Caracterização parcial e atividades farmacológicas do extrato rico em polissacarídeos sulfatatos da angiosperma marinha Halodule wrightii

Sulfated polysaccharides (PS) are biomolecules with a great biotechnological potential. There are few data about PS from high plants. In addition, pharmacological activities of PS from plants have not been carrying out. The aim of this work was extract PS from the angiosperm Halodule wrightii and study their anticoagulant and antioxidant activities. Histological analysis showed the presence of the PS manly in the roots. A polysaccharide-rich extract was obtained from H. wrightii by proteolysis followed by methanol and TCA precipitation. Chemical, infra-red analysis and agarose gel electrophoresis in 1.3 diaminopropane acetate buffer confirmed the presence of sulfated polysaccharides made by glucose, galactose, xylose and sulfate residues in the proportion 1: 0,9: 1: 1. In addition polyacrilamide electrophoresis have shown that extract is mainly compose by 11kDa sulfated polysaccharides. Pharmacological analysis have shown total antioxidant capacity (CAT) that resulted in 15,21 μg for equivalent of ascorbic acid, scavenging activity of the DPPH radical with 41,36 % of scavenging, activity of reducing power with the maximum of 0,290 nm (50 % of vitamin C activity) and scavenging activity superoxide radical (O2-) with a maximum of 32,23 %. Chelating activity of metal less than 4% and scavenging activity of the radical hydroxyl (OH-) less than 2%. Time of activated partial tromboplastin (aPTT) doubling the time of coagulation from 20μg of and protrombin time (PT) was not present. The data indicate that PS from Halodule wrightii could be considered for future applications in medicine, food production or cosmetic industry

Caracterização parcial e atividades farmacológicas do extrato rico em polissacarídeos sulfatados da angiosperma marinha Halodule wrightii

Sulfated polysaccharides (PS) are biomolecules with a great biotechnological potential. There are few data about PS from high plants. In addition, pharmacological activities of PS from plants have not been carrying out. The aim of this work was extract PS from the angiosperm Halodule wrightii and study their anticoagulant and antioxidant activities. Histological analysis showed the presence of the PS manly in the roots. A polysaccharide-rich extract was obtained from H. wrightii by proteolysis followed by methanol and TCA precipitation. Chemical, infra-red analysis and agarose gel electrophoresis in 1.3 diaminopropane acetate buffer confirmed the presence of sulfated polysaccharides made by glucose, galactose, xylose and sulfate residues in the proportion 1: 0,9: 1: 1. In addition polyacrilamide electrophoresis have shown that extract is mainly compose by 11kDa sulfated polysaccharides. Pharmacological analysis have shown total antioxidant capacity (CAT) that resulted in 15,21 μg for equivalent of ascorbic acid, scavenging activity of the DPPH radical with 41,36 % of scavenging, activity of reducing power with the maximum of 0,290 nm (50 % of vitamin C activity) and scavenging activity superoxide radical (O2-) with a maximum of 32,23 %. Chelating activity of metal less than 4% and scavenging activity of the radical hydroxyl (OH-) less than 2%. Time of activated partial tromboplastin (aPTT) doubling the time of coagulation from 20μg of and protrombin time (PT) was not present. The data indicate that PS from Halodule wrightii could be considered for future applications in medicine, food production or cosmetic industry

Atividades Biológicas De Xilana De Sabugo De Milho

The corn cob is an agricultural by-product still little used, this in part due to the low knowledge of the biotechnological potential of their molecules. Xylan from corn cobs (XSM) is a polysaccharide present in greater quantity in the structure of plant and its biotechnology potential is little known. This study aimed to the extraction, chemical characterization and evaluation of biological activities of xylan from corn cobs. To this end, corncobs were cleaned, cut, dried and crushed, resulting in flour. This was subjected to a methodology that combines the use of alkaline conditions with waves of ultrasound. After methanol precipitation, centrifugation and drying was obtained a yield of 40% (g/g flour). Chemical analysis indicated a high percentage of polysaccharides in the sample (60%) and low contamination by protein (0.4%) and phenolic compounds (> 0.01%). Analysis of monosaccharide composition indicated the presence of xylose:glucose:arabinose:galactose:mannose:glucuronic acid in a molar ratio 50:20:15:10:2.5:2.5. The presence of xylan in the sample was confirmed by nuclear magnetic resonance (¹H and ¹³C) and infrared spectroscopy (IR). Tests were conducted to evaluate the antioxidant potential of XSM. This showed a total antioxidant capacity of 48.45 EAA/g sample. However, did not show scavenging activity of superoxide and hydroxyl radical and also reducing power. But, showing a high capacity chelating iron ions with 70% with about 2 mg/mL. The ability to XSM to influence cell proliferation in culture was also evaluated. This polymer did not influence the proliferation of normal fibroblast cells (3T3), however, decreased the rate of proliferation of tumor cells (HeLa) in a dose-dependent, reaching an inhibition of about 50% with a concentration around 2 mg/mL. Analyzing proteins related to cell death, by immunoblotting, XSM increases the amount of Bax, Bcl-2 decrease, increase cytochrome c and AIF, and reduce pro-caspase-3, indicating the induction of cell death induced apoptosis dependent and independent of caspase. XSM did not show anticoagulant activity in the PT test. However, the test of activated partial thromboplastin time (aPTT), XSM increased clotting time at about 5 times with 600 μg of sample compared with the negative control. The presence of sulfate on the XSM was discarded by agarose gel electrophoresis and IR. After carboxyl-reduction of XSM the anticoagulant activity decreased dramatically. The data of this study demonstrate that XSM has potential as antioxidant, antiproliferative and anticoagulant compound. Future studies to characterize these activities of XSM will help to increase knowledge about this molecule extracted from corn and allow their use in functional foods, pharmaceuticals and chemical industries.

Análise proteômica de Chromobacterium violaceum: acúmulo estacionário e diferencial após exposição à luz UVC

Chromobacterium violaceum is a free-living bacillus, Gram-negative commonly found in water and sand of tropical and subtropical regions. One of its main characteristic it's the ability to produce the purple pigment named violacein, that shows countless biological activities. In 2003, the genome of this organism was totally sequenced and revealed important informations about the physiology of this bacteria. However, few post-genomics studies had been accomplished. This work evaluated the protein profile of C. violaceum cultivated in LB medium at 28ºC that allowed the identification and characterization of proteins related to a possible secretion system that wasn't identified and characterized yet in C. violaceum, to the quorum sensing system, to regulatory process of transcription and translation, stress adaptation and biotechnological potential. Moreover, the response of the bacteria to UVC radiation was evaluated. The comparison of the protein profile, analyzed through 2-D electrophoresis, of the control group versus the treatment group allowed the identification of 52 proteins that arose after stress induction. The obtained results enable the elaboration of a stress response pathway in C. violaceum generated by the UVC light. This pathway, that seems to be a general stress response, involves the expression of proteins related to cellular division, purine and pirimidine metabolism, heat chock or chaperones, energy supply, regulation of biofilm formation, transport, regulation of lytic cycle of bacteriophages, besides proteins that show undefined function. Despite the response present similarities with the classic SOS response of E. coli, we still cannot assert that C. violaceum shows a SOS-like response, mainly due to the absence of characterization of a LexA-like protein in this organism

Proteômica diferencial da Chromobacterium violaceum em resposta ao estresse oxidativo induzido por peróxido de hidrogênio

The β-proteobacterium Chromobacterium violaceum is a Gram-negative, free-living, saprophytic and opportunistic pathogen that inhabits tropical and subtropical ecosystems among them, in soil and water of the Amazon. It has great biotechnological potential, and because of this potential, its genome was completely sequenced in 2003. Genome analysis showed that this bacterium has several genes with functions related to the ability to survive under different kinds of environmental stresses. In order to understand the physiological response of C. violaceum under oxidative stress, we applied the tool of shotgun proteomics. Thus, colonies of C. violaceum ATCC 12472 were grown in the presence and absence of 8 mM H2O2 for two hours, total proteins were extracted from bacteria, subjected to SDS-PAGE, stained and hydrolysed. The tryptic peptides generated were subjected to a linear-liquid chromatography (LC) followed by mass spectrometer (LTQ-XL-Orbitrap) to obtain quantitative and qualitative data. A shotgun proteomics allows to compare directly in complex samples, differential expression of proteins and found that in C. Violaceum, 131 proteins are expressed exclusively in the control condition, 177 proteins began to be expressed under oxidative stress and 1175 proteins have expression in both conditions. The results showed that, under the condition of oxidative stress, this bacterium changes its metabolism by increasing the expression of proteins capable of combating oxidative stress and decreasing the expression of proteins related processes bacterial growth and catabolism (transcription, translation, carbon metabolism and fatty acids). A tool with of proteomics as an approach of integrative biology provided an overview of the metabolic pathways involved in the response of C. violaceum to oxidative stress, as well as significantly amplified understanding physiological response to environmental stress. Biochemical and "in silico" assays with the hypothetical ORF CV_0868 found that this is part of an operon. Phylogenetic analysis of superoxide dismutase, protein belonging to the operon also showed that the gene is duplicated in genome of C. violaceum and the second copy was acquired through a horizontal transfer event. Possibly, not only the SOD gene but also all genes comprising this operon were obtained in the same manner. It was concluded that C. violaceum has complex, efficient and versatile mechanisms in oxidative stress response

Metagenômica: busca de novos genes envolvidos com a biodegradação de hidrocarbonetos

Industrial activities, oil spills and its derivatives, as well as the incomplete combustion of fossil fuels have caused a great accumulation of hydrocarbons in the environment. The number of microorganisms on the planet is estimated at 1030 and prokaryotes the most abundant. They colonized diverse environments for thousands of years, including those considered extreme and represent an untapped source of metabolic and genetic diversity with a large biotechnological potential. It is also known that certain microorganisms have the enzymatic capacity to degrade petroleum hydrocarbons and, in many ecosystems, there is an indigenous community capable of performing this function. The metagenomic has revolutionized the microbiology allowing access uncultured microbial communities, being a powerful tool for elucidation of their ecological functions and metabolic profiles, as well as for identification of new biomolecules. Thus, this study applied metagenomic approaches not only for functional selection of genes involved in biodegradation and emulsification processes of the petroleum-derived hydrocarbons, but also to describe the taxonomic and metabolic composition of two metagenomes from aquatic microbiome. We analyzed 123.116 (365 ± 118 bp) and 127.563 sequences (352 ± 120 bp) of marine and estuarine metagenomes, respectively. Eight clones were found, four involved in the petroleum biodegradation and four were able to emulsify kerosene indicating their abilities in biosurfactants synthesis. Therefore, the metagenomic analyses performed were efficient not only in the search of bioproducts of biotechnological interest and in the analysis of the functional and taxonomic profile of the metagenomes studied as well

Prospecção de genes de interesse biotecnológico : uma abordagem metagenômica

The total number of prokaryotic cells on Earth has been estimated at 4 to 6x1030 and only about 1% of microorganisms present in the environment can be cultivated by standard techniques of cultivation and plating. Therefore, it is a huge biological and genetic pool that can be exploited, for the identification and characterization of genes with biotechnological potential. Within this perspective, the metagenomics approach was applied in this work. Functional screening methods were performed aiming to identify new genes related to DNA repair and / or oxidative stress resistance, hydrocarbon degradation and hydrolytic activities (lipase, amylase and protease). Metagenomic libraries were built utilizing DNA extracted from soil samples collected in João Câmara RN. The libraries were analyzed functionally using specific substrate containing solid medium (hydrolytic activity), supplemented with H2O2 (DNA repair and / or resistance to oxidative stress) and liquid medium supplemented with light Arabian oil (activity, degradation of hydrocarbons). After confirmation of activity and exclusion of false-positive results, 49 clones were obtained, being 2 positive for amylase activity, 22 resistant to oxidative stress generated by H2O2 and 25 clones active for hydrocarbons degradation. Analysis of the sequences showed hypothetical proteins, dienelactona hydrolase, DNA polymerase, acetyltransferase, phosphotransferase, methyltransferase, endonucleases, among other proteins. The sequence data obtained matched with the functions tested, highlighting the success of metagenomics approaches combined with functional screening methods, leading to very promising results

Caracterização parcial e atividades farmacológicas do extrato rico em polissacarídeos sulfatatos da angiosperma marinha Halodule wrightii

Sulfated polysaccharides (PS) are biomolecules with a great biotechnological potential. There are few data about PS from high plants. In addition, pharmacological activities of PS from plants have not been carrying out. The aim of this work was extract PS from the angiosperm Halodule wrightii and study their anticoagulant and antioxidant activities. Histological analysis showed the presence of the PS manly in the roots. A polysaccharide-rich extract was obtained from H. wrightii by proteolysis followed by methanol and TCA precipitation. Chemical, infra-red analysis and agarose gel electrophoresis in 1.3 diaminopropane acetate buffer confirmed the presence of sulfated polysaccharides made by glucose, galactose, xylose and sulfate residues in the proportion 1: 0,9: 1: 1. In addition polyacrilamide electrophoresis have shown that extract is mainly compose by 11kDa sulfated polysaccharides. Pharmacological analysis have shown total antioxidant capacity (CAT) that resulted in 15,21 μg for equivalent of ascorbic acid, scavenging activity of the DPPH radical with 41,36 % of scavenging, activity of reducing power with the maximum of 0,290 nm (50 % of vitamin C activity) and scavenging activity superoxide radical (O2-) with a maximum of 32,23 %. Chelating activity of metal less than 4% and scavenging activity of the radical hydroxyl (OH-) less than 2%. Time of activated partial tromboplastin (aPTT) doubling the time of coagulation from 20μg of and protrombin time (PT) was not present. The data indicate that PS from Halodule wrightii could be considered for future applications in medicine, food production or cosmetic industry

Caracterização parcial e atividades farmacológicas do extrato rico em polissacarídeos sulfatados da angiosperma marinha Halodule wrightii

Sulfated polysaccharides (PS) are biomolecules with a great biotechnological potential. There are few data about PS from high plants. In addition, pharmacological activities of PS from plants have not been carrying out. The aim of this work was extract PS from the angiosperm Halodule wrightii and study their anticoagulant and antioxidant activities. Histological analysis showed the presence of the PS manly in the roots. A polysaccharide-rich extract was obtained from H. wrightii by proteolysis followed by methanol and TCA precipitation. Chemical, infra-red analysis and agarose gel electrophoresis in 1.3 diaminopropane acetate buffer confirmed the presence of sulfated polysaccharides made by glucose, galactose, xylose and sulfate residues in the proportion 1: 0,9: 1: 1. In addition polyacrilamide electrophoresis have shown that extract is mainly compose by 11kDa sulfated polysaccharides. Pharmacological analysis have shown total antioxidant capacity (CAT) that resulted in 15,21 μg for equivalent of ascorbic acid, scavenging activity of the DPPH radical with 41,36 % of scavenging, activity of reducing power with the maximum of 0,290 nm (50 % of vitamin C activity) and scavenging activity superoxide radical (O2-) with a maximum of 32,23 %. Chelating activity of metal less than 4% and scavenging activity of the radical hydroxyl (OH-) less than 2%. Time of activated partial tromboplastin (aPTT) doubling the time of coagulation from 20μg of and protrombin time (PT) was not present. The data indicate that PS from Halodule wrightii could be considered for future applications in medicine, food production or cosmetic industry

Atividades biológicas de xilana de sabugo de milho

The corn cob is an agricultural by-product still little used, this in part due to the low knowledge of the biotechnological potential of their molecules. Xylan from corn cobs (XSM) is a polysaccharide present in greater quantity in the structure of plant and its biotechnology potential is little known. This study aimed to the extraction, chemical characterization and evaluation of biological activities of xylan from corn cobs. To this end, corncobs were cleaned, cut, dried and crushed, resulting in flour. This was subjected to a methodology that combines the use of alkaline conditions with waves of ultrasound. After methanol precipitation, centrifugation and drying was obtained a yield of 40% (g/g flour). Chemical analysis indicated a high percentage of polysaccharides in the sample (60%) and low contamination by protein (0.4%) and phenolic compounds (> 0.01%). Analysis of monosaccharide composition indicated the presence of xylose:glucose:arabinose:galactose:mannose:glucuronic acid in a molar ratio 50:20:15:10:2.5:2.5. The presence of xylan in the sample was confirmed by nuclear magnetic resonance (¹H and ¹³C) and infrared spectroscopy (IR). Tests were conducted to evaluate the antioxidant potential of XSM. This showed a total antioxidant capacity of 48.45 EAA/g sample. However, did not show scavenging activity of superoxide and hydroxyl radical and also reducing power. But, showing a high capacity chelating iron ions with 70% with about 2 mg/mL. The ability to XSM to influence cell proliferation in culture was also evaluated. This polymer did not influence the proliferation of normal fibroblast cells (3T3), however, decreased the rate of proliferation of tumor cells (HeLa) in a dose-dependent, reaching an inhibition of about 50% with a concentration around 2 mg/mL. Analyzing proteins related to cell death, by immunoblotting, XSM increases the amount of Bax, Bcl-2 decrease, increase cytochrome c and AIF, and reduce pro-caspase-3, indicating the induction of cell death induced apoptosis dependent and independent of caspase. XSM did not show anticoagulant activity in the PT test. However, the test of activated partial thromboplastin time (aPTT), XSM increased clotting time at about 5 times with 600 μg of sample compared with the negative control. The presence of sulfate on the XSM was discarded by agarose gel electrophoresis and IR. After carboxyl-reduction of XSM the anticoagulant activity decreased dramatically. The data of this study demonstrate that XSM has potential as antioxidant, antiproliferative and anticoagulant compound. Future studies to characterize these activities of XSM will help to increase knowledge about this molecule extracted from corn and allow their use in functional foods, pharmaceuticals and chemical industries.

Análise proteômica de Chromobacterium violaceum: acúmulo estacionário e diferencial após exposição à luz UVC

Chromobacterium violaceum is a free-living bacillus, Gram-negative commonly found in water and sand of tropical and subtropical regions. One of its main characteristic it's the ability to produce the purple pigment named violacein, that shows countless biological activities. In 2003, the genome of this organism was totally sequenced and revealed important informations about the physiology of this bacteria. However, few post-genomics studies had been accomplished. This work evaluated the protein profile of C. violaceum cultivated in LB medium at 28ºC that allowed the identification and characterization of proteins related to a possible secretion system that wasn't identified and characterized yet in C. violaceum, to the quorum sensing system, to regulatory process of transcription and translation, stress adaptation and biotechnological potential. Moreover, the response of the bacteria to UVC radiation was evaluated. The comparison of the protein profile, analyzed through 2-D electrophoresis, of the control group versus the treatment group allowed the identification of 52 proteins that arose after stress induction. The obtained results enable the elaboration of a stress response pathway in C. violaceum generated by the UVC light. This pathway, that seems to be a general stress response, involves the expression of proteins related to cellular division, purine and pirimidine metabolism, heat chock or chaperones, energy supply, regulation of biofilm formation, transport, regulation of lytic cycle of bacteriophages, besides proteins that show undefined function. Despite the response present similarities with the classic SOS response of E. coli, we still cannot assert that C. violaceum shows a SOS-like response, mainly due to the absence of characterization of a LexA-like protein in this organism

Proteômica diferencial da Chromobacterium violaceum em resposta ao estresse oxidativo induzido por peróxido de hidrogênio

The β-proteobacterium Chromobacterium violaceum is a Gram-negative, free-living, saprophytic and opportunistic pathogen that inhabits tropical and subtropical ecosystems among them, in soil and water of the Amazon. It has great biotechnological potential, and because of this potential, its genome was completely sequenced in 2003. Genome analysis showed that this bacterium has several genes with functions related to the ability to survive under different kinds of environmental stresses. In order to understand the physiological response of C. violaceum under oxidative stress, we applied the tool of shotgun proteomics. Thus, colonies of C. violaceum ATCC 12472 were grown in the presence and absence of 8 mM H2O2 for two hours, total proteins were extracted from bacteria, subjected to SDS-PAGE, stained and hydrolysed. The tryptic peptides generated were subjected to a linear-liquid chromatography (LC) followed by mass spectrometer (LTQ-XL-Orbitrap) to obtain quantitative and qualitative data. A shotgun proteomics allows to compare directly in complex samples, differential expression of proteins and found that in C. Violaceum, 131 proteins are expressed exclusively in the control condition, 177 proteins began to be expressed under oxidative stress and 1175 proteins have expression in both conditions. The results showed that, under the condition of oxidative stress, this bacterium changes its metabolism by increasing the expression of proteins capable of combating oxidative stress and decreasing the expression of proteins related processes bacterial growth and catabolism (transcription, translation, carbon metabolism and fatty acids). A tool with of proteomics as an approach of integrative biology provided an overview of the metabolic pathways involved in the response of C. violaceum to oxidative stress, as well as significantly amplified understanding physiological response to environmental stress. Biochemical and "in silico" assays with the hypothetical ORF CV_0868 found that this is part of an operon. Phylogenetic analysis of superoxide dismutase, protein belonging to the operon also showed that the gene is duplicated in genome of C. violaceum and the second copy was acquired through a horizontal transfer event. Possibly, not only the SOD gene but also all genes comprising this operon were obtained in the same manner. It was concluded that C. violaceum has complex, efficient and versatile mechanisms in oxidative stress response