RESUMO
Prostate cancer (PCa) is a common type of cancer affecting male population. PCa treatments have side effects and are temporarily effective, so new therapeutic options are being investigated. Due to the high demand of energy for cell proliferation, an increase in the expression and activity of lipogenic enzymes such as the stearoyl-CoA desaturase (SCD) have been observed in PCa. Sterculic acid, contained in the seed's oil of Malvales, is a natural inhibitor of SCD. The objective of our investigation was to evaluate the effects of sterculic oil (SO) from Sterculia apetala seeds on proliferation, cell cycle and apoptosis in prostate cancer cells. SO was administered to PC3 and LNCaP cells, and to prostate normal cells; cell viability, cell cycle, apoptosis, SCD gene and protein expression and enzymatic activity were analyzed. SO administration (4 mM sterculic acid) diminished cell viability in LNCaP and PC3 cells, arrested cell cycle in G2 and promoted apoptosis. SO diminished SCD enzymatic activity with no effects on gene nor protein expression. Our results suggest that SO might offer benefits as an adjuvant in hormonal and chemotherapy prostate cancer treatments. This is the first study to analyze the effect of SO on cancer cells.
Assuntos
Neoplasias da Próstata , Estearoil-CoA Dessaturase , Apoptose , Linhagem Celular , Proliferação de Células , Humanos , Masculino , Neoplasias da Próstata/tratamento farmacológico , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismoRESUMO
Immobilization of enzymes has many advantages for their application in biotechnological processes. In particular, the cross-linked enzyme aggregates (CLEAs) allow the production of solid biocatalysts with a high enzymatic loading and the advantage of obtaining derivatives with high stability at low cost. The purpose of this study was to produce cross-linked enzymatic aggregates (CLEAs) of LipMatCCR11, a 43 kDa recombinant solvent-tolerant thermoalkaliphilic lipase from Geobacillus thermoleovorans CCR11. LipMatCCR11-CLEAs were prepared using (NH4)2SO4 (40% w/v) as precipitant agent and glutaraldehyde (40 mM) as cross-linker, at pH 9, 20 °C. A U10(56) uniform design was used to optimize CLEA production, varying protein concentration, ammonium sulfate %, pH, glutaraldehyde concentration, temperature, and incubation time. The synthesized CLEAs were also analyzed using scanning electron microscopy (SEM) that showed individual particles of <1 µm grouped to form a superstructure. The cross-linked aggregates showed a maximum mass activity of 7750 U/g at 40 °C and pH 8 and retained more than 20% activity at 100 °C. Greater thermostability, resistance to alkaline conditions and the presence of organic solvents, and better durability during storage were observed for LipMatCCR11-CLEAs in comparison with the soluble enzyme. LipMatCCR11-CLEAs presented good reusability by conserving 40% of their initial activity after 9 cycles of reuse.
Assuntos
Proteínas de Bactérias/química , Geobacillus/enzimologia , Lipase/química , Agregados Proteicos , Proteínas de Bactérias/genética , Reagentes de Ligações Cruzadas/química , Estabilidade Enzimática , Geobacillus/genética , Lipase/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
Bacterial diversity was explored among field samples and cultured isolates from coral reefs within the Veracruz Reef System. Bacterioplankton and bacteriobenthos were characterized by pyrosequencing 16S rRNA genes. Identified sequences belonged to the kingdom Bacteria and classified into 33 phyla. Proteobacteria (likely SAR11 clade) dominated in collective field samples, whereas Firmicutes were the most abundant taxa among cultured isolates. Bioinformatic sorting of sequences to family level revealed 223 bacterial families. Pseudomonadaceae, Exiguobacteraceae and Bacillaceae were dominant among cultured isolates. Vibrionaceae, Alteromonadaceae, and Flavobacteriaceae dominated in reef-associated sediments, whereas Rickettsiaceae and Synechoccaceae were more highly represented in the water column. Bacterial communities from sediments were more diverse than from the water column. This study reveals cryptic bacterial diversity among microenvironmental components of marine microbial reef communities subject to differential influence of anthropogenic stressors. Such investigations are critical for constructing scenarios of environmentally induced shifts in bacterial biodiversity and species composition.
RESUMO
Expression of the regulatory stress rpoS gene controls the transcription of cspA genes, which are involved in survival and adaptation to low temperatures. The purpose of this study was to assess the growth kinetics of naturally occurring V. parahaemolyticus in shellstock oysters and in vitro and the cold-shock-induced expression of the rpoS and cspA gene response in vitro during postharvest refrigeration. Naturally contaminated eastern oysters (Crassostrea virginica) and pathogenic (Vp-tdh) and nonpathogenic (Vp-tlh) isolates were stored at 7 ± 1 °C for 168 h and 216 h, respectively. The regulatory stress (rpos) and cold-shock (cspA) gene expressions were determined by reverse transcription PCR. At 24 h, the (Vp-tdh) strain grew faster (p < 0.05) than the (Vp-tlh) strain in oysters (λ = 0.33, 0.39, respectively) and in vitro (λ = 0.89, 37.65, respectively), indicating a better adaptation to cold shock for the (Vp-tdh) strain in live oysters and in vitro. At 24 h, the (Vp-tdh) strain rpoS and cspA gene expressions were upregulated by 1.9 and 2.3-fold, respectively, but the (Vp-tlh) strain rpoS and cspA gene expressions were repressed and upregulated by -0.024 and 1.9-fold, respectively. The V. parahaemolyticus strains that were isolated from tropical oysters have adaptive expression changes to survive and grow at 7 °C, according to their virulence.
Assuntos
Temperatura Baixa , Crassostrea , Regulação da Expressão Gênica , Ostreidae , Vibrio parahaemolyticus , Animais , Refrigeração , Frutos do Mar/microbiologia , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/patogenicidadeRESUMO
Sterculia apetala (order: Malvales, family: Sterculiaceae) seed oil contains two cyclopropene fatty acids: sterculic and malvalic acid. Both positive and negative effects have been associated with the consumption of sterculic oil. In Mexico, S. apetala seeds are consumed after being boiled or roasted, used as chocolate flavoring, and utilized as animal fodder. Therefore, it is important to evaluate whether the consumption of this seed has a negative impact on the organism. The aim of this study was to evaluate the effect of administration of sterculic oil, during an 8-week period, on anxiety-like behavior and spontaneous locomotor activity in Zucker rats, analyzed through light/dark and open-field tests. The results showed that the consumption of sterculic oil decreased exploration latency in light/dark tests, which suggests an anxiolytic-like effect. Alterations in time spent on rearing and grooming were present in open-field tests, but this was not statistically significant, discarding nonspecific motor alterations. The alterations found in this study are possibly related to intrinsic obesity and metabolic complications present in the Zucker rat model, where leptin plays an important role in animal mood, more so than sterculic oil consumption.
Assuntos
Ansiolíticos/administração & dosagem , Ansiedade/tratamento farmacológico , Ansiedade/psicologia , Óleos de Plantas/administração & dosagem , Sterculia/química , Animais , Ansiolíticos/química , Ansiedade/etiologia , Comportamento Animal/efeitos dos fármacos , Humanos , Masculino , Obesidade/complicações , Obesidade/tratamento farmacológico , Obesidade/psicologia , Óleos de Plantas/química , Ratos , Ratos Zucker , Sementes/químicaRESUMO
A gene encoding a carboxylesterase produced by Geobacillus thermoleovoras CCR11 was cloned in the pET-3b cloning vector, sequenced and expressed in Escherichia coli BL21(DE3). Gene sequence analysis revealed an open reading frame of 750 bp that encodes a polypeptide of 250 amino acid residues (27.3 kDa) named CaesCCR11. The enzyme showed its maximum activity at 50 °C and pH 5-8, with preference for C4 substrates, confirming its esterase nature. It displayed good resistance to temperature, pH, and the presence of organic solvents and detergents, that makes this enzyme biotechnologically applicable in the industries such as fine and oleo-chemicals, cosmetics, pharmaceuticals, organic synthesis, biodiesel production, detergents, and food industries. A 3D model of CaesCCR11 was predicted using the Bacillus sp. monoacyl glycerol lipase bMGL H-257 structure as template (PBD code 3RM3, 99 % residue identity with CaesCCR11). Based on its canonical α/ß hydrolase fold composed of 7 ß-strands and 6 α-helices, the α/ß architecture of the cap domain, the GLSTG pentapeptide, and the formation of distinctive salt bridges, we are proposing CaesCCR11 as a new member of family XV of lipolytic enzymes.
Assuntos
Sequência de Aminoácidos/genética , Geobacillus/enzimologia , Estrutura Secundária de Proteína , Receptores CCR/química , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Geobacillus/química , Modelos Moleculares , Receptores CCR/biossíntese , Receptores CCR/genética , Análise de Sequência de DNA , Especificidade por Substrato , TemperaturaRESUMO
The medium optimization for the production of the Geobacillus thermoleovorans CCR11 thermoalkalophilic lipase was carried out in shake flask cultures using safflower high oleic oil. In the first step of optimization, a two level fractional factorial design allowed the identification of the concentration of nutrient broth and temperature as the main variables significantly affecting lipase production (P<0.05). In a second step, a D-optimal design was applied to determine the variables optimal values, defined as those yielding maximal lipase production in shaken flasks, thus demonstrating that the optimal concentration of nutrient broth was 3.8 g/l and the optimal culture temperature was 39.5°C. The model was experimentally validated, yielding a lipase production of 2283.70 ± 118.36 U/mL which represents a 6.7-fold increase in comparison to the non-optimized medium.
Assuntos
Proteínas de Bactérias/biossíntese , Geobacillus/enzimologia , Geobacillus/crescimento & desenvolvimento , Lipase/biossíntese , Modelos Biológicos , Óleo de Cártamo/química , Óleo de Cártamo/farmacologiaRESUMO
A partially purified lipase produced by the thermophile Geobacillus thermoleovorans CCR11 was immobilized by adsorption on porous polypropylene (Accurel EP-100) in the presence and absence of 0.1% Triton X-100. Lipase production was induced in a 2.5% high oleic safflower oil medium and the enzyme was partially purified by diafiltration (co. 500,000 Da). Immobilization conditions were established at 25 degrees C, pH 6, and a protein concentration of 0.9 mg/mL in the presence and absence of 0.1% Triton X-100. Immobilization increased enzyme thermostability but there was no change in neither the optimum pH nor in pH resistance irrelevant to the presence of the detergent during immobilization. Immobilization with or without Triton X-100 allowed the reuse of the lipase preparation for 11 and 8 cycles, respectively. There was a significant difference between residual activity of immobilized and soluble enzyme after 36 days of storage at 4 degrees C (P < 0.05). With respect to chain length specificity, the immobilized lipase showed less activity over short chain esters than the soluble lipase. The immobilized lipase showed good resistance to desorption with phosphate buffer and NaCl; minor loses with detergents were observed (less than 50% with Triton X-100 and Tween-80), but activity was completely lost with SDS. Immobilization of G. thermoleovorans CCR11 lipase in porous polypropylene is a simple and easy method to obtain a biocatalyst with increased stability, improved performance, with the possibility for re-use, and therefore an interesting potential use in commercial conditions.