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1.
Int J Mol Sci ; 22(15)2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34360759

RESUMO

Salt and osmotic stress are the main abiotic stress factors affecting plant root growth and architecture. We investigated the effect of salt (100 mM NaCl) and osmotic (200 mM mannitol) stress on the auxin metabolome by UHPLC-MS/MS, auxin distribution by confocal microscopy, and transcript levels of selected genes by qRT-PCR in Arabidopsis thaliana ecotype Columbia-0 (Col-0) and DR5rev::GFP (DR5) line. During long-term stress (13 days), a stability of the auxin metabolome and a tendency to increase indole-3-acetic acid (IAA) were observed, especially during salt stress. Short-term stress (3 h) caused significant changes in the auxin metabolome, especially NaCl treatment resulted in a significant reduction of IAA. The data derived from auxin profiling were consistent with gene expressions showing the most striking changes in the transcripts of YUC, GH3, and UGT transcripts, suggesting disruption of auxin biosynthesis, but especially in the processes of amide and ester conjugation. These data were consistent with the auxin distribution observed in the DR5 line. Moreover, NaCl treatment caused a redistribution of auxin signals from the quiescent center and the inner layers of the root cap to the epidermal and cortical cells of the root elongation zone. The distribution of PIN proteins was also disrupted by salt stress; in particular, PIN2 was suppressed, even after 5 min of treatment. Based on our results, the DR5 line was more sensitive to the applied stresses than Col-0, although both lines showed similar trends in root morphology, as well as transcriptome and metabolome parameters under stress conditions.


Assuntos
Proteínas de Arabidopsis/biossíntese , Arabidopsis/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Ácidos Indolacéticos/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Estresse Salino/efeitos dos fármacos , Cloreto de Sódio/farmacologia
2.
Plants (Basel) ; 8(6)2019 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-31174414

RESUMO

Salinity is a major abiotic stress negatively affecting plant growth and consequently crop production. The effects of short-term salt stress were evaluated on seedlings of three globally important Brassica crops-Chinese cabbage (Brassica rapa ssp. pekinensis), white cabbage (Brassica oleracea var. capitata), and kale (Brassica oleracea var. acephala)-with particular focus on phenolic acids. The physiological and biochemical stress parameters in the seedlings and the levels of three main groups of metabolites (total glucosinolates, carotenoids, and phenolics) and individual phenolic acids were determined. The salt treatments caused a dose-dependent reduction in root growth and biomass and an increase in stress parameters (Na+/K+ ratio, reactive oxygen species (ROS) and glutathione (GSH)) in all seedlings but most prominently in Chinese cabbage. Based on PCA, specific metabolites grouped close to the more tolerant species, white cabbage and kale. The highest levels of phenolic acids, particularly hydroxycinnamic acids, were determined in the more tolerant kale and white cabbage. A reduction in caffeic, salicylic, and 4-coumaric acid was found in Chinese cabbage and kale, and an increase in ferulic acid levels was found in kale upon salinity treatments. Phenolic acids are species-specific among Brassicaceae, and some may participate in stress tolerance. Salt-tolerant varieties have higher levels of some phenolic acids and suffer less from metabolic stress disorders under salinity stress.

3.
Front Plant Sci ; 10: 450, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31031786

RESUMO

Soil salinity is severely affecting crop productivity in many countries, particularly in the Mediterranean area. To evaluate early plant responses to increased salinity and characterize tolerance markers, three important Brassica crops - Chinese cabbage (Brassica rapa ssp. pekinensis), white cabbage (B. oleracea var. capitata) and kale (B. oleracea var. acephala) were subjected to short-term (24 h) salt stress by exposing them to NaCl at concentrations of 50, 100, or 200 mM. Physiological (root growth, photosynthetic performance parameters, and Na+/K+ ratio) and biochemical parameters (proline content and lipid peroxidation as indicated by malondialdehyde, MDA, levels) in the plants' roots and leaves were then measured. Photosynthetic parameters such as the total performance index PItotal (describing the overall efficiency of PSI, PSII and the intersystem electron transport chain) appeared to be the most salinity-sensitive parameter and informative stress marker. This parameter was decreased more strongly in Chinese cabbage than in white cabbage and kale. It indicated that salinity reduced the capacity of the photosynthetic system for efficient energy conversion, particularly in Chinese cabbage. In parallel with the photosynthetic impairments, the Na+/K+ ratio was highest in Chinese cabbage leaves and lowest in kale leaves while kale root is able to keep high Na+/K+ ratio without a significant increase in MDA. Thus Na+/K+ ratio, high in root and low in leaves accompanying with low MDA level is an informative marker of salinity tolerance. The crops' tolerance was positively correlated with levels of the stress hormone abscisic acid (ABA) and negatively correlated with levels of jasmonic acid (JA), and jasmonoyl-L-isoleucine (JA-Ile). Furthermore, salinity induced contrasting changes in levels of the growth-promoting hormones brassinosteroids (BRs). The crop's tolerance was positively correlated with levels of BR precursor typhasterol while negatively with the active BR brassinolide. Principal Component Analysis revealed correlations in observed changes in phytohormones, biochemical, and physiological parameters. Overall, the results show that kale is the most tolerant of the three species and Chinese cabbage the most sensitive to salt stress, and provide holistic indications of the spectrum of tolerance mechanisms involved.

4.
Crit Rev Food Sci Nutr ; 59(15): 2411-2422, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-29557674

RESUMO

Kale (Brassica oleracea var. acephala) is a cruciferous vegetable, characterized by leaves along the stem, which, in recent years, have gained a great popularity as a ´superfood´. Consequently, in a popular culture it is listed in many ´lists of the healthiest vegetables´. Without the doubt, a scientific evidences support the fact that cruciferous vegetables included in human diet can positively affect health and well-being, but remains unclear why kale is declared superior in comparison with other cruciferous. It is questionable if this statement about kale is triggered by scientific evidence or by some other factors. Our review aims to bring an overview of kale's botanical characteristics, agronomic requirements, contemporary and traditional use, macronutrient and phytochemical content and biological activity, in order to point out the reasons for tremendous kale popularity.


Assuntos
Brassica/química , Folhas de Planta/química , Animais , Antineoplásicos/análise , Antioxidantes/análise , Brassica/crescimento & desenvolvimento , Sistema Cardiovascular/efeitos dos fármacos , Sistema Cardiovascular/metabolismo , Carotenoides/análise , Linhagem Celular Tumoral , Produtos Agrícolas , Dieta , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/metabolismo , Glucosinolatos/análise , Humanos , Valor Nutritivo , Fenóis/análise , Compostos Fitoquímicos/análise , Extratos Vegetais/análise , Verduras/química
5.
Int J Mol Sci ; 19(10)2018 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-30241414

RESUMO

Drought is one of the major abiotic stresses affecting the productivity of Brassica crops. To understand the role of phytohormones in drought tolerance, we subjected Chinese cabbage (B. rapa ssp. pekinensis), white cabbage (B. oleracea var. capitata), and kale (B. oleracea var. acephala) to drought and examined the stress response on the physiological, biochemical and hormonal levels. The phytohormones abscisic acid (ABA), auxin indole-3-acetic acid (IAA), brassinosteroids (BRs), cytokinins (CKs), jasmonates (JAs), and salicylic acid (SA) were analyzed by ultra-high-performance liquid chromatography⁻tandem mass spectrometry (UHPLC-MS/MS). Based on the physiological and biochemical markers the Chinese cabbage exhibited the lowest tolerance, followed by the white cabbage, while the kale appeared to be the most tolerant to drought. The drought tolerance of the kale correlated with increased levels of SA, ABA, IAA, CKs iP(R) and cZ(R), and typhasterol (TY), a precursor of active BRs. In contrast, the drought sensitivity of the Chinese cabbage correlated with a significant increase in ABA, JAs and the active BRs castasterol (CS) and brassinolide (BL). The moderately tolerant white cabbage, positioned between the kale and Chinese cabbage, showed more similarity in terms of the phytohormone patterns with the kale. We concluded that the drought tolerance in Brassicaceae is mostly determined by the increased endogenous levels of IAA, CKs, ABA and SA and the decreased levels of active BRs.


Assuntos
Brassica/metabolismo , Secas , Regulação da Expressão Gênica de Plantas , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/genética , Estresse Fisiológico , Brassica/classificação , Brassica/genética , Perfilação da Expressão Gênica , Proteínas de Plantas/metabolismo
6.
Food Chem ; 269: 96-102, 2018 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-30100490

RESUMO

Five Brassicaceae sprouts (white cabbage, kale, broccoli, Chinese cabbage, arugula) were comparatively analyzed based on phytochemicals (polyphenols, glucosinolates, carotenoids, chlorophylls, ascorbic acid) content and accompanying enzymes associated with phytochemical stability and bioavailability (peroxidases, myrosinase, and polyphenol-oxidase) that consequently impact food quality. Significantly high content of polyphenols and glucosinolates, as well as a high antioxidant activity were found in white cabbage, followed by kale sprouts. In addition, white cabbage contained higher amount of fibers and lower polyphenol-oxidase activity which potentially indicates prevention of browning and consequently better sprout quality. Arugula and broccoli showed higher activity of myrosinase that may result in higher bioavailability of active glucosinolates forms. According to our data, sprouts are cheap, easy- and fast-growing source of phytochemicals but also they are characterized by different endogenous enzymes activity. Consequently, this parameter should also be taken into consideration in the studies related to the health benefits of the plant-based food.


Assuntos
Brassica/química , Brassica/enzimologia , Qualidade dos Alimentos , Compostos Fitoquímicos/análise , Disponibilidade Biológica , Catecol Oxidase/metabolismo , Análise de Alimentos , Glucosinolatos , Glicosídeo Hidrolases/metabolismo , Humanos , Peroxidases/metabolismo , Compostos Fitoquímicos/farmacocinética
7.
Plant Physiol Biochem ; 125: 74-84, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29427890

RESUMO

Salinity is one of major abiotic stresses affecting Brassica crop production. Here we present investigations into the physiological, biochemical, and hormonal components of the short-term salinity stress response in Chinese cabbage seedlings, with particular emphasis on the biosynthesis and metabolism of auxin indole-3-acetic acid (IAA). Upon salinity treatments (50-200 mM NaCl) IAA level was elevated in a dose dependent manner reaching 1.6-fold increase at the most severe salt treatment in comparison to the control. IAA precursor profiling suggested that salinity activated the indole-3-acetamide and indole-3-acetaldoxime biosynthetic pathways while suppressing the indole-3-pyruvic acid pathway. Levels of the IAA catabolites 2-oxoindole-3-acetic acid and indole-3-acetic acid-aspartate increased 1.7- and 2.0-fold, respectively, under the most severe treatment, in parallel with those of IAA. Conversely, levels of the ester conjugate indole-3-acetyl-1-O-ß-d-glucose and its catabolite 2-oxoindole-3-acetyl-1-O-ß-d-glucose decreased 2.5- and 7.0-fold, respectively. The concentrations of stress hormones including jasmonic acid and jasmonoyl-isoleucine (JA and JA-Ile), salicylic acid (SA) and abscisic acid (ABA) confirmed the stress induced by salt treatment: levels of JA and JA-Ile increased strongly under the mildest treatment, ABA only increased under the most severe treatment, and SA levels decreased dose-dependently. These hormonal changes were related to the observed changes in biochemical stress markers upon salt treatments: reductions in seedling fresh weight and root growth, decreased photosynthesis rate, increased levels of reactive oxygen species, and elevated proline content and the Na+/K+ ratio. Correlations among auxin profile and biochemical stress markers were discussed based on Pearson's coefficients and principal component analysis (PCA).


Assuntos
Brassica rapa/metabolismo , Ácidos Indolacéticos/metabolismo , Plântula/metabolismo , Cloreto de Sódio/farmacologia , Estresse Fisiológico/efeitos dos fármacos
8.
J Colloid Interface Sci ; 508: 95-104, 2017 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-28822865

RESUMO

This study describes and examines the structural and morphological properties of the hierarchically organized, aragonite cuttlebone forms for the common cuttlefish (Sepia officinalis, L.), including its main structural parts, the dorsal shield, and the chambers. Specifically, it complements the mechanism for the self-organized formation of aragonite, identifies the presence, and determines the role of soluble organic matrix (SOM) proteins in the morphogenesis of the cuttlebone's biomineral structures on the nanoscale. The structure and morphology of the cuttlebone were examined using X-ray diffraction (XRD) and field-emission scanning electron microscopy (FESEM), and their thermal properties by thermo-gravimetric analysis (TGA) and differential thermal analysis (DTA). Proteins from the SOM were investigated using two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D-PAGE), matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS), nano liquid chromatography tandem mass spectrometry (nano-LC ESI-MS) and Edman degradation. The results showed that the cuttlebone exhibited several diverse biomineral structures characterized by complex morphologies. Their formation is governed by the organic matrix, particularly proteins, which at the earliest stage of development provide templates for the initial extracellular nucleation of the aragonite nanocrystals. This is followed by a bottom-up morphogenesis, based on the nanoscale oriented aggregation and coalescence of primarily formed aragonite nanograins, which results in the hierarchically organized, nanostructured, aragonite forms. The molecular masses of the most pronounced SOM proteins from the dorsal shield were about 10, 15, 40 and 60kDa, while from the chambers they were 10, 20, 25, 30 and 45kDa. Peptide fragments corresponding to Sep7, Sep8, chitin synthase 1, ficoline-2, polyubiquitin and the ubiquitin carboxyl-terminal hydrolase 32-like protein were detected in the SOM, with these proteins having functional properties related to the biomineralization processes. In general, there are mostly acidic proteins present in alternatively glycosylated forms, which are common attributes of biomineralization-related proteins.

9.
Biol Chem ; 398(1): 101-112, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-27467751

RESUMO

In a search for plant homologues of dipeptidyl peptidase III (DPP III) family, we found a predicted protein from the moss Physcomitrella patens (UniProt entry: A9TLP4), which shared 61% sequence identity with the Arabidopsis thaliana uncharacterized protein, designated Nudix hydrolase 3. Both proteins contained all conserved regions of the DPP III family, but instead of the characteristic hexapeptide HEXXGH zinc-binding motif, they possessed a pentapeptide HEXXH, and at the N-terminus, a Nudix box, a hallmark of Nudix hydrolases, known to act upon a variety of nucleoside diphosphate derivatives. To investigate their biochemical properties, we expressed heterologously and purified Physcomitrella (PpND) and Arabidopsis (AtND) protein. Both hydrolyzed, with comparable catalytic efficiency, the isopentenyl diphosphate (IPP), a universal precursor for the biosynthesis of isoprenoid compounds. In addition, PpND dephosphorylated four purine nucleotides (ADP, dGDP, dGTP, and 8-oxo-dATP) with strong preference for oxidized dATP. Furthermore, PpND and AtND showed DPP III activity against dipeptidyl-2-arylamide substrates, which they cleaved with different specificity. This is the first report of a dual activity enzyme, highly conserved in land plants, which catalyzes the hydrolysis of a peptide bond and of a phosphate bond, acting both as a dipeptidyl peptidase III and an atypical Nudix hydrolase.


Assuntos
Arabidopsis/enzimologia , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Pirofosfatases/metabolismo , Sequência de Aminoácidos , Dipeptidil Peptidases e Tripeptidil Peptidases/química , Cinética , Modelos Moleculares , Domínios Proteicos , Pirofosfatases/química , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade
10.
Phys Chem Chem Phys ; 18(13): 8890-900, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-26959939

RESUMO

Brassica rapa auxin amidohydrolase (BrILL2) participates in the homeostasis of the plant hormones auxins by hydrolyzing the amino acid conjugates of auxins, thereby releasing the free active form of hormones. Herein, the potential role of the two conserved Cys residues of BrILL2 (at sequence positions 139 and 320) has been investigated by using interdisciplinary approaches and methods of molecular biology, biochemistry, biophysics and molecular modelling. The obtained results show that both Cys residues participate in the regulation of enzyme activity. Cys320 located in the satellite domain of the enzyme is mainly responsible for protein stability and regulation of enzyme activity through polymer formation, as has been revealed by enzyme kinetics and differential scanning calorimetry analysis of the BrILL2 wild type and mutants C320S and C139S. Cys139 positioned in the active site of the catalytic domain is involved in the coordination of one Mn(2+) ion of the bimetal center and is crucial for the enzymatic activity. Although the point mutation Cys139 to Ser causes the loss of enzyme activity, it does not affect the metal binding to the BrILL2 enzyme, as has been shown by isothermal titration calorimetry, circular dichroism spectropolarimetry and differential scanning calorimetry data. MD simulations (200 ns) revealed a different active site architecture of the BrILL2C139S mutant in comparison to the wild type enzyme. Additional possible reasons for the inactivity of the BrILL2C139S mutant have been discussed based on MD simulations and MM-PBSA free energy calculations of BrILL2 enzyme complexes (wt and C139S mutant) with IPA-Ala as a substrate.


Assuntos
Amidoidrolases/metabolismo , Brassica rapa/enzimologia , Cisteína/química , Ácidos Indolacéticos/metabolismo , Calorimetria , Estabilidade Enzimática , Espectrometria de Massas , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida
11.
Food Chem ; 194: 828-34, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26471624

RESUMO

The worldwide established strawberry cultivar 'Albion' and three recently introduced cultivars in Europe: 'Monterey', 'Capri', and 'Murano', grown hydroponically, were studied to ascertain the influence of cultivar and harvesting date on the physical, chemical, antioxidant and phytochemical properties of their fruits. Interrelationships of investigated parameters and these cultivars were investigated by the statistical approach of principal component analysis (PCA). Results indicated that cultivar had a more significant effect on the analyzed parameters than harvesting date. Thus grouping of the variables in a PCA plot indicated that each cultivar has specific characteristics important for consumer or industrial use. Cultivar 'Monterey' was the richest in phytochemical contents and consequently in antioxidant activity, 'Albion' showed the highest contents of total soluble solids, titratable acidity content and ascorbic acid, 'Capri' had the highest value of firmness, while 'Murano' had lighter color in comparison to others. Potential use of these cultivars has been assessed according to these important measured attributes.


Assuntos
Fragaria/química , Frutas/química , Fenóis/análise , Compostos Fitoquímicos/análise , Análise de Componente Principal/métodos
12.
Food Chem ; 186: 298-305, 2015 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-25976825

RESUMO

Teucrium arduini L., an Ilyric-Balcanic endemic species, has been reported for decades as a valuable plant used in traditional medicine for treating digestive disorders. The present study evaluated genetic and phytochemical variability of six T. arduini populations in order to determine factors that influence an accumulation of polyphenolic compounds. Results strongly suggest that a phytochemical variation was caused by environmental rather than genetic factors. T. arduini leaf extract from the locality Ucka, which accumulated significantly more polyphenolic phytochemicals in comparison to others, showed antioxidant activity in DNA and lipid bioassays. Furthermore, the same extract exhibited prooxidant behaviour at protein level and induce formation of reactive oxygen species in human laryngeal carcinoma cells causing cytotoxic activity, in a dose dependent manner. All the results of the present study suggested that T. arduini extract could be responsible for antioxidative/prooxidative mechanisms and would help in determination of optimal conditions for their ethnopharmacological use.


Assuntos
Antioxidantes/química , Oxidantes/química , Teucrium/química , Teucrium/genética , Antioxidantes/análise , Linhagem Celular Tumoral , Croácia , Etnofarmacologia , Humanos , Compostos Fitoquímicos/análise , Fitoterapia , Extratos Vegetais/química , Folhas de Planta/química , Espécies Reativas de Oxigênio/análise
13.
Nat Prod Res ; 29(18): 1770-4, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25597227

RESUMO

Phytochemical composition and antioxidant activity of three wild populations of endemic Illyric-Balcanic species Micromeria croatica (Pers.) Schott have been evaluated with respect to plant organ and growing location. Multivariate analysis (principal component analysis) was performed to visualise (dis-)similarity among samples and identify the correlations between phytochemical variables that explain the most variability. The tested leaf extract from BaCic kuk locality exhibited protective effects against reactive oxygen species-induced damage of DNA and inhibition of lipid peroxidation, while it caused oxidative degradation of protein in the bovine serum albumin assay at higher concentrations. This extract also exhibited cytotoxic activity and facilitated the formation of reactive oxygen species in the HEp2 cell line, in a dose-dependent manner.


Assuntos
Antioxidantes/química , Dano ao DNA/efeitos dos fármacos , Lamiaceae/química , Peroxidação de Lipídeos/efeitos dos fármacos , Extratos Vegetais/química , Polifenóis/química , Linhagem Celular Tumoral , Humanos , Estresse Oxidativo/efeitos dos fármacos , Folhas de Planta/química , Espécies Reativas de Oxigênio/química
14.
Plant Cell Rep ; 32(7): 1031-42, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23508255

RESUMO

KEY MESSAGE : Stress hormones, particularly jasmonic acid, influenced root growth, auxin levels, and transcription of auxin amidohydrolase BrIAR3 in Brassica rapa seedlings, while auxin conjugate synthetases BrGH3.1 and BrGH3.9 were down-regulated by all treatments. The influence of stress hormones: jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA) on 1-day-old seedlings of Chinese cabbage (Brassica rapa L. ssp. pekinensis) was investigated with particular focus on auxin levels and the regulation of reversible auxin conjugation as a mechanism of auxin homeostasis. At the physiological level, stress hormones inhibited root growth, where JA was the most prominent inhibitor with an IC50 value 3.1 µM, which is one and two orders of magnitude lower than that found for ABA and SA, respectively. JA treatment significantly increased the total auxin content, by induction of free and conjugated forms. Also, the stress hormones affected the transcription of genes involved in the process of the reversible auxin conjugation: auxin amidohydrolases BrIAR3 and BrILL2, and auxin conjugate synthetases BrGH3.1 and BrGH3.9. JA treatment increased the transcript level of BrIAR3 two-fold, while it did not affect the transcription of BrILL2. SA and ABA down-regulated the transcription of both auxin amidohydrolase genes by 30 %. Transcription of both auxin conjugate synthetases was significantly down-regulated by all treatments by 30-70 %. Among the investigated biochemical stress markers, glutathione along with protein carbonylation appeared the most affected upon treatments. The redox status of the seedlings was shifted to the more oxidized state upon JA and ABA treatments, whereas SA caused more reduced redox state in comparison to the control. The principal component analysis visualized relationship among auxin and stress parameters upon treatments. Accordingly, the role of auxin in stress response of Brassica seedlings was discussed.


Assuntos
Brassica/metabolismo , Ácidos Indolacéticos/metabolismo , Plântula/metabolismo , Ácido Abscísico/metabolismo , Ciclopentanos/metabolismo , Glutationa/metabolismo , Peroxidação de Lipídeos , Oxilipinas/metabolismo , Fenóis/metabolismo , Raízes de Plantas/metabolismo , Ácido Salicílico/metabolismo
15.
Biol Chem ; 393(1-2): 37-46, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22628297

RESUMO

Dipeptidyl peptidase III (DPP III), a member of the metallopeptidase family M49, was considered as an exclusively eukaryotic enzyme involved in intracellular peptide catabolism and pain modulation. In 2003, new data on genome sequences revealed the first prokaryotic orthologs, which showed low sequence similarity to eukaryotic ones and a cysteine (Cys) residue in the zinc-binding motif HEXXGH. Here we report the cloning and heterologous expression of DPP III from the human gut symbiont Bacteroides thetaiotaomicron. The catalytic efficiency of bacterial DPP III for preferred synthetic substrate hydrolysis was very similar to that of the human host enzyme. Substitution of Cys450 from the active-site motif by serine did not substantially change the enzymatic activity. However, this residue was wholly responsible for the inactivation effect of sulfhydryl reagents. Molecular modeling indicated seven basic amino acid residues in the local environment of Cys450 as a possible cause for its high reactivity. Sequence analysis of 81 bacterial M49 peptidases showed conservation of the HECLGH motif in 68 primary structures with the majority of proteins lacking an active-site Cys originated from aerobic bacteria. Data obtained suggest that Cys450 of B. thetaiotaomicron DPP III is a regulatory residue for the enzyme activity.


Assuntos
Bacteroides/enzimologia , Cisteína/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/química , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Motivos de Aminoácidos , Domínio Catalítico , Cisteína/química , Dipeptidil Peptidases e Tripeptidil Peptidases/isolamento & purificação , Ativação Enzimática , Estrutura Secundária de Proteína , Desdobramento de Proteína , Temperatura
16.
Org Biomol Chem ; 10(26): 5063-72, 2012 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-22622806

RESUMO

A novel activity of halohydrin dehalogenases towards spiroepoxides has been found. The enzyme from Arthrobacter sp. (HheA) catalysed highly regioselective azidolysis of spiroepoxides containing 5, 6 and 7-membered cycloalkane rings, while the enzyme from Agrobacterium radiobacter (HheC), besides high regioselectivity, also displayed moderate to high enantioselectivity (E up to >200) that can be applied for the kinetic resolution of chiral spiroepoxides. The orientations of spiroepoxides in the active site of halohydrin dehalogenases were studied by quantum-chemical calculations and docking simulations. Analyses of the complexes obtained revealed the origins of diastereoselectivity and enantioselectivity of the investigated biotransformations.


Assuntos
Arthrobacter/enzimologia , Compostos de Epóxi/metabolismo , Hidrolases/metabolismo , Compostos de Espiro/metabolismo , Agrobacterium/enzimologia , Arthrobacter/química , Azidas/metabolismo , Domínio Catalítico , Hidrolases/química , Modelos Moleculares , Estereoisomerismo , Especificidade por Substrato
17.
J Colloid Interface Sci ; 354(1): 181-9, 2011 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-21130464

RESUMO

This study describes the morphological properties and discusses the colloid-chemical mechanisms of the formation of hierarchically structured aragonite fibers in the exoskeleton structure of the Mediterranean zooxanthellate scleractinian coral Cladocora caespitosa. The study is based on a detailed structural and morphological examination of the coral exoskeleton and on a preliminary biochemical and molecular identification of the isolated soluble proteinaceus organic matrix. The biomineral structure was examined by X-ray diffraction (XRD), field-emission scanning electron microscopy (FESEM) and atomic-force microscopy (AFM), while the isolated protein organic constituents were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MALDI-TOF-MS). The SDS-PAGE analysis of the soluble protein matrix showed three major protein bands at 15, 41, and 80kDa. Based on the MALDI-TOF-MS analyses, the identified peptides tend to exhibit an acidic character. The results obtained confirm and complement the existing hypotheses relating to the significant role of the soluble acidic protein matrix and the biologically induced colloid-chemical processes in the phase formation and growth of scleractinian submicrometer fibrous aragonite units. It was also shown that the general strategy for the morphogenesis of fibrous structured aragonite lies in the nanoscale aggregation and subsequent coalescence processes that occur simultaneously. The subsequent morphological conversion of the initially formed submicrometer fibrous aragonite units into well-defined, micrometer-sized, prismatic facets in the skeletal structures of the corals is demonstrated.


Assuntos
Antozoários/química , Carbonato de Cálcio/síntese química , Coloides/química , Animais , Antozoários/crescimento & desenvolvimento , Carbonato de Cálcio/química , Microscopia Eletrônica de Varredura
18.
J Biochem Mol Toxicol ; 24(3): 165-73, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20583293

RESUMO

Auxins, of which indole-3-acetic acid (IAA) is the most widespread representative, are plant hormones. In addition to plants, IAA also naturally occurs in humans in micromolar concentrations. In the presence of peroxidase, indolic auxins are converted to cytotoxic oxidation products and have thus been proposed for use in gene-directed enzyme/prodrug tumor therapy. Since data on the genotoxicity of IAA and its derivatives are not consistent, here we investigate the early DNA damaging effects (2-h treatment) of the auxins, IAA, and 2-methyl-indole-3-acetic acid (2-Me-IAA) by the alkaline comet assay and compare them with their free radical-scavenging activity measured by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Human neutrophils are chosen as the test system since they possess inherent peroxidase activity. The results of the comet assay indicate an increase in DNA damage in a dose-dependent manner up to 1.00 mM of both auxins. Generally, IAA applied in the same concentration had greater potential to damage DNA in human neutrophils than did 2-Me-IAA. The genotoxicities of the two examined auxins are negatively correlated with their antioxidant activities, as measured by the DPPH assay; 2-Me-IAA showed a higher antioxidant capacity than did IAA. We assume that differences in the molecular structure of the tested auxins contributed to differences in their metabolism, in particular, with respect to interactions with peroxidases and other oxidative enzymes in neutrophils. However, the exact mechanisms have to be elucidated in future studies.


Assuntos
Ensaio Cometa/métodos , Dano ao DNA , Sequestradores de Radicais Livres/farmacologia , Ácidos Indolacéticos/farmacologia , Neutrófilos/efeitos dos fármacos , Adulto , Relação Dose-Resposta a Droga , Humanos , Masculino , Neutrófilos/metabolismo
19.
Talanta ; 80(2): 651-5, 2009 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-19836533

RESUMO

An analytical protocol for the isolation and quantification of indole-3-acetic acid (IAA) and its amino acid conjugates was developed. IAA is an important phytohormone and formation of its conjugates plays a crucial role in regulating IAA levels in plants. The developed protocol combines a highly specific immunoaffinity extraction with a sensitive and selective LC-MS/MS analysis. By using internal standards for each of the studied compounds, IAA and seven amino acid conjugates were analyzed in quantities of fresh plant material as low as 30 mg. In seeds of Helleborus niger, physiological levels of these compounds were found to range from 7.5 nmol g(-1) fresh weight (IAA) to 0.44 pmol g(-1) fresh weight (conjugate with Ala). To our knowledge, the identification of IAA conjugates with Gly, Phe and Val from higher plants is reported here for the first time.


Assuntos
Anticorpos Monoclonais/imunologia , Ácidos Indolacéticos/imunologia , Ácidos Indolacéticos/isolamento & purificação , Especificidade de Anticorpos , Cromatografia de Afinidade/instrumentação , Cromatografia de Afinidade/métodos , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Glicina/química , Helleborus/química , Ácidos Indolacéticos/química , Fenilalanina/química , Reguladores de Crescimento de Plantas/imunologia , Reguladores de Crescimento de Plantas/isolamento & purificação , Reguladores de Crescimento de Plantas/normas , Padrões de Referência , Sementes/química , Espectrofotometria Ultravioleta , Espectrometria de Massas em Tandem , Valina/química
20.
Plant Cell Physiol ; 50(9): 1587-99, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19602499

RESUMO

Two auxin amidohydrolases, BrIAR3 and BrILL2, from Chinese cabbage [Brassica rapa L. ssp. pekinensis (Lour.) Hanelt] were produced by heterologous expression in Escherichia coli, purified, and screened for activity towards N-(indol-3-ylacetyl)-L-alanine (IAA-Ala) and the long-chain auxin-amino acid conjugates, N-[3-(indol-3-yl)propionyl]-L-alanine (IPA-Ala) and N-[4-(indol-3-yl)butyryl]-L-alanine (IBA-Ala). IPA-Ala was shown to be the favored substrate of both enzymes, but BrILL2 was approximately 15 times more active than BrIAR3. Both enzymes cleaved IBA-Ala and IAA-Ala to a lesser extent. The enzyme kinetics were measured for BrILL2 and the obtained parameters suggested similar binding affinities for the long-chain auxin-amino acid conjugates (IPA-Ala and IBA-Ala). The velocity of the hydrolyzing reaction decreased in the order IPA-Ala > IBA-Ala > IAA-Ala. In a root growth bioassay, higher growth inhibition was caused by IPA-Ala and IBA-Ala in comparison with IAA-Ala. Neither these conjugates nor the corresponding free auxins affected the expression of the BrILL2 gene. A modeling study revealed several possible modes of IPA-Ala binding to BrILL2. Based on these results, two possible scenarios for substrate hydrolysis are proposed. In one the metal binding water is activated by the carboxyl group of the substrate itself, and in the other by a glutamate residue from the active site of the enzyme.


Assuntos
Amidoidrolases/metabolismo , Brassica rapa/enzimologia , Ácidos Indolacéticos/metabolismo , Indóis/metabolismo , Proteínas de Plantas/metabolismo , Brassica rapa/genética , Regulação da Expressão Gênica de Plantas , Hidrólise , Cinética , Modelos Moleculares , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Estrutura Secundária de Proteína , Especificidade por Substrato
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