RESUMEN
BACKGROUND: Broomcorn millet is highly tolerant to drought and barren soil. Changes in chlorophyll content directly affect leaf color, which subsequently leadsleading to poor photosynthetic performance and reduced crop yield. Herein, we isolated a yellow leaf mutant (YX-yl) using a forward genetics approach and evaluated its agronomic traits, photosynthetic pigment content, chloroplast ultrastructure, and chlorophyll precursors. Furthermore, the molecular mechanism of yellowing was explored using transcriptome sequencing. RESULTS: The YX-yl mutant showed significantly decreased plant height and low yield. The leaves exhibited a yellow-green phenotype and poor photosynthetic capacity during the entire growth period. The content of chlorophyll a, chlorophyll b, and carotenoids in YX-yl leaves was lower than that in wild-type leaves. Chlorophyll precursor analysis results showed that chlorophyll biosynthesis in YX-yl was hindered by the conversion of porphobilinogen to protoporphyrin IX. Examination of chloroplast ultrastructure in the leaves revealed that the chloroplasts of YX-yl accumulated on one side of the cell. Moreover, the chloroplast structure of YX-yl was degraded. The inner and outer membranes of the chloroplasts could not be distinguished well. The numbers of grana and grana thylakoids in the chloroplasts were low. The transcriptome of the yellowing mutant YX-yl was sequenced and compared with that of the wild type. Nine chlorophyll-related genes with significantly different expression profiles were identified: PmUROD, PmCPO, PmGSAM, PmPBDG, PmLHCP, PmCAO, PmVDE, PmGluTR, and PmPNPT. The proteins encoded by these genes were located in the chloroplast, chloroplast membrane, chloroplast thylakoid membrane, and chloroplast matrix and were mainly involved in chlorophyll biosynthesis and redox-related enzyme regulation. CONCLUSIONS: YX-yl is an ideal material for studying pigment metabolism mechanisms. Changes in the expression patterns of some genes between YX-yl and the wild type led to differences in chloroplast structures and enzyme activities in the chlorophyll biosynthesis pathway, ultimately resulting in a yellowing phenotype in the YX-yl mutant. Our findings provide an insight to the molecular mechanisms of leaf color formation and chloroplast development in broomcorn millet.
Asunto(s)
Panicum , Carotenoides/metabolismo , Clorofila/metabolismo , Clorofila A/metabolismo , Regulación de la Expresión Génica de las Plantas , Panicum/metabolismo , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Porfobilinógeno/metabolismo , SueloRESUMEN
Hydroxymethylbilane synthase (HMBS), which is involved in the heme biosynthesis pathway, has a dipyrromethane cofactor and combines four porphobilinogen (PBG) molecules to form a linear tetrapyrrole, hydroxymethylbilane. Enzyme kinetic study of human HMBS using a PBG-derivative, 2-iodoporphobilinogen (2-I-PBG), exhibited noncompetitive inhibition with the inhibition constant being 5.4 ± 0.3â µM. To elucidate the reaction mechanism of HMBS in detail, crystal structure analysis of 2-I-PBG-bound holo-HMBS and its reaction intermediate possessing two PBG molecules (ES2), and inhibitor-free ES2 was performed at 2.40, 2.31, and 1.79â Å resolution, respectively. Their overall structures are similar to that of inhibitor-free holo-HMBS, and the differences are limited near the active site. In both 2-I-PBG-bound structures, 2-I-PBG is located near the terminus of the cofactor or the tetrapyrrole chain. The propionate group of 2-I-PBG interacts with the side chain of Arg173, and its acetate group is associated with the side chains of Arg26 and Ser28. Furthermore, the aminomethyl group and pyrrole nitrogen of 2-I-PBG form hydrogen bonds with the side chains of Gln34 and Asp99, respectively. These amino acid residues form a single substrate-binding site, where each of the four PBG molecules covalently binds to the cofactor (or oligopyrrole chain) consecutively, ultimately forming a hexapyrrole chain. Molecular dynamics simulation of the ES2 intermediate suggested that the thermal fluctuation of the lid and cofactor-binding loops causes substrate recruitment and oligopyrrole chain shift needed for consecutive condensation. Finally, the hexapyrrole chain is hydrolyzed self-catalytically to produce hydroxymethylbilane.
Asunto(s)
Hidroximetilbilano Sintasa/química , Hidroximetilbilano Sintasa/metabolismo , Porfobilinógeno/metabolismo , Uroporfirinógenos/metabolismo , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Simulación de Dinámica Molecular , Conformación Proteica , Dominios Proteicos , Especificidad por SustratoRESUMEN
BACKGROUND: 5-Aminolevulinic acid dehydratase (ALAD) porphyria (ADP) is an ultrarare autosomal recessive disease, with only eight documented cases, all of whom were males. Although classified as an acute hepatic porphyria (AHP), induction of the rate limiting hepatic enzyme 5-aminolevulinic acid synthase-1 (ALAS1) has not been demonstrated, and the marrow may also contribute excess 5-aminolevulinic acid (ALA). Two patients have died and reported follow up for the others is limited, so the natural history of this disease is poorly understood and treatment experience limited. METHODS: We report new molecular findings and update the clinical course and treatment of the sixth reported ADP patient, now 31 years old and the only known case in the Americas, and review published data regarding genotype-phenotype correlation and treatment. RESULTS: Circulating hepatic 5-aminolevulinic acid synthase-1 (ALAS1) mRNA was elevated in this case, as in other AHPs. Gain of function mutation of erythroid specific ALAS2 - an X-linked modifying gene in some other porphyrias - was not found. Seven reported ADP cases had compound heterozygous ALAD mutations resulting in very low residual ALAD activity and symptoms early in life or adolescence. One adult with a germline ALAD mutant allele developed ADP in association with a clonal myeloproliferative disorder, polycythemia vera. CONCLUSIONS: Elevation in circulating hepatic ALAS1 and response to treatment with hemin indicate that the liver is an important source of excess ALA in ADP, although the marrow may also contribute. Intravenous hemin was effective in most reported cases for treatment and prevention of acute attacks of neurological symptoms.
Asunto(s)
5-Aminolevulinato Sintetasa/genética , Porfobilinógeno Sintasa/deficiencia , Porfobilinógeno Sintasa/genética , Porfiria Intermitente Aguda/genética , Porfirias Hepáticas/genética , 5-Aminolevulinato Sintetasa/sangre , Adolescente , Adulto , Niño , Preescolar , Femenino , Hemo/genética , Hemina/administración & dosificación , Humanos , Lactante , Recién Nacido , Hígado/metabolismo , Hígado/patología , Masculino , Persona de Mediana Edad , Mutación/genética , Porfobilinógeno/metabolismo , Porfobilinógeno Sintasa/sangre , Porfiria Intermitente Aguda/sangre , Porfiria Intermitente Aguda/tratamiento farmacológico , Porfiria Intermitente Aguda/patología , Porfirias Hepáticas/sangre , Porfirias Hepáticas/tratamiento farmacológico , Porfirias Hepáticas/patología , ARN Mensajero/sangre , Adulto JovenRESUMEN
Senescence-associated diseases have severely diminished the quality of life and health of patients. However, a sensitive assay of these diseases remains limited due to a lack of straightforward methods. Considering that senescence-associated ß-galactosidase (SA-ß-Gal) is overexpressed in senescent cells, the detection of SA-ß-Gal in senescent cells and tissues might be a feasible strategy for the early diagnosis of SA diseases. In this study, a ß-galactosidase-activatable nanoprobe BOD-L-ßGal-NPs was developed for the imaging of senescent cells and vasculature in atherosclerotic mice via real-time monitoring of ß-Gal. BOD-L-ßGal-NPs was fabricated by encapsulating a newly designed NIR ratiometric probe BOD-L-ßGal within a poly(lactic-co-glycolic) acid (PLGA) core. Nanoprobe BOD-L-ßGal-NPs showed good accumulation in arteries, thus successfully visualizing senescent cells and vasculature in atherosclerotic mice by tail vein injection. Our findings indicated that nanoprobe BOD-L-ßGal-NPs holds great potential for the early diagnosis and therapy of atherosclerosis and other aging-associated diseases.
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Aterosclerosis/diagnóstico , Boro/química , Colorantes Fluorescentes/química , Nanopartículas/química , Porfobilinógeno/análogos & derivados , beta-Galactosidasa/análisis , Animales , Aterosclerosis/metabolismo , Boro/metabolismo , Senescencia Celular , Colorantes Fluorescentes/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Nanopartículas/metabolismo , Porfobilinógeno/química , Porfobilinógeno/metabolismo , Ratas , Ratas Sprague-Dawley , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoAsunto(s)
Betacoronavirus/patogenicidad , Biomarcadores , Infecciones por Coronavirus/metabolismo , Hemo/biosíntesis , Neumonía Viral/metabolismo , Ácido Aminolevulínico/metabolismo , Biomarcadores/análisis , COVID-19 , Infecciones por Coronavirus/virología , Interacciones Huésped-Patógeno , Humanos , Interleucina-6/sangre , Pandemias , Neumonía Viral/virología , Porfobilinógeno/metabolismo , Porfobilinógeno/orina , SARS-CoV-2 , Trombosis/etiología , Proteínas Virales/metabolismoRESUMEN
Garlic has attracted considerable attention because of its bactericidal and anticancer effects. However, the greening of garlic purees greatly affects the product quality. This study investigated the influence of light colors and power on the greening of garlic, and determined the key substances of garlic puree greening, including γ-glutamyl transpeptidase (γ-GT), thiosulfinate, and alliinase. Results showed that purple light source greatly affects greening power, γ-GT, and thiosulfinate. Illumination using a 3-W power lamp could reduce the production of thiosulfinate and alliinase and inhibit the green transformation reaction. Illumination using a 5-W power lamp greatly affected the thiosulfinate content and greening power, whereas that using a 7-W power lamp greatly influenced the γ-GT activity, porphobilinogen content, and alliinase content. Results showed that the green color of garlic puree is greatly affected by the illumination color and intensity, which provides theoretical support for the anti-greening of light garlic puree. PRACTICAL APPLICATION: Because garlic puree easily turns green during processing, which affects the product quality and economic value, this study uses controllable light source radiation to influence the greening of garlic puree, hoping to delay or even solve this problem and provide a new simple method to prevent garlic puree from turning greening.
Asunto(s)
Liasas de Carbono-Azufre/metabolismo , Ajo/enzimología , Ajo/efectos de la radiación , Proteínas de Plantas/metabolismo , gamma-Glutamiltransferasa/metabolismo , Color , Ajo/química , Ajo/crecimiento & desarrollo , Luz , Pigmentos Biológicos/análisis , Pigmentos Biológicos/metabolismo , Porfobilinógeno/análisis , Porfobilinógeno/metabolismoRESUMEN
Human porphobilinogen deaminase (PBGD), the third enzyme in the heme pathway, catalyzes four times a single reaction to convert porphobilinogen into hydroxymethylbilane. Remarkably, PBGD employs a single active site during the process, with a distinct yet chemically equivalent bond formed each time. The four intermediate complexes of the enzyme have been biochemically validated and they can be isolated but they have never been structurally characterized other than the apo- and holo-enzyme bound to the cofactor. We present crystal structures for two human PBGD intermediates: PBGD loaded with the cofactor and with the reaction intermediate containing two additional substrate pyrrole rings. These results, combined with SAXS and NMR experiments, allow us to propose a mechanism for the reaction progression that requires less structural rearrangements than previously suggested: the enzyme slides a flexible loop over the growing-product active site cavity. The structures and the mechanism proposed for this essential reaction explain how a set of missense mutations result in acute intermittent porphyria.
Asunto(s)
Hidroximetilbilano Sintasa/química , Hidroximetilbilano Sintasa/metabolismo , Pirroles/química , Pirroles/metabolismo , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Polimerizacion , Porfobilinógeno/química , Porfobilinógeno/metabolismo , Conformación Proteica , Uroporfirinógenos/química , Uroporfirinógenos/metabolismoRESUMEN
BACKGROUND: Acute intermittent porphyria (AIP) is caused by diminished activity of porphobilinogen deaminase (PBGD). The purpose of this study was to validate and compare two assays for PBGD activity. The clinical sensitivity of the PBGD activity assays in AIP diagnosis was also evaluated. METHODS: This study included 74 subjects from 18 Taiwanese families including symptomatic patients with AIP, asymptomatic carriers, and healthy family members. The specific mutations in AIP patients were identified by DNA sequencing. PBGD activity was measured in erythrocytes by quantifying formation of coproporphyrin or uroporphyrin by the enzyme using porphobilinogen (PBG) as a substrate and fluorimetry for detection. RESULTS: The calibration curves obtained with pure coproporphyrin or uroporphyrin were linear with correlation coefficients >0.99 in the range of 0-200nM for coproporphyrin and 0-150nM for uroporphyrin. The coefficients of variation for within-run and between-day imprecision were <9.8% for both assays. The three groups of subjects were used to establish the best cut-off of PBGD activity for identifying symptomatic AIP patients by using area under receiver operating characteristic curve analysis. The symptomatic AIP patients and asymptomatic carriers had significantly lower PBGD activity compared with the healthy family members (all p<.001). CONCLUSION: Two different PBGD activity assays were validated. The best cut-off for coproporphyrin was derived as 46.4nmol/h/mL RBC with corresponding sensitivity of 100% and specificity of 100% and the best cut-off for uroporphyrin was derived as 43.7nkat/L RBC with corresponding sensitivity of 100% and specificity of 97.4%.
Asunto(s)
Hidroximetilbilano Sintasa/metabolismo , Porfiria Intermitente Aguda/diagnóstico , Adolescente , Adulto , Anciano , Niño , ADN/análisis , Femenino , Fluorometría , Hematócrito , Hemoglobinas/análisis , Humanos , Hidroximetilbilano Sintasa/sangre , Hidroximetilbilano Sintasa/genética , Masculino , Persona de Mediana Edad , Estructura Molecular , Mutación , Porfobilinógeno/análisis , Porfobilinógeno/metabolismo , Porfiria Intermitente Aguda/sangre , Porfiria Intermitente Aguda/metabolismo , Especificidad por Sustrato , Taiwán , Adulto JovenRESUMEN
Catechol is one of phenolic metabolites of benzene that is a general occupational hazard and a ubiquitous environmental air pollutant. Catechol also occurs naturally in fruits, vegetables and cigarettes. Previous studies have revealed that 72h exposure to catechol improved hemin-induced erythroid differentiation of K562 cells accompanied with elevated methylation in erythroid specific genes. In present study, K562 cells were treated with 0, 10 or 20µM catechol for 1-4weeks, hemin-induced hemoglobin synthesis increased in a concentration- and time-dependent manner and the enhanced hemoglobin synthesis was relatively stable. The mRNA expression of α-, ß- and γ-globin genes, erythroid heme synthesis enzymes PBGD and ALAS2, transcription factor GATA-1 and NF-E2 showed a significant increase in K562 cells exposed to 20µM catechol for 3w, and catechol enhanced hemin-induced mRNA expression of these genes. Quantitative MassARRAY methylation analysis also confirmed that the exposure to catechol changed DNA methylation levels at several CpG sites in several erythroid-specific genes and their far upstream of regulatory elements. These results demonstrated that long-term exposure to low concentration of catechol enhanced the hemin-induced erythroid differentiation of K562 cells, in which DNA methylation played a role by up-regulating erythroid specific genes.
Asunto(s)
Contaminantes Atmosféricos/toxicidad , Catecoles/toxicidad , Metilación de ADN/efectos de los fármacos , 5-Aminolevulinato Sintetasa/genética , Factor de Transcripción GATA1/genética , Globinas/genética , Globinas/metabolismo , Hemina , Humanos , Células K562 , Subunidad p45 del Factor de Transcripción NF-E2/genética , Porfobilinógeno/metabolismo , ARN Mensajero/metabolismoRESUMEN
Recently, development of fluorescent nanoparticle-based probes for various bioimaging applications has attracted great attention. This work aims to develop a new type fluorescent nanoparticle conjugate and evaluate its cytotoxic effects on A549 and BEAS 2B cell lines. Throughout the study, ionically crosslinked chitosan nanoparticles (CNs) were conjugated with carboxylated 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY-COOH). The results of conjugates (BODIPY-CNs) were investigated with regard to their physic-chemical, optical, cytotoxic properties and cellular internalization. The morphology of BODIPY-CNs was found to be spherical in shape and quite uniform having average diameter of 70.25 ± 11.99 nm. Cytotoxicty studies indicated that although BODIPY-COOH itself was quite toxic on both A549- and BEAS 2B-treated cells, CNs increased the cell viability of both cell lines via conjugation to BODIPY-COOH fluorescent molecule up to 67% for A549 and 74% for BEAS 2B cells. These results may suggest a possible utilization of the new fluorescent nanoparticle-based probe for bioimaging in biology and medicine.
Asunto(s)
Bronquios/metabolismo , Quitosano/metabolismo , Colorantes Fluorescentes/metabolismo , Nanopartículas/metabolismo , Porfobilinógeno/análogos & derivados , Mucosa Respiratoria/metabolismo , Absorción Fisiológica , Bronquios/citología , Bronquios/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Quitosano/efectos adversos , Quitosano/química , Diagnóstico por Imagen/efectos adversos , Dispersión Dinámica de Luz , Colorantes Fluorescentes/efectos adversos , Colorantes Fluorescentes/química , Humanos , Microscopía de Fuerza Atómica , Microscopía Confocal , Microscopía Electrónica de Rastreo , Nanopartículas/efectos adversos , Nanopartículas/química , Nanopartículas/ultraestructura , Tamaño de la Partícula , Porfobilinógeno/efectos adversos , Porfobilinógeno/química , Porfobilinógeno/metabolismo , Mucosa Respiratoria/citología , Mucosa Respiratoria/efectos de los fármacos , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
In this article we present the results of the studies on interactions between the VC1 domain of the Receptor for Advanced Glycation End Products (RAGE) and its ligand, the S100B protein, performed by contact angle measurements. Histidine-tagged (His6) VC1-RAGE domain was covalently bonded to Cu(II) or Ni(II) complexes with dipyrromethene (DPM) self-assembled on gold surface. The method based on the theory of van Oss was used for the purpose of determining the Lifshitz-van der Waals (γ(LW)) component as well as the electron acceptor-electron donor (the Lewis acid-base, γ(+)-γ(-)) parameters of the VC1-RAGE-S100B complex. Moreover, the surface free energies of the interactions between the VC1 domain attached to the surface and the ligand present in the aqueous phase were determined. The specificity of the VC1- RAGE interactions with the ligand studied was also proved.
Asunto(s)
Mapeo de Interacción de Proteínas/métodos , Receptor para Productos Finales de Glicación Avanzada/química , Subunidad beta de la Proteína de Unión al Calcio S100/química , Agua/química , Algoritmos , Cobre/química , Cobre/metabolismo , Histidina/química , Modelos Químicos , Modelos Moleculares , Níquel/química , Níquel/metabolismo , Porfobilinógeno/análogos & derivados , Porfobilinógeno/química , Porfobilinógeno/metabolismo , Unión Proteica , Dominios Proteicos , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Subunidad beta de la Proteína de Unión al Calcio S100/metabolismo , Propiedades de SuperficieRESUMEN
Lipid-based nanoparticles are frequently used for drug or DNA delivery into mammalian cells. However it is difficult to determine whether such particles are taken up via endocytosis or fusion to the plasma membrane. Here, we propose a simple and reliable analytical method to do so based on the unique spectral properties of the fluorescent tracer BODIPY FL. At high local concentrations, this dye displays an additional red-shifted emission peak that is absent at low concentrations. In dye-loaded liposomes taken up by endocytosis, the local dye concentration did not significantly change upon internalization. Accordingly, unchanged fluorescence spectra were detected. When cells were incubated with liposomes able to fuse with the plasma membrane of mammalian cells, a reduction of local dye concentration and much weaker emission in the red-shifted peak were observed. The ratio of intensities in both fluorescence channels was shown to be a reliable indicator of the cellular uptake mechanism.
Asunto(s)
Bioensayo , Membrana Celular/metabolismo , Citometría de Flujo/métodos , Colorantes Fluorescentes/química , Liposomas/metabolismo , Porfobilinógeno/análogos & derivados , Animales , Células CHO , Membrana Celular/química , Cricetulus , Endocitosis , Fibroblastos/citología , Fibroblastos/metabolismo , Colorantes Fluorescentes/metabolismo , Células HEK293 , Células HeLa , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Fusión de Membrana , Ratones , Microscopía Confocal , Porfobilinógeno/química , Porfobilinógeno/metabolismo , Cultivo Primario de CélulasRESUMEN
γ-Glutamyltranspeptidase (GGT) is a tumor biomarker that selectively catalyzes the cleavage of glutamate overexpressed on the plasma membrane of tumor cells. Here, we developed two novel fluorescent inâ situ targeting (FIST) probes that specifically target GGT in tumor cells, which comprise 1)â a GGT-specific substrate unit (GSH), and 2)â a boron-dipyrromethene (BODIPY) moiety for fluorescent signalling. In the presence of GGT, sulfur-substituted BODIPY was converted to amino-substituted BODIPY, resulting in dramatic fluorescence variations. By exploiting this enzyme-triggered photophysical property, we employed these FIST probes to monitor the GGT activity in living cells, which showed remarkable differentiation between ovarian cancer cells and normal cells. These probes represent two first-generation chemodosimeters featuring enzyme-mediated rapid, irreversible aromatic hydrocarbon transfer between the sulfur and nitrogen atoms accompanied by switching of photophysical properties.
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Compuestos de Boro/química , Colorantes Fluorescentes/química , Imagen Óptica/métodos , Neoplasias Ováricas/diagnóstico , Neoplasias Ováricas/enzimología , Porfobilinógeno/análogos & derivados , gamma-Glutamiltransferasa/análisis , Compuestos de Boro/metabolismo , Línea Celular Tumoral , Pruebas de Enzimas/métodos , Femenino , Colorantes Fluorescentes/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Microscopía Confocal/métodos , Ovario/enzimología , Porfobilinógeno/química , Porfobilinógeno/metabolismo , gamma-Glutamiltransferasa/metabolismoRESUMEN
Antibiotic resistance (AR) is increasingly prevalent in low and middle income countries (LMICs), but the extent of the problem is poorly understood. This lack of knowledge is a critical deficiency, leaving local health authorities essentially blind to AR outbreaks and crippling their ability to provide effective treatment guidelines. The crux of the problem is the lack of microbiology laboratory capacity available in LMICs. To address this unmet need, we demonstrate a rapid and simple test of ß -lactamase resistance (the most common form of AR) that uses a modified ß -lactam structure decorated with two fluorophores quenched due to their close proximity. When the ß -lactam core is cleaved by ß -lactamase, the fluorophores dequench, allowing assay speeds of 20 min to be obtained with a simple, streamlined protocol. Furthermore, by testing in competition with antibiotics, the ß -lactamase-associated antibiotic susceptibility can also be extracted. This assay can be easily implemented into standard lab work flows to provide near real-time information of ß -lactamase resistance, both for epidemiological purposes as well as individualized patient care.
Asunto(s)
Bacterias/enzimología , Colorantes Fluorescentes/química , Espectrometría de Fluorescencia/métodos , Resistencia betalactámica , beta-Lactamasas/análisis , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Técnicas Bacteriológicas/métodos , Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/metabolismo , Técnicas de Sonda Molecular , Porfobilinógeno/análogos & derivados , Porfobilinógeno/análisis , Porfobilinógeno/química , Porfobilinógeno/metabolismo , beta-Lactamasas/metabolismo , beta-Lactamas/farmacologíaRESUMEN
The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses an early step of the tetrapyrrole-biosynthesis pathway in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. The enzyme possesses a dipyrromethane cofactor, which is covalently linked by a thioether bridge to an invariant cysteine residue (Cys241 in the Bacillus megaterium enzyme). The cofactor is extended during the reaction by the sequential addition of the four substrate molecules, which are released as a linear tetrapyrrole product. Expression in Escherichia coli of a His-tagged form of B. megaterium PBGD has permitted the X-ray analysis of the enzyme from this species at high resolution, showing that the cofactor becomes progressively oxidized to the dipyrromethene and dipyrromethanone forms. In previously solved PBGD structures, the oxidized cofactor is in the dipyromethenone form, in which both pyrrole rings are approximately coplanar. In contrast, the oxidized cofactor in the B. megaterium enzyme appears to be in the dipyrromethanone form, in which the C atom at the bridging α-position of the outer pyrrole ring is very clearly in a tetrahedral configuration. It is suggested that the pink colour of the freshly purified protein is owing to the presence of the dipyrromethene form of the cofactor which, in the structure reported here, adopts the same conformation as the fully reduced dipyrromethane form.
Asunto(s)
Bacillus megaterium/enzimología , Hidroximetilbilano Sintasa/química , Porfobilinógeno/análogos & derivados , Secuencia de Aminoácidos , Bacillus megaterium/metabolismo , Cristalización , Cristalografía por Rayos X , Hidroximetilbilano Sintasa/metabolismo , Datos de Secuencia Molecular , Oxidación-Reducción , Porfobilinógeno/química , Porfobilinógeno/metabolismoRESUMEN
BACKGROUND: Garlic (Allium sativum L.) bulb is processed into various forms such as crushed garlic, garlic juice, granules, dehydrated garlic pieces and garlic powder. However, greening is often a major problem when garlic is crushed, since it affects the appearance and quality of the resulting product. Therefore study of the formation mechanism of garlic green pigments is very important for garlic processing. RESULTS: The effect of porphobilinogen (PBG) on the formation of garlic green pigments was investigated in this study. As the storage time increased, there was a significant positive correlation between garlic greening and PBG content at low temperature (4 °C). PBG content decreased significantly during the garlic greening process. When treated with respiration inhibitor, both garlic greening strength and PBG content decreased as the concentration of respiration inhibitor increased. The green colour was generated when extracted PBG and allicin mixed thoroughly. CONCLUSION: There was a clear relationship between PBG content and garlic greening. As a provider of pyrrolyl compounds, PBG plays an important role in the formation of garlic green pigments.
Asunto(s)
Respiración de la Célula , Frío , Almacenamiento de Alimentos/métodos , Ajo/metabolismo , Pigmentos Biológicos , Raíces de Plantas/metabolismo , Porfobilinógeno/metabolismo , Dieta , Disulfuros , Humanos , Ácidos SulfínicosRESUMEN
The Arabidopsis rugosa1 (rug1) mutant has irregularly shaped leaves and reduced growth. In the absence of pathogens, leaves of rug1 plants have spontaneous lesions reminiscent of those seen in lesion-mimic mutants; rug1 plants also express cytological and molecular markers associated with defence against pathogens. These rug1 phenotypes are made stronger by dark/light transitions. The rug1 mutant also has delayed flowering time, upregulation of the floral repressor FLOWERING LOCUS C (FLC) and downregulation of the flowering promoters FT and SOC1/AGL20. Vernalization suppresses the late flowering phenotype of rug1 by repressing FLC. Microarray analysis revealed that 280 nuclear genes are differentially expressed between rug1 and wild type; almost a quarter of these genes are involved in plant defence. In rug1, the auxin response is also affected and several auxin-responsive genes are downregulated. We identified the RUG1 gene by map-based cloning and found that it encodes porphobilinogen deaminase (PBGD), also known as hydroxymethylbilane synthase, an enzyme of the tetrapyrrole biosynthesis pathway, which produces chlorophyll, heme, siroheme and phytochromobilin in plants. PBGD activity is reduced in rug1 plants, which accumulate porphobilinogen. Our results indicate that Arabidopsis PBGD deficiency impairs the porphyrin pathway and triggers constitutive activation of plant defence mechanisms leading to leaf lesions and affecting vegetative and reproductive development.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Hidroximetilbilano Sintasa/genética , Desarrollo de la Planta/genética , Hojas de la Planta/genética , Reproducción/genética , Arabidopsis/efectos de los fármacos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Biliverdina/análogos & derivados , Biliverdina/metabolismo , Clorofila/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Hemo/metabolismo , Hidroximetilbilano Sintasa/metabolismo , Ácidos Indolacéticos/farmacología , Proteínas de Dominio MADS/genética , Proteínas de Dominio MADS/metabolismo , Mutación , Fenotipo , Desarrollo de la Planta/efectos de los fármacos , Reguladores del Crecimiento de las Plantas/farmacología , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/metabolismo , Plantas Modificadas Genéticamente/efectos de los fármacos , Plantas Modificadas Genéticamente/metabolismo , Porfobilinógeno/metabolismo , Reproducción/efectos de los fármacosRESUMEN
The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses a key early step of the haem-biosynthesis pathway in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. The enzyme possesses a dipyrromethane cofactor which is covalently linked by a thioether bridge to an invariant cysteine residue. Since PBGD catalyses a reaction which is common to the biosynthesis of both haem and chlorophyll, structural studies of a plant PBGD enzyme offer great potential for the discovery of novel herbicides. Until recently, structural data have only been available for the Escherichia coli and human forms of the enzyme. Expression in E. coli of a codon-optimized gene for Arabidopsis thaliana PBGD has permitted for the first time the crystallization and preliminary X-ray analysis of the enzyme from a plant species at high resolution.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/enzimología , Hidroximetilbilano Sintasa/química , Tetrapirroles/biosíntesis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cristalización , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Hidroximetilbilano Sintasa/metabolismo , Modelos Moleculares , Porfobilinógeno/química , Porfobilinógeno/metabolismo , Conformación Proteica , Tetrapirroles/químicaRESUMEN
Using cell based screening assay, we identified a novel anti-tubulin agent (Z)-5-((5-(4-bromo-3-chlorophenyl)furan-2-yl)methylene)-2-thioxothiazolidin-4-one (BCFMT) that inhibited proliferation of human cervical carcinoma (HeLa) (IC(50), 7.2 ± 1.8 µM), human breast adenocarcinoma (MCF-7) (IC(50), 10.0 ± 0.5 µM), highly metastatic breast adenocarcinoma (MDA-MB-231) (IC(50), 6.0 ± 1 µM), cisplatin-resistant human ovarian carcinoma (A2780-cis) (IC(50), 5.8 ± 0.3 µM) and multi-drug resistant mouse mammary tumor (EMT6/AR1) (IC(50), 6.5 ± 1 µM) cells. Using several complimentary strategies, BCFMT was found to inhibit cancer cell proliferation at G2/M phase of the cell cycle apparently by targeting microtubules. In addition, BCFMT strongly suppressed the dynamics of individual microtubules in live MCF-7 cells. At its half maximal proliferation inhibitory concentration (10 µM), BCFMT reduced the rates of growing and shortening phases of microtubules in MCF-7 cells by 37 and 40%, respectively. Further, it increased the time microtubules spent in the pause (neither growing nor shortening detectably) state by 135% and reduced the dynamicity (dimer exchange per unit time) of microtubules by 70%. In vitro, BCFMT bound to tubulin with a dissociation constant of 8.3 ± 1.8 µM, inhibited tubulin assembly and suppressed GTPase activity of microtubules. BCFMT competitively inhibited the binding of BODIPY FL-vinblastine to tubulin with an inhibitory concentration (K(i)) of 5.2 ± 1.5 µM suggesting that it binds to tubulin at the vinblastine site. In cultured cells, BCFMT-treatment depolymerized interphase microtubules, perturbed the spindle organization and accumulated checkpoint proteins (BubR1 and Mad2) at the kinetochores. BCFMT-treated MCF-7 cells showed enhanced nuclear accumulation of p53 and its downstream p21, which consequently activated apoptosis in these cells. The results suggested that BCFMT inhibits proliferation of several types of cancer cells including drug resistance cells by suppressing microtubule dynamics and indicated that the compound may have chemotherapeutic potential.
Asunto(s)
Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Neoplasias/patología , Tiazolidinas/farmacología , Tionas/farmacología , Moduladores de Tubulina/farmacología , Animales , Proteínas de Unión al Calcio/metabolismo , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ciclina B1/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Cinetocoros/efectos de los fármacos , Proteínas Mad2 , Ratones , Índice Mitótico , Polimerizacion/efectos de los fármacos , Porfobilinógeno/análogos & derivados , Porfobilinógeno/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Represoras/metabolismo , Rodanina/farmacología , Tiazolidinas/química , Tionas/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química , Proteína p53 Supresora de Tumor/metabolismo , Vinblastina/farmacologíaRESUMEN
Near-infrared (NIR) fluorescence probes are especially useful for simple and noninvasive in vivo imaging inside the body because of low autofluorescence and high tissue transparency in the NIR region compared with other wavelength regions. However, existing NIR fluorescence probes for matrix metalloproteinases (MMPs), which are tumor, atherosclerosis, and inflammation markers, have various disadvantages, especially as regards sensitivity. Here, we report a novel design strategy to obtain a NIR fluorescence probe that is rapidly internalized by free diffusion and well retained intracellularly after activation by extracellular MMPs. We designed and synthesized four candidate probes, each consisting of a cell permeable or nonpermeable NIR fluorescent dye as a Förster resonance energy transfer (FRET) donor linked to the NIR dark quencher BHQ-3 as a FRET acceptor via a MMP substrate peptide. We applied these probes for detection of the MMP activity of cultured HT-1080 cells, which express MMP2 and MT1-MMP, by fluorescence microscopy. Among them, the probe incorporating BODIPY650/665, BODIPY-MMP, clearly visualized the MMP activity as an increment of fluorescence inside the cells. We then applied this probe to a mouse xenograft tumor model prepared with HT-1080 cells. Following intratumoral injection of the probe, MMP activity could be visualized for much longer with BODIPY-MMP than with the probe containing SulfoCy5, which is cell impermeable and consequently readily washed out of the tissue. This simple design strategy should be applicable to develop a range of sensitive, rapidly responsive NIR fluorescence probes not only for MMP activity, but also for other proteases.