Recent progress on the effects of structured lipids and fluidity of plasma membrane on tumor metastasis / 药学学报

The lipid composition of cell plasma membranes of aggressive tumors is significantly altered from normal, affecting the membrane fluidity and function. Plasma membrane fluidity involves multiple steps in tumor invasion and metastasis, including cell movement, adhesion, lateral diffusion of membrane molecules, signal transduction, material exchange and so on. This review highlights the difference in plasma membrane lipid composition and fluidity between normal and cancer cells, as well as the correlation with the invasion and metastasis potential of cancer. We also point out that the proliferation, invasion and metastasis of tumors can be inhibited by improving membrane fluidity or interfering with the membrane structured lipid composition, this focusing more on changing the biophysical properties of cancer cell membranes, and providing a novel strategy that works for treatment of tumor metastasis.

Fluidez sexual: contextualização histórica e contributos teóricos / Fluidez sexual: contextualización histórico y aportaciones teóricas / Sexual fluidity: historical contextualization and theoretical contributions

RESUMO. Neste estudo, tivemos como objetivo aprofundar o conceito de fluidez sexual pela análise crítica da literatura e de uma síntese integrativa sobre o tema. Nele, analisamos estudos que se debruçam sobre a fluidez sexual, exploramos o conceito, sob orientação do modo como Lisa Diamond o introduz na literatura científica, o define e operacionaliza. Por fim, debruçamo-nos sobre estudos no âmbito da fluidez sexual, a fim de perceber os significados atribuídos, e questionando o potencial viés de gênero que acompanha um conceito situado num tempo e num contexto heteronormativos. Como contributo para o aprofundamento do conceito e reforço das suas potencialidades, são apresentadas reflexões sobre a relação entre a fluidez sexual e a masculinidade hegemônica, uma relação pouco valorizada na literatura científica. Globalmente, este trabalho promove, criticamente e de modo interseccional, o questionamento sobre a fixação no sexo/gênero das pessoas envolvidas numa situação sexual e são discutidas as fronteiras do conceito de fluidez sexual e a sua (im)permeabilidade.

RESUMEN. En este estudio, buscamos profundizar el concepto de fluidez sexual a través de un análisis crítico de la literatura y de una síntesis integradora sobre el tema. Para tal, se analizaron estudios enfocados en la fluidez sexual y se exploró este mismo concepto según la guía de cómo Lisa Diamond lo introduce en la literatura científica, lo define y lo concretiza. El análisis de los estudios en torno de la fluidez sexual se realizó teniendo como objetivos comprender los significados atribuidos al concepto y cuestionar el posible sesgo de género que acompaña un concepto ubicado en un tiempo y en un contexto heteronormativos. De cara a profundizar el concepto y reforzar su potencial, se presentan reflexiones sobre la relación entre la fluidez sexual y la masculinidad hegemónica, una relación que no se tiene en cuenta habitualmente en la literatura científica. En general, este trabajo contribuye, de manera crítica e interseccional, al cuestionamiento sobre la fijación en el sexo/género de las personas involucradas en una situación sexual y discute los límites del concepto de fluidez sexual y su (im)permeabilidad.

ABSTRACT. In this study, we sought to deepen the concept of sexual fluidity through a critical analysis of the literature and an integrative synthesis on the topic. In it, we analyzed studies that focus on sexual fluidity, explored the concept, under the guidance of how Lisa Diamond introduces it into the scientific literature, defines it, and operationalizes it. Finally, we focused on studies on sexual fluidity, trying to understand the meanings attributed to the concept, questioning the potential gender bias that accompanies a concept located in a heteronormative time and context. As a contribution to the deepening of the concept and reinforcement of its potential, reflections are presented on the relationship between sexual fluidity and hegemonic masculinity, a relationship that is little valued in the scientific literature. Overall, this work promotes, critically and in an intersectional way, the questioning about the fixation in sex/gender of people involved in a sexual situation and discussed the boundaries of the concept of sexual fluidity and its (im)permeability.

Improving compactibility properties of Lonicera Japonica Flos by Plasdone S-630 / 药学学报

To improve the fluidity and compactibility properties of raw powders of traditional Chinese medicine by particle modification technology, Lonicera Japonica Flos was used as a model drug, fluidized bed bottom spray technology was used, and Plasdone S-630 was used as a modifier to prepare modified particles. The powder properties, tablet compactibility parameters, disintegration time and dissolution were measured. The surface morphology of the powder particles before and after modification and compressed tablets were characterized by combining with scanning electron microscopy technology. The results showed that the particle size of Lonicera Japonica powder has been increased after particle modification, the fluidity, compressibility and compactibility of the powder have been improved to some extent, the disintegration time has also been reduced, and the dissolution in vitro is not affected. Therefore, this study can provide reference and ideas for the common problem that raw powder of traditional Chinese medicine that cannot meet the needs of preparation production due to poor powder properties such as fluidity and compressibility.

Effect of fish oil on oxidative stress markers in patients with probable Alzheimer´s disease

High intake of omega-3 fatty acids has been associated with synaptic plasticity, neurogenesis and memory in several experimental models. To assess the efficacy of fish oil supplementation on oxidative stress markers in patients diagnosed with probable Alzheimer´s disease (AD) we conducted a double blind, randomized, placebo controlled clinical trial. AD patients who met the inclusive criteria were given fish oil (containing 0.45 g eicosapentaenoic acid and 1 g docosahexaenoic acid) or placebo daily for 12 months. Oxidative stress markers [lipoperoxides, nitric oxide catabolites levels, oxidized/reduced glutathione ratio, and membrane fluidity] and fatty acid profile in erythrocytes were assessed at enrollment, and 6 and 12 months after the start of the testing period. At the end of the trial, in patients who received fish oil, we detected a decrease in the omega 6/omega 3 ratio in erythrocyte membrane phospholipids. This change was parallel with decreases in plasma levels of lipoperoxides and nitric oxide catabolites. Conversely, the ratio of reduced to oxidized glutathione was significantly increased. In addition, membrane fluidity was increased significantly in plasma membrane samples. In conclusion fish oil administration has a beneficial effect in decreasing the levels of oxidative stress markers and improving the membrane fluidity in plasma(AU)

El alto consumo de ácidos grasos omega-3 se asocia con la plasticidad sináptica, neurogénesis y memoria en varios modelos experimentales. Para evaluar la eficacia de la suplementación con aceite de pescado en los marcadores de estrés oxidativo en pacientes con diagnóstico de la enfermedad de Alzheimer (EA) probable realizamos un ensayo clínico doble ciego, aleatorizado, controlado con placebo. A los pacientes con la EA que cumplían los criterios de inclusión se les administró aceite de pescado (que contenía 0,45 g de ácido eicosapentaenoico y 1 g de ácido docosahexaenoico) o placebo diariamente durante 12 meses. Los marcadores de estrés oxidativo plasmático [niveles de lipoperóxidos y catabolitos del óxido nítrico, cociente de glutatión reducido a glutatiónoxidado) y fluidez de la membrana] y el perfil de ácidos grasos en los eritrocitos se evaluaron al inicio, 6 meses y alos 12 meses. Al final del ensayo, en pacientes que recibieron aceite de pescado detectamos una disminución en el cociente de ácidos grasos omega 6/omega 3 en los fosfolípidos de la membrana eritrocitaria. Este cambio ocurrió en paralelo a la disminución de los niveles plasmáticos de lipoperóxidos y catabolitos del óxido nítrico. Por el contrario, el cociente de glutatión reducido a glutatión oxidado se incrementó significativamente. Además, la fluidez de la membrana aumentó significativamente en las muestras analizadas. En conclusión, la administración de aceite de pescado tiene un efecto beneficioso al disminuir los niveles de marcadores de estrés oxidativo plasmático y mejorar la fluidez de la membrana plasmática(AU)

Effects of source difference of sodium carboxymethyl starch on its properties and function / 药学学报

During the process of consistency evaluation, it was found that the consistency of drug release between generic and original brands in vitro was not sufficient to demonstrate their same release in vivo. The disintegration of tablets, as a premise for the release of tablet drugs, greatly affects the release of drugs, depending on the structure and properties of disintegrants. Hence the systematic research on disintegrants would be very important for the evaluation of generic consistency. In this experiment, the physicochemical properties and application of 11 different sodium carboxymethyl starch (CMS-Na) from 9 manufacturers were investigated. This provides the reference for selection of excipients for consistency evaluation. The particle morphology of CMS-Na was observed by scanning electron microscope. The particle size distribution was determined by dry particle size analyzer. The determination of pH and loss on drying was carried out according to the 2015 edition of Chinese pharmacopoeia method. The powder fluidity was evaluated with Carr's index, Hausner ratio and angle of repose. The disintegration performance of CMS-Na was evaluated through determining the water absorption and swelling. The disintegration effect of CMS-Na tablets was studied using lactose and microcrystalline cellulose as fillers. The results showed that pH values and weight loss on drying of all samples met the requirements, whereas the particle morphology, fluidity, water swell-ability and disintegration time had a large variation, which leads to the large differences the properties of CMS-Na depending on the sources. Therefore in order to ensure that the reproducibility of generic drugs from their name brand, our studies indicate that only a sizable choice of disintegrants could ensure good inter batch reproducibility.

Fluidez de gênero e gênero como força interpelativa: discussão com Jacqueline Rose / Gender fluidity and gender as interpellative force: a discussion with Jacqueline Rose / Fluidez de género y género como fuerza interpelativa: discusión con Jacqueline Rose / Fluidité de genre et le genre comme force interpellative: discussion avec Jacqueline Rose

Este estudo examina as implicações da fluidez de gênero e da permanência de gênero para fins de experiência clínica e social. Consideram-se as crises de desenvolvimento em torno da separação tanto para desenvolvimento masculino quanto para o feminino. Teorias de desenvolvimento, teoria de sistemas dinâmicos não lineares e interseccionalidade são propostas como modelos úteis, uma vez que contemplam a identificação no desenvolvimento.

This essay examine the implications of gender fluidity and gender fixity for clinical and social experience. The developmental crises around separation are considered for both male and female development. Developmental theories, nonlinear dynamic systems theory and intersectionality are proposed as useful templates for considering identification in development.

Este estudio examina las implicaciones de la fluidez de género y de la permanencia de género para fines de experiencia clínica y social. Se consideran las crisis de desarrollo en relación a la separación tanto para el desarrollo masculino como para el femenino. Teorías de desarrollo, teoría de sistemas dinámicos no lineales e interseccionalidad son propuestas como modelos útiles, pues contemplan la identificación en el desarrollo.

Cette étude examine les implications de la fluidité et de la permanence de genre, ayant pour but une expérience clinique et social. On a pris en compte les crises de développement au tour de la séparation, soit pour le développement masculin, soit pour le féminin. On propose des théories de développement, la théorie de systèmes dynamiques non linéaires et de l'intersectionnalité en tant que modèles utiles, étant donné qu'ils traitent de l'identification dans le développement.

The preparation of supported lipid bilayers and its functional study / 生物医学工程学杂志

Supported lipid bilayers (SLBs) have been widely used in biomedical and bioengineering research because its structure and function are similar to natural cell membrane. A fluorescence recovery after photobleaching (FRAP) technique was used to measure the lateral diffusion of the SLBs composed of 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1, 2-dioleoyl-sn-glycero-3-［(N-(5-amino-1-carboxyp-entyl) iminodiacetic acid)］ (DGS-NTA) on the glass slide, and the effects of the DOPC-to-DGS-NTA ratio, small unilamellar vesicles (SUV) producing method, sizes of bleaching areas and concentrations of loading proteins on the SLBs fluidity and diffusion coefficient were studied systematically in this paper. The results demonstrated that: (1) SUV made by probe sonication exhibited more uniform and smaller size compared with that made by film extrusion, but the whole process of SLBs formation must not be exposed to air. (2) The fluorescence recovery rate and diffusion coefficient of the SLBs decreased with the increasing bleaching area size. With the mole ratio of DOPC to DGS-NTA decreasing from 98∶2 to 84∶16, the fluidity and fluorescence recovery degree decreased gradually, and the SLBs would lose its fluidity if the ratio reached to 82∶18. (3) The average fluorescence intensity of SLBs increased linearly with the loading protein concentration (10-40 nmol·L ), and the protein showed good mobility on the SLBs. The study would provide a good platform of bio-membrane for further research on interactions among cell membrane molecules and subsequent signals response.

Effect of paeoniflorin and menthol on membrane fluidity, Na⁺-K⁺-ATPase activity and Ca²⁺-ATPase activity during transport of puerarin in Calu-3 cell / 中国中药杂志

The aim of this research is to investigate the effects of paeoniflorin and menthol on the physiological function of Calu-3 cell membrane during the transport of puerarin. Calu-3 cell was used as the cell model to simulate nasal mucosa tissues, and the cell membrane fluidity, Na⁺-K⁺-ATPase activity and Ca²⁺-ATPase activity were detected by fluorescence recovery after photobleaching(FRAP) and ultramicro enzyme activity testing, in order to explore the mechanism of compatible drugs on promoting puerarin transport. The results showed that when puerarin associated with low, middle and high concentration of menthol or both paeoniflorin and menthol, the fluorescence recovery rate was increased significantly, while Na⁺-K⁺-ATPase activity had no significant change and Ca²⁺-ATPase activity was enhanced significantly as compared with puerarin alone. Therefore, it was concluded that menthol had the abilit of promoting the transport and the mechanism might be related to increasing membrane fluidity and activating Ca²⁺-ATPase.

Study on mechanism of enhanced HaCaT cellular uptake of tetrahydropalmatine by asarum essential oil and sinapine in Sanfu Patch / 中草药

Objective To study the mechanism of enhanced HaCaT cellular uptake of tetrahydropalmatine (THP) by asarum essential oil (AEO) and sinapine thiocyanate (SPT) in Sanfu Patch. Methods Effect of SPT, AEO, and THP on cell viability of HaCaT were determined by MTT. HaCaT cellular uptake of THP and the enhancing effects of AEO and SPT on THP uptake were visualized with confocal laser scanning microscope (CLSM) based on the green autofluorescence of THP, and the THP uptake content by HaCaT was further determined with HPLC. Moreover, HaCaT cells were labeled with diphenylhexatriene (DPH). After the labeled cells were treated with AEO, SPT, and THP, respectively, the cellular membrane fluidity was determined with fluorescence polarization technology. Results THP fluorescence intensity within HaCaT cells was significantly increased when THP was co-delivered with AEO or SPT respectively, and the THP content with each group within the cells was also significantly higher than that of THP delivered alone. In addition, AEO was superior to SPT in enhancing THP uptake by HaCaT cells. The fluorescence polarization and membrane micro-viscosity of HaCaT cells were significantly decreased after AEO treatment, which indicated that membrane fluidity was increased by the treatment with AEO. However, SPT or THP did not present the character of increasing the membrane fluidity.Conclusion HaCaT cellular uptake of THP can be enhanced by AEO and SPT of Sanfu Patch, in which AEO enhances the cellular uptake of THP through increasing the cellular membrane fluidity, while the mechanism of SPT in enhancing THP cellular uptake remains further clarification.

Effects of microemulsion on transport and mechanism of puerarin in MDCK-MDR1 cells / 中草药

Objective To explore the effect of microemulsion on the transport and mechanism of puerarin in blood brain barrier (BBB) cell model MDCK-MDR1. Methods MTT assay was used to evaluate the cytotoxicity of puerarin microemulsion and solution, and determine the appropriate concentration of administration. The bilateral transport characteristics of puerarin solution-microemulsion was investigated in MDCK-MDR1 monolayer. Immunohistochemical staining was used to study the expression of tight junction proteins, and the changes in cell membrane fluidity was studied by fluorescence bleaching recovery, and the changes of membrane potential was measured by anion probe combined with flow cytometry. The mechanism of the effect of microemulsion on puerarin transport was clarified. Results The MDCK-MDR1 showed no significant toxicity when the mass concentration of puerarin solution ranged from 50 to 300 μg/mL and the microemulsion dilution was over 500 times. The Papp value in absorption direction of puerarin solution on MDCK-MDR1 monolayer was 1.04 × 10-6 cm/s, and the Papp value of excretion direction was 1.05 × 10-6 cm/s. The Papp value of puerarin in microemulsion was significantly increased compared with that in solution (P < 0.05). Microemulsification could reduce the expression of Claudin-1, Occludin, ZO-1, and F-actin in MDCK-MDR1, promote cell membrane flow, and decrease cell membrane potential. Conclusion Microemulsion can promote the bilateral transport of puerarin in the BBB cell model MDCK-MDR1. The mechanism is closely connected with the opening of tight junctions, increasing the cell membrane fluidity, making the cell depolarizing and reducing membrane potential, and increasing the permeation of paracellular.

Study on prescription and process of Fritillariae Thunbergii Bulbus formula granules based on characterization of powdery / 中草药

Objective: To study the prescription and process of Fritillariae Thunbergii Bulbus (FTB) formula granules. Methods: FTB was extracted by decocting method. FTB extract powder was prepared by spray drying method. Wet-extruding granulating, extruded-rolling granulating, and one-step granulating were adopted for preparing the formula granules. A comprehensive evaluation method was based on the powder fluidity parameters such as the rest angle, the final volume reduction degree (a), the filling velocity constant (b, k), and the yield of the particles, to optimize the optimum preparation process, prescription excipients and their proportion. An HPLC method was used to determine the contents of peimine and peiminine. The chromatographic column was the Agilent Zorbax Eclipse XDB-C18 column (150 mm × 4.6 mm, 5 μm), and the flow phase was Acetonitrile-water-diethylamine (70:30:0.03); The volume flow was 1.0 mL/min; The column temperature was 30 ℃. Results: The synthesis score of the FTB formula granules prepared by one-step granulating method was the highest. The best prescription was the extract powder-dextrin- 95% ethanol solution (100:100:160). The particle yield was 91.3%, the rest angle was 30.73°, the value of a was 0.109 1, the value of b was 0.025 5 and the value of k was 0.030 1, the fluidity of the granules was good and the yield was high. The content of peimine was 0.305% and the content of peiminine was 0.098% in the particles by HPLC. Conclusion: In this experiment, the formulation and process of FTB formula granules were designed to met the design requirements, which could be used in the production of the technology.

Mitochondrial ATPase activity and membrane fluidity changes in rat liver in response to intoxication with Buckthorn (Karwinskia humboldtiana)

BACKGROUND: Karwinskia humboldtiana (Kh) is a poisonous plant of the rhamnacea family. To elucidate some of the subcellular effects of Kh toxicity, membrane fluidity and ATPase activities as hydrolytic and as proton-pumping activity were assessed in rat liver submitochondrial particles. Rats were randomly assigned into control non-treated group and groups that received 1,1.5 and 2 g/Kg body weight of dry powder of Kh fruit, respectively. Rats were euthanized at day 1 and 7 after treatment. RESULTS: Rats under Kh treatment at all dose levels tested, does not developed any neurologic symptoms. However, we detected alterations in membrane fluidity and ATPase activity. Lower dose of Kh on day 1 after treatment induced higher mitochondrial membrane fluidity than control group. This change was strongly correlated with increased ATPase activity and pH gradient driven by ATP hydrolysis. On the other hand, membrane fluidity was hardly affected on day 7 after treatment with Kh. Surprisingly, the pH gradient driven by ATPase activity was significantly higher than controls despite an diminution of the hydrolytic activity of ATPase. CONCLUSIONS: The changes in ATPase activity and pH gradient driven by ATPase activity suggest an adaptive condition whereby the fluidity of the membrane is altered.

Effects of Borneol on Membrane Fluidity and Membrane Potential of HaCaT Cell / 中国中医药信息杂志

Objective To investigate the action mode of borneol on activity of epidermal skin;To investigate action mode of borneol as penetration enhancer. Methods The well-established and standard penetration enhancer Azone was employed as a positive control in this study. The cytotoxicities of borneol and Azone on HaCaT cells were detected by CCK-8 assay, and their half 50% inhibitory concentrations (IC50) were calculated. The fluorescence recovery after photo bleaching was employed to investigate the effect of borneol and Azone on membrane fluidity, and the flow cytometer was used to monitor the changes of membrane potential of HaCaT cell after treated with these penetration enhancers. Results The IC50 values of borneol and Azone were 2.826 , 0.172 mmol/L, respectively. Borneol could significantly improve the membrane fluidity in a concentration-dependent manner, and effectively decrease the membrane potential of HaCaT cell, which exhibited the performances similar to those of Azone. Conclusion The penetration enhancement mechanism of borneol was associated with the concentrations of Ca2+ in keratinocytes, which changes the membrane fluidity and membrane potential of HaCaT cell.

Functionality of GalenIQ 721 as excipient for direct compression tablets.

The purpose of the work is the comparative evaluation of GalenIQ 721, against known excipients such as Pharmatose M200 and Alfacel type 102. The evaluated parameters included compactibility curves, tablet ejection pressure, disintegration time and flowability of individual powders and mixtures of the excipients and amoxicillin. The surrogate and explicit compactibility of Alfacel 102 (492 N; 7.9 N) is superior, followed by GalenIQ 721 (310 N; 0.93 N) and Pharmatose M200 (203 N; -2.8 N). The lubricity of Alfacel 102 is superior (ejection pressure - Pe=0.590 MPa), followed by GalenIQ 721 (Pe=6.45 MPa) and Pharmatose M200 (Pe=6.51 MPa). Disintegrability of tablets was better by GalenIQ (0.33 s/N), followed by Alfacel 102 (2.48 s/N) and Pharmatose M200 (4.47 s/N).GalenIQ 721 displays a powder fluidity of (14.4 g/s), followed by Alfacel 102 (7.88 g/s) and Pharmatose M200 (0.99 g/s). The tableting functionality of GalenIQ 721 is better than that of Pharmatose M200 but inferior of that of Alfacel 102. Although the GalenIQ 721 characteristics, predominantly brittle, are expected to be more stable to changes in formula composition and process conditions than those of Alfacel 102, plastic behavior.

Gender differences in alcohol-induced oxidative stress and altered membrane properties in erythrocytes of rats.

Alcohol-induced oxidative stress leads to imbalance between reactive oxygen species (ROS) and the antioxidant defense system, resulting in oxidative damage to membrane components such as lipids and proteins, ultimately altering membrane properties. In this study, we assessed oxidative stress status and alterations in erythrocyte membrane properties in alcohol-administered rats with respect to gender difference. Alcohol (20% v/v) administered rats of both genders showed significant changes in plasma lipid profile with elevated nitrite/nitrate levels. Furthermore, alcohol-administration significantly decreased erythrocyte antioxidant enzymes and enhanced erythrocyte membrane lipid peroxidation, cholesterol/phospholipid (C/P) ratio and Na+/K+-ATPase activity in both males and females. Besides, anisotropic studies revealed that alcohol-administration significantly decreased erythrocyte membrane fluidity. In conclusion, alcohol- administration significantly increased oxidative stress by decreasing antioxidant status, and subsequent generation of ROS altered membrane properties by altering fluidity and Na+/K+-ATPase activity. Female rats were more vulnerable to alcohol-induced biochemical and biophysical changes in plasma and erythrocyte including oxidative stress than male rats.

Caracterização da superexpressão do fator sigma ECF σx em Pseudomonas aeruginosa PA14 / Characterization ofthe ECF sigma fator σx overexpression in Pseudomonas aeruginosa PA14

Pseudomonas aeruginosa é uma proteobactéria do grupo gama muito versátil, capaz de colonizar ambientes variados e infectar hospedeiros filogeneticamente distintos, incluindo humanos imunocomprometidos. Os fatores sigma de função extracitoplasmática (ECF) são membros de sistemas de sinalização de superfície celular (CSS), abundantes em P. aeruginosa. Vinte genes codificando fatores sigma ECF estão presentes nos genomas sequenciados de P. aeruginosa, a maioria fazendo parte de sistemas TonB relacionados à captação de ferro. Neste trabalho, seis fatores sigma pobremente caracterizados foram superexpressos na linhagem PA14 a partir de um promotor induzível por arabinose para investigar seu papel na expressão dos sistemas de dois componentes PvrSR e RcsCB, que atuam na regulação da fímbria CupD, além de sua influência no crescimento de culturas de P. aeruginosa. Não foi observado efeito positivo de nenhum dos fatores sigma testados na expressão dos sistemas de dois componentes e a superexpressão de cinco deles tampouco levou a qualquer alteração no crescimento, porém a produção de piocianina foi alterada na superexpressão de PA14_55550 e a superexpressão de PA14_26600 e PA14_46810 levou a um discreto aumento no início da formação de biofilme em PA14. Por outro lado, culturas superexpressando σx (ALB04) apresentaram um perfil alterado de lipopolissacarídeo e uma curva de crescimento bifásica, alcançando precocemente uma fase estacionária seguida de uma recuperação do crescimento até uma segunda fase estacionária. Durante a primeira fase estacionária, a maior parte das células aumenta de tamanho e morre, mas as células remanescentes retornam à morfologia selvagem e seguem para a segunda fase de crescimento exponencial. Isso não acontece devido a mutações compensatórias, uma vez que células coletadas de pontos tardios da curva e diluídas em meio novo repetem este comportamento. Apesar de trabalhos com a linhagem PAO1 associarem σx à transcrição de oprF, que codifica a principal porina não específica de Pseudomonas, nas condições dos nossos ensaios em PA14 a expressão dessa porina não foi induzida pela superexpressão de σx. Assim, os efeitos observados nessa superexpressão também não podem ser atribuídos a OprF. A transcrição de oprF em PA14 mostrou-se majoritariamente dependente da região promotora a que se atribui a ligação de σ70, ao contrário dos relatos na literatura da dependência da região de ligação a σx. Análises proteômicas foram realizadas para investigar os elementos envolvidos nesses efeitos de superexpressão de σx, o que revelou a indução de diversas enzimas envolvidas na via de biossíntese de ácidos graxos. As células superexpressando σx apresentam uma maior proporção de ácidos hexadecanoico (C16) e hexadecenoico (C16:1) e dados de anisotropia mostram uma maior fluidez da(s) membrana(s). Este trabalho é o primeiro relato de um fator sigma ECF envolvido em biossíntese de lipídeos em P. aeruginosa

Pseudomonas aeruginosa is a very versatile gammaproteobacteria, able to colonize different environments and to infect phylogenetically distinct hosts, including immunocompromised humans. The extracytoplasmic function sigma factors (ECFs) are members of cell signaling systems (CSS), abundant in P. aeruginosa. Twenty genes coding for ECF sigma factors are present in the sequenced genomes of P. aeruginosa, most of them being part of TonB systems related to iron uptake. In this work, six poorly characterized sigma factors were overexpressed in strain PA14 from an arabinose inducible promoter to investigate their role in the expression of the two-component systems PvrSR and RcsCB, which regulates CupD fimbria, and their influence in P. aeruginosa cultures growth. None of the tested sigma factors led to two-component systems upregulation and overexpression of five of them caused no change in the growth profile, but pyocyanin production was altered in PA14_55550 overexpression and PA14_26600 and PA14_46810 overexpression led to a slight increase in biofilm initiation in PA14. By the other side, cultures overexpressing σx (ALB04) presented an altered lipopolysaccharide profile and a biphasic growth curve, reaching an early stationary phase followed by a growth resuming untill a second stationary phase. During the early stationary phase, most cells swells and dies, but the remaining cells return to wild type morphology and proceed to the second exponential phase of growth. This is not due to compensatory mutations, since cells collected from late points of the curve and diluted in fresh medium repeat this behavior. Although studies with strain PAO1 associate σx with transcription of oprF, encoding the major nonspecific porin of Pseudomonas, under our experiments conditions with PA14, this porin expression is not induced by σx overexpression. Thus, the effects observed in this overexpression cannot be attributed to OprF. Transcription of oprF in PA14 proved to be mainly controlled by the σ70-dependent promoter region instead of the σx-dependent promoter region reported in the literature. Proteomic analyses were performed to investigate the elements involved in these effects of σx overexpression, which revealed the induction of several enzymes involved in fatty acids biosynthesis. Cells overexpressing σx exhibit a greater proportion of hexadecanoic (C16) and hexadecenoic (C16: 1) acids and anisotropy data show higher fluidity of the membrane (s). This work is the first report of an ECF sigma factor involved in lipid biosynthesis in P. aeruginosa

F2α-isoprostane, Na+-K+ ATPase and membrane fluidity of placental syncytiotrophoblast cell in preeclamptic women with vitamin E supplementation.

Background: The aim of our study was to analyze F2α-isoprostane level, Na+-K+ ATPase activity and placental syncytiotrophoblast cell membrane fluidity in preeclamptic women who received vitamin E supplementation. Methods: The study was conducted between September 2003 and February 2005 at Budi Kemuliaan Maternity Hospital, Central Jakarta. Samples were 6 preeclamptic women with vitamin E supplementation, 6 preeclamptic women without vitamin E supplementation and 6 normal pregnant women. The dose of vitamin E was 200 mg daily. F2α-isoprostane was measured with ELISA reader at λ of 450 nm. Cell membrane fluidity was measured by comparing the molar ratio of total cholesterol and cell membrane phospholipid concentration. The cholesterol was measured by Modular C800 using Roche reagent. Phospholipid was measured by Shimadzu RF5301PC spectrofluorometer (excitation 267 nm, emission 307 nm). Na+-K+ ATPase activity was inhibited by ouabain. Pi production was measured with Fiske and Subbarow method using spectrophotometer at λ of 660 nm. Data was analyzed using F test with one-way ANOVA. Results: Vitamin E supplementation in preeclamptic women decreased the oxidative stress, indicated by significantly lower level of F2α-isoprostane compared to those without vitamin E (26.72 ± 11.21 vs 41.85 ± 7.09 ng/mL, respectively, p = 0.017). Membrane fluidity in syncytiotrophoblast cell of preeclampsia with vitamin E group was maintained at 0.39 ± 0.08 while in those without vitamin E was 0.53 ± 0.14 (p = 0.04). Na+-K+ ATPase activity in syncytiotrophoblast cell membrane was not affected by vitamin E (p = 0.915). Conclusion: Vitamin E supplementation in preeclamptic women decreases F2α-isoprostane level and maintains cell membrane fluidity of syncytiotrophoblast cells; however, it does not increase Na+-K+ ATPase enzyme activity.

Radiomodulation by Hoechst 33258 against radiation- induced damage in murine splenocytes.

In this study modulatory effect of Hoechst 33258 on radiation induced membrane related signaling events which ultimately leads to apoptosis has been investigated. Splenocytes from swiss albino mice were irradiated in air at room temperature in a gamma chamber (240 TBq 60Co Model 4000 A) at the dose-rate of 0.052 Gys-1. Membrane lipid peroxidation, fluidity, specific activities of antioxidant enzymes, levels of nitric oxide, glutathione and apoptosis in presence and absence of different concentrations of Hoechst 33258 has been assayed. DNA binding activity of nuclear factor kappa B and activator protein–1 was also assayed by electrophoretic mobility shift assay. Modulatory effect of Hoechst 33258 was examined at 3 and 5 Gy using different concentrations (10, 20 and 30 µM). Hoechst 33258 was found to inhibit radiation induced peroxidative damage and fluidity and lowered the level of nitric oxide and apoptosis - as evident by DNA ladder assay and FACS, indicating free radicals scavenging potential. Dot plot diagramme clearly showed that 30 µM Hoechst 33258 caused 14% and 19% decrease in apoptotic cells at 3 Gy and 5 Gy of radiation respectively (compared to irradiated control group). Further DNA binding activity of nuclear factor kappa B and activator protein–1 was also inhibited but the antioxidant potential of the cells was enhanced. These findings support that Hoechst 33258 protects the cell from undergoing apoptosis. Hoechst 33258 may have interacted and has an ability to protect splenocytes against radiation induced apoptosis through modulation of membrane-related signaling events and antioxidant status.

Electron paramagnetic resonance study of lipid and protein membrane components of erythrocytes oxidized with hydrogen peroxide

Electron paramagnetic resonance (EPR) spectroscopy of spin labels was used to monitor membrane dynamic changes in erythrocytes subjected to oxidative stress with hydrogen peroxide (H2O2). The lipid spin label, 5-doxyl stearic acid, responded to dramatic reductions in membrane fluidity, which was correlated with increases in the protein content of the membrane. Membrane rigidity, associated with the binding of hemoglobin (Hb) to the erythrocyte membrane, was also indicated by a spin-labeled maleimide, 5-MSL, covalently bound to the sulfhydryl groups of membrane proteins. At 2% hematocrit, these alterations in membrane occurred at very low concentrations of H2O2 (50 µM) after only 5 min of incubation at 37°C in azide phosphate buffer, pH 7.4. Lipid peroxidation, suggested by oxidative hemolysis and malondialdehyde formation, started at 300 µM H2O2 (for incubation of 3 h), which is a concentration about six times higher than those detected with the probes. Ascorbic acid and α-tocopherol protected the membrane against lipoperoxidation, but did not prevent the binding of proteins to the erythrocyte membrane. Moreover, the antioxidant (+)-catechin, which also failed to prevent the cross-linking of cytoskeletal proteins with Hb, was very effective in protecting erythrocyte ghosts from lipid peroxidation induced by the Fenton reaction. This study also showed that EPR spectroscopy can be useful to assess the molecular dynamics of red blood cell membranes in both the lipid and protein domains and examine oxidation processes in a system that is so vulnerable to oxidation.

Growth, fatty acid profile in major lipid classes and lipid fluidity of Aurantiochytrium mangrovei Sk-02 as a function of growth temperature

Aurantiochytrium mangrovei Sk-02 was grown in a medium containing glucose (40 g/l), yeast extract (10 g/L) and sea salts (15 g/L) at temperatures ranging from 12 to 35°C. The fastest growth (µmax= 0.15 h-1) and highest fatty acid content of 415 mg/g-dry cell weight were found in the cells grown at 30°C. However, the cells grown at 12°C showed the highest percentage of polyunsaturated fatty acid (PUFA) (48.6% of total fatty acid). The percentage of docosahexaenoic acid (DHA) and pentadecanoic acid (C15:0) decreased with an increase in the growth temperature, whereas, palmitic acid (C16:0), stearic acid (C18:0) and DPA (C22:5n6) increased with an increase in the growth temperature. The composition of the major lipid class (%w/w) was slightly affected by the growth temperature. The fluidity of the organelle membrane or intracellular lipid (by DPH measurement) decreased with an increase in the growth temperatures, while the plasma membrane fluidity (by TMA-DPH measurement) could still maintain its fluidity in a wide range of temperatures (15 - 37°C). Furthermore, the distribution of DHA was found to be higher (36 - 54%) in phospholipid (PL) as compared to neutral lipid (NL) (20 - 41%).