB and isoxaben substantially reduced CSC movement, but had no measureable effect on CSC density in the plasma membrane. These results suggest that DCB and isoxaben have similar effects on CSC movement in P. patens and Arabidopsis, but have different effects on CSC intracellular trafficking, cell growth and cell integrity in these divergent plant lineages.

Desleucyl-Oritavancin with a Damaged d-Ala-d-Ala Binding Site Inhibits the Transpeptidation Step of Cell-Wall Biosynthesis in Whole Cells of Staphylococcus aureus.

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Kim, Sung Joon; Singh, Manmilan; Sharif, Shasad; Schaefer, Jacob

2017-03-14

We have used solid-state nuclear magnetic resonance to characterize the exact nature of the dual mode of action of oritavancin in preventing cell-wall assembly in Staphylococcus aureus. Measurements performed on whole cells labeled selectively in vivo have established that des-N-methylleucyl-N-4-(4-fluorophenyl)benzyl-chloroeremomycin, an Edman degradation product of [ 19 F]oritavancin, which has a damaged d-Ala-d-Ala binding aglycon, is a potent inhibitor of the transpeptidase activity of cell-wall biosynthesis. The desleucyl drug binds to partially cross-linked peptidoglycan by a cleft formed between the drug aglycon and its biphenyl hydrophobic side chain. This type of binding site is present in other oritavancin-like glycopeptides, which suggests that for these drugs a similar transpeptidase inhibition occurs.

Pathways and Subcellular Compartmentation of NAD Biosynthesis in Human Cells

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Nikiforov, Andrey; Dölle, Christian; Niere, Marc; Ziegler, Mathias

2011-01-01

NAD is a vital redox carrier, and its degradation is a key element of important regulatory pathways. NAD-mediated functions are compartmentalized and have to be fueled by specific biosynthetic routes. However, little is known about the different pathways, their subcellular distribution, and regulation in human cells. In particular, the route(s) to generate mitochondrial NAD, the largest subcellular pool, is still unknown. To visualize organellar NAD changes in cells, we targeted poly(ADP-ribose) polymerase activity into the mitochondrial matrix. This activity synthesized immunodetectable poly(ADP-ribose) depending on mitochondrial NAD availability. Based on this novel detector system, detailed subcellular enzyme localizations, and pharmacological inhibitors, we identified extracellular NAD precursors, their cytosolic conversions, and the pathway of mitochondrial NAD generation. Our results demonstrate that, besides nicotinamide and nicotinic acid, only the corresponding nucleosides readily enter the cells. Nucleotides (e.g. NAD and NMN) undergo extracellular degradation resulting in the formation of permeable precursors. These precursors can all be converted to cytosolic and mitochondrial NAD. For mitochondrial NAD synthesis, precursors are converted to NMN in the cytosol. When taken up into the organelles, NMN (together with ATP) serves as substrate of NMNAT3 to form NAD. NMNAT3 was conclusively localized to the mitochondrial matrix and is the only known enzyme of NAD synthesis residing within these organelles. We thus present a comprehensive dissection of mammalian NAD biosynthesis, the groundwork to understand regulation of NAD-mediated processes, and the organismal homeostasis of this fundamental molecule. PMID:21504897

HOG MAP kinase regulation of alternariol biosynthesis in Alternaria alternata is important for substrate colonization.

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Graf, Eva; Schmidt-Heydt, Markus; Geisen, Rolf

2012-07-16

Strains of the genus Alternaria are ubiquitously present and frequently found on fruits, vegetables and cereals. One of the most commonly found species from this genus is A. alternata which is able to produce the mycotoxin alternariol among others. To date only limited knowledge is available about the regulation of the biosynthesis of alternariol, especially under conditions relevant to food. Tomatoes are a typical substrate of A. alternata and have a high water activity. On the other hand cereals with moderate water activity are also frequently colonized by A. alternata. In the current analysis it was demonstrated that even minor changes in the osmotic status of the substrate affect the alternariol biosynthesis of strains from vegetables resulting in nearly complete inhibition. High osmolarity in the environment is usually transmitted to the transcriptional level of downstream regulated genes by the HOG signal cascade (high osmolarity glycerol cascade) which is a MAP kinase transduction pathway. The phosphorylation status of the A. alternata HOG (AaHOG) was determined. Various concentrations of NaCl induce the phosphorylation of AaHOG in a concentration, time and strain dependent manner. A strain with a genetically inactivated aahog gene was no longer able to produce alternariol indicating that the activity of the aahog gene is required for alternariol biosynthesis. Further experiments revealed that the biosynthesis of alternariol is important for the fungus to colonize tomato tissue. The tight water activity dependent regulation of alternariol biosynthesis ensures alternariol biosynthesis at conditions which indicate an optimal colonization substrate for the fungus. Copyright © 2012 Elsevier B.V. All rights reserved.

Engineering Escherichia coli Nicotinic Acid Mononucleotide Adenylyltransferase for Fully Active Amidated NAD Biosynthesis.

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Wang, Xueying; Zhou, Yongjin J; Wang, Lei; Liu, Wujun; Liu, Yuxue; Peng, Chang; Zhao, Zongbao K

2017-07-01

NAD and its reduced form NADH function as essential redox cofactors and have major roles in determining cellular metabolic features. NAD can be synthesized through the deamidated and amidated pathways, for which the key reaction involves adenylylation of nicotinic acid mononucleotide (NaMN) and nicotinamide mononucleotide (NMN), respectively. In Escherichia coli , NAD de novo biosynthesis depends on the protein NadD-catalyzed adenylylation of NaMN to nicotinic acid adenine dinucleotide (NaAD), followed by NAD synthase-catalyzed amidation. In this study, we engineered NadD to favor NMN for improved amidated pathway activity. We designed NadD mutant libraries, screened by a malic enzyme-coupled colorimetric assay, and identified two variants, 11B4 (Y84V/Y118D) and 16D8 (A86W/Y118N), with a high preference for NMN. Whereas in the presence of NMN both variants were capable of enabling the viability of cells of E. coli BW25113-derived NAD-auxotrophic strain YJE003, for which the last step of the deamidated pathway is blocked, the 16D8 expression strain could grow without exogenous NMN and accumulated a higher cellular NAD(H) level than BW25113 in the stationary phase. These mutants established fully active amidated NAD biosynthesis and offered a new opportunity to manipulate NAD metabolism for biocatalysis and metabolic engineering. IMPORTANCE Adenylylation of nicotinic acid mononucleotide (NaMN) and adenylylation of nicotinamide mononucleotide (NMN), respectively, are the key steps in the deamidated and amidated pathways for NAD biosynthesis. In most organisms, canonical NAD biosynthesis follows the deamidated pathway. Here we engineered Escherichia coli NaMN adenylyltransferase to favor NMN and expressed the mutant enzyme in an NAD-auxotrophic E. coli strain that has the last step of the deamidated pathway blocked. The engineered strain survived in M9 medium, which indicated the implementation of a functional amidated pathway for NAD biosynthesis. These results enrich

ODORANT1 Regulates Fragrance Biosynthesis in Petunia FlowersW⃞

Science.gov (United States)

Verdonk, Julian C.; Haring, Michel A.; van Tunen, Arjen J.; Schuurink, Robert C.

2005-01-01

Floral scent is important to plant reproduction because it attracts pollinators to the sexual organs. Therefore, volatile emission is usually tuned to the foraging activity of the pollinators. In Petunia hybrida, volatile benzenoids determine the floral aroma. Although the pathways for benzenoid biosynthesis have been characterized, the enzymes involved are less well understood. How production and emission are regulated is unknown. By targeted transcriptome analyses, we identified ODORANT1 (ODO1), a member of the R2R3-type MYB family, as a candidate for the regulation of volatile benzenoids in Petunia hybrida cv W115 (Mitchell) flowers. These flowers are only fragrant in the evening and at night. Transcript levels of ODO1 increased before the onset of volatile emission and decreased when volatile emission declined. Downregulation of ODO1 in transgenic P. hybrida Mitchell plants strongly reduced volatile benzenoid levels through decreased synthesis of precursors from the shikimate pathway. The transcript levels of several genes in this pathway were reduced by suppression of ODO1 expression. Moreover, ODO1 could activate the promoter of the 5-enol-pyruvylshikimate-3-phosphate synthase gene. Flower pigmentation, which is furnished from the same shikimate precursors, was not influenced because color and scent biosynthesis occur at different developmental stages. Our studies identify ODO1 as a key regulator of floral scent biosynthesis. PMID:15805488

MRE: a web tool to suggest foreign enzymes for the biosynthesis pathway design with competing endogenous reactions in mind

KAUST Repository

Kuwahara, Hiroyuki; Alazmi, Meshari; Cui, Xuefeng; Gao, Xin

2016-01-01

To rationally design a productive heterologous biosynthesis system, it is essential to consider the suitability of foreign reactions for the specific endogenous metabolic infrastructure of a host. We developed a novel web server, called MRE, which, for a given pair of starting and desired compounds in a given chassis organism, ranks biosynthesis routes from the perspective of the integration of new reactions into the endogenous metabolic system. For each promising heterologous biosynthesis pathway, MRE suggests actual enzymes for foreign metabolic reactions and generates information on competing endogenous reactions for the consumption of metabolites. These unique, chassis-centered features distinguish MRE from existing pathway design tools and allow synthetic biologists to evaluate the design of their biosynthesis systems from a different angle. By using biosynthesis of a range of high-value natural products as a case study, we show that MRE is an effective tool to guide the design and optimization of heterologous biosynthesis pathways. The URL of MRE is http://www.cbrc.kaust.edu.sa/mre/.

MRE: a web tool to suggest foreign enzymes for the biosynthesis pathway design with competing endogenous reactions in mind

KAUST Repository

Kuwahara, Hiroyuki

2016-04-29

To rationally design a productive heterologous biosynthesis system, it is essential to consider the suitability of foreign reactions for the specific endogenous metabolic infrastructure of a host. We developed a novel web server, called MRE, which, for a given pair of starting and desired compounds in a given chassis organism, ranks biosynthesis routes from the perspective of the integration of new reactions into the endogenous metabolic system. For each promising heterologous biosynthesis pathway, MRE suggests actual enzymes for foreign metabolic reactions and generates information on competing endogenous reactions for the consumption of metabolites. These unique, chassis-centered features distinguish MRE from existing pathway design tools and allow synthetic biologists to evaluate the design of their biosynthesis systems from a different angle. By using biosynthesis of a range of high-value natural products as a case study, we show that MRE is an effective tool to guide the design and optimization of heterologous biosynthesis pathways. The URL of MRE is http://www.cbrc.kaust.edu.sa/mre/.

Molecular analysis of "de novo" purine biosynthesis in solanaceous species and in Arabidopsis thaliana

DEFF Research Database (Denmark)

van der Graaff, Eric; Hooykaas, Paul; Lein, Wolfgang

2004-01-01

Purine nucleotides are essential components to sustain plant growth and development. In plants they are either synthesized "de novo" during the process of purine biosynthesis or are recycled from purine bases and purine nucleosides throughout the salvage pathway. Comparison between animals...... biosynthesis pathway in plants, and the in planta functional analysis of PRPP (5-phosphoribosyl-1-pyrophoshate) amidotransferase (ATase), catalyzing the first committed step of the "de novo" purine biosynthesis. The cloning of the genes involved in the purine biosynthesis pathway was attained by a screening...... strategy with heterologous cDNA probes and by using S. cerevisiae mutants for complementation. Southern hybridization showed a complex genomic organization for these genes in solanaceous species and their organ- and developmental specific expression was analyzed by Northern hybridization. The specific role...

BIOSYNTHESIS AND ACTION OF JASMONATES IN PLANTS.

Science.gov (United States)

Creelman, Robert A.; Mullet, John E.

1997-06-01

Jasmonic acid and its derivatives can modulate aspects of fruit ripening, production of viable pollen, root growth, tendril coiling, and plant resistance to insects and pathogens. Jasmonate activates genes involved in pathogen and insect resistance, and genes encoding vegetative storage proteins, but represses genes encoding proteins involved in photosynthesis. Jasmonic acid is derived from linolenic acid, and most of the enzymes in the biosynthetic pathway have been extensively characterized. Modulation of lipoxygenase and allene oxide synthase gene expression in transgenic plants raises new questions about the compartmentation of the biosynthetic pathway and its regulation. The activation of jasmonic acid biosynthesis by cell wall elicitors, the peptide systemin, and other compounds will be related to the function of jasmonates in plants. Jasmonate modulates gene expression at the level of translation, RNA processing, and transcription. Promoter elements that mediate responses to jasmonate have been isolated. This review covers recent advances in our understanding of how jasmonate biosynthesis is regulated and relates this information to knowledge of jasmonate modulated gene expression.

Triterpene biosynthesis in plants.

Science.gov (United States)

Thimmappa, Ramesha; Geisler, Katrin; Louveau, Thomas; O'Maille, Paul; Osbourn, Anne

2014-01-01

The triterpenes are one of the most numerous and diverse groups of plant natural products. They are complex molecules that are, for the most part, beyond the reach of chemical synthesis. Simple triterpenes are components of surface waxes and specialized membranes and may potentially act as signaling molecules, whereas complex glycosylated triterpenes (saponins) provide protection against pathogens and pests. Simple and conjugated triterpenes have a wide range of applications in the food, health, and industrial biotechnology sectors. Here, we review recent developments in the field of triterpene biosynthesis, give an overview of the genes and enzymes that have been identified to date, and discuss strategies for discovering new triterpene biosynthetic pathways.

A directed-overflow and damage-control N-glycosidase in riboflavin biosynthesis

Science.gov (United States)

Frelin, Océane; Huang, Lili; Hasnain, Ghulam; Jeffryes, James G.; Ziemak, Michael J.; Rocca, James R.; Wang, Bing; Rice, Jennifer; Roje, Sanja; Yurgel, Svetlana N.; Gregory, Jesse F.; Edison, Arthur S.; Henry, Christopher S.; deCrécy-Lagard, Valérie; Hanson, Andrew D.

2015-01-01

Plants and bacteria synthesize the essential human micronutrient riboflavin (vitamin B2) via the same multistep pathway. The early intermediates of this pathway are notoriously reactive, and may be overproduced in vivo because riboflavin biosynthesis enzymes lack feedback controls. Here we demonstrate disposal of riboflavin intermediates by COG3236 (DUF1768), a protein of previously unknown function that is fused to two different riboflavin pathway enzymes in plants and bacteria (RIBR and RibA, respectively). We present cheminformatic, biochemical, genetic, and genomic evidence to show that: (i) plant and bacterial COG3236 proteins cleave the N-glycosidic bond of the first two intermediates of riboflavin biosynthesis, yielding relatively innocuous products; (ii) certain COG3236 proteins are in a multienzyme riboflavin biosynthesis complex that gives them privileged access to riboflavin intermediates; and (iii) COG3236 action in Arabidopsis thaliana and Escherichia coli helps maintain flavin levels. COG3236 proteins thus illustrate two emerging principles in chemical biology: directed overflow metabolism, in which excess flux is diverted out of a pathway, and the pre-emption of damage from reactive metabolites. PMID:25431972

Biosynthesis of gold nanoparticles by actinomycete Streptomyces viridogens strain HM10.

Science.gov (United States)

Balagurunathan, R; Radhakrishnan, M; Rajendran, R Babu; Velmurugan, D

2011-10-01

Biosynthesis of gold nanoparticles by Streptomycetes from Himalayan Mountain was undertaken for the first time. Out of 10 actinomycete strains tested, four strains (D10, HM10, ANS2 and MSU) showed evidence for the intracellular biosynthesis of gold nanoparticles, among which the strain HM10 showed high potency. Presence of spherical and rod shaped gold nanoparticles in mycelium of the strain HM10 was determined by transmission electron microscopy (TEM) and X-ray diffraction analysis. The average particle size ranged from 18-20 nm. UV spectral analysis indicated that the reduction of chloroauric acid (HAuCl4) occurred within 24 h of reaction period. Further, the strain HM10 showed enhanced growth at 1 and 10 mM concentration of HAuCl4. The gold nanoparticles synthesized by the strain HM10 showed good antibacterial activity against S. aureus and E. coli in well-diffusion method. The potential actinomycete HM10 strain was phenotypically characterized and identified as Streptomyces viridogens (HM10). Thus, actinomycete strain HM10 reported in this study is a newly added source for the biosynthesis of gold nanoparticles.

Inhibitors

Science.gov (United States)

... JM, and the Hemophilia Inhibitor Research Study Investigators. Validation of Nijmegen-Bethesda assay modifications to allow inhibitor ... webinars on blood disorders Language: English (US) Español (Spanish) File Formats Help: How do I view different ...

Macromolecule biosynthesis assay and fluorescence spectroscopy methods to explore antimicrobial peptide mode(s) of action

DEFF Research Database (Denmark)

Jana, Bimal; Baker, Kristin Renee; Guardabassi, Luca

2017-01-01

the biosynthesis rate of macromolecules (e.g., DNA, RNA, protein, and cell wall) and the cytoplasmic membrane proton motive force (PMF) energy can help to unravel the diverse modes of action of AMPs. Here, we present an overview of macromolecule biosynthesis rate measurement and fluorescence spectroscopy methods...

Pharmacophore modeling and in silico / in vitro screening for human cytochrome P450 11B1 & cytochrome P450 11B2 inhibitors

Science.gov (United States)

Akram, Muhammad; Waratchareeyakul, Watcharee; Haupenthal, Joerg; Hartmann, Rolf W.; Schuster, Daniela

2017-12-01

Cortisol synthase (CYP11B1) is the main enzyme for the endogenous synthesis of cortisol and its inhibition is a potential way for the treatment of diseases associated with increased cortisol levels, such as Cushing’s syndrome, metabolic diseases, and delayed wound healing. Aldosterone synthase (CYP11B2) is the key enzyme for aldosterone biosynthesis and its inhibition is a promising approach for the treatment of congestive heart failure, cardiac fibrosis, and certain forms of hypertension. Both CYP11B1 and CYP11B2 are structurally very similar and expressed in the adrenal cortex. To facilitate the identification of novel inhibitors of these enzymes, ligand-based pharmacophore models of CYP11B1 and CYP11B2 inhibition were developed. A virtual screening of the SPECS database was performed with our pharmacophore queries. Biological evaluation of the selected hits lead to the discovery of three potent novel inhibitors of both CYP11B1 and CYP11B2 in the submicromolar range (compounds 8-10), one selective CYP11B1 inhibitor (Compound 11, IC50 = 2.5 µM), and one selective CYP11B2 inhibitor (compound 12, IC50 = 1.1 µM), respectively. The overall success rate of this prospective virtual screening experiment is 20.8% indicating good predictive power of the pharmacophore models.

Biosynthesis and therapeutic properties of Lavandula essential oil constituents.

Science.gov (United States)

Woronuk, Grant; Demissie, Zerihun; Rheault, Mark; Mahmoud, Soheil

2011-01-01

Lavenders and their essential oils have been used in alternative medicine for several centuries. The volatile compounds that comprise lavender essential oils, including linalool and linalyl acetate, have demonstrative therapeutic properties, and the relative abundance of these metabolites is greatly influenced by the genetics and environment of the developing plants. With the rapid progress of molecular biology and the genomic sciences, our understanding of essential oil biosynthesis has greatly improved over the past few decades. At the same time, there is a recent surge of interest in the use of natural remedies, including lavender essential oils, in alternative medicine and aromatherapy. This article provides a review of recent developments related to the biosynthesis and medicinal properties of lavender essential oils. © Georg Thieme Verlag KG Stuttgart · New York.

[A systematic review of biosynthesis of poly (3-hydroxypropionate)].

Science.gov (United States)

Chang, Le; Zhan, Yuanlong; Liu, Changli

2018-04-25

Poly (3-hydroxypropionate) (P3HP), a new member of thermoplastic of family polyhydroxyalkanoates (PHAs), has excellent characteristics of biodegradability and biocompatibility. By now no reports can be found about wild-type bacteria that naturally synthesize P3HP, so the main way to produce P3HP is chemical and biological methods. Chemical method by adding high cost 3-HP monomers or their structural analogs as precursors, has the drawbacks of toxicity, low effectiveness and high cost. Biological method using engineered strain may utilize inexpensive and renewable carbon source to produce P3HP and has gradually become more and more popular. We systematically review here the biosynthesis of P3HP research progress. The advantages and disadvantages of biosynthesis pathways of glycerol pathway, malonyl-CoA pathway and β-alanine pathway were analyzed.

The Fatty Acid Biosynthesis Enzyme FabI Plays a Key Role In the Development of Liver Stage Malarial Parasites

Science.gov (United States)

Yu, Min; Santha Kumar, T. R.; Nkrumah, Louis J.; Coppi, Alida; Retzlaff, Silke; Li, Celeste D.; Kelly, Brendan J.; Moura, Pedro A.; Lakshmanan, Viswanathan; Freundlich, Joel S.; Valderramos, Juan-Carlos; Vilcheze, Catherine; Siedner, Mark; Tsai, Jennifer H.-C.; Falkard, Brie; Sidhu, Amar bir Singh; Purcell, Lisa A.; Gratraud, Paul; Kremer, Laurent; Waters, Andy P.; Schiehser, Guy; Jacobus, David P.; Janse, Chris J.; Ager, Arba; Jacobs, William R.; Sacchettini, James C.; Heussler, Volker; Sinnis, Photini; Fidock, David A.

2008-01-01

SUMMARY Fatty acid biosynthesis has been viewed as an important biological function of and therapeutic target for Plasmodium falciparum asexual blood stage infection. This apicoplast-resident type II pathway, distinct from the mammalian type I process, includes FabI. Here, we report synthetic chemistry and transfection studies concluding that Plasmodium FabI is not the target of the antimalarial activity of the bacterial FabI inhibitor triclosan. Disruption of fabI in P. falciparum or the rodent parasite P. berghei does not impede blood stage growth. In contrast, mosquito-derived fabI-deficient P. berghei sporozoites are markedly less infective for mice and typically fail to complete liver stage development in vitro. This is characterized by an inability to form intra-hepatic merosomes that normally initiate blood stage infections. These data illuminate key differences between liver and blood stage parasites in their requirements for host versus de novo synthesized fatty acids, and create new prospects for stage-specific antimalarial interventions. PMID:19064257

Production of New Cladosporin Analogues by Reconstitution of the Polyketide Synthases Responsible for the Biosynthesis of this Antimalarial Agent.

Science.gov (United States)

Cochrane, Rachel V K; Sanichar, Randy; Lambkin, Gareth R; Reiz, Béla; Xu, Wei; Tang, Yi; Vederas, John C

2016-01-11

The antimalarial agent cladosporin is a nanomolar inhibitor of the Plasmodium falciparum lysyl-tRNA synthetase, and exhibits activity against both blood- and liver-stage infection. Cladosporin can be isolated from the fungus Cladosporium cladosporioides, where it is biosynthesized by a highly reducing (HR) and a non-reducing (NR) iterative type I polyketide synthase (PKS) pair. Genome sequencing of the host organism and subsequent heterologous expression of these enzymes in Saccharomyces cerevisiae produced cladosporin, confirming the identity of the putative gene cluster. Incorporation of a pentaketide intermediate analogue indicated a 5+3 assembly by the HR PKS Cla2 and the NR PKS Cla3 during cladosporin biosynthesis. Advanced-intermediate analogues were synthesized and incorporated by Cla3 to furnish new cladosporin analogues. A putative lysyl-tRNA synthetase resistance gene was identified in the cladosporin gene cluster. Analysis of the active site emphasizes key structural features thought to be important in resistance to cladosporin. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Monomethylarsonous acid inhibited endogenous cholesterol biosynthesis in human skin fibroblasts

Energy Technology Data Exchange (ETDEWEB)

Guo, Lei [Environmental Toxicology Graduate Program, University of California, Riverside, CA 92521-0403 (United States); Xiao, Yongsheng [Department of Chemistry, University of California, Riverside, CA 92521-0403 (United States); Wang, Yinsheng, E-mail: yinsheng.wang@ucr.edu [Environmental Toxicology Graduate Program, University of California, Riverside, CA 92521-0403 (United States); Department of Chemistry, University of California, Riverside, CA 92521-0403 (United States)

2014-05-15

Human exposure to arsenic in drinking water is a widespread public health concern, and such exposure is known to be associated with many human diseases. The detailed molecular mechanisms about how arsenic species contribute to the adverse human health effects, however, remain incompletely understood. Monomethylarsonous acid [MMA(III)] is a highly toxic and stable metabolite of inorganic arsenic. To exploit the mechanisms through which MMA(III) exerts its cytotoxic effect, we adopted a quantitative proteomic approach, by coupling stable isotope labeling by amino acids in cell culture (SILAC) with LC-MS/MS analysis, to examine the variation in the entire proteome of GM00637 human skin fibroblasts following acute MMA(III) exposure. Among the ∼ 6500 unique proteins quantified, ∼ 300 displayed significant changes in expression after exposure with 2 μM MMA(III) for 24 h. Subsequent analysis revealed the perturbation of de novo cholesterol biosynthesis, selenoprotein synthesis and Nrf2 pathways evoked by MMA(III) exposure. Particularly, MMA(III) treatment resulted in considerable down-regulation of several enzymes involved in cholesterol biosynthesis. In addition, real-time PCR analysis showed reduced mRNA levels of select genes in this pathway. Furthermore, MMA(III) exposure contributed to a distinct decline in cellular cholesterol content and significant growth inhibition of multiple cell lines, both of which could be restored by supplementation of cholesterol to the culture media. Collectively, the present study demonstrated that the cytotoxicity of MMA(III) may arise, at least in part, from the down-regulation of cholesterol biosynthesis enzymes and the resultant decrease of cellular cholesterol content. - Highlights: • MMA(III)-induced perturbation of the entire proteome of GM00637 cells is studied. • Quantitative proteomic approach revealed alterations of multiple cellular pathways. • MMA(III) inhibits de novo cholesterol biosynthesis. • MMA

Effects of inhibiting CoQ10 biosynthesis with 4-nitrobenzoate in human fibroblasts.

Directory of Open Access Journals (Sweden)

Catarina M Quinzii

Full Text Available Coenzyme Q(10 (CoQ(10 is a potent lipophilic antioxidant in cell membranes and a carrier of electrons in the mitochondrial respiratory chain. We previously characterized the effects of varying severities of CoQ(10 deficiency on ROS production and mitochondrial bioenergetics in cells harboring genetic defects of CoQ(10 biosynthesis. We observed a unimodal distribution of ROS production with CoQ(10 deficiency: cells with <20% of CoQ(10 and 50-70% of CoQ(10 did not generate excess ROS while cells with 30-45% of CoQ(10 showed increased ROS production and lipid peroxidation. Because our previous studies were limited to a small number of mutant cell lines with heterogeneous molecular defects, here, we treated 5 control and 2 mildly CoQ(10 deficient fibroblasts with varying doses of 4-nitrobenzoate (4-NB, an analog of 4-hydroxybenzoate (4-HB and inhibitor of 4-para-hydroxybenzoate:polyprenyl transferase (COQ2 to induce a range of CoQ(10 deficiencies. Our results support the concept that the degree of CoQ(10 deficiency in cells dictates the extent of ATP synthesis defects and ROS production and that 40-50% residual CoQ(10 produces maximal oxidative stress and cell death.

Transcriptome Analysis of Genes Involved in Lipid Biosynthesis in the Developing Embryo of Pecan (Carya illinoinensis).

Science.gov (United States)

Huang, Ruimin; Huang, Youjun; Sun, Zhichao; Huang, Jianqin; Wang, Zhengjia

2017-05-24

Pecan (Carya illinoinensis) is an important woody tree species because of the high content of healthy oil in its nut. Thus far, the pathways and key genes related to oil biosynthesis in developing pecan seeds remain largely unclear. Our analyses revealed that mature pecan embryo accumulated more than 80% oil, in which 90% was unsaturated fatty acids with abundant oleic acid. RNA sequencing generated 84,643 unigenes in three cDNA libraries prepared from pecan embryos collected at 105, 120, and 165 days after flowering (DAF). We identified 153 unigenes associated with lipid biosynthesis, including 107 unigenes for fatty acid biosynthesis, 34 for triacylglycerol biosynthesis, 7 for oil bodies, and 5 for transcription factors involved in oil synthesis. The genes associated with fatty acid synthesis were the most abundantly expressed genes at 120 DAF. Additionally, the biosynthesis of oil began to increase while crude fat contents increased from 16.61 to 74.45% (165 DAF). We identified four SAD, two FAD2, one FAD6, two FAD7, and two FAD8 unigenes responsible for unsaturated fatty acid biosynthesis. However, FAD3 homologues were not detected. Consequently, we inferred that the linolenic acid in developing pecan embryos is generated by FAD7 and FAD8 in plastids rather than FAD3 in endoplasmic reticula. During pecan embryo development, different unigenes are expressed for plastidial and cytosolic glycolysis. Plastidial glycolysis is more relevant to lipid synthesis than cytosolic glycolysis. The 18 most important genes associated with lipid biosynthesis were evaluated in five stages of developing embryos using quantitative PCR (qPCR). The qPCR data were well consistent with their expression in transcriptomic analyses. Our data would be important for the metabolic engineering of pecans to increase oil contents and modify fatty acid composition.

Abiraterone acetate: oral androgen biosynthesis inhibitor for treatment of castration-resistant prostate cancer

Directory of Open Access Journals (Sweden)

Rosenberg JE

2012-01-01

Full Text Available Yasser Rehman1, Jonathan E Rosenberg21Division of Hospital Medicine, UMass Memorial Healthcare, Worcester, MA, USA; 2Lank Center for Genitourinary Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, USAAbstract: Prostate cancer is the second leading cause of cancer death in men in the US and Europe. The treatment of advanced-stage prostate cancer has been androgen deprivation. Medical castration leads to decreased production of testosterone and dihydrotestosterone by the testes, but adrenal glands and even prostate cancer tissue continue to produce androgens, which eventually leads to continued prostate cancer growth despite castrate level of androgens. This stage is known as castrate-resistant prostate cancer (CRPC, which continues to be a challenge to treat. Addition of androgen antagonists to hormonal deprivation has been successful in lowering the prostate-specific antigen levels further, but has not actually translated into life-prolonging options. The results of several contemporary studies have continued to demonstrate activation of the androgen receptor as being the key factor in the continued growth of prostate cancer. Blockade of androgen production by nongonadal sources has led to clinical benefit in this setting. One such agent is abiraterone acetate, which significantly reduces androgen production by blocking the enzyme, cytochrome P450 17 alpha-hydroxylase (CYP17. This has provided physicians with another treatment option for patients with CRPC. The landscape for prostate cancer treatment has changed with the approval of cabazitaxel, sipuleucel-T and abiraterone. Here we provide an overview of abiraterone acetate, its mechanism of action, and its potential place for therapy in CRPC.Keywords: CRPC, abiraterone, CYP17, inhibitors, androgens, castration resistant prostate cancer

Distinct Prominent Roles for Enzymes of Plasmodium berghei Heme Biosynthesis in Sporozoite and Liver Stage Maturation

Science.gov (United States)

Matuschewski, Kai; Haussig, Joana M.

2016-01-01

Malarial parasites have evolved complex regulation of heme supply and disposal to adjust to heme-rich and -deprived host environments. In addition to its own pathway for heme biosynthesis, Plasmodium likely harbors mechanisms for heme scavenging from host erythrocytes. Elaborate compartmentalization of de novo heme synthesis into three subcellular locations, including the vestigial plastid organelle, indicates critical roles in life cycle progression. In this study, we systematically profile the essentiality of heme biosynthesis by targeted gene deletion of enzymes in early steps of this pathway. We show that disruption of endogenous heme biosynthesis leads to a first detectable defect in oocyst maturation and sporogony in the Anopheles vector, whereas blood stage propagation, colonization of mosquito midguts, or initiation of oocyst development occurs indistinguishably from that of wild-type parasites. Although sporozoites are produced by parasites lacking an intact pathway for heme biosynthesis, they are absent from mosquito salivary glands, indicative of a vital role for heme biosynthesis only in sporozoite maturation. Rescue of the first defect in sporogony permitted analysis of potential roles in liver stages. We show that liver stage parasites benefit from but do not strictly depend upon their own aminolevulinic acid synthase and that they can scavenge aminolevulinic acid from the host environment. Together, our experimental genetics analysis of Plasmodium enzymes for heme biosynthesis exemplifies remarkable shifts between the use of endogenous and host resources during life cycle progression. PMID:27600503

A model for evolution and regulation of nicotine biosynthesis regulon in tobacco.

Science.gov (United States)

Kajikawa, Masataka; Sierro, Nicolas; Hashimoto, Takashi; Shoji, Tsubasa

2017-06-03

In tobacco, the defense alkaloid nicotine is produced in roots and accumulates mainly in leaves. Signaling mediated by jasmonates (JAs) induces the formation of nicotine via a series of structural genes that constitute a regulon and are coordinated by JA-responsive transcription factors of the ethylene response factor (ERF) family. Early steps in the pyrrolidine and pyridine biosynthesis pathways likely arose through duplication of the polyamine and nicotinamide adenine dinucleotide (NAD) biosynthetic pathways, respectively, followed by recruitment of duplicated primary metabolic genes into the nicotine biosynthesis regulon. Transcriptional regulation of nicotine biosynthesis by ERF and cooperatively-acting MYC2 transcription factors is implied by the frequency of cognate cis-regulatory elements for these factors in the promoter regions of the downstream structural genes. Indeed, a mutant tobacco with low nicotine content was found to have a large chromosomal deletion in a cluster of closely related ERF genes at the nicotine-controlling NICOTINE2 (NIC2) locus.

Bioactive Mushroom Polysaccharides: A Review on Monosaccharide Composition, Biosynthesis and Regulation.

Science.gov (United States)

Wang, Qiong; Wang, Feng; Xu, Zhenghong; Ding, Zhongyang

2017-06-13

Mushrooms are widely distributed around the world and are heavily consumed because of their nutritional value and medicinal properties. Polysaccharides (PSs) are an important component of mushrooms, a major factor in their bioactive properties, and have been intensively studied during the past two decades. Monosaccharide composition/combinations are important determinants of PS bioactivities. This review summarizes: (i) monosaccharide composition/combinations in various mushroom PSs, and their relationships with PS bioactivities; (ii) possible biosynthetic pathways of mushroom PSs and effects of key enzymes on monosaccharide composition; (iii) regulation strategies in PS biosynthesis, and prospects for controllable biosynthesis of PSs with enhanced bioactivities.

Biosynthesis of silver nanoparticles and its antibacterial activity ...

African Journals Online (AJOL)

In the present research work, biosynthesis of silver nanoparticles and its activity on bacterial pathogens were investigated. Silver nanoparticles were rapidly synthesized using Urospora sp. and the formation of nanoparticles was observed within 30 min. The results recorded from UV–vis spectrum, Fourier Transform Infrared ...

Biosynthesis of silver nanoparticles by Leishmania tropica | Rahi ...

African Journals Online (AJOL)

A novel biosynthesis route for Silver Nanoparticles (Ag-NPs) was attempted in the present study using Leishmania tropica the causative agent of cutaneous leishmaniasis in different countries, particularly in Mediterranean region in Iraq. Silver nanoparticles were successfully synthesized from AgNO3 by reduction of ...

Biosynthesis of silver nanoparticles and its antibacterial activity ...

African Journals Online (AJOL)

Dr.Rajasekar

2012-07-19

Jul 19, 2012 ... Available online at http://www.academicjournals.org/AJB ... Transmission Electron Microscopy (HRTEM) support the biosynthesis and characterization of silver nanoparticles. ... nanoparticle from seaweed is a green chemical method ... operating at a voltage of 80 kV and a current of 30 mA (Chandran.

Control of biotin biosynthesis in mycobacteria by a pyruvate carboxylase dependent metabolic signal.

Science.gov (United States)

Lazar, Nathaniel; Fay, Allison; Nandakumar, Madhumitha; Boyle, Kerry E; Xavier, Joao; Rhee, Kyu; Glickman, Michael S

2017-12-01

Biotin is an essential cofactor utilized by all domains of life, but only synthesized by bacteria, fungi and plants, making biotin biosynthesis a target for antimicrobial development. To understand biotin biosynthesis in mycobacteria, we executed a genetic screen in Mycobacterium smegmatis for biotin auxotrophs and identified pyruvate carboxylase (Pyc) as required for biotin biosynthesis. The biotin auxotrophy of the pyc::tn strain is due to failure to transcriptionally induce late stage biotin biosynthetic genes in low biotin conditions. Loss of bioQ, the repressor of biotin biosynthesis, in the pyc::tn strain reverted biotin auxotrophy, as did reconstituting the last step of the pathway through heterologous expression of BioB and provision of its substrate DTB. The role of Pyc in biotin regulation required its catalytic activities and could be supported by M. tuberculosis Pyc. Quantitation of the kinetics of depletion of biotinylated proteins after biotin withdrawal revealed that Pyc is the most rapidly depleted biotinylated protein and metabolomics revealed a broad metabolic shift in wild type cells upon biotin withdrawal which was blunted in cell lacking Pyc. Our data indicate that mycobacterial cells monitor biotin sufficiency through a metabolic signal generated by dysfunction of a biotinylated protein of central metabolism. © 2017 John Wiley & Sons Ltd.

Gene expression analyses in tomato near isogenic lines provide evidence for ethylene and abscisic acid biosynthesis fine-tuning during arbuscular mycorrhiza development.

Science.gov (United States)

Fracetto, Giselle Gomes Monteiro; Peres, Lázaro Eustáquio Pereira; Lambais, Marcio Rodrigues

2017-07-01

Plant responses to the environment and microorganisms, including arbuscular mycorrhizal fungi, involve complex hormonal interactions. It is known that abscisic acid (ABA) and ethylene may be involved in the regulation of arbuscular mycorrhiza (AM) and that part of the detrimental effects of ABA deficiency in plants is due to ethylene overproduction. In this study, we aimed to determine whether the low susceptibility to mycorrhizal colonization in ABA-deficient mutants is due to high levels of ethylene and whether AM development is associated with changes in the steady-state levels of transcripts of genes involved in the biosynthesis of ethylene and ABA. For that, tomato (Solanum lycopersicum) ethylene overproducer epinastic (epi) mutant and the ABA-deficient notabilis (not) and sitiens (sit) mutants, in the same Micro-Tom (MT) genetic background, were inoculated with Rhizophagus clarus, and treated with the ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG). The development of AM, as well as the steady-state levels of transcripts involved in ethylene (LeACS2, LeACO1 and LeACO4) and ABA (LeNCED) biosynthesis, was determined. The intraradical colonization in epi, not and sit mutants was significantly reduced compared to MT. The epi mutant completely restored the mycorrhizal colonization to the levels of MT with the application of 10 µM of AVG, probably due to the inhibition of the ACC synthase gene expression. The steady-state levels of LeACS2 and LeACO4 transcripts were induced in mycorrhizal roots of MT, whereas the steady-state levels of LeACO1 and LeACO4 transcripts were significantly induced in sit, and the steady-state levels of LeNCED transcripts were significantly induced in all genotypes and in mycorrhizal roots of epi mutants treated with AVG. The reduced mycorrhizal colonization in sit mutants seems not to be limited by ethylene production via ACC oxidase regulation. Both ethylene overproduction and ABA deficiency impaired AM fungal

Thioridazine affects transcription of genes involved in cell wall biosynthesis in methicillin-resistant Staphylococcus aureus

DEFF Research Database (Denmark)

Bonde, Mette; Højland, Dorte Heidi; Kolmos, Hans Jørn

2011-01-01

have previously shown that the expression of some resistance genes is abolished after treatment with thioridazine and oxacillin. To further understand the mechanism underlying the reversal of resistance, we tested the expression of genes involved in antibiotic resistance and cell wall biosynthesis...... in response to thioridazine in combination with oxacillin. We observed that the oxacillin-induced expression of genes belonging to the VraSR regulon is reduced by the addition of thioridazine. The exclusion of such key factors involved in cell wall biosynthesis will most likely lead to a weakened cell wall...... reversal of resistance by thioridazine relies on decreased expression of specific genes involved in cell wall biosynthesis....

Towards a Molecular Understanding of the Biosynthesis of Amaryllidaceae Alkaloids in Support of Their Expanding Medical Use

Directory of Open Access Journals (Sweden)

Adam M. Takos

2013-05-01

Full Text Available The alkaloids characteristically produced by the subfamily Amaryllidoideae of the Amaryllidaceae, bulbous plant species that include well know genera such as Narcissus (daffodils and Galanthus (snowdrops, are a source of new pharmaceutical compounds. Presently, only the Amaryllidaceae alkaloid galanthamine, an acetylcholinesterase inhibitor used to treat symptoms of Alzheimer’s disease, is produced commercially as a drug from cultivated plants. However, several Amaryllidaceae alkaloids have shown great promise as anti-cancer drugs, but their further clinical development is restricted by their limited commercial availability. Amaryllidaceae species have a long history of cultivation and breeding as ornamental bulbs, and phytochemical research has focussed on the diversity in alkaloid content and composition. In contrast to the available pharmacological and phytochemical data, ecological, physiological and molecular aspects of the Amaryllidaceae and their alkaloids are much less explored and the identity of the alkaloid biosynthetic genes is presently unknown. An improved molecular understanding of Amaryllidaceae alkaloid biosynthesis would greatly benefit the rational design of breeding programs to produce cultivars optimised for the production of pharmaceutical compounds and enable biotechnology based approaches.

Ascorbate Biosynthesis in Mitochondria Is Linked to the Electron Transport Chain between Complexes III and IV1

Science.gov (United States)

Bartoli, Carlos G.; Pastori, Gabriela M.; Foyer, Christine H.

2000-01-01

Ascorbic acid is synthesized from galactono-γ-lactone (GL) in plant tissues. An improved extraction procedure involving ammonium sulfate precipitation of membrane proteins from crude leaf homogenates yielded a simple, quick method for determining tissue activities of galactono-γ-lactone dehydrogenase (GLDH). Total foliar ascorbate and GLDH activity decreased with leaf age. Subcellular fractionation experiments using marker enzymes demonstrated that 80% of the total GLDH activity was located on the inner mitochondrial membrane, and 20% in the microsomal fraction. Specific antibody raised against potato (Solanum tuberosum L.) tuber GLDH recognized a 56-kD polypeptide in extracts from the mitochondrial membranes but failed to detect the equivalent polypeptide in microsomes. We demonstrate that isolated intact mitochondria synthesize ascorbate in the presence of GL. GL stimulated mitochondrial electron transport rates. The respiration inhibitor antimycin A stimulated ascorbate biosynthesis, while cyanide inhibited both respiration and ascorbate production. GL-dependent oxygen uptake was observed in isolated intact mitochondria. This evidence suggests that GLDH delivers electrons to the mitochondrial electron transport chain between complexes III and IV. PMID:10806250

Potent host-directed small-molecule inhibitors of myxovirus RNA-dependent RNA-polymerases.

Directory of Open Access Journals (Sweden)

Stefanie A Krumm

Full Text Available Therapeutic targeting of host cell factors required for virus replication rather than of pathogen components opens new perspectives to counteract virus infections. Anticipated advantages of this approach include a heightened barrier against the development of viral resistance and a broadened pathogen target spectrum. Myxoviruses are predominantly associated with acute disease and thus are particularly attractive for this approach since treatment time can be kept limited. To identify inhibitor candidates, we have analyzed hit compounds that emerged from a large-scale high-throughput screen for their ability to block replication of members of both the orthomyxovirus and paramyxovirus families. This has returned a compound class with broad anti-viral activity including potent inhibition of different influenza virus and paramyxovirus strains. After hit-to-lead chemistry, inhibitory concentrations are in the nanomolar range in the context of immortalized cell lines and human PBMCs. The compound shows high metabolic stability when exposed to human S-9 hepatocyte subcellular fractions. Antiviral activity is host-cell species specific and most pronounced in cells of higher mammalian origin, supporting a host-cell target. While the compound induces a temporary cell cycle arrest, host mRNA and protein biosynthesis are largely unaffected and treated cells maintain full metabolic activity. Viral replication is blocked at a post-entry step and resembles the inhibition profile of a known inhibitor of viral RNA-dependent RNA-polymerase (RdRp activity. Direct assessment of RdRp activity in the presence of the reagent reveals strong inhibition both in the context of viral infection and in reporter-based minireplicon assays. In toto, we have identified a compound class with broad viral target range that blocks host factors required for viral RdRp activity. Viral adaptation attempts did not induce resistance after prolonged exposure, in contrast to rapid

Identification and characterization of an archaeal ketopantoate reductase and its involvement in regulation of coenzyme A biosynthesis.

Science.gov (United States)

Tomita, Hiroya; Imanaka, Tadayuki; Atomi, Haruyuki

2013-10-01

Coenzyme A (CoA) biosynthesis in bacteria and eukaryotes is regulated primarily by feedback inhibition towards pantothenate kinase (PanK). As most archaea utilize a modified route for CoA biosynthesis and do not harbour PanK, the mechanisms governing regulation of CoA biosynthesis are unknown. Here we performed genetic and biochemical studies on the ketopantoate reductase (KPR) from the hyperthermophilic archaeon Thermococcus kodakarensis. KPR catalyses the second step in CoA biosynthesis, the reduction of 2-oxopantoate to pantoate. Gene disruption of TK1968, whose product was 20-29% identical to previously characterized KPRs from bacteria/eukaryotes, resulted in a strain with growth defects that were complemented by addition of pantoate. The TK1968 protein (Tk-KPR) displayed reductase activity specific for 2-oxopantoate and preferred NADH as the electron donor, distinct to the bacterial/eukaryotic NADPH-dependent enzymes. Tk-KPR activity decreased dramatically in the presence of CoA and KPR activity in cell-free extracts was also inhibited by CoA. Kinetic studies indicated that CoA inhibits KPR by competing with NADH. Inhibition of ketopantoate hydroxymethyltransferase, the first enzyme of the pathway, by CoA was not observed. Our results suggest that CoA biosynthesis in T. kodakarensis is regulated by feedback inhibition of KPR, providing a feasible regulation mechanism of CoA biosynthesis in archaea. © 2013 John Wiley & Sons Ltd.

Essential oil biosynthesis and regulation in the genus Cymbopogon.

Science.gov (United States)

Ganjewala, Deepak; Luthra, Rajesh

2010-01-01

Essential oils distilled from Cymbopogon species are of immense commercial value as flavors and fragrances in the perfumery, cosmetics, soaps, and detergents and in pharmaceutical industries. Two major constituents of the essential oil, geraniol and citral, due to their specific rose and lemon like aromas are widely used as flavors, fragrances and cosmetics. Citral is also used for the synthesis of vitamin A and ionones (for example, beta-ionone, methyl ionone). Moreover, Cymbopogon essential oils and constituents possess many useful biological activities including cytotoxic, anti-inflammatory and antioxidant. Despite the immense commercial and biological significance of the Cymbopogon essential oils, little is known about their biosynthesis and regulatory mechanisms. So far it is known that essential oils are biosynthesized via the classical acetate-MVA route and existence of a newly discovered MEP pathway in Cymbopogon remains as a topic for investigation. The aim of the present review is to discuss the biosynthesis and regulation of essential oils in the genus Cymbopogon with given emphasis to two elite members, lemongrass (C. flexuosus Nees ex Steud) and palmarosa (C. martinii Roxb.). This article highlights the work done so far towards understanding of essential oil biosynthesis and regulation in the genus Cymbopogon. Also, based on our experiences with Cymbopogon species, we would like to propose C. flexuosus as a model system for the study of essential oil metabolism beyond the much studied plant family Lamiaceae.

Pharmacophore Modeling and in Silico/in Vitro Screening for Human Cytochrome P450 11B1 and Cytochrome P450 11B2 Inhibitors.

Science.gov (United States)

Akram, Muhammad; Waratchareeyakul, Watcharee; Haupenthal, Joerg; Hartmann, Rolf W; Schuster, Daniela

2017-01-01

Cortisol synthase (CYP11B1) is the main enzyme for the endogenous synthesis of cortisol and its inhibition is a potential way for the treatment of diseases associated with increased cortisol levels, such as Cushing's syndrome, metabolic diseases, and delayed wound healing. Aldosterone synthase (CYP11B2) is the key enzyme for aldosterone biosynthesis and its inhibition is a promising approach for the treatment of congestive heart failure, cardiac fibrosis, and certain forms of hypertension. Both CYP11B1 and CYP11B2 are structurally very similar and expressed in the adrenal cortex. To facilitate the identification of novel inhibitors of these enzymes, ligand-based pharmacophore models of CYP11B1 and CYP11B2 inhibition were developed. A virtual screening of the SPECS database was performed with our pharmacophore queries. Biological evaluation of the selected hits lead to the discovery of three potent novel inhibitors of both CYP11B1 and CYP11B2 in the submicromolar range (compounds 8 - 10 ), one selective CYP11B1 inhibitor (Compound 11 , IC 50 = 2.5 μM), and one selective CYP11B2 inhibitor (compound 12 , IC 50 = 1.1 μM), respectively. The overall success rate of this prospective virtual screening experiment is 20.8% indicating good predictive power of the pharmacophore models.

Pharmacophore Modeling and in Silico/in Vitro Screening for Human Cytochrome P450 11B1 and Cytochrome P450 11B2 Inhibitors

Directory of Open Access Journals (Sweden)

Muhammad Akram

2017-12-01

Full Text Available Cortisol synthase (CYP11B1 is the main enzyme for the endogenous synthesis of cortisol and its inhibition is a potential way for the treatment of diseases associated with increased cortisol levels, such as Cushing's syndrome, metabolic diseases, and delayed wound healing. Aldosterone synthase (CYP11B2 is the key enzyme for aldosterone biosynthesis and its inhibition is a promising approach for the treatment of congestive heart failure, cardiac fibrosis, and certain forms of hypertension. Both CYP11B1 and CYP11B2 are structurally very similar and expressed in the adrenal cortex. To facilitate the identification of novel inhibitors of these enzymes, ligand-based pharmacophore models of CYP11B1 and CYP11B2 inhibition were developed. A virtual screening of the SPECS database was performed with our pharmacophore queries. Biological evaluation of the selected hits lead to the discovery of three potent novel inhibitors of both CYP11B1 and CYP11B2 in the submicromolar range (compounds 8–10, one selective CYP11B1 inhibitor (Compound 11, IC50 = 2.5 μM, and one selective CYP11B2 inhibitor (compound 12, IC50 = 1.1 μM, respectively. The overall success rate of this prospective virtual screening experiment is 20.8% indicating good predictive power of the pharmacophore models.

Inhibitory effect of luteolin on estrogen biosynthesis in human ovarian granulosa cells by suppression of aromatase (CYP19).

Science.gov (United States)

Lu, Dan-feng; Yang, Li-juan; Wang, Fei; Zhang, Guo-lin

2012-08-29

Inhibition of aromatase, the key enzyme in estrogen biosynthesis, is an important strategy in the treatment of breast cancer. Several dietary flavonoids show aromatase inhibitory activity, but their tissue specificity and mechanism remain unclear. This study found that the dietary flavonoid luteolin potently inhibited estrogen biosynthesis in a dose- and time-dependent manner in KGN cells derived from human ovarian granulosa cells, the major source of estrogens in premenopausal women. Luteolin decreased aromatase mRNA and protein expression in KGN cells. Luteolin also promoted aromatase protein degradation and inhibited estrogen biosynthesis in aromatase-expressing HEK293A cells, but had no effect on recombinant expressed aromatase. Estrogen biosynthesis in KGN cells was inhibited with differing potencies by extracts of onion and bird chili and by four other dietary flavonoids: kaempferol, quercetin, myricetin, and isorhamnetin. The present study suggests that luteolin inhibits estrogen biosynthesis by decreasing aromatase expression and destabilizing aromatase protein, and it warrants further investigation as a potential treatment for estrogen-dependent cancers.

Radiographic features of the skeleton in disorders of post-squalene cholesterol biosynthesis

Energy Technology Data Exchange (ETDEWEB)

Rossi, Massimiliano; Edery, Patrick [Hospices Civils de Lyon, Genetic Department, Referral Centre for Developmental Abnormalities, Femme-Mere-Enfant Hospital, Bron (France); INSERM U1028 UMR CNRS 5,292, UCBL, CRNL TIGER Team, CH le Vinater, Bron (France); Hall, Christine M. [Retired from Department of Radiology, Great Ormond Street Hospital, London (United Kingdom); Bouvier, Raymonde; Collardeau-Frachon, Sophie [Hospices Civils de Lyon, Department of Pathology, CBPE, Bron (France); Le Breton, Frederique [Hospices Civils de Lyon, Department of Pathology, Croix-Rousse Hospital, Lyon (France); Bucourt, Martine [AP-HP, Foetopathology Unit, Jean Verdier Hospital, Bondy (France); Cordier, Marie Pierre [Hospices Civils de Lyon, Genetic Department, Referral Centre for Developmental Abnormalities, Femme-Mere-Enfant Hospital, Bron (France); Vianey-Saban, Christine [Hospices Civils de Lyon, Department of Inborn Errors of Metabolism and Neonatal Screening, CBPE, Bron (France); Parenti, Giancarlo; Andria, Generoso [Federico II University, Department of Translational Medical Sciences, Section of Pediatrics, Naples (Italy); Le Merrer, Martine [AP-HP, Genetic Department, Referal Centre for Skeletal Dysplasias, Institut Imagine, Necker-Enfants Malades Hospital, Paris (United Kingdom); Offiah, Amaka C. [Stephenson Wing Sheffield Children' s NHS Foundation Trust Western Bank, Radiology Department, Children' s Hospital, Academic Unit of Child Health Room C4, Sheffield (United Kingdom)

2015-07-15

Disorders of post-squalene cholesterol biosynthesis are inborn errors of metabolism characterised by multiple congenital abnormalities, including significant skeletal involvement. The most frequent and best-characterised example is the Smith-Lemli-Opitz syndrome. Nine other disorders are known, namely autosomal-recessive Antley-Bixler syndrome, Greenberg dysplasia, X-linked dominant chondrodysplasia punctata, X-linked recessive male emopamil-binding protein deficiency, CHILD syndrome, CK syndrome, sterol C4 methyloxidase-like deficiency, desmosterolosis and lathosterolosis. This study provides an overview of the radiologic features observed in these diseases. A common pattern of limb abnormalities is recognisable, including polydactyly, which is typically post-axial and rarely interdigital and can involve all four limbs, and syndactyly of the toes. Chondrodysplasia punctata is specifically associated with a subgroup of disorders of cholesterol biosynthesis (Greenberg dysplasia, CHILD syndrome, X-linked dominant chondrodysplasia punctata, male emopamil-binding protein deficiency). The possible occurrence of epiphyseal stippling in the Smith-Lemli-Opitz syndrome, initially reported, does not appear to be confirmed. Stippling is also associated with other congenital disorders such as chromosomal abnormalities, brachytelephalangic chondrodysplasia punctata (X-linked recessive chondrodysplasia punctata, disruptions of vitamin K metabolism, maternal autoimmune diseases), rhizomelic chondrodysplasia punctata (peroxisomal disorders) and lysosomal storage disorders. In the differential diagnosis of epiphyseal stippling, a moth-eaten appearance of bones, asymmetry, or presence of a common pattern of limb abnormalities indicate inborn errors of cholesterol biosynthesis. We highlight the specific differentiating radiologic features of disorders of post-squalene cholesterol biosynthesis. (orig.)

Radiographic features of the skeleton in disorders of post-squalene cholesterol biosynthesis

International Nuclear Information System (INIS)

Rossi, Massimiliano; Edery, Patrick; Hall, Christine M.; Bouvier, Raymonde; Collardeau-Frachon, Sophie; Le Breton, Frederique; Bucourt, Martine; Cordier, Marie Pierre; Vianey-Saban, Christine; Parenti, Giancarlo; Andria, Generoso; Le Merrer, Martine; Offiah, Amaka C.

2015-01-01

Disorders of post-squalene cholesterol biosynthesis are inborn errors of metabolism characterised by multiple congenital abnormalities, including significant skeletal involvement. The most frequent and best-characterised example is the Smith-Lemli-Opitz syndrome. Nine other disorders are known, namely autosomal-recessive Antley-Bixler syndrome, Greenberg dysplasia, X-linked dominant chondrodysplasia punctata, X-linked recessive male emopamil-binding protein deficiency, CHILD syndrome, CK syndrome, sterol C4 methyloxidase-like deficiency, desmosterolosis and lathosterolosis. This study provides an overview of the radiologic features observed in these diseases. A common pattern of limb abnormalities is recognisable, including polydactyly, which is typically post-axial and rarely interdigital and can involve all four limbs, and syndactyly of the toes. Chondrodysplasia punctata is specifically associated with a subgroup of disorders of cholesterol biosynthesis (Greenberg dysplasia, CHILD syndrome, X-linked dominant chondrodysplasia punctata, male emopamil-binding protein deficiency). The possible occurrence of epiphyseal stippling in the Smith-Lemli-Opitz syndrome, initially reported, does not appear to be confirmed. Stippling is also associated with other congenital disorders such as chromosomal abnormalities, brachytelephalangic chondrodysplasia punctata (X-linked recessive chondrodysplasia punctata, disruptions of vitamin K metabolism, maternal autoimmune diseases), rhizomelic chondrodysplasia punctata (peroxisomal disorders) and lysosomal storage disorders. In the differential diagnosis of epiphyseal stippling, a moth-eaten appearance of bones, asymmetry, or presence of a common pattern of limb abnormalities indicate inborn errors of cholesterol biosynthesis. We highlight the specific differentiating radiologic features of disorders of post-squalene cholesterol biosynthesis. (orig.)

Accumulation of Charantin and Expression of Triterpenoid Biosynthesis Genes in Bitter Melon (Momordica charantia).

Science.gov (United States)

Cuong, Do Manh; Jeon, Jin; Morgan, Abubaker M A; Kim, Changsoo; Kim, Jae Kwang; Lee, Sook Young; Park, Sang Un

2017-08-23

Charantin, a natural cucurbitane type triterpenoid, has been reported to have beneficial pharmacological functions such as anticancer, antidiabetic, and antibacterial activities. However, accumulation of charantin in bitter melon has been little studied. Here, we performed a transcriptome analysis to identify genes involved in the triterpenoid biosynthesis pathway in bitter melon seedlings. A total of 88,703 transcripts with an average length of 898 bp were identified in bitter melon seedlings. On the basis of a functional annotation, we identified 15 candidate genes encoding enzymes related to triterpenoid biosynthesis and analyzed their expression in different organs of mature plants. Most genes were highly expressed in flowers and/or fruit from the ripening stages. An HPLC analysis confirmed that the accumulation of charantin was highest in fruits from the ripening stage, followed by male flowers. The accumulation patterns of charantin coincide with the expression pattern of McSE and McCAS1, indicating that these genes play important roles in charantin biosynthesis in bitter melon. We also investigated optimum light conditions for enhancing charantin biosynthesis in bitter melon and found that red light was the most effective wavelength.

Benchmarking of Processes for the Biosynthesis of Natural Products

DEFF Research Database (Denmark)

Seita, Catarina Sanches

putida GS1. (R)-perillic acid is a monoterpenoic acid with antimicrobial properties. It has a strong inhibitory effect on bacteria and fungus, which makes it an attractive compound to be used as a preservative for instance in cosmetic industry, but on the other hand makes the biosynthesis a complicated....... These biological activities can be of interest for use in different sectors of chemical industry, in particular pharmaceutical industry where several drugs are derived or inspired by natural products structure. However, the large scale production of natural products is hindered by its relatively poor abundance...... of the process in comparison with other sweeteners. The main benefit of this early-stage evaluation is putting the biosynthesis of natural products into context in relation to demands of an industrially feasible chemical process. Moreover, it can give very meaningful insight into process development and provides...

The plant cuticle is required for osmotic stress regulation of abscisic acid biosynthesis and osmotic stress tolerance in Arabidopsis

KAUST Repository

Wang, Zhenyu

2011-05-01

Osmotic stress activates the biosynthesis of abscisic acid (ABA). One major step in ABA biosynthesis is the carotenoid cleavage catalyzed by a 9-cis epoxycarotenoid dioxygenase (NCED). To understand the mechanism for osmotic stress activation of ABA biosynthesis, we screened for Arabidopsis thaliana mutants that failed to induce the NCED3 genee xpression in response to osmotic stress treatments. The ced1 (for 9-cis epoxycarotenoid dioxy genase defective 1) mutant isolated in this study showed markedly reduced expression of NCED3 in response to osmotic stress (polyethylene glycol)treatments compared with the wild type. Other ABA biosynthesis genes are also greatly reduced in ced1 under osmotic stress. ced1 mutant plants are very sensitive to even mild osmotic stress. Map-based cloning revealed unexpectedly thatCED1 encodes a putative a/b hydrolase domain-containing protein and is allelic to the BODYGUARD gene that was recently shown to be essential for cuticle biogenesis. Further studies discovered that other cut in biosynthesis mutants are also impaired in osmotic stress induction of ABA biosynthesis genes and are sensitive to osmotic stress. Our work demonstrates that the cuticle functions not merely as a physical barrier to minimize water loss but also mediates osmotic stress signaling and tolerance by regulating ABA biosynthesis and signaling. © 2011 American Society of Plant Biologists. All rights reserved.

Muscle type-specific responses to NAD+ salvage biosynthesis promote muscle function in Caenorhabditis elegans.

Science.gov (United States)

Vrablik, Tracy L; Wang, Wenqing; Upadhyay, Awani; Hanna-Rose, Wendy

2011-01-15

Salvage biosynthesis of nicotinamide adenine dinucleotide (NAD(+)) from nicotinamide (NAM) lowers NAM levels and replenishes the critical molecule NAD(+) after it is hydrolyzed. This pathway is emerging as a regulator of multiple biological processes. Here we probe the contribution of the NAM-NAD(+) salvage pathway to muscle development and function using Caenorhabditis elegans. C. elegans males with mutations in the nicotinamidase pnc-1, which catalyzes the first step of this NAD(+) salvage pathway, cannot mate due to a spicule muscle defect. Multiple muscle types are impaired in the hermaphrodites, including body wall muscles, pharyngeal muscles and vulval muscles. An active NAD(+) salvage pathway is required for optimal function of each muscle cell type. However, we found surprising muscle-cell-type specificity in terms of both the timing and relative sensitivity to perturbation of NAD(+) production or NAM levels. Active NAD(+) biosynthesis during development is critical for function of the male spicule protractor muscles during adulthood, but these muscles can surprisingly do without salvage biosynthesis in adulthood under the conditions examined. The body wall muscles require ongoing NAD(+) salvage biosynthesis both during development and adulthood for maximum function. The vulval muscles do not function in the presence of elevated NAM concentrations, but NAM supplementation is only slightly deleterious to body wall muscles during development or upon acute application in adults. Thus, the pathway plays distinct roles in different tissues. As NAM-NAD(+) biosynthesis also impacts muscle differentiation in vertebrates, we propose that similar complexities may be found among vertebrate muscle cell types. Copyright © 2010 Elsevier Inc. All rights reserved.

Pre-termination in aflR of Aspergillus sojae inhibits aflatoxin biosynthesis.

Science.gov (United States)

Matsushima, K; Chang, P K; Yu, J; Abe, K; Bhatnagar, D; Cleveland, T E

2001-05-01

The aflR gene product is the main transcriptional regulator of aflatoxin biosynthesis in Aspergillus parasiticus and Aspergillus flavus. Although A. sojae strains do not produce aflatoxins, they do have an aflR homologue. When compared with the aflR of A. parasiticus, the A. sojae gene contains two mutations: an HAHA motif and a premature stop codon. To investigate the functionality of the A. sojae aflR gene product, we used a GAL4 one-hybrid system in yeast. The transcription-activating activity of AflR from A. sojae was 15% of that from A. parasiticus. The introduction of an additional aflR from A. sojae into an A. parasiticus strain did not affect aflatoxin productivity. A hybrid aflR comprising the amino-terminal region of A. sojae aflR and the carboxy-terminal region of A. parasiticus aflR suppressed the effect associated with pre-termination of the A. sojae AflR. We conclude that the premature stop codon of the A. sojae aflR is the key to its functionality and leads to prevention of aflatoxin biosynthesis through loss of the transcription of aflatoxin biosynthesis-related genes.

Inhibition of Lipid A Biosynthesis as the Primary Mechanism of CHIR-090 Antibiotic Activity in Escherichia coli

Science.gov (United States)

Barb, Adam W.; McClerren, Amanda L.; Snehelatha, Karnem; Reynolds, C. Michael; Zhou, Pei; Raetz, Christian R.H.

2009-01-01

The deacetylation of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine (UDP-3-O-acyl-GlcNAc) by LpxC is the committed reaction of lipid A biosynthesis. CHIR-090, a novel N-aroyl-l-threonine hydroxamic acid, is a potent, slow, tight-binding inhibitor of the LpxC deacetylase from the hyperthermophile Aquifex aeolicus, and it has excellent antibiotic activity against P. aeruginosa and E. coli, as judged by disk diffusion assays. We now report that CHIR-090 is also a two-step slow, tight-binding inhibitor of Escherichia coli LpxC with Ki = 4.0 nM, Ki* = 0.5 nM, k5 = 1.9 min-1 and k6 = 0.18 min-1. CHIR-090 at low nM levels inhibits LpxC orthologues from diverse Gram-negative pathogens, including Pseudomonas aeruginosa, Neisseria meningitidis, and Helicobacter pylori. In contrast, CHIR-090 is a relatively weak competitive and conventional inhibitor (lacking slow, tight-binding kinetics) of LpxC from Rhizobium leguminosarum (Ki = 340 nM), a Gram-negative plant endosymbiont that is resistant to this compound. The KM (4.8 μM) and the kcat (1.7 s-1) of R. leguminosarum LpxC with UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine as the substrate are similar to values reported for E. coli LpxC. R. leguminosarum LpxC therefore provides a useful control for validating LpxC as the primary target of CHIR-090 in vivo. An E. coli construct in which the chromosomal lpxC gene is replaced by R. leguminosarum lpxC is resistant to CHIR-090 up to 100 μg/mL, or 400 times above the minimal inhibitory concentration for wild-type E. coli. Given its relatively broad spectrum and potency against diverse Gram-negative pathogens, CHIR-090 is an excellent lead for the further development of new antibiotics targeting the lipid A pathway. PMID:17335290

Biosynthesis of flavonoids in bilberry and blueberry - possibilities of the gene level information for the future

OpenAIRE

Jaakola, Laura

2007-01-01

We have studied the biosynthesis of flavonoids in various tissues of naturally growing European blueberry (bilberry) and the blueberry cultivar 'Northblue'. Focus has also been on the biosynthesis of flavonoids in developing bilberry fruits as well as on the control genes regulating fruit development.

Phosphatidylserine biosynthesis in cultured Chinese hamster ovary cells. I. Inhibition of de novo phosphatidylserine biosynthesis by exogenous phosphatidylserine and its efficient incorporation

International Nuclear Information System (INIS)

Nishijima, M.; Kuge, O.; Akamatsu, Y.

1986-01-01

The effect of phosphatidylserine exogenously added to the medium on de novo biosynthesis of phosphatidylserine was investigated in cultured Chinese hamster ovary cells. When cells were cultured for several generations in medium supplemented with phosphatidylserine and 32 Pi, the incorporation of 32 Pi into cellular phosphatidylserine was remarkably inhibited, the degree of inhibition being dependent upon the concentration of added phosphatidylserine. 32 Pi uptake into cellular phosphatidylethanolamine was also partly reduced by the addition of exogenous phosphatidylserine, consistent with the idea that phosphatidylethanolamine is biosynthesized via decarboxylation of phosphatidylserine. However, incorporation of 32 Pi into phosphatidylcholine, sphingomyelin, and phosphatidylinositol was not significantly affected. In contrast, the addition of either phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, or phosphatidylinositol to the medium did not inhibit endogenous biosynthesis of the corresponding phospholipid. Radiochemical and chemical analyses of the cellular phospholipid composition revealed that phosphatidylserine in cells grown with 80 microM phosphatidylserine was almost entirely derived from the added phospholipid. Phosphatidylserine uptake was also directly determined by using [ 3 H]serine-labeled phospholipid. Pulse and pulse-chase experiments with L-[U- 14 C] serine showed that when cells were cultured with 80 microM phosphatidylserine, the rate of synthesis of phosphatidylserine was reduced 3-5-fold. Enzyme assaying of extracts prepared from cells grown with and without phosphatidylserine indicated that the inhibition of de novo phosphatidylserine biosynthesis by the added phosphatidylserine appeared not to be caused by a reduction in the level of the enzyme involved in the base-exchange reaction between phospholipids and serine

Fatty acid biosynthesis in pea root plastids

International Nuclear Information System (INIS)

Stahl, R.J.; Sparace, S.A.

1989-01-01

Fatty acid biosynthesis from [1- 14 C]acetate was optimized in plastids isolated from primary root tips of 7-day-old germinating pea seeds. Fatty acid synthesis was maximum at approximately 80 nmoles/hr/mg protein in the presence of 200 μM acetate, 0.5 mM each of NADH, NADPH and CoA, 6 mM each of ATP and MgCl 2 , 1 mM each of the MnCl 2 and glycerol-3-phosphate, 15 mM KHCO 3 , and 0.1M Bis-tris-propane, pH 8.0 incubated at 35C. At the standard incubation temperature of 25C, fatty acid synthesis was linear from up to 6 hours with 80 to 100 μg/mL plastid protein. ATP and CoA were absolute requirements, whereas KHCO 3 , divalent cations and reduced nucleotides all improved activity by 80 to 85%. Mg 2+ and NADH were the preferred cation and nucleotide, respectively. Dithiothreitol and detergents were generally inhibitory. The radioactive products of fatty acid biosynthesis were approximately 33% 16:0, 10% 18:0 and 56% 18:1 and generally did not vary with increasing concentrations of each cofactor

Molecular Regulation of Antibiotic Biosynthesis in Streptomyces

Science.gov (United States)

Liu, Gang; Chandra, Govind; Niu, Guoqing

2013-01-01

SUMMARY Streptomycetes are the most abundant source of antibiotics. Typically, each species produces several antibiotics, with the profile being species specific. Streptomyces coelicolor, the model species, produces at least five different antibiotics. We review the regulation of antibiotic biosynthesis in S. coelicolor and other, nonmodel streptomycetes in the light of recent studies. The biosynthesis of each antibiotic is specified by a large gene cluster, usually including regulatory genes (cluster-situated regulators [CSRs]). These are the main point of connection with a plethora of generally conserved regulatory systems that monitor the organism's physiology, developmental state, population density, and environment to determine the onset and level of production of each antibiotic. Some CSRs may also be sensitive to the levels of different kinds of ligands, including products of the pathway itself, products of other antibiotic pathways in the same organism, and specialized regulatory small molecules such as gamma-butyrolactones. These interactions can result in self-reinforcing feed-forward circuitry and complex cross talk between pathways. The physiological signals and regulatory mechanisms may be of practical importance for the activation of the many cryptic secondary metabolic gene cluster pathways revealed by recent sequencing of numerous Streptomyces genomes. PMID:23471619

Essences in Metabolic Engineering of Lignan Biosynthesis

Directory of Open Access Journals (Sweden)

Honoo Satake

2015-05-01

Full Text Available Lignans are structurally and functionally diverse phytochemicals biosynthesized in diverse plant species and have received wide attentions as leading compounds of novel drugs for tumor treatment and healthy diets to reduce of the risks of lifestyle-related non-communicable diseases. However, the lineage-specific distribution and the low-amount of production in natural plants, some of which are endangered species, hinder the efficient and stable production of beneficial lignans. Accordingly, the development of new procedures for lignan production is of keen interest. Recent marked advances in the molecular and functional characterization of lignan biosynthetic enzymes and endogenous and exogenous factors for lignan biosynthesis have suggested new methods for the metabolic engineering of lignan biosynthesis cascades leading to the efficient, sustainable, and stable lignan production in plants, including plant cell/organ cultures. Optimization of light conditions, utilization of a wide range of elicitor treatments, and construction of transiently gene-transfected or transgenic lignan-biosynthesizing plants are mainly being attempted. This review will present the basic and latest knowledge regarding metabolic engineering of lignans based on their biosynthetic pathways and biological activities, and the perspectives in lignan production via metabolic engineering.

The effect of MEP pathway and other inhibitors on the intracellular localization of a plasma membrane-targeted, isoprenylable GFP reporter protein in tobacco BY-2 cells [v1; ref status: indexed, http://f1000r.es/yx

Directory of Open Access Journals (Sweden)

Michael Hartmann

2013-08-01

Full Text Available We have established an in vivo visualization system for the geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, based on the expression of a dexamethasone-inducible GFP fused to the carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL. By using pathway-specific inhibitors it was demonstrated that inhibition of the methylerythritol phosphate (MEP pathway with known inhibitors like oxoclomazone and fosmidomycin, as well as inhibition of the protein geranylgeranyltransferase type 1 (PGGT-1, shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA pathway with mevinolin did not affect the localization. During the present work, this test system has been used to examine the effect of newly designed inhibitors of the MEP pathway and inhibitors of sterol biosynthesis such as squalestatin, terbinafine and Ro48-8071. In addition, we also studied the impact of different post-prenylation inhibitors or those suspected to affect the transport of proteins to the plasma membrane on the localization of the geranylgeranylable fusion protein GFP-BD-CVIL.

The cathepsin B inhibitor z-FA-CMK induces cell death in leukemic T cells via oxidative stress.

Science.gov (United States)

Liow, K Y; Chow, Sek C

2018-01-01

The cathepsin B inhibitor benzyloxycarbonyl-phenylalanine-alanine-chloromethyl ketone (z-FA-CMK) was recently found to induce apoptosis at low concentrations in Jurkat T cells, while at higher concentrations, the cells die of necrosis. In the present study, we showed that z-FA-CMK readily depletes intracellular glutathione (GSH) with a concomitant increase in reactive oxygen species (ROS) generation. The toxicity of z-FA-CMK in Jurkat T cells was completely abrogated by N-acetylcysteine (NAC), suggesting that the toxicity mediated by z-FA-CMK is due to oxidative stress. We found that L-buthionine sulfoximine (BSO) which depletes intracellular GSH through the inhibition of GSH biosynthesis in Jurkat T cells did not promote ROS increase or induce cell death. However, NAC was still able to block z-FA-CMK toxicity in Jurkat T cells in the presence of BSO, indicating that the protective effect of NAC does not involve GSH biosynthesis. This is further corroborated by the protective effect of the non-metabolically active D-cysteine on z-FA-CMK toxicity. Furthermore, in BSO-treated cells, z-FA-CMK-induced ROS increased which remains unchanged, suggesting that the depletion of GSH and increase in ROS generation mediated by z-FA-CMK may be two separate events. Collectively, our results demonstrated that z-FA-CMK toxicity is mediated by oxidative stress through the increase in ROS generation.

Exploration of natural product ingredients as inhibitors of human HMG-CoA reductase through structure-based virtual screening.

Science.gov (United States)

Lin, Shih-Hung; Huang, Kao-Jean; Weng, Ching-Feng; Shiuan, David

2015-01-01

Cholesterol plays an important role in living cells. However, a very high level of cholesterol may lead to atherosclerosis. HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase is the key enzyme in the cholesterol biosynthesis pathway, and the statin-like drugs are inhibitors of human HMG-CoA reductase (hHMGR). The present study aimed to virtually screen for potential hHMGR inhibitors from natural product to discover hypolipidemic drug candidates with fewer side effects and lesser toxicities. We used the 3D structure 1HWK from the PDB (Protein Data Bank) database of hHMGR as the target to screen for the strongly bound compounds from the traditional Chinese medicine database. Many interesting molecules including polyphenolic compounds, polisubstituted heterocyclics, and linear lipophilic alcohols were identified and their ADMET (absorption, disrtibution, metabolism, excretion, toxicity) properties were predicted. Finally, four compounds were obtained for the in vitro validation experiments. The results indicated that curcumin and salvianolic acid C can effectively inhibit hHMGR, with IC50 (half maximal inhibitory concentration) values of 4.3 µM and 8 µM, respectively. The present study also demonstrated the feasibility of discovering new drug candidates through structure-based virtual screening.

The Arabidopsis transcription factor ANAC032 represses anthocyanin biosynthesis in response to high sucrose and oxidative and abiotic stresses

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Kashif Mahmood

2016-10-01

Full Text Available Production of anthocyanins is one of the adaptive responses employed by plants during stress conditions. During stress, anthocyanin biosynthesis is mainly regulated at the transcriptional level via a complex interplay between activators and repressors of anthocyanin biosynthesis genes. In this study, we investigated the role of a NAC transcription factor, ANAC032, in the regulation of anthocyanin biosynthesis during stress conditions. ANAC032 expression was found to be induced by exogenous sucrose as well as high light stress. Using biochemical, molecular and transgenic approaches, we show that ANAC032 represses anthocyanin biosynthesis in response to sucrose treatment, high light and oxidative stress. ANAC032 was found to negatively affect anthocyanin accumulation and the expression of anthocyanin biosynthesis (DFR, ANS/LDOX and positive regulatory (TT8 genes as demonstrated in overexpression line (35S:ANAC032 compared to wild-type under high light stress. The chimeric repressor line (35S:ANAC032-SRDX exhibited the opposite expression patterns for these genes. The negative impact of ANAC032 on the expression of DFR, ANS/LDOX and TT8 was found to be correlated with the altered expression of negative regulators of anthocyanin biosynthesis, AtMYBL2 and SPL9. In addition to this, ANAC032 also repressed the MeJA- and ABA-induced anthocyanin biosynthesis. As a result, transgenic lines overexpressing ANAC032 (35S:ANAC032 produced drastically reduced levels of anthocyanin pigment compared to wild-type when challenged with salinity stress. However, transgenic chimeric repressor lines (35S:ANAC032-SRDX exhibited the opposite phenotype. Our results suggest that ANAC032 functions as a negative regulator of anthocyanin biosynthesis in Arabidopsis thaliana during stress conditions.

The Arabidopsis Transcription Factor ANAC032 Represses Anthocyanin Biosynthesis in Response to High Sucrose and Oxidative and Abiotic Stresses.

Science.gov (United States)

Mahmood, Kashif; Xu, Zhenhua; El-Kereamy, Ashraf; Casaretto, José A; Rothstein, Steven J

2016-01-01

Production of anthocyanins is one of the adaptive responses employed by plants during stress conditions. During stress, anthocyanin biosynthesis is mainly regulated at the transcriptional level via a complex interplay between activators and repressors of anthocyanin biosynthesis genes. In this study, we investigated the role of a NAC transcription factor, ANAC032, in the regulation of anthocyanin biosynthesis during stress conditions. ANAC032 expression was found to be induced by exogenous sucrose as well as high light (HL) stress. Using biochemical, molecular and transgenic approaches, we show that ANAC032 represses anthocyanin biosynthesis in response to sucrose treatment, HL and oxidative stress. ANAC032 was found to negatively affect anthocyanin accumulation and the expression of anthocyanin biosynthesis ( DFR, ANS/LDOX) and positive regulatory ( TT8) genes as demonstrated in overexpression line (35S:ANAC032) compared to wild-type under HL stress. The chimeric repressor line (35S:ANAC032-SRDX) exhibited the opposite expression patterns for these genes. The negative impact of ANAC032 on the expression of DFR, ANS/LDOX and TT8 was found to be correlated with the altered expression of negative regulators of anthocyanin biosynthesis, AtMYBL2 and SPL9 . In addition to this, ANAC032 also repressed the MeJA- and ABA-induced anthocyanin biosynthesis. As a result, transgenic lines overexpressing ANAC032 (35S:ANAC032) produced drastically reduced levels of anthocyanin pigment compared to wild-type when challenged with salinity stress. However, transgenic chimeric repressor lines (35S:ANAC032-SRDX) exhibited the opposite phenotype. Our results suggest that ANAC032 functions as a negative regulator of anthocyanin biosynthesis in Arabidopsis thaliana during stress conditions.

Antitumor activity of a novel and orally available inhibitor of serine palmitoyltransferase

International Nuclear Information System (INIS)

Yaguchi, Masahiro; Shibata, Sachio; Satomi, Yoshinori; Hirayama, Megumi; Adachi, Ryutaro; Asano, Yasutomi; Kojima, Takuto; Hirata, Yasuhiro; Mizutani, Akio; Kiba, Atsushi; Sagiya, Yoji

2017-01-01

Metabolic reprogramming is an essential hallmark of neoplasia. Therefore, targeting cancer metabolism, including lipid synthesis, has attracted much interest in recent years. Serine palmitoyltransferase (SPT) plays a key role in the initial and rate-limiting step of de novo sphingolipid biosynthesis, and inhibiting SPT activity prevents the proliferation of certain cancer cells. Here, we identified a novel and orally available SPT inhibitor, compound-2. Compound-2 showed an anti-proliferative effect in several cancer cell models, reducing the levels of the sphingolipids ceramide and sphingomyelin. In the presence of compound-2, exogenously added S1P partially compensated the intracellular sphingolipid levels through the salvage pathway by partially rescuing compound-2-induced cytotoxicity. This suggested that the mechanism underlying the anti-proliferative effect of compound-2 involved the reduction of sphingolipid levels. Indeed, compound-2 promoted multinuclear formation with reduced endogenous sphingomyelin levels specifically in a compound-2-sensitive cell line, indicating that the effect was induced by sphingolipid reduction. Furthermore, compound-2 showed potent antitumor activity without causing significant body weight loss in the PL-21 acute myeloid leukemia mouse xenograft model. Therefore, SPT may be an attractive therapeutic anti-cancer drug target for which compound-2 may be a promising new drug. - Highlights: • We discovered compound-2, a novel and orally available SPT inhibitor. • Compound-2 was cytotoxic against PL-21 acute myeloid leukemia cells. • Compound-2 showed antitumor activity in the PL-21 mouse xenograft model.

The HAP Complex Governs Fumonisin Biosynthesis and Maize Kernel Pathogenesis in Fusarium verticillioides.

Science.gov (United States)

Ridenour, John B; Smith, Jonathon E; Bluhm, Burton H

2016-09-01

Contamination of maize ( Zea mays ) with fumonisins produced by the fungus Fusarium verticillioides is a global concern for food safety. Fumonisins are a group of polyketide-derived secondary metabolites linked to esophageal cancer and neural tube birth defects in humans and numerous toxicoses in livestock. Despite the importance of fumonisins in global maize production, the regulation of fumonisin biosynthesis during kernel pathogenesis is poorly understood. The HAP complex is a conserved, heterotrimeric transcriptional regulator that binds the consensus sequence CCAAT to modulate gene expression. Recently, functional characterization of the Hap3 subunit linked the HAP complex to the regulation of secondary metabolism and stalk rot pathogenesis in F. verticillioides . Here, we determine the involvement of HAP3 in fumonisin biosynthesis and kernel pathogenesis. Deletion of HAP3 suppressed fumonisin biosynthesis on both nonviable and live maize kernels and impaired pathogenesis in living kernels. Transcriptional profiling via RNA sequencing indicated that the HAP complex regulates at least 1,223 genes in F. verticillioides , representing nearly 10% of all predicted genes. Disruption of the HAP complex caused the misregulation of biosynthetic gene clusters underlying the production of secondary metabolites, including fusarins. Taken together, these results reveal that the HAP complex is a central regulator of fumonisin biosynthesis and kernel pathogenesis and works as both a positive and negative regulator of secondary metabolism in F. verticillioides .

Molecular characterization of genes encoding leucoanthocyanidin reductase involved in proanthocyanidin biosynthesis in apple

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Yuepeng eHan

2015-04-01

Full Text Available Proanthocyanidins (PAs are the major component of phenolics in apple, but mechanisms involved in PA biosynthesis remain unclear. Here, the relationship between the PA biosynthesis and the expression of genes encoding leucoanthocyanidin reductase (LAR and anthocyanidin reductase (ANR was investigated in fruit skin of one apple cultivar and three crabapples. Transcript levels of LAR1 and ANR2 genes were significantly correlated with the contents of catechin and epicatechin, respectively, which suggests their active roles in PA synthesis. Surprisingly, transcript levels for both LAR1 and LAR2 genes were almost undetectable in two crabapples that accumulated both flavan-3-ols and PAs. This contradicts the previous finding that LAR1 gene is a strong candidate regulating the accumulation of metabolites such as epicatechin and PAs in apple. Ectopic expression of apple MdLAR1 gene in tobacco suppresses expression of the late genes in anthocyanin biosynthetic pathway, resulting in loss of anthocyanin in flowers. Interestingly, a decrease in PA biosynthesis was also observed in flowers of transgenic tobacco plants overexpressing the MdLAR1 gene, which could be attributed to decreased expression of both the NtANR1 and NtANR2 genes. Our study not only confirms the in vivo function of apple LAR1 gene, but it is also helpful for understanding the mechanism of PA biosynthesis.

Reconstitution of a fungal meroterpenoid biosynthesis reveals the involvement of a novel family of terpene cyclases

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Itoh, Takayuki; Tokunaga, Kinya; Matsuda, Yudai; Fujii, Isao; Abe, Ikuro; Ebizuka, Yutaka; Kushiro, Tetsuo

2010-10-01

Meroterpenoids are hybrid natural products of both terpenoid and polyketide origin. We identified a biosynthetic gene cluster that is responsible for the production of the meroterpenoid pyripyropene in the fungus Aspergillus fumigatus through reconstituted biosynthesis of up to five steps in a heterologous fungal expression system. The cluster revealed a previously unknown terpene cyclase with an unusual sequence and protein primary structure. The wide occurrence of this sequence in other meroterpenoid and indole-diterpene biosynthetic gene clusters indicates the involvement of these enzymes in the biosynthesis of various terpenoid-bearing metabolites produced by fungi and bacteria. In addition, a novel polyketide synthase that incorporated nicotinyl-CoA as the starter unit and a prenyltransferase, similar to that in ubiquinone biosynthesis, was found to be involved in the pyripyropene biosynthesis. The successful production of a pyripyropene analogue illustrates the catalytic versatility of these enzymes for the production of novel analogues with useful biological activities.

Hacking an Algal Transcription Factor for Lipid Biosynthesis.

Science.gov (United States)

Chen, Xiulai; Hu, Guipeng; Liu, Liming

2018-03-01

Transcriptional engineering is a viable means for engineering microalgae to produce lipid, but it often results in a trade-off between production and growth. A recent study shows that engineering a single transcriptional regulator enables efficient carbon partitioning to lipid biosynthesis with high biomass productivity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Regulation of Isoprenoid Pheromone Biosynthesis in Bumblebee Males

Czech Academy of Sciences Publication Activity Database

Prchalová, Darina; Buček, Aleš; Brabcová, Jana; Žáček, Petr; Kindl, Jiří; Valterová, Irena; Pichová, Iva

2016-01-01

Roč. 17, č. 3 (2016), s. 260-267 ISSN 1439-4227 R&D Projects: GA MŠk LO1302; GA ČR GA15-06569S Institutional support: RVO:61388963 Keywords : biosynthesis * Bombus spp. * gene expression * isoprenoid s * pheromones * transcriptional regulation Subject RIV: CE - Biochemistry Impact factor: 2.847, year: 2016

Rapid extra-/intracellular biosynthesis of gold nanoparticles by the fungus Penicillium sp.

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Du, Liangwei; Xian, Liang; Feng, Jia-Xun

2011-03-01

In this work, the fungus Penicillium was used for rapid extra-/intracellular biosynthesis of gold nanoparticles. AuCl4 - ions reacted with the cell filtrate of Penicillium sp. resulting in extracellular biosynthesis of gold nanoparticles within 1 min. Intracellular biosynthesis of gold nanoparticles was obtained by incubating AuCl4 - solution with fungal biomass for 8 h. The gold nanoparticles were characterized by means of visual observation, UV-Vis absorption spectroscopy, X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDX). The extracellular nanoparticles exhibited maximum absorbance at 545 nm in UV-Vis spectroscopy. The XRD spectrum showed Bragg reflections corresponding to the gold nanocrystals. TEM exhibited the formed spherical gold nanoparticles in the size range from 30 to 50 nm with an average size of 45 nm. SEM and TEM revealed that the intracellular gold nanoparticles were well dispersed on the cell wall and within the cell, and they are mostly spherical in shape with an average diameter of 50 nm. The presence of gold was confirmed by EDX analysis.

Biosynthesis of the fungal cell wall polysaccharide galactomannan requires intraluminal GDP-mannose.

Science.gov (United States)

Engel, Jakob; Schmalhorst, Philipp S; Routier, Françoise H

2012-12-28

Fungal cell walls frequently contain a polymer of mannose and galactose called galactomannan. In the pathogenic filamentous fungus Aspergillus fumigatus, this polysaccharide is made of a linear mannan backbone with side chains of galactofuran and is anchored to the plasma membrane via a glycosylphosphatidylinositol or is covalently linked to the cell wall. To date, the biosynthesis and significance of this polysaccharide are unknown. The present data demonstrate that deletion of the Golgi UDP-galactofuranose transporter GlfB or the GDP-mannose transporter GmtA leads to the absence of galactofuran or galactomannan, respectively. This indicates that the biosynthesis of galactomannan probably occurs in the lumen of the Golgi apparatus and thus contrasts with the biosynthesis of other fungal cell wall polysaccharides studied to date that takes place at the plasma membrane. Transglycosylation of galactomannan from the membrane to the cell wall is hypothesized because both the cell wall-bound and membrane-bound polysaccharide forms are affected in the generated mutants. Considering the severe growth defect of the A. fumigatus GmtA-deficient mutant, proving this paradigm might provide new targets for antifungal therapy.

Discovery of PF-04620110, a Potent, Selective, and Orally Bioavailable Inhibitor of DGAT-1.

Science.gov (United States)

Dow, Robert L; Li, Jian-Cheng; Pence, Michael P; Gibbs, E Michael; LaPerle, Jennifer L; Litchfield, John; Piotrowski, David W; Munchhof, Michael J; Manion, Tara B; Zavadoski, William J; Walker, Gregory S; McPherson, R Kirk; Tapley, Susan; Sugarman, Eliot; Guzman-Perez, Angel; DaSilva-Jardine, Paul

2011-05-12

Acyl-CoA:diacylglycerol acyltransferase-1 (DGAT-1) catalyzes the final committed step in the biosynthesis of triglycerides. DGAT-1 knockout mice have been shown to be resistant to diet-induced obesity and have increased insulin sensitivity. Thus, inhibition of DGAT-1 may represent an attractive target for the treatment of obesity or type II diabetes. Herein, we report the discovery and characterization of a potent and selective DGAT-1 inhibitor PF-04620110 (3). Compound 3 inhibits DGAT-1 with an IC50 of 19 nM and shows high selectivity versus a broad panel of off-target pharmacologic end points. In vivo DGAT-1 inhibition has been demonstrated through reduction of plasma triglyceride levels in rodents at doses of ≥0.1 mg/kg following a lipid challenge. On the basis of this pharmacologic and pharmacokinetic profile, compound 3 has been advanced to human clinical studies.

Bacterial cellulose biosynthesis: diversity of operons, subunits, products and functions

Science.gov (United States)

Römling, Ute; Galperin, Michael Y.

2015-01-01

Summary Recent studies of bacterial cellulose biosynthesis, including structural characterization of a functional cellulose synthase complex, provided the first mechanistic insight into this fascinating process. In most studied bacteria, just two subunits, BcsA and BcsB, are necessary and sufficient for the formation of the polysaccharide chain in vitro. Other subunits – which differ among various taxa – affect the enzymatic activity and product yield in vivo by modulating expression of biosynthesis apparatus, export of the nascent β-D-glucan polymer to the cell surface, and the organization of cellulose fibers into a higher-order structure. These auxiliary subunits play key roles in determining the quantity and structure of the resulting biofilm, which is particularly important for interactions of bacteria with higher organisms that lead to rhizosphere colonization and modulate virulence of cellulose-producing bacterial pathogens inside and outside of host cells. Here we review the organization of four principal types of cellulose synthase operons found in various bacterial genomes, identify additional bcs genes that encode likely components of the cellulose biosynthesis and secretion machinery, and propose a unified nomenclature for these genes and subunits. We also discuss the role of cellulose as a key component of biofilms formed by a variety of free-living and pathogenic bacteria and, for the latter, in the choice between acute infection and persistence in the host. PMID:26077867

RNAi down-regulation of cinnamate-4-hydroxylase increases artemisinin biosynthesis in Artemisia annua

OpenAIRE

Kumar, Ritesh; Vashisth, Divya; Misra, Amita; Akhtar, Md Qussen; Jalil, Syed Uzma; Shanker, Karuna; Gupta, Madan Mohan; Rout, Prashant Kumar; Gupta, Anil Kumar; Shasany, Ajit Kumar

2016-01-01

Cinnamate-4-hydroxylase (C4H) converts trans-cinnamic acid (CA) to p-coumaric acid (COA) in the phenylpropanoid/lignin biosynthesis pathway. Earlier we reported increased expression of AaCYP71AV1 (an important gene of artemisinin biosynthesis pathway) caused by CA treatment in Artemisia annua. Hence, AaC4H gene was identified, cloned, characterized and silenced in A. annua with the assumption that the elevated internal CA due to knock down may increase the artemisinin yield. Accumulation of t...

Heparan sulfate C5-epimerase is essential for heparin biosynthesis in mast cells.

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Feyerabend, Thorsten B; Li, Jin-Ping; Lindahl, Ulf; Rodewald, Hans-Reimer

2006-04-01

Biosynthesis of heparin, a mast cell-derived glycosaminoglycan with widespread importance in medicine, has not been fully elucidated. In biosynthesis of heparan sulfate (HS), a structurally related polysaccharide, HS glucuronyl C5-epimerase (Hsepi) converts D-glucuronic acid (GlcA) to L-iduronic acid (IdoA) residues. We have generated Hsepi-null mouse mutant mast cells, and we show that the same enzyme catalyzes the generation of IdoA in heparin and that 'heparin' lacking IdoA shows a distorted O-sulfation pattern.

Engineering fatty acid biosynthesis in microalgae for sustainable biodiesel.

Science.gov (United States)

Blatti, Jillian L; Michaud, Jennifer; Burkart, Michael D

2013-06-01

Microalgae are a promising feedstock for biodiesel and other liquid fuels due to their fast growth rate, high lipid yields, and ability to grow in a broad range of environments. However, many microalgae achieve maximal lipid yields only under stress conditions hindering growth and providing compositions not ideal for biofuel applications. Metabolic engineering of algal fatty acid biosynthesis promises to create strains capable of economically producing fungible and sustainable biofuels. The algal fatty acid biosynthetic pathway has been deduced by homology to bacterial and plant systems, and much of our understanding is gleaned from basic studies in these systems. However, successful engineering of lipid metabolism in algae will necessitate a thorough characterization of the algal fatty acid synthase (FAS) including protein-protein interactions and regulation. This review describes recent efforts to engineer fatty acid biosynthesis toward optimizing microalgae as a biodiesel feedstock. Copyright © 2013 Elsevier Ltd. All rights reserved.

Biosynthesis and function of simple amides in Xenorhabdus doucetiae.

Science.gov (United States)

Bode, Edna; He, Yue; Vo, Tien Duy; Schultz, Roland; Kaiser, Marcel; Bode, Helge B

2017-11-01

Xenorhabdus doucetiae, the bacterial symbiont of the entomopathogenic nematode Steinernema diaprepesi produces several different fatty acid amides. Their biosynthesis has been studied using a combination of analysis of gene deletions and promoter exchanges in X. doucetiae and heterologous expression of candidate genes in E. coli. While a decarboxylase is required for the formation of all observed phenylethylamides and tryptamides, the acyltransferase XrdE encoded in the xenorhabdin biosynthesis gene cluster is responsible for the formation of short chain acyl amides. Additionally, new, long-chain and cytotoxic acyl amides were identified in X. doucetiae infected insects and when X. doucetiae was grown in Galleria Instant Broth (GIB). When the bioactivity of selected amides was tested, a quorum sensing modulating activity was observed for the short chain acyl amides against the two different quorum sensing systems from Chromobacterium and Janthinobacterium. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Characterization of the regulatory network of BoMYB2 in controlling anthocyanin biosynthesis in purple cauliflower.

Science.gov (United States)

Chiu, Li-Wei; Li, Li

2012-10-01

Purple cauliflower (Brassica oleracea L. var. botrytis) Graffiti represents a unique mutant in conferring ectopic anthocyanin biosynthesis, which is caused by the tissue-specific activation of BoMYB2, an ortholog of Arabidopsis PAP2 or MYB113. To gain a better understanding of the regulatory network of anthocyanin biosynthesis, we investigated the interaction among cauliflower MYB-bHLH-WD40 network proteins and examined the interplay of BoMYB2 with various bHLH transcription factors in planta. Yeast two-hybrid studies revealed that cauliflower BoMYBs along with the other regulators formed the MYB-bHLH-WD40 complexes and BobHLH1 acted as a bridge between BoMYB and BoWD40-1 proteins. Different BoMYBs exhibited different binding activity to BobHLH1. Examination of the BoMYB2 transgenic lines in Arabidopsis bHLH mutant backgrounds demonstrated that TT8, EGL3, and GL3 were all involved in the BoMYB2-mediated anthocyanin biosynthesis. Expression of BoMYB2 in Arabidopsis caused up-regulation of AtTT8 and AtEGL3 as well as a subset of anthocyanin structural genes encoding flavonoid 3'-hydroxylase, dihydroflavonol 4-reductase, and leucoanthocyanidin dioxygenase. Taken together, our results show that MYB-bHLH-WD40 network transcription factors regulated the bHLH gene expression, which may represent a critical feature in the control of anthocyanin biosynthesis. BoMYB2 together with various BobHLHs specifically regulated the late anthocyanin biosynthetic pathway genes for anthocyanin biosynthesis. Our findings provide additional information for the complicated regulatory network of anthocyanin biosynthesis and the transcriptional regulation of transcription factors in vegetable crops.

Terpenoid biosynthesis in Euphorbia lathyris and Copaifera spp

Energy Technology Data Exchange (ETDEWEB)

Skrukrud, C.L.

1987-07-01

Biosynthesis of triterpenoids by isolated latex of Euphorbia lathyris was investigated. The rate of in vitro incorporation of mevalonic acid into triterpenoids was thirty times greater than acetate incorporation indicating that the rate-limiting step in the pathway occurs prior to mevalonate. Both HMG-CoA reductase (EC 1.1.1.34) and HMG-CoA lyase (EC 4.1.3.4) activities were detected in isolated latex. HMG-CoA reductase was localized to a membrane-bound fraction of a 5000g pellet of latex. The rate of conversion of HMG-CoA to mevalonate by this enzyme is comparable to the overall rate of acetate incorporation into the triterpenoids suggesting that this enzyme is rate-determining in the biosynthesis of triterpenoids in E. lathyris latex. HMG-CoA reductase of E. lathyris vegetative tissue was localized to the membrane-bound portion of a particulate fraction (18,000g), and was solubilized by treatment with 2% polyoxyethylene ether W-1. Differences in the optimal pH for activity of HMG-CoA reductase from the latex and vegetative tissue suggest that isozymes of the enzyme may be present in the two tissue types. Studies of the incorporation of various precursors into leaf discs and cuttings taken from Copaifera spp. show differences in the rate of incorporation into Copaifera sesquiterpenes suggesting that the site of sesquiterpene biosynthesis may differ in its accessibility to the different substrates and/or reflecting the metabolic controls on carbon allocation to the terpenes. Mevalonate incorporation by Copaifera langsdorfii cuttings into sesquiterpenes was a hundred-fold greater than either acetate or glucose incorporation, however, its incorporation into squalene and triterpenoids was also a hundred-fold greater than the incorporation into sesquiterpenes. 119 refs., 58 figs., 16 tabs.

Terpenoid biosynthesis in Euphorbia lathyris and Copaifera spp

International Nuclear Information System (INIS)

Skrukrud, C.L.

1987-07-01

Biosynthesis of triterpenoids by isolated latex of Euphorbia lathyris was investigated. The rate of in vitro incorporation of mevalonic acid into triterpenoids was thirty times greater than acetate incorporation indicating that the rate-limiting step in the pathway occurs prior to mevalonate. Both HMG-CoA reductase (EC 1.1.1.34) and HMG-CoA lyase (EC 4.1.3.4) activities were detected in isolated latex. HMG-CoA reductase was localized to a membrane-bound fraction of a 5000g pellet of latex. The rate of conversion of HMG-CoA to mevalonate by this enzyme is comparable to the overall rate of acetate incorporation into the triterpenoids suggesting that this enzyme is rate-determining in the biosynthesis of triterpenoids in E. lathyris latex. HMG-CoA reductase of E. lathyris vegetative tissue was localized to the membrane-bound portion of a particulate fraction (18,000g), and was solubilized by treatment with 2% polyoxyethylene ether W-1. Differences in the optimal pH for activity of HMG-CoA reductase from the latex and vegetative tissue suggest that isozymes of the enzyme may be present in the two tissue types. Studies of the incorporation of various precursors into leaf discs and cuttings taken from Copaifera spp. show differences in the rate of incorporation into Copaifera sesquiterpenes suggesting that the site of sesquiterpene biosynthesis may differ in its accessibility to the different substrates and/or reflecting the metabolic controls on carbon allocation to the terpenes. Mevalonate incorporation by Copaifera langsdorfii cuttings into sesquiterpenes was a hundred-fold greater than either acetate or glucose incorporation, however, its incorporation into squalene and triterpenoids was also a hundred-fold greater than the incorporation into sesquiterpenes. 119 refs., 58 figs., 16 tabs

A chloroplast pathway for the de novo biosynthesis of triacylglycerol in Chlamydomonas reinhardtii

Energy Technology Data Exchange (ETDEWEB)

Fan, J.; Xu, C.; Andre, C.

2011-06-23

Neutral lipid metabolism has been extensively studied in yeast, plants and mammals. In contrast, little information is available regarding the biochemical pathway, enzymes and regulatory factors involved in the biosynthesis of triacylglycerol (TAG) in microalgae. In the conventional TAG biosynthetic pathway widely accepted for yeast, plants and mammals, TAG is assembled in the endoplasmic reticulum (ER) from its immediate precursor diacylglycerol (DAG) made by ER-specific acyltransferases, and is deposited exclusively in lipid droplets in the cytosol. Here, we demonstrated that the unicellular microalga Chlamydomonas reinhardtii employs a distinct pathway that uses DAG derived almost exclusively from the chloroplast to produce TAG. This unique TAG biosynthesis pathway is largely dependent on de novo fatty acid synthesis, and the TAG formed in this pathway is stored in lipid droplets in both the chloroplast and the cytosol. These findings have wide implications for understanding TAG biosynthesis and storage and other areas of lipid metabolism in microalgae and other organisms.

Gibberellins regulate iron deficiency-response by influencing iron transport and translocation in rice seedlings (Oryza sativa).

Science.gov (United States)

Wang, Baolan; Wei, Haifang; Xue, Zhen; Zhang, Wen-Hao

2017-04-01

Gibberellins (GAs) are a class of plant hormones with diverse functions. However, there has been little information on the role of GAs in response to plant nutrient deficiency. To evaluate the roles of GAs in regulation of Fe homeostasis, the effects of GA on Fe accumulation and Fe translocation in rice seedlings were investigated using wild-type, a rice mutant ( eui1 ) displaying enhnaced endogenous GA concentrations due to a defect in GA deactivation, and transgenic rice plants overexpressing OsEUI . Exposure to Fe-deficient medium significantly reduced biomass of rice plants. Both exogenous application of GA and an endogenous increase of bioactive GA enhanced Fe-deficiency response by exaggerating foliar chlorosis and reducing growth. Iron deficiency significantly suppressed production of GA 1 and GA 4 , the biologically active GAs in rice. Exogenous application of GA significantly decreased leaf Fe concentration regardless of Fe supply. Iron concentration in shoot of eui1 mutants was lower than that of WT plants under both Fe-sufficient and Fe-deficient conditions. Paclobutrazol, an inhibitor of GA biosynthesis, alleviated Fe-deficiency responses, and overexpression of EUI significantly increased Fe concentration in shoots and roots. Furthermore, both exogenous application of GA and endogenous increase in GA resulting from EUI mutation inhibited Fe translocation within shoots by suppressing OsYSL2 expression, which is involved in Fe transport and translocation. The novel findings provide compelling evidence to support the involvement of GA in mediation of Fe homeostasis in strategy II rice plants by negatively regulating Fe transport and translocation. © The Author 2017. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Salicylic Acid Alleviates Aluminum Toxicity in Soybean Roots through Modulation of Reactive Oxygen Species Metabolism

Directory of Open Access Journals (Sweden)

Ning Liu

2017-11-01

Full Text Available As an important signal molecule, salicylic acid (SA improves plant tolerance to aluminum (Al stress. The objective of this study was to investigate the effects of exogenous SA application on the dynamics of endogenous SA and reactive oxygen species in soybean (Glycine max L. exposed to Al stress. The roots of soybean seedlings were exposed to a combination of AlCl3 (30 μM and SA (10 μM/PAC (100 μM, paclobutrazol, SA biosynthesis inhibitor for 3, 6, 9, and 12 h. Al stress induced an increase in endogenous SA concentration in a time-dependent manner, also verified by the up-regulated expression of GmNPR1, an SA-responsive gene. Al stress increased the activities of phenylalanine ammonia-lyase (PAL and benzoic acid 2-hydroxylase (BA2H, and the contents of SA, O2- and malondialdehyde (MDA in the root apex. The application of exogenous SA increased PAL and BA2H, and reduced O2- and MDA contents in soybean roots under Al stress. PAC inhibited the SA induced increase in BA2H activity. In addition, the SA application resulted in a rapid increase in hydrogen peroxide (H2O2 concentration under Al stress, followed by a sharp decrease. Compared with the plants exposed to Al alone, Al+SA plants possessed higher activities of superoxide dismutase, peroxidase, and ascorbate peroxidase, and lower catalase activity, indicating that SA alleviated Al-induced oxidative damage. These results suggested that PAL and BA2H were involved in Al-induced SA production and showed that SA alleviated the adverse effects of Al toxicity by modulating the cellular H2O2 level and the antioxidant enzyme activities in the soybean root apex.

Salicylic acid alleviates aluminum toxicity in soybean roots through modulation of reactive oxygen species metabolism

Science.gov (United States)

Liu, Ning; Song, Fengbin; Zhu, Xiancan; You, Jiangfeng; Yang, Zhenming; Li, Xiangnan

2017-11-01

As an important signal molecule, salicylic acid (SA) improves plant tolerance to aluminum (Al) stress. The objective of this study was to investigate the effects of exogenous SA application on the dynamics of endogenous SA and reactive oxygen species in soybean (Glycine max L.) exposed to Al stress. The roots of soybean seedlings were exposed to a combination of AlCl3 (30 μM) and SA (10 μM)/PAC (100 μM, paclobutrazol, SA biosynthesis inhibitor) for 3, 6, 9 and 12 h. Al stress induced an increase in endogenous SA concentration in a time-dependent manner, also verified by the up-regulated expression of GmNPR1, an SA-responsive gene. Al stress increased the activities of phenylalanine ammonia-lyase (PAL) and benzoic acid 2-hydroxylase (BA2H), and the contents of SA, O2- and malondialdehyde (MDA) in the root apex. The application of exogenous SA increased PAL and BA2H, and reduced O2- and MDA contents in soybean roots under Al stress. PAC inhibited the SA induced increase in BA2H activity. In addition, the SA application resulted in a rapid increase in hydrogen peroxide (H2O2) concentration under Al stress, followed by a sharp decrease. Compared with the plants exposed to Al alone, Al+SA plants possessed higher activities of superoxide dismutase, peroxidase and ascorbate peroxidase, and lower catalase activity, indicating that SA alleviated Al-induced oxidative damage. These results suggested that PAL and BA2H were involved in Al-induced SA production and showed that SA alleviated the adverse effects of Al toxicity by modulating the cellular H2O2 level and the antioxidant enzyme activities in the soybean root apex.

Primary Metabolism during Biosynthesis of Secondary Wall Polymers of Protoxylem Vessel Elements1[OPEN

Science.gov (United States)

Morisaki, Keiko; Sawada, Yuji; Sano, Ryosuke; Yamamoto, Atsushi; Kurata, Tetsuya; Suzuki, Shiro; Matsuda, Mami; Hasunuma, Tomohisa; Hirai, Masami Yokota

2016-01-01

Xylem vessels, the water-conducting cells in vascular plants, undergo characteristic secondary wall deposition and programmed cell death. These processes are regulated by the VASCULAR-RELATED NAC-DOMAIN (VND) transcription factors. Here, to identify changes in metabolism that occur during protoxylem vessel element differentiation, we subjected tobacco (Nicotiana tabacum) BY-2 suspension culture cells carrying an inducible VND7 system to liquid chromatography-mass spectrometry-based wide-target metabolome analysis and transcriptome analysis. Time-course data for 128 metabolites showed dynamic changes in metabolites related to amino acid biosynthesis. The concentration of glyceraldehyde 3-phosphate, an important intermediate of the glycolysis pathway, immediately decreased in the initial stages of cell differentiation. As cell differentiation progressed, specific amino acids accumulated, including the shikimate-related amino acids and the translocatable nitrogen-rich amino acid arginine. Transcriptome data indicated that cell differentiation involved the active up-regulation of genes encoding the enzymes catalyzing fructose 6-phosphate biosynthesis from glyceraldehyde 3-phosphate, phosphoenolpyruvate biosynthesis from oxaloacetate, and phenylalanine biosynthesis, which includes shikimate pathway enzymes. Concomitantly, active changes in the amount of fructose 6-phosphate and phosphoenolpyruvate were detected during cell differentiation. Taken together, our results show that protoxylem vessel element differentiation is associated with changes in primary metabolism, which could facilitate the production of polysaccharides and lignin monomers and, thus, promote the formation of the secondary cell wall. Also, these metabolic shifts correlate with the active transcriptional regulation of specific enzyme genes. Therefore, our observations indicate that primary metabolism is actively regulated during protoxylem vessel element differentiation to alter the cell’s metabolic

Unraveling Additional O-Methylation Steps in Benzylisoquinoline Alkaloid Biosynthesis in California Poppy (Eschscholzia californica).

Science.gov (United States)

Purwanto, Ratmoyo; Hori, Kentaro; Yamada, Yasuyuki; Sato, Fumihiko

2017-09-01

California poppy (Eschscholzia californica), a member of the Papaveraceae family, produces many biologically active benzylisoquinoline alkaloids (BIAs), such as sanguinarine, macarpine and chelerythrine. Sanguinarine biosynthesis has been elucidated at the molecular level, and its biosynthetic genes have been isolated and used in synthetic biology approaches to produce BIAs in vitro. However, several genes involved in the biosynthesis of macarpine and chelerythrine have not yet been characterized. In this study, we report the isolation and characterization of a novel O-methyltransferase (OMT) involved in the biosynthesis of partially characterized BIAs, especially chelerythrine. A search of the RNA sequence database from NCBI and PhytoMetaSyn for the conserved OMT domain identified 68 new OMT-like sequences, of which the longest 22 sequences were selected based on sequence similarity. Based on their expression in cell lines with different macarpine/chelerythrine profiles, we selected three OMTs (G2, G3 and G11) for further characterization. G3 expression in Escherichia coli indicated O-methylation activity of the simple benzylisoquinolines, including reticuline and norreticuline, and the protoberberine scoulerine with dual regio-reactivities. G3 produced 7-O-methylated, 3'-O-methylated and dual O-methylated products from reticuline and norreticuline, and 9-O-methylated tetrahydrocolumbamine, 2-O-methylscoulerine and tetrahydropalmatine from scoulerine. Further enzymatic analyses suggested that G3 is a scoulerine-9-O-methyltransferase for the biosynthesis of chelerythrine in California poppy. In the present study, we discuss the physiological role of G3 in BIA biosynthesis. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

[Age and characteristics of cholesterol biosynthesis in rat liver under normal conditions and during atherogenic loading].

Science.gov (United States)

Chaialo, P P

1977-02-01

Intraperitoneal injection of C14CH3COONa to normal rats aged 6--8 and 28--32 months revealed a slower dynamics of cholesterol biosynthesis in the liver of old rats at the maximum of the tracer incorporation was lower than in the young ones. Atherogenic diet (0.25 g of cholesterol per 100 g of animal weight for a period of 20 days) was accompanied by an increase in the total cholesterol content and depressio of its biosynthesis in the liver, more pronounced in the young rats. Continued cholesterol administration caused further depression of its biosynthesis, most pronounced (in this case) in the old animals.

Methionine salvage pathway in relation to ethylene biosynthesis

International Nuclear Information System (INIS)

Miyazaki, J.H.

1987-01-01

The recycling of methionine during ethylene biosynthesis (the methionine cycle) was studied. During ethylene biosynthesis, the H 3 CS-group of S-adenosylmethionine (SAM) is released at 5'-methylthioadenosine (MTA), which is recycled to methionine via 5'-methylthioribose (MTS). In mungbean hypocotyls and cell-free extracts of avocado fruit, [ 14 C]MTR was converted to labeled methionine via 2-keto-4-methylthiobutyric acid (KMB) and 2-hydroxy-4-methylthiobutyric acid (HMB) as intermediates. Radioactive tracer studies showed that KMB was converted readily in vivo and in vitro to methionine, while HMB was converted much more slowly. The conversion of KMB to methionine by dialyzed avocado extract required an amino group donor. Among several potential donors tested, L-glutamine was the most efficient. Incubation of [ribose-U- 14 C]MTR with avocado extract resulted in the production of [ 14 C]formate, with little evolution of other 14 C-labeled one-carbon compounds, indicating that the conversion of MTR to KMB involves a loss of formate, presumably from C-1 of MTR

Structure of S. aureus HPPK and the discovery of a new substrate site inhibitor.

Directory of Open Access Journals (Sweden)

Sandeep Chhabra

Full Text Available The first structural and biophysical data on the folate biosynthesis pathway enzyme and drug target, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (SaHPPK, from the pathogen Staphylococcus aureus is presented. HPPK is the second essential enzyme in the pathway catalysing the pyrophosphoryl transfer from cofactor (ATP to the substrate (6-hydroxymethyl-7,8-dihydropterin, HMDP. In-silico screening identified 8-mercaptoguanine which was shown to bind with an equilibrium dissociation constant, K(d, of ∼13 µM as measured by isothermal titration calorimetry (ITC and surface plasmon resonance (SPR. An IC(50 of ∼41 µM was determined by means of a luminescent kinase assay. In contrast to the biological substrate, the inhibitor has no requirement for magnesium or the ATP cofactor for competitive binding to the substrate site. The 1.65 Å resolution crystal structure of the inhibited complex showed that it binds in the pterin site and shares many of the key intermolecular interactions of the substrate. Chemical shift and (15N heteronuclear NMR measurements reveal that the fast motion of the pterin-binding loop (L2 is partially dampened in the SaHPPK/HMDP/α,β-methylene adenosine 5'-triphosphate (AMPCPP ternary complex, but the ATP loop (L3 remains mobile on the µs-ms timescale. In contrast, for the SaHPPK/8-mercaptoguanine/AMPCPP ternary complex, the loop L2 becomes rigid on the fast timescale and the L3 loop also becomes more ordered--an observation that correlates with the large entropic penalty associated with inhibitor binding as revealed by ITC. NMR data, including (15N-(1H residual dipolar coupling measurements, indicate that the sulfur atom in the inhibitor is important for stabilizing and restricting important motions of the L2 and L3 catalytic loops in the inhibited ternary complex. This work describes a comprehensive analysis of a new HPPK inhibitor, and may provide a foundation for the development of novel antimicrobials targeting

Molecular and biochemical studies of fragrance biosynthesis in rose

NARCIS (Netherlands)

Sun, P.

2017-01-01

Roses are one of the most popular ornamental plants, whose floral volatiles are not only involved in environmental interactions but also widely used by industries. The biosynthesis of many of these volatiles in roses is not well understood. This thesis describes alternative pathways for the

Different biosynthesis patterns among flavonoid 3-glycosides with distinct effects on accumulation of other flavonoid metabolites in pears (Pyrus bretschneideri Rehd..

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Rui Zhai

Full Text Available Flavonoid biosynthesis profile was clarified by fruit bagging and re-exposure treatments in the green Chinese pear 'Zaosu' (Pyrus bretschneideri Rehd. and its red mutant 'Red Zaosu'. Two distinct biosynthesis patterns of flavonoid 3-glycosides were found in 'Zaosu' pear. By comparison with 'Red Zaosu', the biosynthesis of flavonoid 3-galactosides and flavonoid 3-arabinosides were inhibited by bagging and these compounds only re-accumulated to a small degree in the fruit peel of 'Zaosu' after the bags were removed. In contrast, the biosynthesis of flavonoid 3-gluctosides and flavonoid 3-rutinosides was reduced by bagging and then increased when the fruits were re-exposed to sunlight. A combination of correlation, multicollinearity test and partial-correlation analyses among major flavonoid metabolites indicated that biosynthesis of each phenolic compound was independent in 'Zaosu' pear, except for the positive correlation between flavonoid 3-rutincosides and flavanols. In contrast with the green pear cultivar, almost all phenolic compounds in the red mutant had similar biosynthesis patterns except for arbutin. However, only the biosynthesis of flavonoid 3-galactosides was relatively independent and strongly affected the synthesis of the other phenolic compounds. Therefore, we propose a hypothesis that the strong accumulation of flavonoid 3-galactosides stimulated the biosynthesis of other flavonoid compounds in the red mutant and, therefore, caused systemic variation of flavonoid biosynthesis profiles between 'Zaosu' and its red mutant. This hypothesis had been further demonstrated by the enzyme activity of UFGT, and transcript levels of flavonoid biosynthetic genes and been well tested by a stepwise linear regression forecasting model. The gene that encodes flavonoid 3-galacosyltransferase was also identified and isolated from the pear genome.

CPC, a single-repeat R3 MYB, is a negative regulator of anthocyanin biosynthesis in Arabidopsis.

Science.gov (United States)

Zhu, Hui-Fen; Fitzsimmons, Karen; Khandelwal, Abha; Kranz, Robert G

2009-07-01

Single-repeat R3 MYB transcription factors like CPC (CAPRICE) are known to play roles in developmental processes such as root hair differentiation and trichome initiation. However, none of the six Arabidopsis single-repeat R3 MYB members has been reported to regulate flavonoid biosynthesis. We show here that CPC is a negative regulator of anthocyanin biosynthesis. In the process of using CPC to test GAL4-dependent driver lines, we observed a repression of anthocyanin synthesis upon GAL4-mediated CPC overexpression. We demonstrated that this is not due to an increase in nutrient uptake because of more root hairs. Rather, CPC expression level tightly controls anthocyanin accumulation. Microarray analysis on the whole genome showed that, of 37 000 features tested, 85 genes are repressed greater than three-fold by CPC overexpression. Of these 85, seven are late anthocyanin biosynthesis genes. Also, anthocyanin synthesis genes were shown to be down-regulated in 35S::CPC overexpression plants. Transient expression results suggest that CPC competes with the R2R3-MYB transcription factor PAP1/2, which is an activator of anthocyanin biosynthesis genes. This report adds anthocyanin biosynthesis to the set of programs that are under CPC control, indicating that this regulator is not only for developmental programs (e.g. root hairs, trichomes), but can influence anthocyanin pigment synthesis.

Comparative proteomic analysis provides insight into 10-hydroxy-2-decenoic acid biosynthesis in honey bee workers.

Science.gov (United States)

Yang, Xiao-Hui; Yang, Shi-Fa; Wang, Rui-Ming

2017-07-01

10-Hydroxy-2-decenoic acid (10-HDA) is the major compound produced from the mandibular glands (MGs) of honey bee workers. However, little information is available on the molecular mechanisms of 10-HDA biosynthesis. In our study, based on investigating the 10-HDA secretion pattern and the morphological characteristics of MGs from honey bee workers of different ages, a comparative proteomic analysis was performed in the MGs of workers with different 10-HDA production. In total, 59 up-regulated protein species representing 45 unique proteins were identified in high 10-HDA-producing workers by 2-DE-MALDI-TOF/TOF MS. These proteins were involved in carbohydrate/energy metabolism, fatty acid metabolism, protein metabolism and folding, antioxidation, cytoskeleton, development and cell signaling. Proteins related to fatty acid metabolism, including fatty acid synthase and β-oxidation enzymes, are potentially crucial proteins involved in 10-HDA biosynthesis pathway. And RNA interference (RNAi) results demonstrated that knockdown of electron transfer flavoprotein subunit beta (ETF-β), one of the protein related to fatty acid metabolism, decreased 10-HDA production of worker bees, suggesting that ETF-β was necessary for 10-HDA biosynthesis. This study reveals the characteristics of MGs of worker bees at different developmental stages and proteins associated with 10-HDA biosynthesis, which provides the first insight into the molecular mechanism of 10-HDA biosynthesis.

Extrinsic functions of lectin domains in O-N-acetylgalactosamine glycan biosynthesis

DEFF Research Database (Denmark)

Lorenz, Virginia; Ditamo, Yanina; Cejas, Romina B

2016-01-01

during O-GalNAc glycan biosynthesis. The presence of lectin domain T3lec or T4lec during ppGalNAc-T2 and ppGalNAc-T3 catalytic reaction had a clear inhibitory effect on GalNAc-T activity. Interaction of T3lec or T4lec with ppGalNAc-T2 catalytic domain was not mediated by carbohydrate. T3lec, but not T2......Glycan biosynthesis occurs mainly in Golgi. Molecular organization and functional regulation of this process are not well understood. We evaluated the extrinsic effect of lectin domains (β-trefoil fold) of polypeptide GalNAc-transferases (ppGalNAc-Ts) on catalytic activity of glycosyltransferases...

OleA Glu117 is key to condensation of two fatty-acyl coenzyme A substrates in long-chain olefin biosynthesis

Energy Technology Data Exchange (ETDEWEB)

Jensen, Matthew R.; Goblirsch, Brandon R.; Christenson, James K.; Esler, Morgan A.; Mohamed, Fatuma A.; Wackett, Lawrence P.; Wilmot, Carrie M. (UMM)

2017-10-12

In the interest of decreasing dependence on fossil fuels, microbial hydrocarbon biosynthesis pathways are being studied for renewable, tailored production of specialty chemicals and biofuels. One candidate is long-chain olefin biosynthesis, a widespread bacterial pathway that produces waxy hydrocarbons. Found in three- and four-gene clusters, oleABCD encodes the enzymes necessary to produce cis-olefins that differ by alkyl chain length, degree of unsaturation, and alkyl chain branching. The first enzyme in the pathway, OleA, catalyzes the Claisen condensation of two fatty acyl-coenzyme A (CoA) molecules to form a β-keto acid. In this report, the mechanistic role of Xanthomonas campestris OleA Glu117 is investigated through mutant enzymes. Crystal structures were determined for each mutant as well as their complex with the inhibitor cerulenin. Complemented by substrate modeling, these structures suggest that Glu117 aids in substrate positioning for productive carbon–carbon bond formation. Analysis of acyl-CoA substrate hydrolysis shows diminished activity in all mutants. When the active site lacks an acidic residue in the 117 position, OleA cannot form condensed product, demonstrating that Glu117 has a critical role upstream of the essential condensation reaction. Profiling of pH dependence shows that the apparent pKa for Glu117 is affected by mutagenesis. Taken together, we propose that Glu117 is the general base needed to prime condensation via deprotonation of the second, non-covalently bound substrate during turnover. This is the first example of a member of the thiolase superfamily of condensing enzymes to contain an active site base originating from the second monomer of the dimer.

Efeitos de reguladores de crescimento na elongação do colmo de trigo = Effect of growth regulators on wheat stem elongation

Directory of Open Access Journals (Sweden)

Marcelo Curitiba Espindula

2010-01-01

Full Text Available Objetivou-se com este trabalho avaliar o efeito de doses e épocas deaplicação de três reguladores de crescimento na elongação do colmo de plantas de trigo. O experimento, conduzido em Viçosa, Estado de Minas Gerais, de maio a setembro de 2005, foi instalado com tratamentos em esquema fatorial e hierárquico com uma testemunha, no delineamento em blocos casualizados com quatro repetições. Os tratamentos foram 500; 1.000 e 1.500 g ha-1 de clormequat; 62,5; 125,0 e 187,5 g ha-1 detrinexapac-etil e 40; 80 e 120 g ha-1 de paclobutrazol, aplicados no estádio 6 ou 8 da escala Feeks e Large, e uma testemunha. O comprimento do colmo com trinexapac-etil foi menor do que com o clormequat, que, por sua vez, foi menor do que com o paclobutrazol. A aplicação de reguladores na época 1 promoveu maior redução dosentrenós basais, enquanto a aplicação na época 2 proporcionou maior influência no pedúnculo. O aumento das doses de clormequat e trinexapac-etil promoveu redução do comprimento do colmo e das partes que o formam. O aumento das doses de paclobutrazol promoveu respostas pouco expressivas no colmo e seus componentes.The objective of this work was to evaluate the effect of doses and times of application of three growth regulators on wheat stem elongation. The experiments were conducted in Viçosa, Minas Gerais State, from May to September 2005, in a factorial and hierarchicalrandomized block design with four repetitions and a control treatment. Treatments consisted of 500, 1000 and 1500 g ha-1 of chlormequat; 62.5, 125.0 and 187.5 g ha-1 of trinexapac-ethyl and 40, 80 and 120 g ha-1 of paclobutrazol, applied either at the 6 or 8stage of the Feeks and Large scale, with a control. Stem length with trinexapac-ethyl was smaller than with chlormequat, which was in its turn smaller than with paclobutrazol. Application of growth regulators at stage 1 produced shorter basal internodes, whereas the stage 2 application caused greater reduction

pH induced protein-scaffold biosynthesis of tunable shape gold nanoparticles

International Nuclear Information System (INIS)

Zhang Xiaorong; He Xiaoxiao; Wang Kemin; Ren Fang; Qin Zhihe

2011-01-01

In this paper, a pH-inductive protein-scaffold biosynthesis of shape-tunable crystalline gold nanoparticles at room temperature has been developed. By simple manipulation of the reaction solution's pH, anisotropic gold nanoparticles including spheres, triangles and cubes could be produced by incubating an aqueous solution of sodium tetrachloroaurate with Dolichomitriopsis diversiformis biomasses after immersion in ultrapure Millipore water overnight. A moss protein with molecular weight of about 71 kDa and pI of 4.9 was the primary biomolecule involved in the biosynthesis of gold nanoparticles. The secondary configuration of the proteins by CD spectrum implied that the moss protein could display different secondary configurations including random coil, α-helix and intermediate conformations between random coil and α-helix for the experimental pH solution. The growth process of gold nanoparticles further showed that the moss protein with different configurations provided the template scaffold for the shape-controlled biosynthesis of gold nanoparticles. The constrained shape of the gold nanoparticles, however, disappeared in boiled moss extract. The gold nanoparticles with designed morphology were successfully reconstructed using the moss protein purified from the gold nanoparticles. Structural characterizations by SEM, TEM and SAED showed that the triangular and cubic gold nanoparticles were single crystalline.

Wybutosine biosynthesis: Structural and mechanistic overview

Science.gov (United States)

Perche-Letuvée, Phanélie; Molle, Thibaut; Forouhar, Farhad; Mulliez, Etienne; Atta, Mohamed

2014-01-01

Over the last 10 years, significant progress has been made in understanding the genetics, enzymology and structural components of the wybutosine (yW) biosynthetic pathway. These studies have played a key role in expanding our understanding of yW biosynthesis and have revealed unexpected evolutionary ties, which are presently being unraveled. The enzymes catalyzing the 5 steps of this pathway, from genetically encoded guanosine to wybutosine base, provide an ensemble of amazing reaction mechanisms that are to be discussed in this review article. PMID:25629788

Antioxidant capacity changes and phenolic profile of Echinacea purpurea, nettle (Urtica dioica L.), and dandelion (Taraxacum officinale) after application of polyamine and phenolic biosynthesis regulators.

Science.gov (United States)

Hudec, Jozef; Burdová, Mária; Kobida, L'ubomír; Komora, Ladislav; Macho, Vendelín; Kogan, Grigorij; Turianica, Ivan; Kochanová, Radka; Lozek, Otto; Habán, Miroslav; Chlebo, Peter

2007-07-11

The changes of the antioxidant (AOA) and antiradical activities (ARA) and the total contents of phenolics, anthocyanins, flavonols, and hydroxybenzoic acid in roots and different aerial sections of Echinacea purpurea, nettle, and dandelion, after treatment with ornithine decarboxylase inhibitor, a polyamine inhibitor (O-phosphoethanolamine, KF), and a phenol biosynthesis stimulator (carboxymethyl chitin glucan, CCHG) were analyzed spectrophotometrically; hydroxycinnamic acids content was analyzed by RP-HPLC with UV detection. Both regulators increased the AOA measured as inhibition of peroxidation (IP) in all herb sections, with the exception of Echinacea stems after treatment with KF. In root tissues IP was dramatically elevated mainly after CCHG application: 8.5-fold in Echinacea, 4.14-fold in nettle, and 2.08-fold in dandelion. ARA decrease of Echinacea leaves treated with regulators was in direct relation only with cichoric acid and caftaric acid contents. Both regulators uphold the formation of cinnamic acid conjugates, the most expressive being that of cichoric acid after treatment with CCHG in Echinacea roots from 2.71 to 20.92 mg g(-1). There was a strong relationship between increase of the total phenolics in all sections of Echinacea, as well as in the studied sections of dandelion, and the anthocyanin content.

The Arabidopsis Transcription Factor ANAC032 Represses Anthocyanin Biosynthesis in Response to High Sucrose and Oxidative and Abiotic Stresses

OpenAIRE

Mahmood, Kashif; Xu, Zhenhua; El-Kereamy, Ashraf; Casaretto, Jos? A.; Rothstein, Steven J.

2016-01-01

Production of anthocyanins is one of the adaptive responses employed by plants during stress conditions. During stress, anthocyanin biosynthesis is mainly regulated at the transcriptional level via a complex interplay between activators and repressors of anthocyanin biosynthesis genes. In this study, we investigated the role of a NAC transcription factor, ANAC032, in the regulation of anthocyanin biosynthesis during stress conditions. ANAC032 expression was found to be induced by exogenous su...

Virus-Induced Silencing of Key Genes Leads to Differential Impact on Withanolide Biosynthesis in the Medicinal Plant, Withania somnifera.

Science.gov (United States)

Agarwal, Aditya Vikram; Singh, Deeksha; Dhar, Yogeshwar Vikram; Michael, Rahul; Gupta, Parul; Chandra, Deepak; Trivedi, Prabodh Kumar

2018-02-01

Withanolides are a collection of naturally occurring, pharmacologically active, secondary metabolites synthesized in the medicinally important plant, Withania somnifera. These bioactive molecules are C28-steroidal lactone triterpenoids and their synthesis is proposed to take place via the mevalonate (MVA) and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways through the sterol pathway using 24-methylene cholesterol as substrate flux. Although the phytochemical profiles as well as pharmaceutical activities of Withania extracts have been well studied, limited genomic information and difficult genetic transformation have been a major bottleneck towards understanding the participation of specific genes in withanolide biosynthesis. In this study, we used the Tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS) approach to study the participation of key genes from MVA, MEP and triterpenoid biosynthesis for their involvement in withanolide biosynthesis. TRV-infected W. somnifera plants displayed unique phenotypic characteristics and differential accumulation of total Chl as well as carotenoid content for each silenced gene suggesting a reduction in overall isoprenoid synthesis. Comprehensive expression analysis of putative genes of withanolide biosynthesis revealed transcriptional modulations conferring the presence of complex regulatory mechanisms leading to withanolide biosynthesis. In addition, silencing of genes exhibited modulated total and specific withanolide accumulation at different levels as compared with control plants. Comparative analysis also suggests a major role for the MVA pathway as compared with the MEP pathway in providing substrate flux for withanolide biosynthesis. These results demonstrate that transcriptional regulation of selected Withania genes of the triterpenoid biosynthetic pathway critically affects withanolide biosynthesis, providing new horizons to explore this process further, in planta.

Biosynthesis of Anthocyanins and Their Regulation in Colored Grapes

Directory of Open Access Journals (Sweden)

Guo-Liang Yan

2010-12-01

Full Text Available Anthocyanins, synthesized via the flavonoid pathway, are a class of crucial phenolic compounds which are fundamentally responsible for the red color of grapes and wines. As the most important natural colorants in grapes and their products, anthocyanins are also widely studied for their numerous beneficial effects on human health. In recent years, the biosynthetic pathway of anthocyanins in grapes has been thoroughly investigated. Their intracellular transportation and accumulation have also been further clarified. Additionally, the genetic mechanism regulating their biosynthesis and the phytohormone influences on them are better understood. Furthermore, due to their importance in the quality of wine grapes, the effects of the environmental factors and viticulture practices on anthocyanin accumulation are being investigated increasingly. The present paper summarizes both the basic information and the most recent advances in the study of the anthocyanin biosynthesis in red grapes, emphasizing their gene structure, the transcriptional factors and the diverse exterior regulation factors.

Biosynthesis of anthocyanins and their regulation in colored grapes.

Science.gov (United States)

He, Fei; Mu, Lin; Yan, Guo-Liang; Liang, Na-Na; Pan, Qiu-Hong; Wang, Jun; Reeves, Malcolm J; Duan, Chang-Qing

2010-12-09

Anthocyanins, synthesized via the flavonoid pathway, are a class of crucial phenolic compounds which are fundamentally responsible for the red color of grapes and wines. As the most important natural colorants in grapes and their products, anthocyanins are also widely studied for their numerous beneficial effects on human health. In recent years, the biosynthetic pathway of anthocyanins in grapes has been thoroughly investigated. Their intracellular transportation and accumulation have also been further clarified. Additionally, the genetic mechanism regulating their biosynthesis and the phytohormone influences on them are better understood. Furthermore, due to their importance in the quality of wine grapes, the effects of the environmental factors and viticulture practices on anthocyanin accumulation are being investigated increasingly. The present paper summarizes both the basic information and the most recent advances in the study of the anthocyanin biosynthesis in red grapes, emphasizing their gene structure, the transcriptional factors and the diverse exterior regulation factors.

Genome-wide Expression Analysis and Metabolite Profiling Elucidate Transcriptional Regulation of Flavonoid Biosynthesis and Modulation under Abiotic Stresses in Banana.

Science.gov (United States)

Pandey, Ashutosh; Alok, Anshu; Lakhwani, Deepika; Singh, Jagdeep; Asif, Mehar H; Trivedi, Prabodh K

2016-08-19

Flavonoid biosynthesis is largely regulated at the transcriptional level due to the modulated expression of genes related to the phenylpropanoid pathway in plants. Although accumulation of different flavonoids has been reported in banana, a staple fruit crop, no detailed information is available on regulation of the biosynthesis in this important plant. We carried out genome-wide analysis of banana (Musa acuminata, AAA genome) and identified 28 genes belonging to 9 gene families associated with flavonoid biosynthesis. Expression analysis suggested spatial and temporal regulation of the identified genes in different tissues of banana. Analysis revealed enhanced expression of genes related to flavonol and proanthocyanidin (PA) biosynthesis in peel and pulp at the early developmental stages of fruit. Genes involved in anthocyanin biosynthesis were highly expressed during banana fruit ripening. In general, higher accumulation of metabolites was observed in the peel as compared to pulp tissue. A correlation between expression of genes and metabolite content was observed at the early stage of fruit development. Furthermore, this study also suggests regulation of flavonoid biosynthesis, at transcriptional level, under light and dark exposures as well as methyl jasmonate (MJ) treatment in banana.

Genome-wide identification of GLABRA3 downstream genes for anthocyanin biosynthesis and trichome formation in Arabidopsis.

Science.gov (United States)

Gao, Chenhao; Li, Dong; Jin, Changyu; Duan, Shaowei; Qi, Shuanghui; Liu, Kaige; Wang, Hanchen; Ma, Haoli; Hai, Jiangbo; Chen, Mingxun

2017-04-01

GLABRA3 (GL3), a bHLH transcription factor, has previously proved to be involved in anthocyanin biosynthesis and trichome formation in Arabidopsis, however, its downstream targeted genes are still largely unknown. Here, we found that GL3 was widely present in Arabidopsis vegetative and reproductive organs. New downstream targeted genes of GL3 for anthocyanin biosynthesis and trichome formation were identified in young shoots and expanding true leaves by RNA sequencing. GL3-mediated gene expression was tissue specific in the two biological processes. This study provides new clues to further understand the GL3-mediated regulatory network of anthocyanin biosynthesis and trichome formation in Arabidopsis. Copyright © 2017 Elsevier Inc. All rights reserved.

Regulation of melanin biosynthesis via the dihydroxynaphthalene pathway is dependent on sexual development in the ascomycete Sordaria macrospora.

Science.gov (United States)

Engh, Ines; Nowrousian, Minou; Kück, Ulrich

2007-10-01

The filamentous ascomycete Sordaria macrospora accumulates melanin during sexual development. The four melanin biosynthesis genes pks, teh, sdh and tih were isolated and their homology to genes involved in 1,8 dihydroxynaphthalene (DHN) melanin biosynthesis was shown. The presence of DHN melanin in S. macrospora was further confirmed by disrupting the pks gene encoding a putative polyketide synthase and by RNA interference-mediated silencing of the sdh gene encoding a putative scytalone dehydratase. Because melanin occurs in fruiting bodies that develop through several intermediate stages within 7 days of growth, a Northern analysis of a developmental time-course was conducted. These data revealed a time-dependent regulation of teh and sdh transcript levels. Comparing the transcriptional expression by real-time PCR of melanin biosynthesis genes in the wild type under conditions allowing or repressing sexual development, a significant downregulation during vegetative growth was detected. Quantitative real-time PCR and Northern blot analysis of melanin biosynthesis gene expression in different developmental mutants confirmed that melanin biosynthesis is linked to fruiting body development and is under the control of specific regulatory genes that participate in sexual differentiation.

Two Cycloartenol Synthases for Phytosterol Biosynthesis in Polygala tenuifolia Willd.

Science.gov (United States)

Jin, Mei Lan; Lee, Woo Moon; Kim, Ok Tae

2017-11-15

Oxidosqualene cyclases (OSCs) are enzymes that play a key role in control of the biosynthesis of phytosterols and triterpene saponins. In order to uncover OSC genes from Polygala tenuifolia seedlings induced by methyl jasmonate (MeJA), RNA-sequencing analysis was performed using the Illumina sequencing platform. A total of 148,488,632 high-quality reads from two samples (control and the MeJA treated) were generated. We screened genes related to phytosterol and triterpene saponin biosynthesis and analyzed the transcriptional changes of differentially expressed unigene (DEUG) values calculated by fragments per kilobase million (FPKM). In our datasets, two full-length cDNAs of putative OSC genes, PtCAS1 , and PtCAS2 , were found, in addition to the PtBS (β-amyrin synthase) gene reported in our previous studies and the two cycloartenol synthase genes of P. tenuifolia . All genes were isolated and characterized in yeast cells. The functional expression of the two PtCAS genes in yeast cells showed that the genes all produce a cycloartenol as the sole product. When qRT-PCR analysis from different tissues was performed, the expressions of PtCAS1 and PtCAS2 were highest in flowers and roots, respectively. After MeJA treatment, the transcripts of PtCAS1 and PtCAS2 genes increased by 1.5- and 2-fold, respectively. Given these results, we discuss the potential roles of the two PtCAS genes in relation to triterpenoid biosynthesis.

Two Cycloartenol Synthases for Phytosterol Biosynthesis in Polygala tenuifolia Willd

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Mei Lan Jin

2017-11-01

Full Text Available Oxidosqualene cyclases (OSCs are enzymes that play a key role in control of the biosynthesis of phytosterols and triterpene saponins. In order to uncover OSC genes from Polygala tenuifolia seedlings induced by methyl jasmonate (MeJA, RNA-sequencing analysis was performed using the Illumina sequencing platform. A total of 148,488,632 high-quality reads from two samples (control and the MeJA treated were generated. We screened genes related to phytosterol and triterpene saponin biosynthesis and analyzed the transcriptional changes of differentially expressed unigene (DEUG values calculated by fragments per kilobase million (FPKM. In our datasets, two full-length cDNAs of putative OSC genes, PtCAS1, and PtCAS2, were found, in addition to the PtBS (β-amyrin synthase gene reported in our previous studies and the two cycloartenol synthase genes of P. tenuifolia. All genes were isolated and characterized in yeast cells. The functional expression of the two PtCAS genes in yeast cells showed that the genes all produce a cycloartenol as the sole product. When qRT-PCR analysis from different tissues was performed, the expressions of PtCAS1 and PtCAS2 were highest in flowers and roots, respectively. After MeJA treatment, the transcripts of PtCAS1 and PtCAS2 genes increased by 1.5- and 2-fold, respectively. Given these results, we discuss the potential roles of the two PtCAS genes in relation to triterpenoid biosynthesis.

Mur Ligase Inhibitors as Anti-bacterials: A Comprehensive Review.

Science.gov (United States)

Sangshetti, Jaiprakash N; Joshi, Suyog S; Patil, Rajendra H; Moloney, Mark G; Shinde, Devanand B

2017-01-01

Exploring a new target for antibacterial drug discovery has gained much attention because of the emergence of Multidrug Resistance (MDR) strains of bacteria. To overcome this problem the development of novel antibacterial was considered as highest priority task and was one of the biggest challenge since multiple factors were involved. The bacterial peptidoglycan biosynthetic pathway has been well documented in the last few years and has been found to be imperative source for the development of novel antibacterial agents with high target specificity as they are essential for bacterial survival and have no homologs in humans. We have therefore reviewed the process of peptidoglycan biosynthesis which involves various steps like formation of UDP-Nacetylglucosamine (GlcNAc), UDP-N-acetylmuramic acid (MurNAc) and lipid intermediates (Lipid I and Lipid II) which are controlled by various enzymes like GlmS, GlmM, GlmU enzyme, followed by Mur Ligases (MurAMurF) and finally by MraY and MurG respectively. These four amide ligases MurC-MurF can be used as the source for the development of novel multi-target antibacterial agents as they shared and conserved amino acid regions, catalytic mechanisms and structural features. This review begins with the need for novel antibacterial agents and challenges in their development even after the development of bacterial genomic studies. An overview of the peptidoglycan monomer formation, as a source of disparity in this process is presented, followed by detailed discussion of structural and functional aspects of all Mur enzymes and different chemical classes of their inhibitors along with their SAR studies and inhibitory potential. This review finally emphasizes on different patents and novel Mur inhibitors in the development phase. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Filling gaps in bacterial amino acid biosynthesis pathways with high-throughput genetics.

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Morgan N Price

2018-01-01

Full Text Available For many bacteria with sequenced genomes, we do not understand how they synthesize some amino acids. This makes it challenging to reconstruct their metabolism, and has led to speculation that bacteria might be cross-feeding amino acids. We studied heterotrophic bacteria from 10 different genera that grow without added amino acids even though an automated tool predicts that the bacteria have gaps in their amino acid synthesis pathways. Across these bacteria, there were 11 gaps in their amino acid biosynthesis pathways that we could not fill using current knowledge. Using genome-wide mutant fitness data, we identified novel enzymes that fill 9 of the 11 gaps and hence explain the biosynthesis of methionine, threonine, serine, or histidine by bacteria from six genera. We also found that the sulfate-reducing bacterium Desulfovibrio vulgaris synthesizes homocysteine (which is a precursor to methionine by using DUF39, NIL/ferredoxin, and COG2122 proteins, and that homoserine is not an intermediate in this pathway. Our results suggest that most free-living bacteria can likely make all 20 amino acids and illustrate how high-throughput genetics can uncover previously-unknown amino acid biosynthesis genes.

Diphthamide biosynthesis requires an organic radical generated by an iron-sulphur enzyme

Energy Technology Data Exchange (ETDEWEB)

Zhang, Yang; Zhu, Xuling; Torelli, Andrew T; Lee, Michael; Dzikovski, Boris; Koralewski, Rachel M; Wang, Eileen; Freed, Jack; Krebs, Carsten; Ealick, Steve E; Lin, Hening [Cornell; (Penn)

2010-08-30

Archaeal and eukaryotic translation elongation factor 2 contain a unique post-translationally modified histidine residue called diphthamide, which is the target of diphtheria toxin. The biosynthesis of diphthamide was proposed to involve three steps, with the first being the formation of a C-C bond between the histidine residue and the 3-amino-3-carboxypropyl group of S-adenosyl-l-methionine (SAM). However, further details of the biosynthesis remain unknown. Here we present structural and biochemical evidence showing that the first step of diphthamide biosynthesis in the archaeon Pyrococcus horikoshii uses a novel iron-sulphur-cluster enzyme, Dph2. Dph2 is a homodimer and each of its monomers can bind a [4Fe-4S] cluster. Biochemical data suggest that unlike the enzymes in the radical SAM superfamily, Dph2 does not form the canonical 5'-deoxyadenosyl radical. Instead, it breaks the C_γ,Met-S bond of SAM and generates a 3-amino-3-carboxypropyl radical. Our results suggest that P. horikoshii Dph2 represents a previously unknown, SAM-dependent, [4Fe-4S]-containing enzyme that catalyses unprecedented chemistry.

Expression profiles of genes involved in tanshinone biosynthesis of ...

Indian Academy of Sciences (India)

Expression profiles of genes involved in tanshinone biosynthesis of two. Salvia miltiorrhiza genotypes with different tanshinone contents. Zhenqiao Song, Jianhua Wang and Xingfeng Li. J. Genet. 95, 433–439. Table 1. S. miltiorrhiza genes and primer pairs used for qRT-PCR. Gene. GenBank accession. Primer name.

Cyclopiazonic Acid Biosynthesis of Aspergillus flavus and Aspergillus oryzae

Science.gov (United States)

Cyclopiazonic acid (CPA) is an indole-tetramic acid neurotoxin produced by some of the same strains of A. flavus that produce aflatoxins and by some Aspergillus oryzae strains. Despite its discovery 40 years ago, few reviews of its toxicity and biosynthesis have been reported. This review examines w...

Three-dimensional structures of Plasmodium falciparum spermidine synthase with bound inhibitors suggest new strategies for drug design

International Nuclear Information System (INIS)

Sprenger, Janina; Svensson, Bo; Hålander, Jenny; Carey, Jannette; Persson, Lo; Al-Karadaghi, Salam

2015-01-01

In this work, X-ray crystallography was used to examine ligand complexes of spermidine synthase from the malaria parasite Plasmodium falciparum (PfSpdS). The enzymes of the polyamine-biosynthesis pathway have been proposed to be promising drug targets in the treatment of malaria. Spermidine synthase (SpdS; putrescine aminopropyltransferase) catalyzes the transfer of the aminopropyl moiety from decarboxylated S-adenosylmethionine to putrescine, leading to the formation of spermidine and 5′-methylthioadenosine (MTA). In this work, X-ray crystallography was used to examine ligand complexes of SpdS from the malaria parasite Plasmodium falciparum (PfSpdS). Five crystal structures were determined of PfSpdS in complex with MTA and the substrate putrescine, with MTA and spermidine, which was obtained as a result of the enzymatic reaction taking place within the crystals, with dcAdoMet and the inhibitor 4-methylaniline, with MTA and 4-aminomethylaniline, and with a compound predicted in earlier in silico screening to bind to the active site of the enzyme, benzimidazol-(2-yl)pentan-1-amine (BIPA). In contrast to the other inhibitors tested, the complex with BIPA was obtained without any ligand bound to the dcAdoMet-binding site of the enzyme. The complexes with the aniline compounds and BIPA revealed a new mode of ligand binding to PfSpdS. The observed binding mode of the ligands, and the interplay between the two substrate-binding sites and the flexible gatekeeper loop, can be used in the design of new approaches in the search for new inhibitors of SpdS

Three-dimensional structures of Plasmodium falciparum spermidine synthase with bound inhibitors suggest new strategies for drug design

Energy Technology Data Exchange (ETDEWEB)

Sprenger, Janina [Lund University, SE-221 00 Lund (Sweden); Lund University, SE-221 84 Lund (Sweden); Svensson, Bo [Lund University, SE-221 00 Lund (Sweden); SARomics Biostructures AB, Box 724, SE-220 07 Lund (Sweden); Hålander, Jenny [Lund University, SE-221 00 Lund (Sweden); Carey, Jannette [Princeton University, Princeton, New Jersey (United States); Persson, Lo [Lund University, SE-221 84 Lund (Sweden); Al-Karadaghi, Salam, E-mail: salam.al-karadaghi@biochemistry.lu.se [Lund University, SE-221 00 Lund (Sweden)

2015-03-01

In this work, X-ray crystallography was used to examine ligand complexes of spermidine synthase from the malaria parasite Plasmodium falciparum (PfSpdS). The enzymes of the polyamine-biosynthesis pathway have been proposed to be promising drug targets in the treatment of malaria. Spermidine synthase (SpdS; putrescine aminopropyltransferase) catalyzes the transfer of the aminopropyl moiety from decarboxylated S-adenosylmethionine to putrescine, leading to the formation of spermidine and 5′-methylthioadenosine (MTA). In this work, X-ray crystallography was used to examine ligand complexes of SpdS from the malaria parasite Plasmodium falciparum (PfSpdS). Five crystal structures were determined of PfSpdS in complex with MTA and the substrate putrescine, with MTA and spermidine, which was obtained as a result of the enzymatic reaction taking place within the crystals, with dcAdoMet and the inhibitor 4-methylaniline, with MTA and 4-aminomethylaniline, and with a compound predicted in earlier in silico screening to bind to the active site of the enzyme, benzimidazol-(2-yl)pentan-1-amine (BIPA). In contrast to the other inhibitors tested, the complex with BIPA was obtained without any ligand bound to the dcAdoMet-binding site of the enzyme. The complexes with the aniline compounds and BIPA revealed a new mode of ligand binding to PfSpdS. The observed binding mode of the ligands, and the interplay between the two substrate-binding sites and the flexible gatekeeper loop, can be used in the design of new approaches in the search for new inhibitors of SpdS.

A protein interaction map of the kalimantacin biosynthesis assembly line

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Birgit Uytterhoeven

2016-11-01

Full Text Available The antimicrobial secondary metabolite kalimantacin is produced by a hybrid polyketide/ non-ribosomal peptide system in Pseudomonas fluorescens BCCM_ID9359. In this study, the kalimantacin biosynthesis gene cluster is analyzed by yeast two-hybrid analysis, creating a protein-protein interaction map of the entire assembly line. In total, 28 potential interactions were identified, of which 13 could be confirmed further. These interactions include the dimerization of ketosynthase domains, a link between assembly line modules 9 and 10, and a specific interaction between the trans-acting enoyl reductase BatK and the carrier proteins of modules 8 and 10. These interactions reveal fundamental insight into the biosynthesis of secondary metabolites.This study is the first to reveal interactions in a complete biosynthetic pathway. Similar future studies could build a strong basis for engineering strategies in such clusters.

Engineering Microbial Cells for the Biosynthesis of Natural Compounds of Pharmaceutical Significance

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Philippe Jeandet

2013-01-01

Full Text Available Microbes constitute important platforms for the biosynthesis of numerous molecules of pharmaceutical interest such as antitumor, anticancer, antiviral, antihypertensive, antiparasitic, antioxidant, immunological agents, and antibiotics as well as hormones, belonging to various chemical families, for instance, terpenoids, alkaloids, polyphenols, polyketides, amines, and proteins. Engineering microbial factories offers rich opportunities for the production of natural products that are too complex for cost-effective chemical synthesis and whose extraction from their originating plants needs the use of many solvents. Recent progresses that have been made since the millennium beginning with metabolic engineering of microorganisms for the biosynthesis of natural products of pharmaceutical significance will be reviewed.

Chloroplast SRP43 acts as a chaperone for glutamyl-tRNA reductase, the rate-limiting enzyme in tetrapyrrole biosynthesis.

Science.gov (United States)

Wang, Peng; Liang, Fu-Cheng; Wittmann, Daniel; Siegel, Alex; Shan, Shu-Ou; Grimm, Bernhard

2018-04-10

Assembly of light-harvesting complexes requires synchronization of chlorophyll (Chl) biosynthesis with biogenesis of light-harvesting Chl a/b-binding proteins (LHCPs). The chloroplast signal recognition particle (cpSRP) pathway is responsible for transport of nucleus-encoded LHCPs in the stroma of the plastid and their integration into the thylakoid membranes. Correct folding and assembly of LHCPs require the incorporation of Chls, whose biosynthesis must therefore be precisely coordinated with membrane insertion of LHCPs. How the spatiotemporal coordination between the cpSRP machinery and Chl biosynthesis is achieved is poorly understood. In this work, we demonstrate a direct interaction between cpSRP43, the chaperone that mediates LHCP targeting and insertion, and glutamyl-tRNA reductase (GluTR), a rate-limiting enzyme in tetrapyrrole biosynthesis. Concurrent deficiency for cpSRP43 and the GluTR-binding protein (GBP) additively reduces GluTR levels, indicating that cpSRP43 and GBP act nonredundantly to stabilize GluTR. The substrate-binding domain of cpSRP43 binds to the N-terminal region of GluTR, which harbors aggregation-prone motifs, and the chaperone activity of cpSRP43 efficiently prevents aggregation of these regions. Our work thus reveals a function of cpSRP43 in Chl biosynthesis and suggests a striking mechanism for posttranslational coordination of LHCP insertion with Chl biosynthesis.

The magnesium chelation step in chlorophyll biosynthesis. Progress report 1993

Energy Technology Data Exchange (ETDEWEB)

Weinstein, J.D.

1993-12-31

Progress is reported on the identification and fractionation of Magnesium chealatase, an enzyme involved in addition of Mg to chlorophyll during the later`s biosynthesis. Progress is documented as a series of synopsis of published and unpublished papers by the author.

Lincosamides: Chemical structure, biosynthesis, mechanism of action, resistance, and applications

Czech Academy of Sciences Publication Activity Database

Spížek, Jaroslav; Řezanka, Tomáš

2017-01-01

Roč. 133, June 1 SI (2017), s. 20-28 ISSN 0006-2952 Institutional support: RVO:61388971 Keywords : Lincosamides * Chemical structure * Biosynthesis and mechanism of action Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 4.581, year: 2016

Temporal expression of genes involved in the biosynthesis of ...

African Journals Online (AJOL)

Gibberellins (GAs) are a large family of endogenous plant growth regulators. Bioactive GAs influence nearly all processes during plant growth and development. In the present study, we cloned and identified 10 unique genes that are potentially involved in the biosynthesis of GAs, including one BpGGDP gene, two BpCPS ...

Estrogen Replacement Therapy in Ovariectomized Nonpregnant Ewes Stimulates Uterine Artery Hydrogen Sulfide Biosynthesis by Selectively Up-Regulating Cystathionine β-Synthase Expression.

Science.gov (United States)

Lechuga, Thomas J; Zhang, Hong-hai; Sheibani, Lili; Karim, Muntarin; Jia, Jason; Magness, Ronald R; Rosenfeld, Charles R; Chen, Dong-bao

2015-06-01

Estrogens dramatically dilate numerous vascular beds with the greatest response in the uterus. Endogenous hydrogen sulfide (H2S) is a potent vasodilator and proangiogenic second messenger, which is synthesized from L-cysteine by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). We hypothesized that estrogen replacement therapy (ERT) selectively stimulates H2S biosynthesis in uterine artery (UA) and other systemic arteries. Intact and endothelium-denuded UA, mesenteric artery (MA), and carotid artery (CA) were obtained from ovariectomized nonpregnant ewes (n = 5/group) receiving vehicle or estradiol-17β replacement therapy (ERT). Total RNA and protein were extracted for measuring CBS and CSE, and H2S production was determined by the methylene blue assay. Paraffin-embedded UA rings were used to localize CBS and CSE proteins by immunofluorescence microscopy. ERT significantly stimulated CBS mRNA and protein without altering CSE mRNA or protein in intact and denuded UA. Quantitative immunofluorescence microscopic analyses showed CBS and CSE protein localization in endothelium and smooth muscle and confirmed that ERT stimulated CBS but not CSE protein expression in UA endothelium and smooth muscle. ERT also stimulated CBS, but not CSE, mRNA and protein expression in intact and denuded MA but not CA in ovariectomized ewes. Concomitantly, ERT stimulated UA and MA but not CA H2S production. ERT-stimulated UA H2S production was completely blocked by a specific CBS but not CSE inhibitor. Thus, ERT selectively stimulates UA and MA but not CA H2S biosynthesis by specifically up-regulating CBS expression, implicating a role of H2S in estrogen-induced vasodilation and postmenopausal women's health.

Cysteine Biosynthesis Controls Serratia marcescens Phospholipase Activity.

Science.gov (United States)

Anderson, Mark T; Mitchell, Lindsay A; Mobley, Harry L T

2017-08-15

Serratia marcescens causes health care-associated opportunistic infections that can be difficult to treat due to a high incidence of antibiotic resistance. One of the many secreted proteins of S. marcescens is the PhlA phospholipase enzyme. Genes involved in the production and secretion of PhlA were identified by screening a transposon insertion library for phospholipase-deficient mutants on phosphatidylcholine-containing medium. Mutations were identified in four genes ( cyaA , crp , fliJ , and fliP ) that are involved in the flagellum-dependent PhlA secretion pathway. An additional phospholipase-deficient isolate harbored a transposon insertion in the cysE gene encoding a predicted serine O -acetyltransferase required for cysteine biosynthesis. The cysE requirement for extracellular phospholipase activity was confirmed using a fluorogenic phospholipase substrate. Phospholipase activity was restored to the cysE mutant by the addition of exogenous l-cysteine or O -acetylserine to the culture medium and by genetic complementation. Additionally, phlA transcript levels were decreased 6-fold in bacteria lacking cysE and were restored with added cysteine, indicating a role for cysteine-dependent transcriptional regulation of S. marcescens phospholipase activity. S. marcescens cysE mutants also exhibited a defect in swarming motility that was correlated with reduced levels of flhD and fliA flagellar regulator gene transcription. Together, these findings suggest a model in which cysteine is required for the regulation of both extracellular phospholipase activity and surface motility in S. marcescens IMPORTANCE Serratia marcescens is known to secrete multiple extracellular enzymes, but PhlA is unusual in that this protein is thought to be exported by the flagellar transport apparatus. In this study, we demonstrate that both extracellular phospholipase activity and flagellar function are dependent on the cysteine biosynthesis pathway. Furthermore, a disruption of cysteine

Comparative metabolomics in vanilla pod and vanilla bean revealing the biosynthesis of vanillin during the curing process of vanilla.

Science.gov (United States)

Gu, Fenglin; Chen, Yonggan; Hong, Yinghua; Fang, Yiming; Tan, Lehe

2017-12-01

High-performance liquid chromatography-mass spectrometry (LC-MS) was used for comprehensive metabolomic fingerprinting of vanilla fruits prepared from the curing process. In this study, the metabolic changes of vanilla pods and vanilla beans were characterized using MS-based metabolomics to elucidate the biosynthesis of vanillin. The vanilla pods were significantly different from vanilla beans. Seven pathways of vanillin biosynthesis were constructed, namely, glucovanillin, glucose, cresol, capsaicin, vanillyl alcohol, tyrosine, and phenylalanine pathways. Investigations demonstrated that glucose, cresol, capsaicin, and vanillyl alcohol pathway were detected in a wide range of distribution in microbial metabolism. Thus, microorganisms might have participated in vanillin biosynthesis during vanilla curing. Furthermore, the ion strength of glucovanillin was stable, which indicated that glucovanillin only participated in the vanillin biosynthesis during the curing of vanilla.

Transcriptomic analysis reveals key genes related to betalain biosynthesis in pulp coloration of Hylocereus polyrhizus

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Hua eQingzhu

2016-01-01

Full Text Available Betalains have high nutritional value and bioactivities. Red pulp pitaya (Hylocereus polyrhizus is the only fruit containing abundant betalains for consumer. However, no information is available about genes involved in betalain biosynthesis in H. polyrhizus. Herein, two cDNA libraries of pitaya pulps with two different coloration stages (white and red pulp stages of Guanhuahong (H. polyrhizus were constructed. A total of about 12 Gb raw RNA-Seq data was generated and was de novo assembled into 122,677 transcripts with an average length of 1,183 bp and an N50 value of 2008. Approximately 99.99% of all transcripts were annotated based on seven public databases. A total of 8,871 transcripts were significantly regulated. Thirty-three candidate transcripts related to betalain biosynthesis were obtained from the transcriptome data. Transcripts encoding enzymes involved in betalain biosynthesis were analyzed using RT-qPCR at the whole pulp coloration stages of H. Polyrhizus (7-1 and H. Undatus (132-4. Nine key transcripts of betalain biosynthesis were identified. They were assigned to four kinds of genes in betalain biosynthetic pathway, including tyrosinase, 4, 5-DOPA dioxygenase extradiol, cytochrome P450 and glucosyltransferase. Ultimately, a preliminary betalain biosynthetic pathway for pitaya was proposed based on betalain analyses and gene expression profiles.

Biosynthesis of silver nanoparticles using Ocimum sanctum (Tulsi) leaf extract and screening its antimicrobial activity

Science.gov (United States)

Singhal, Garima; Bhavesh, Riju; Kasariya, Kunal; Sharma, Ashish Ranjan; Singh, Rajendra Pal

2011-07-01

Development of green nanotechnology is generating interest of researchers toward ecofriendly biosynthesis of nanoparticles. In this study, biosynthesis of stable silver nanoparticles was done using Tulsi ( Ocimum sanctum) leaf extract. These biosynthesized nanoparticles were characterized with the help of UV-vis spectrophotometer, Atomic Absorption Spectroscopy (AAS), Dynamic light scattering (DLS), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and Transmission electron microscopy (TEM). Stability of bioreduced silver nanoparticles was analyzed using UV-vis absorption spectra, and their antimicrobial activity was screened against both gram-negative and gram-positive microorganisms. It was observed that O. sanctum leaf extract can reduce silver ions into silver nanoparticles within 8 min of reaction time. Thus, this method can be used for rapid and ecofriendly biosynthesis of stable silver nanoparticles of size range 4-30 nm possessing antimicrobial activity suggesting their possible application in medical industry.

Biosynthesis of silver nanoparticles using Ocimum sanctum (Tulsi) leaf extract and screening its antimicrobial activity

International Nuclear Information System (INIS)

Singhal, Garima; Bhavesh, Riju; Kasariya, Kunal; Sharma, Ashish Ranjan; Singh, Rajendra Pal

2011-01-01

Development of green nanotechnology is generating interest of researchers toward ecofriendly biosynthesis of nanoparticles. In this study, biosynthesis of stable silver nanoparticles was done using Tulsi (Ocimum sanctum) leaf extract. These biosynthesized nanoparticles were characterized with the help of UV–vis spectrophotometer, Atomic Absorption Spectroscopy (AAS), Dynamic light scattering (DLS), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and Transmission electron microscopy (TEM). Stability of bioreduced silver nanoparticles was analyzed using UV–vis absorption spectra, and their antimicrobial activity was screened against both gram-negative and gram-positive microorganisms. It was observed that O. sanctum leaf extract can reduce silver ions into silver nanoparticles within 8 min of reaction time. Thus, this method can be used for rapid and ecofriendly biosynthesis of stable silver nanoparticles of size range 4–30 nm possessing antimicrobial activity suggesting their possible application in medical industry.

Anthocyanin biosynthesis in pears is regulated by a R2R3-MYB transcription factor PyMYB10.

Science.gov (United States)

Feng, Shouqian; Wang, Yanling; Yang, Song; Xu, Yuting; Chen, Xuesen

2010-06-01

Skin color is an important factor in pear breeding programs. The degree of red coloration is determined by the content and composition of anthocyanins. In plants, many MYB transcriptional factors are involved in regulating anthocyanin biosynthesis. In this study, a R2R3-MYB transcription factor gene, PyMYB10, was isolated from Asian pear (Pyrus pyrifolia) cv. 'Aoguan'. Sequence analysis suggested that the PyMYB10 gene was an ortholog of MdMYB10 gene, which regulates anthocyanin biosynthesis in red fleshed apple (Malus x domestica) cv. 'Red Field'. PyMYB10 was identified at the genomic level and had three exons, with its upstream sequence containing core sequences of cis-acting regulatory elements involved in light responsiveness. Fruit bagging showed that light could induce expression of PyMYB10 and anthocyanin biosynthesis. Quantitative real-time PCR revealed that PyMYB10 was predominantly expressed in pear skins, buds, and young leaves, and the level of transcription in buds was higher than in skin and young leaves. In ripening fruits, the transcription of PyMYB10 in the skin was positively correlated with genes in the anthocyanin pathway and with anthocyanin biosynthesis. In addition, the transcription of PyMYB10 and genes of anthocyanin biosynthesis were more abundant in red-skinned pear cultivars compared to blushed cultivars. Transgenic Arabidopsis plants overexpressing PyMYB10 exhibited ectopic pigmentation in immature seeds. The study suggested that PyMYB10 plays a role in regulating anthocyanin biosynthesis and the overexpression of PyMYB10 was sufficient to induce anthocyanin accumulation.

Identification of Candidate Genes and Biosynthesis Pathways Related to Fertility Conversion by Wheat KTM3315A Transcriptome Profiling

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Lingli Zhang

2017-04-01

Full Text Available The Aegilops kotschyi thermo-sensitive cytoplasmic male sterility (K-TCMS system may facilitate hybrid wheat (Triticum aestivum L. seed multiplication and production. The K-TCMS line is completely male sterile during the normal wheat-growing season, whereas its fertility can be restored in a high-temperature environment. To elucidate the molecular mechanisms responsible for male sterility/fertility conversion and candidate genes involved with pollen development in K-TCMS, we employed RNA-seq to sequence the transcriptomes of anthers from K-TCMS line KTM3315A during development under sterile and fertile conditions. We identified 16840 differentially expressed genes (DEGs in different stages including15157 known genes (15135 nuclear genes and 22 plasmagenes and 1683 novel genes. Bioinformatics analysis identified possible metabolic pathways involved with fertility based on KEGG pathway enrichment of the DEGs expressed in fertile and sterile plants. We found that most of the genes encoding key enzyme in the phenylpropanoid biosynthesis and jasmonate biosynthesis pathways were significant upregulated in uninucleate, binuclate or trinucleate stage, which both interact with MYB transcription factors, and that link between all play essential roles in fertility conversion. The relevant DEGs were verified by quantitative RT-PCR. Thus, we suggested that phenylpropanoid biosynthesis and jasmonate biosynthesis pathways were involved in fertility conversion of K-TCMS wheat. This will provide a new perspective and an effective foundation for the research of molecular mechanisms of fertility conversion of CMS wheat. Fertility conversion mechanism in thermo-sensitive cytoplasmic male sterile/fertile wheat involves the phenylpropanoid biosynthesis pathway, jasmonate biosynthesis pathway, and MYB transcription factors.

[Expression of saponin biosynthesis related genes in different tissues of Panax quinquefolius].

Science.gov (United States)

Wang, Kang-Yu; Liu, Wei-Can; Zhang, Mei-Ping; Zhao, Ming-Zhu; Wang, Yan-Fang; Li, Li; Sun, Chun-Yu; Hu, Ke-Xin; Cong, Yue-Yi; Wang, Yi

2018-01-01

The relationship between saponin content of Panax quinquefolius in different parts of the organization and expression of ginsenoside biosynthesis related gene was obtained by the correlation analysis between saponin content and gene expression. The 14 tissue parts of P. quinquefolius were studied, six saponins in P. quinquefolius. Samples (ginsenoside Rg₁, Re, Rb₁, Rc, Rb₂ and Rd), group saponins and total saponins were determined by high performance liquid chromatography and vanillin-sulfuric acid colorimetric method. Simultaneously, the expression levels of 7 ginsenoside biosynthesis related genes ( SQS, OSC, DS, β-AS, SQE, P450 and FPS ) in different tissues of P. quinquefolius were determined by Real-time fluorescence quantitative PCR. Although 7 kinds of ginsenoside biosynthesis related enzyme gene in the P. quinquefolius involved in ginsenoside synthesis, the expression of β-AS and P450 genes had no significant effect on the content of monosodium saponins, grouping saponins and total saponins, FPS, SQS, OSC, DS and SQE had significant or extremely significant on the contents of single saponins Re, Rg1, Rb1, Rd, group saponin PPD and PPT, total saponin TMS and total saponin TS ( P saponins, grouping saponins and total saponins in P. quinquefolius was affected by the interaction of multiple enzyme genes in the saponin synthesis pathway, the content of saponins in different tissues of P. quinquefolius was determined by the differences in the expression of key enzymes in the biosynthetic pathway. Therefore, this study further clarified that FPS, SQS, OSC, DS and SQE was the key enzyme to control the synthesis of saponins in P. quinquefolius by correlation analysis, the biosynthesis of ginsenosides in P. quinquefolius was regulated by these five kind of enzymes in cluster co-expression of interaction mode. Copyright© by the Chinese Pharmaceutical Association.

Biosynthesis and Application of Silver and Gold Nanoparticles

OpenAIRE

Sadowski, Zygmunt

2010-01-01

A green chemistry synthetic route has been used for both silver and gold nanoparticles synthesis. The reaction occurred at ambient temperature. Among the nanoparticles biological organism, some microorganisms such as bacteria, fungi, and yeast have been exploited for nanoparticles synthesis. Several plant biomass or plant extracts have been successfully used for extracellular biosynthesis of silver and gold nanoparticles. Analytical techniques, such as ultraviolet-visible spectroscopy (UV-vis...

The plant cuticle is required for osmotic stress regulation of abscisic acid biosynthesis and osmotic stress tolerance in Arabidopsis

KAUST Repository

Wang, Zhenyu; Xiong, Liming; Li, Wenbo; Zhu, Jian-Kang; Zhu, Jianhua

2011-01-01

Osmotic stress activates the biosynthesis of abscisic acid (ABA). One major step in ABA biosynthesis is the carotenoid cleavage catalyzed by a 9-cis epoxycarotenoid dioxygenase (NCED). To understand the mechanism for osmotic stress activation of ABA