RESUMO
Solieria filiformis has been reported to have molecules with various biological activities. In this study we used environmentally friendly extraction methods, such as enzyme-assisted extraction (EAE), as a first step to obtain bioactive compounds from this species. Five combinations of protease (PRO) and carbohydrase (AMG) were utilized (1:0, 0:1, 2:1, 1:1, 1:2 PRO:AMG) to obtain Water Soluble Enzymatic Hydrolysates (WSEHs). Extraction yields, biochemical and structural characterization, as well as in vitro activity against Herpes simplex virus type 1 (HSV-1) and antioxidant capacities were determined. All PRO:AMG combinations significantly improved yields. EAE yielded heterogeneous extracts rich in iota-carrageenan and phenols, as confirmed by FTIR spectra. The highest antiherpetic activity (EC50 4.5 ± 0.4 µg mL-1) was found in the WSEHs obtained under 2:1 PRO:AMG. At this combination high antioxidant capacity was also obtained for ABTS (2,2'-Azino-Bis-3-ethylbenzoThiazoline-6-Sulfonic acid) radical scavenging activity and Ferric Reducing Antioxidant Power (FRAP). These could probably play a synergistic role associated to the strong antiviral activity obtained. These results suggest that 2:1 PRO:AMG could be effective in promoting the hydrolytic breakdown of high MW polysaccharides, contributing to the improvement of WSEHs bioactivity. Although Solieria filiformis WSEHs showed promising results, further research, including separation and purification techniques are needed.
Assuntos
Carragenina/química , Carragenina/farmacologia , Rodófitas/enzimologia , Antioxidantes/química , Antivirais/química , Compostos de Bifenilo/química , Herpesvirus Humano 1/efeitos dos fármacos , Fenóis/química , Picratos/química , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Polissacarídeos/química , Polissacarídeos/isolamento & purificação , Rodófitas/químicaRESUMO
BACKGROUND: The orderly progression through mitosis is regulated by the Anaphase-Promoting Complex (APC), a large multiprotein E3 ubiquitin ligase that targets key cell-cycle regulators for destruction by the 26 S proteasome. The APC is composed of at least 11 subunits and associates with additional regulatory activators during mitosis and interphase cycles. Despite extensive research on APC and activator functions in the cell cycle, only a few components have been functionally characterized in plants. RESULTS: Here, we describe an in-depth search for APC subunits and activator genes in the Arabidopsis, rice and poplar genomes. Also, searches in other genomes that are not completely sequenced were performed. Phylogenetic analyses indicate that some APC subunits and activator genes have experienced gene duplication events in plants, in contrast to animals. Expression patterns of paralog subunits and activators in rice could indicate that this duplication, rather than complete redundancy, could reflect initial specialization steps. The absence of subunit APC7 from the genome of some green algae species and as well as from early metazoan lineages, could mean that APC7 is not required for APC function in unicellular organisms and it may be a result of duplication of another tetratricopeptide (TPR) subunit. Analyses of TPR evolution suggest that duplications of subunits started from the central domains. CONCLUSIONS: The increased complexity of the APC gene structure, tied to the diversification of expression paths, suggests that land plants developed sophisticated mechanisms of APC regulation to cope with the sedentary life style and its associated environmental exposures.
Assuntos
Evolução Molecular , Proteínas de Plantas/genética , Plantas/genética , Complexos Ubiquitina-Proteína Ligase/genética , Sequência de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase , Arabidopsis/genética , Sequência de Bases , Clorófitas/enzimologia , Clorófitas/genética , Mapeamento Cromossômico , Cromossomos de Plantas/genética , Regulação da Expressão Gênica de Plantas , Variação Genética , Genoma de Planta/genética , Dados de Sequência Molecular , Oryza/genética , Filogenia , Proteínas de Plantas/classificação , Plantas/enzimologia , Populus/genética , Subunidades Proteicas/classificação , Subunidades Proteicas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rodófitas/enzimologia , Rodófitas/genética , Especificidade da Espécie , Sintenia , Complexos Ubiquitina-Proteína Ligase/classificaçãoRESUMO
We investigated the wound response of the commercially important red alga, Gracilaria chilensis, in order to obtain insight into its interaction with epiphytic pests. After wounding, the host releases free fatty acids as well as the hydroxylated eicosanoids, 8R-hydroxy eicosatetraenoic acid (8-HETE) and 7S,8R-dihydroxy eicosatetraenoic acid (7,8-di-HETE). While the release of free arachidonic acid and subsequent formation of 8-HETE is controlled by phospholipase A, 7,8-di-HETE production is independent of this lipase. This dihydroxylated fatty acid might be directly released from galactolipids. Physiologically relevant concentrations of oxylipins reduced spore settlement of Acrochaetium sp. (Rhodophyta, Acrochaetiaceae) and suppressed the development of hapteria in Ceramium rubrum (Rhodophyta, Ceramiaceae) when these model epiphytes were exposed to artificial surfaces that contained 8-HETE or 7,8-di-HETE. Thus, the immediate release of oxylipins can be seen as G. chilensis defence against epiphytes.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Fosfolipases/metabolismo , Rodófitas/fisiologia , Cromatografia em Camada Fina , Eicosanoides/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Espectroscopia de Ressonância Magnética , Compostos Orgânicos/metabolismo , Rodófitas/enzimologiaRESUMO
Algal cells have developed different strategies to cope with the common environmentally promoted generation of H(2)O(2), which include induction of catalase (CAT) and ascorbate peroxidase (APX), massive H(2)O(2) release in seawater, and synthesis of volatile halocarbons by specific peroxidases. The antioxidant adaptability of the economically important carrageenophyte Kappaphycus alvarezii (Doty) Doty (Gigartinales: Rhodophyta) was tested here against exposure to clofibrate (CFB), a known promoter of peroxisomal beta-oxidation in mammals and plants. Possibly as a consequence of CFB-induced H2O2 peroxisomal production, the maximum concentration of H(2)O(2) in the seawater of red algae cultures was found to occur (120+/-17 min) after the addition of CFB, which was followed by a significant decrease in the photosynthetic activity of PSII after 24 h. Interestingly, 4 h after the addition of CFB, the total SOD activity was about 2.5-fold higher than in the control, whereas no significant changes were observed in lipoperoxidation levels (TBARS) or in CAT and APX activities. The two H(2)O(2)-scavenging enzymes were only induced later (after 72 h), whereupon CAT showed a dose-dependent response with increasing concentrations of CFB. A more pronounced increase of TBARS concentration than in the controls was evidenced when a 50 microM Fe(2+/3+) solution (3:2 ratio) was added to CFB-treated cultures, suggesting that the combination of exacerbated H(2)O(2) levels in the seawater-in this work, caused by CFB exposure-and Fenton-reaction catalyst (ferric/ferrous ions), imposes harsh oxidative conditions on algal cultures. The bulk of data suggests that K. alvarezii possesses little ability to promptly induce CAT and APX compared to the immediately responsive antioxidant enzyme SOD and, to avoid harmful accumulation of H(2)O(2), the red alga presumably releases H(2)O(2) into the surrounding medium as an alternative mechanism.
Assuntos
Clofibrato/farmacologia , Peróxido de Hidrogênio/metabolismo , Peroxidases/biossíntese , Rodófitas/efeitos dos fármacos , Rodófitas/enzimologia , Água do Mar/análise , Superóxido Dismutase/biossíntese , Adaptação Fisiológica/fisiologia , Ascorbato Peroxidases , Células Cultivadas , Relação Dose-Resposta a Droga , Ativação Enzimática , Homeostase/fisiologia , Peróxido de Hidrogênio/análise , Ferro/farmacologia , Rodófitas/metabolismo , Estatística como Assunto , Fatores de TempoRESUMO
A daily rhythm in the activity of nitrate reductase (NR: EC 1.6.6.1) isolated from the marine red algae Gracilaria tenuistipitata is shown to be attributable to changes in amounts of the protein. The enzyme was purified in four steps: ion exchange Q-Sepharose separation, ammonium sulfate precipitation, gel filtration on Sephacryl S-300, and affinity chromatography on Affigel-blue resin. This purification procedure yielded an active purified NR of about 500-fold with a recovery of 85%. The SDS-PAGE silver staining of purified NR revealed a 110 kDa single band. Non-denaturated protein showed a molecular mass of 440 kDa on gel filtration comparing with SDS-PAGE, the enzyme is apparently composed of four identical subunits. In extracts of algae grown under either constant dim light or a light-dark cycle, the activity of NR exhibited a daily rhythm, peaking at midday phase as does photosynthesis. Staining with monoclonal antibodies, raised against NR from Porphyra yezoensis, showed that the amount of protein changes by a factor of about 12, with a maximum occurring in the midday phase.
Assuntos
Nitrato Redutases/metabolismo , Rodófitas/enzimologia , Anticorpos Monoclonais/farmacologia , Ritmo Circadiano , Inibidores Enzimáticos/farmacologia , Cinética , Nitrato Redutase , Nitrato Redutases/imunologia , Nitrato Redutases/isolamento & purificaçãoRESUMO
Liagora farinosa is a red alga found in tropical around the world. The topical antiedematous activity of its organic extract was studied by means of two experimental models: the in vivo mouse ear edema and the in vitro inhibition of bee venom-derived PLA2. The results showed that the polar and apolar fractions of L. farinosa extract were able to inhibir respectively 52.53 and 63.61 percent of the ear edema induced by croton oil. In vitro tests showed thath both fractions also inhibited the activity of PLA2, indicating that the possible machanism of action for the topical antiedematous activity is through the inactivation of this enzyme that releases arachidonic acid during the beginning of the infalmatory mediator cascade.
Assuntos
Animais , Camundongos , Enzimas/metabolismo , Técnicas In Vitro , Rodófitas/enzimologia , Venenos de Abelha/enzimologia , Otopatias/induzido quimicamente , Edema/induzido quimicamente , Óleo de Cróton/efeitos adversosRESUMO
We present here the first report of a group of alpha-1,4-glucan lyases (EC 4.2.2.13) and their genes. The lyases produce 1, 5-anhydro-D-fructose from starch and related oligomers and polymers. The enzymes were isolated from the red alga Gracilariopsis lemaneiformis from the Pacific coasts of China and USA, and the Atlantic Coast of Venezuela. Three lyase isozymes (GLq1, GLq2 and GLq3) from the Chinese subspecies, two lyase isozymes (GLs1 and GLs2) from the USA subspecies and one lyase (GLa1) from the Venezuelan subspecies were identified and investigated. GLq1, GLq3, GLs1 and GLa1 were purified and partially sequenced. Based on the amino acid sequences obtained, three lyase genes or their cDNAs (GLq1, GLq2 and GLs1) were cloned and completely sequenced and two other genes (GLq3 and GLs2) were partially sequenced. The coding sequences of the lyase genes GLq1, GLq2 and GLs1 are 3267, 3276 and 3279 bp, encoding lyases of 1088, 1091 and 1092 amino acids, respectively. The deduced molecular masses of the mature lyases from the coding sequences are 117030, 117667 and 117790 Da, respectively, close to those determined by mass spectrometry using purified lyases. The amino acid sequence identity is more than 70% among the six algal lyase isozymes. The algal GLq1 gene was expressed in Pichia pastoris and Aspergillus niger, and the expression product was identical to the wild-type enzyme.