RESUMO
The purpose of this study was to determine the origin, presence, and fate of the endocrine disruptor di-ethylhexil phthalate (DEHP) during tequila production. For this, three tequila factories (small, medium, and large) were monitored. DEHP concentrations in water, agave, additives, lubricating greases, neoprene seals, and materials of each stage process were analyzed using gas chromatography/mass spectrometry. DEHP mass balances were performed to identify the processes with significant changes in the inputs/outputs. DEHP was detected in agave at up to 0.08 ± 0.03 mg kg-1, water 0.02 ± 0.01 mg kg-1, lubricant greases 131.05 ± 2.80 mg kg-1, and neoprene seals 369.11 ± 22.52 mg kg-1. Whereas, tequila produced in the large, medium, and small factories contained 0.05 ± 0.01, 0.24 ± 0.04, and 1.43 ± 0.48 mg kg-1 DEHP, respectively. Furthermore, in waste materials (vinasses and bagasse) released, 534.26 ± 349.02, 947.18 ± 65.84, and 5222.60 ± 2836.94 mg of DEHP was detected for every 1000 L of tequila produced. The most significant increase in DEHP occurred during the sugar extraction and distillation stages. Results demonstrate that main raw materials, such as agave and water, contain DEHP, but lubricant greases and neoprene seals are the major sources of DEHP contamination. Identification of the contamination sources can help the tequila industry to take actions to reduce it, protect consumer health and the environment, and prevent circular contamination.
RESUMO
Amaranth has been recognized as a nutraceutical food because it contains high-quality proteins due to its adequate amino acid composition that covers the recommended requirements for children and adults. Since pre-Hispanic times, amaranth has been consumed as popped grain; the popping process improves its nutritive quality and improves its digestibility. Popped amaranth consumption has been associated with the recovery of malnourished children. However, there is no information on the impact that popped amaranth consumption has on gut microbiota composition. A non-randomized pilot trial was conducted to evaluate the changes in composition, structure, and function of the gut microbiota of stunted children who received four grams of popped amaranth daily for three months. Stool and serum were collected at the beginning and at the end of the trial. Short-chain fatty acids (SCFA) were quantified, and gut bacterial composition was analyzed by 16S rRNA gene sequencing. Biometry and hematology results showed that children had no pathology other than low height-for-age. A decrease in the relative abundance of Alistipes putredinis, Bacteroides coprocola, and Bacteroides stercoris bacteria related to inflammation and colitis, and an increase in the relative abundance of Akkermansia muciniphila and Streptococcus thermophiles bacteria associated with health and longevity, was observed. The results demonstrate that popped amaranth is a nutritious food that helps to combat childhood malnutrition through gut microbiota modulation.
RESUMO
OBJECTIVE: The aim of this work was to characterize Lippia graveolens oleoresins, obtained by Supercritical Fluid Extraction (SFE), from crops collected at different locations in Mexico. The antimicrobial effect of oleoresins was tested in reference strains and clinical isolates of susceptible and multidrug-resistant (MDR) strains of Enterococcus faecalis and Staphylococcus aureus. SIGNIFICANCE: The increasing of MDR strains is becoming a global public health problem that has led to the search for new treatments, and essential oils have resurged as a source of compounds with bactericidal functions. Oregano essential oil has attracted attention recently, however, this oil is mainly obtained by hydro-distillation (uses large amounts of water) or solvents extraction (potential contaminant). SFE has gained popularity as it represents an environmentally friendly technology. METHODS: L. graveolens oleoresins were obtained by SFE, total phenol contents were quantified by Folin-Ciocalteu method, the identification of compounds and thymol and carvacrol quantification was carried out by GC-MS. The antimicrobial activity was tested by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). RESULTS: SFE showed higher yields compared with the hydro-distillation process. L. graveolens grown in different Mexican locations showed differences in oleoresin composition and a slightly different antimicrobial capacity against clinical isolates. CONCLUSIONS: It was demonstrated that SFE is an efficient technology for extracting L. graveolens oleoresins. Additionally, the solvent-free extraction method and the observed antimicrobial effect increase the applications of these oleoresins in fields, such as cosmetics, food industry, medicine, amongst others.
Assuntos
Anti-Infecciosos , Lippia , Óleos Voláteis , Antibacterianos/farmacologia , Anti-Infecciosos/farmacologia , Resistência a Múltiplos Medicamentos , Enterococcus faecalis , Testes de Sensibilidade Microbiana , Óleos Voláteis/farmacologia , Extratos Vegetais , Staphylococcus aureusRESUMO
The goal of this work was the autodisplay of the endo ß-1,4-xylanase (XynA) from Clostridium cellulovorans in Escherichia coli using the AIDA system to carry out whole-cell biocatalysis and hydrolysate xylans. For this, pAIDA-xynA vector containing a synthetic xynA gene was fused to the signal peptide of the toxin subunit B Vibro cholere (ctxB) and the auto-transporter of the synthetic aida gene, which encodes for the connector peptide and ß-barrel of the auto-transporter (AT-AIDA). E. coli TOP10 cells were transformed and the biocatalyst was characterized using beechwood xylans as substrate. Optimal operational conditions were temperature of 55 °C and pH 6.5, and the Michaelis-Menten catalytic constants Vmax and Km were 149 U/gDCW and 6.01 mg/mL, respectively. Xylanase activity was inhibited by Cu2+, Zn2+ and Hg2+ as well as EDTA, detergents, and organic acids, and improved by Ca2+, Co2+ and Mn2+ ions. Ca2+ ion strongly enhanced the xylanolytic activity up to 2.4-fold when 5 mM CaCl2 were added. Also, Ca2+ improved enzyme stability at 60 and 70 °C. Results suggest that pAIDA-xynA vector has the ability to express functional xylanase to perform whole-cell biocatalysis in order to hydrolysate xylans from hemicellulose feedstock.
Assuntos
Clostridium cellulovorans , Xilanos , Clostridium cellulovorans/metabolismo , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , TemperaturaRESUMO
Liometopum apiculatum is a species of ants widely distributed in arid and semi-arid ecosystems where there is a relative food shortage compared with tropical ecosystems. L. apiculatum has established an ecological balance involving symbiotic interactions, which have allowed them to survive through mechanisms that are still unknown. Therefore, the aim of this study was to explore the metabolic potential of isolated bacteria from L. apiculatum using enzymatic activity assay and substrate assimilation. Results revealed a complex bacteria consortium belonging to Proteobacteria, Firmicutes, and Actinobacteria phylum. Most of the isolated bacteria showed activities associated with biopolymers degradation, from them Exiguobacterium and B. simplex showed the highest amylolytic activity (27 U/mg protein), while A. johnsonii and B. pumulis showed the highest cellulolytic and xylanolytic activities (1 and 2.9 U/mg protein, respectively). By other hand, some microorganisms such as S. ficaria, E. asburiae, P. agglomerans, A. johnsonii, S. rubidaea, S. marcescens, S. warneri, and M. hydrocarbonoxydans were able to grow up to 1000 mg/L of phthalates esters. These results not only revealed the important contribution of the symbionts in L apiculatum ants feeding habits, but also have shown a promising source of enzymes with potential biotechnological applications such as lignocellulosic biomass hydrolysis and bioremediation processes.
Assuntos
Formigas/microbiologia , Bactérias/isolamento & purificação , Bactérias/metabolismo , Biodegradação Ambiental , Microbiota/fisiologia , Animais , Bactérias/classificação , Bactérias/enzimologia , Biomassa , Celulose/metabolismo , Hábitos , Hidrólise , Larva/microbiologia , Lignina/metabolismo , Polissacarídeos/metabolismo , Simbiose , Xilanos/metabolismoRESUMO
The presence of diethyl-phthalate (DEP), dibutyl-phthalate (DBP), butylbenzyl-phthalate (BBP), diethylhexyl-phthalate (DEHP) and diisononyl-phthalate (DINP) was determined in 295 tequila samples. They were grouped by age of maturation (white, aged, extra aged or ultra aged) and year of production (between 2013 and 2018). Gas Chromatography coupled with Mass Spectrometry was used for identification and quantification. The results showed that 65 samples (22% of the total) were phthalate free. DEP (0.13-0.27 mg/kg), BBP (0.05-2.91 mg/kg) and DINP (1.64-3.43 mg/kg) were detected in 11 (3.73%), 37 (12.54%) and 5 (1.69%) samples, respectively. But, these concentrations did not exceed the maximum permitted limits (MPL) of phthalates for alcoholic beverages. DBP (0.01-2.20 mg/kg) and DEHP (0.03-4.64 mg/kg) were detected in 96 (32.54%) and 224 (75.93%) samples, from them only 10 (3.39%) and 15 (5.08%) samples, respectively, exceeded the MPL for alcoholic beverages and they were few tequilas produced in the year 2014 or before. DEHP was the most frequent phthalate found in tequila and observed DEHP concentrations were 2-times higher in ultra aged tequilas compared to those in white tequilas. We concluded that all tequilas produced in 2015 and after, satisfied the international standards for these compounds.
Assuntos
Bebidas Alcoólicas/análise , Contaminação de Alimentos/análise , Ácidos Ftálicos/análise , Dibutilftalato/análise , Dietilexilftalato/análise , Análise de Alimentos , Cromatografia Gasosa-Espectrometria de Massas/métodos , México , Fatores de TempoRESUMO
Phthalates are esters of phthalic acid used industrially as plastic additives, however, these are not covalently bound to the polymer matrix and therefore can be released to the environment. The aim of this study was to evaluate the effect of four phthalates: dibutyl phthalate (DBP), benzyl butyl phthalate (BBP), diethyl phthalate (DEP) and diethylhexyl phthalate (DEHP) on the in vitro expansion of human hematopoietic cells from umbilical cord blood. For this, 0.5 × 106 cells/mL were exposure to concentrations ranging from 0.1 to 100 µg/mL and the total cell expansion was determined after 14 days of culture in IMDM-cytokines medium. The control cultures attained 1.31 ± 0.21 × 106 cell/mL, whereas the cultures exposed to DBP, BBP and DEHP showed a reduction from 23 to 81%, 17 to 69% and 15 to 93.5%, respectively. DEP did not affect the total cell expansion. The most significant decrease on total cell expansion was observed at 0.1 µg/mL DBP, 100 µg/mL BBP and 10 µg/mL DEHP (p < 0.05). Additionally, the effect of these compounds on the expansion of hematopoietic progenitors was analyzed by clonogenic assays as colony forming units (CFU). The CFU decreased considerably compared with respect to the control cultures. The reduction was 74.6 and 99.1% at 10 and 100 µg/mL DBP respectively, whereas 100 µg/mL BBP and 100 µg/mL DEHP reduced the CFU expansion in 97.1% and 81%, respectively. Cultures exposed to DEP did not show significant differences. The results demonstrate the toxicity of DBP, BBP and DEHP on the human hematopoietic stem cells.
RESUMO
Edible insects, due to their high nutritive value, are currently considered as a potential renewable source for food and feed production. Liometopum apiculatum ants are widely distributed in arid and semi-arid ecosystems and their larvae (escamoles) are considered as a delicacy, however the microbial importance in L. apiculatum nutritional ecology is unknown. The aim of this research was to characterize the microorganisms associated with both L. apiculatum larvae and the reproductive adult ants using the 16S rRNA gene sequencing and culturomics approaches. The obligate endosymbionts were also investigated through microscopic analysis. The most abundant Phylum identified by sequencing in the larvae was Firmicutes while in adult ants was Proteobacteria. Interestingly, the culturomics results showed 15 genera corresponding to the bacteria identified by sequencing analysis. Particularly, it was observed a large population of nitrogen-fixing bacteria, which could be linked with the high protein content in escamoles. Endosymbionts were detected in bacteoriocytes, these bacteria are related with vitamins and essential amino acids biosynthesis, and both compounds contributing to the high nutritional value of escamoles. This is the first report of the microorganisms present in the escamolera ant ensuring their safety as food and opening new areas of nutritional ecological and food processing.
Assuntos
Formigas/microbiologia , Bactérias/isolamento & purificação , Microbiota , Valor Nutritivo , Animais , Bactérias/classificação , Bactérias/genética , Feminino , Análise de Alimentos/métodos , Interações Hospedeiro-Patógeno , Larva/microbiologia , Masculino , Metagenômica , Ribotipagem , SimbioseRESUMO
Vasostatin 30 (Vs30) is an active fragment derived from the N-terminal region (135-164 aa) of human calreticulin and has the ability to inhibit angiogenesis. In this work, the expression of Vs30 was performed using a protease-deficient strain of the methylotrophic yeast Pichia pastoris. The vs30 gene was optimized for P. pastoris preferential codon usage and inserted into constitutive expression vector pGAPZαA. In addition, a plasmid with four copies of the expression cassette was obtained and transformed into P. pastoris. The flask fermentation conditions were: culture volume of 25 mL in 250 mL baffled flasks at 28 °C, pH 6 and harvest time of 48 h. Up to 21.07 mg/L Vs30 were attained and purified by ultrafiltration with a 30-kDa cut-off membrane and the recovery was 49.7%. Bioactivity of Vs30 was confirmed by the inhibition of cell proliferation, as well as the inhibition of the capillary-like structures formation of EA.hy926 cells in vitro. This work constitutes the first report on the expression of Vs30 in Pichia pastoris using a constitutive promoter and multi-copy approach such as strategies to improve the recombinant Vs30 expression.
Assuntos
Calreticulina/genética , Clonagem Molecular/métodos , Fragmentos de Peptídeos/genética , Calreticulina/isolamento & purificação , Linhagem Celular , Expressão Gênica , Humanos , Fragmentos de Peptídeos/isolamento & purificação , Pichia/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Obesity and type 2 diabetes(T2D) are the most prevalent and serious metabolic diseases affecting people worldwide. However racial and ethnic disparities seems to be a risk factor for their development. Mexico has been named as one of the largest populations with the highest prevalence of diabetes and obesity. The aim of this study was to identify novel T2D-associated proteins in Mexican patients. Blood samples were collected from 62 Mexican patients with T2D and they were grouped according to their body mass index(BMI). A panel of 10 diabetes and obesity serum markers was determined using MAGPIX. A comparative proteomics study was performed using two-dimensional difference in-gel electrophoresis(2D-DIGE) followed by mass spectrometry(LC-MS/MS). We detected 113 spots differentially accumulated, in which 64 unique proteins were identified, proteins that were involved in metabolism pathways, molecular transport, and cellular signalling. Four proteins(14-3-3, ApoH, ZAG, and OTO3) showing diabetes-related variation and also changes in relation to obesity were selected for further validation by western blotting. Our results reveal new diabetes related proteins present in the Mexican population. These could provide additional insight into the understanding of diabetes development in Mexican population and may also be useful candidate biomarkers.
Assuntos
Biomarcadores/sangue , Índice de Massa Corporal , Diabetes Mellitus Tipo 2/sangue , Eletroforese em Gel Diferencial Bidimensional/métodos , Idoso , Cromatografia Líquida , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Humanos , Masculino , México , Pessoa de Meia-Idade , Obesidade/sangue , Obesidade/diagnóstico , Obesidade/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Massas em TandemRESUMO
A novel Cu/ZnSOD from Amaranthus hypochondriacus was cloned, expressed, and characterized. Nucleotide sequence analysis showed an open reading frame (ORF) of 456 bp, which was predicted to encode a 15.6-kDa molecular weight protein with a pI of 5.4. Structural analysis showed highly conserved amino acid residues involved in Cu/Zn binding. Recombinant amaranth superoxide dismutase (rAhSOD) displayed more than 50 % of catalytic activity after incubation at 100 °C for 30 min. In silico analysis of Amaranthus hypochondriacus SOD (AhSOD) amino acid sequence for globularity and disorder suggested that this protein is mainly disordered; this was confirmed by circular dichroism, which showed the lack of secondary structure. Intrinsic fluorescence studies showed that rAhSOD undergoes conformational changes in two steps by the presence of Cu/Zn, which indicates the presence of two binding sites displaying different affinities for metals ions. Our results show that AhSOD could be classified as an intrinsically disordered protein (IDP) that is folded when metals are bound and with high thermal stability.
Assuntos
Amaranthus/enzimologia , Proteínas Intrinsicamente Desordenadas/metabolismo , Superóxido Dismutase/metabolismo , Sequência de Aminoácidos , Cromatografia em Gel , Dicroísmo Circular , Estabilidade Enzimática/efeitos dos fármacos , Fluorescência , Peróxido de Hidrogênio/farmacologia , Proteínas Intrinsicamente Desordenadas/química , Cinética , Metais/farmacologia , Modelos Moleculares , Dados de Sequência Molecular , Multimerização Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Cloreto de Sódio/farmacologia , Superóxido Dismutase/química , TemperaturaRESUMO
We present here the structural modeling and biochemical characterization of a recombinant superoxide dismutase (SOD) from Deschampsia antarctica E. Desv. [Poaceae] produced in Escherichia coli. The recombinant protein was purified by affinity chromatography nickel-nitrilotriacetic acid (Ni-NTA), and its identity was demonstrated by immunoblotting and inhibition by H2O2 and KCN. Inductively coupled plasma optical emission spectroscopy (ICP-OES) analysis confirmed the presence of Cu and Zn. Modeling of the D. antarctica Cu/Zn-SOD (DaSOD) amino acid sequence using the SWISS-MODEL and 2Q2L_B monomer of the psychrophilic Cu/Zu-SOD from Potentilla atrosanguinea (PaSOD) as template produced a structure similar to that of the typical eukaryotic Cu/Zn-SODs. Activity assays using the p-nitro blue tetrazolium chloride (NBT) solution method showed that the purified DaSOD had a specific activity of 5818 U/mg at 25 °C and pH 7.2 and that it was active in a pH interval of 5-8 and a temperature interval of 0-40 °C. Furthermore, DaSOD was still active at -20 °C as observed by a zymogram assay. We found 100 % activity when it was heated at 80 °C for 60 min, indicating a high thermostability. DaSOD properties suggest that this enzyme could be useful for preventing the oxidation of refrigerated or frozen foods, as well as in the preparation of cosmetic and pharmaceutical products.
Assuntos
Poaceae/enzimologia , Proteínas Recombinantes/biossíntese , Superóxido Dismutase/biossíntese , Sequência de Aminoácidos , Clonagem Molecular , Temperatura Baixa , Escherichia coli/genética , Regulação da Expressão Gênica de Plantas , Peróxido de Hidrogênio , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Superóxido Dismutase/química , Superóxido Dismutase/genética , Superóxido Dismutase/isolamento & purificaçãoRESUMO
The effect of salt stress was analyzed in chloroplasts of Amaranthus cruentus var. Amaranteca, a plant NAD-malic enzyme (NAD-ME) type. Morphology of chloroplasts from bundle sheath (BSC) and mesophyll (MC) was observed by transmission electron microscopy (TEM). BSC and MC from control plants showed similar morphology, however under stress, changes in BSC were observed. The presence of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) was confirmed by immunohistochemical staining in both types of chloroplasts. Proteomic profiles of thylakoid protein complexes from BSC and MC, and their changes induced by salt stress were analyzed by blue-native polyacrylamide gel electrophoresis followed by SDS-PAGE (2-D BN/SDS-PAGE). Differentially accumulated protein spots were analyzed by LC-MS/MS. Although A. cruentus photosynthetic tissue showed the Kranz anatomy, the thylakoid proteins showed some differences at photosystem structure level. Our results suggest that A. cruentus var. Amaranteca could be better classified as a C3-C4 photosynthetic plant.
Assuntos
Adaptação Fisiológica , Amaranthus/metabolismo , Cloroplastos/metabolismo , Proteínas de Plantas/metabolismo , Proteômica , Cromatografia Líquida , Bases de Dados de Proteínas , Eletroforese em Gel de Poliacrilamida , Complexos de Proteínas Captadores de Luz , Células do Mesofilo , Microscopia Eletrônica de Transmissão , Complexos Multiproteicos , Fotossíntese , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Folhas de Planta/metabolismo , Feixe Vascular de Plantas/metabolismo , Cloreto de Sódio/farmacologia , Estresse Fisiológico , Espectrometria de Massas em Tandem , Tilacoides/metabolismoRESUMO
The impact of nitrogen source on hydrogen production by Escherichia coli WDHL (ΔhycAΔlacI) strain using cheese whey as a substrate was evaluated. To improve the assimilation of complex proteins such as lactalbumin, we assessed treatment with a protease. Also, five external nitrogen sources were tested: NH4Cl, (NH4)2SO4, urea, yeast extract, and tryptone. The treatments in 120 mL serological bottles with pancreatin 1,000 mg/L produced 1.75-fold more hydrogen than the cultures without pancreatin. In the bottle cultures supplemented with yeast extract or tryptone 5 g/L, hydrogen production increased up to 3.2- and 3.5-fold, respectively, whereas inorganic salts and urea had no statistical difference with respect to the control cultures. In 1-L bioreactors, the use of tryptone improved 2.1-fold hydrogen production. Tryptone or yeast extract enable the total consumption of lactose in 40 h, whereas in the control assay the lactose was not completely consumed. Our results demonstrate that it is necessary to select an adequate nitrogen source, which allows both carbon source consumption and high hydrogen production.
Assuntos
Reatores Biológicos , Queijo , Escherichia coli/crescimento & desenvolvimento , Hidrogênio/metabolismo , Escherichia coli/genética , Nitrogênio/metabolismo , Pancreatina/farmacologiaRESUMO
The primary structure of amaranth 11S globulin (Ah11S) was engineered with the aim to improve its functional properties. Four continuous methionines were inserted in variable region V, obtaining the Ah11Sr+4M construction. Changes on protein structure and surface characteristics were analyzed in silico. Solubility and heat-induced gelation of recombinant amaranth 11S proglobulin (Ah11Sr and Ah11Sr+4M) were compared with the native protein (Ah11Sn) purified from amaranth seed flour. The Ah11Sr+4 M showed the highest surface hydrophobicity, but as consequence the solubility was reduced. At low ionic strength (µ = 0.2) and acidic pH (<4.1), the recombinant proteins Ah11Sr and Ah11Sr+4 M had the highest and lowest solubility values, respectively. All globulins samples formed gels at 90 °C and low ionic strength, but Ah11Sn produced the weakest and Ah11Sr the strongest gels. Differential scanning calorimetry analysis under gel forming conditions revealed only exothermic transitions for all amaranth 11S globulins analyzed. In conclusion, the 3D structure analysis has revealed interesting molecular features that could explain the thermal resistance and gel forming ability of amaranth 11S globulins. The incorporation of four continuous methionines in amaranth increased the hydrophobicity, and self-supporting gels formed had intermediate hardness between Ah11Sn and Ah11Sr. These functional properties could be used in the food industry for the development of new products based on amaranth proteins.
Assuntos
Amaranthus/química , Proteínas Alimentares/química , Globulinas/química , Proteínas de Armazenamento de Sementes/química , Sementes/química , Proteínas Alimentares/metabolismo , Suplementos Nutricionais , Alimentos Formulados , Géis , Globulinas/genética , Globulinas/metabolismo , Temperatura Alta , Transição de Fase , Conformação Proteica , Engenharia de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas de Armazenamento de Sementes/genética , Proteínas de Armazenamento de Sementes/metabolismo , Solubilidade , Propriedades de SuperfícieRESUMO
Angiogenesis has been considered an important target for cancer therapy. The inhibition of angiogenesis represents a promising strategy for anti-cancer treatment, tumor growth inhibition, and metastasis. Vasostatin 30 (Vs30), and the 14.1 kDa vasoinhibin (Vi-II-14.1) are two peptides with remarkable anti-tumor and anti-angiogenic effect. The aim of this study was to produce a novel fusion protein between Vs30 and Vi-II-14.1, denominated VS_VI, to obtain a new protein with higher biological activity. The protein fusion genes were cloned into a T7 promoter-based vector, expressed in Escherichia coli BL21-SI and purified by affinity column chromatography. In vitro assays showed that the recombinant fusion protein inhibited rat coronary endothelial cell proliferation at 65.5 % at 10 nM, whereas recombinant Vs30 and Vi-II-14.1 inhibited at 33 and 50.5 % respectively, at the same concentration. The results showed that VS_VI is significantly more active than the Vs30 and Vi-II-14.1 separately. In addition, a practical classification of the vasoinhibins based on the peptide origin and theoretical molecular weight is proposed. This is the first study to produce a new fusion protein derived from Vs30 and Vi-II-14.1, both of them proposed as promising therapeutic agents.
Assuntos
Inibidores da Angiogênese/farmacologia , Calreticulina/farmacologia , Células Endoteliais/efeitos dos fármacos , Prolactina/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Sequência de Aminoácidos , Inibidores da Angiogênese/química , Inibidores da Angiogênese/genética , Inibidores da Angiogênese/metabolismo , Animais , Calreticulina/química , Calreticulina/genética , Calreticulina/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/citologia , Escherichia coli/genética , Vetores Genéticos/genética , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Prolactina/química , Prolactina/genética , Prolactina/metabolismo , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Fermentations of lactose, glucose and galactose using Escherichia coli WDHL, a hydrogen over producer strain, were performed. With glucose as substrate pyruvate was mainly routed to the lactate pathway, resulting in hydrogen production and yield of 1037 mL and 0.30 mol H(2)/mol of glucose, respectively. When galactose was the substrate, the pyruvate formate lyase pathway was the main route for pyruvate and a fermentation yield of 1.12 mol H(2)/mol of galactose and a hydrogen production of 2080 mL were obtained. The fermentation of lactose or glucose plus galactose showed a similar yield of 1.02 mol H(2)/mol of hexose consumed. This work clearly demonstrated that the kinetics of hydrogen and metabolites production as well as the hydrogen yield were affected by the type of sugar used as substrate as reflected by the deviations from the metabolic hydrogen-production pathway.
Assuntos
Metabolismo dos Carboidratos , Escherichia coli/metabolismo , Fermentação , Hidrogênio/metabolismo , Lactose/metabolismoRESUMO
Papaya (Carica papaya L.) is a climacteric fruit susceptible to postharvest losses due to the ethylene-induced ripening. The inhibitor of ethylene action, 1-methylcyclopropene (1-MCP), has been used worldwide as a safe postharvest non-toxic agent, but the physiological and biochemical modifications induced by 1-MCP are not well understood. Using the 2-DE analysis, we report the changes in the protein profiles after 6 and 18 days of postharvest and the effect of the effect of 1-MCP treatment on fruits. Twenty seven protein spots showing differences in abundance during ripening were successfully identified by nano-LC-ESI/MS/MS. Some spots corresponded to the cell wall degrading enzymes related to fruit ripening; others were involved in oxidative damage protection, protein folding, and cell growth and survival that were induced by 1-MCP. This is the first proteomic report analyzing the effect of 1-MCP in papaya ripening. The present data will help to shed light on papaya fruit ripening process.
Assuntos
Carica/metabolismo , Ciclopropanos/farmacologia , Frutas/metabolismo , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Fatores de TempoRESUMO
BACKGROUND: Cervical cancer (CC) is a common malignancy in women worldwide. Cervical tumorigenesis involves a multistep process in which accumulations of genetic alterations are present. Homeotic genes, such as HOX gene re-expression, have been reported in a wide variety of tumors. METHODS: In order to know the role of HOX B4 gene expression in CC, in the present study, two-dimensional polyacrylamide gel electrophoresis, matrix-assisted laser desorption/ionization, and time-of-flight mass spectrometry were used for differential screening of protein expression in CC. Immunohistochemical analysis was performed on the cervical tissue microarray (TMA) to detect the Hox B4 protein. RESULTS: Hox B4 peptide was detected among 15 increased spots differentially observed in CC. Using TMA, Hox B4 protein was also immunodetected in the nuclei of cervical epithelial tumor cells, while in normal cervical epithelium, it was absent. Interestingly, it was possible to detect the Hox B4 protein in the precursor lesions. CONCLUSIONS: Hox B4 protein is present in the precursor lesions as CC cells, suggesting that Hox B4 could be a protein related to the neoplastic state (non-differentiated cells) of human cervical epithelium.
Assuntos
Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Diferenciação Celular/genética , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Sequência de Aminoácidos , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Eletroforese em Gel Bidimensional/métodos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Genes Homeobox , Humanos , Dados de Sequência Molecular , Proteoma/genética , Proteoma/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Neoplasias do Colo do Útero/patologiaRESUMO
Calcium (Ca(2+)) is a critical ion for the growth and development of plants and plays an important role in signal transduction pathways in response to biotic and abiotic stresses. We investigated the Ca(2+) stress responsive-genes in amaranth leaves by using the suppression subtractive hybridization technique. Screening of the libraries generated 420 up-regulated transcripts and 199 down-regulated transcripts. The differentially expressed transcripts were associated with general stress response, transcription factors, gene regulation, signal transduction, and some other with unknown function. Selected genes were used to study their differential regulation by sqRT-PCR. Among the up-regulated transcripts, a fragment containing the motif of C3HC4-type RING-Zinc family was further characterized. The ORF of amaranth zinc finger protein (AhZnf) has a closer relationship with its ortholog from Ricinus communis while is distantly related to the Arabidopsis thaliana C3HC4-type ortholog. We have identified a novel putative zinc finger protein along with other novel proteins such as the wall associated kinase, phosphoinositide binding protein, and rhomboid protease involved in response to Ca(2+) stress in amaranth leaves.