ABSTRACT
Background: A major impediment in treatment for cancers is resistance to chemotherapy and is primarily attributed to over-expression of efflux pumps. This study aimed to establish the cytotoxicity of malabaricone-A (MAL-A) in P-glycoprotein/multidrug resistance (P-gp/MDR) over-expressing hematopoietic cancer cell lines.Methods: Leukemia and multiple myeloma cell lines were indirectly evaluated for their P-gp/MDR status by examining Calcein-AM fluorescence and cell viability was assessed by the MTS-PMS assay.Results: The fluorescence of calcein was significantly decreased in three cell lines LP-1, RPMI-8226 and CEM-ADR 5000 and reversal with verapamil endorsed their P-gp/MDR activity. The mean IC50 of MAL-A in these MDR+ cell lines (5.40±1.41 to 12.33±0.78 µg/ml) was comparable with the MDR- leukemic (9.72±1.08 to 19.26±0.75 µg/ml) and multiple myeloma cell lines (9.65±0.39 to 18.05±0.17 ?g/ml).Conclusions: Irrespective of their P-gp activity, the cytotoxicity of MAL-A was comparable, making it worthy of future pharmacological consideration in multidrug resistance.
ABSTRACT
Objective To observe the effect of varying intensities of water-jet force on autologous fat graft viability.Methods Lipoaspirate was taken from 12 female patients undergoing waterjet assisted abdominal liposuction at our department.According to the intensity of water-jet force,the experimental group was divided into four subgroups:R1 (pressure,30 bar),R2 (pressure,50 bar),R3 (pressure,70 bar) and R4 (pressure,90 bar).Hand-held suction was taken as the control group C.Adipose tissue was filtered with cotton cushion and centrifuged at low speed,and the composition ratio of water and fat tissue from each group was observed.Calcein-AM/Hoechst 33342 staining was used to detect the viability of adipocytes.Results Fat aspirates was divided into four layers:oil layer,pure fat tissue,liquid and bottom sediment.Oil ratios of R1,R2,R3,R4 and C were (8.9 ± 2.3) ％,(9.6±2.1)％,(10.3±1.3)％,(14.2±1.6)％ and (9.5±1.8)％,respectively.There was no statistically significant difference between R1,R2,R3 and C (P＞0.05).Statistically significant difference was found between R4 and other groups (P＜0.001).Viability of adipocytes from R1,R2,R3,R4 and C groups were (88.1±2.8)％,(89.9±1.9)％,(84.8±2.3)％,(78.0±1.7)％ and (91.1±2.9)％ respectively.There was no statistically significant difference between R1,R2 and C (P＞ 0.05).Statistically significant difference was found between R3,R4 and C (P ＜ 0.05).Conclusions Viability of fat graft harvested under lower intensity of water-jet force (R1,R2) is higher than that harvested under higher intensity of water-jet force (R3,R4).
ABSTRACT
Objective To prepare the targeted ursolic acid liposome and evaluate its targeting and inhibitory effects on HeLa cells in vitro. Methods Using folate receptors as cell targets, the new functional targeted material Folate-CONH-PEG-NH-Cholesterol conjugate was synthesized chemically. Ursolic acid liposome was developed by modifying Folate-CONH-PEG-NH-Cholesterol using film dispersed method. The liposome was constructed with calcein as a fluorescent probe marker, and the penetrating ability of targeted liposome on HeLa cells was observed by fluorescence confocal microscopy. The uptake efficiency of targeted liposome was investigated by flow cytometry. Meanwhile, the growth inhibitory effect of ursolic acid liposome on HeLa cells was investigated. Results The targeted ursolic acid liposome prepared by thin dispersion method had obvious targeting effect on HeLa cells and have significant anti-proliferative effect. Conclusion The targeted ursolic acid liposome can effectively penetrate HeLa cells with great killing power on the activity of cervical cancer cells.
ABSTRACT
Age validation is the first step to determine shellfish species age determination. This information is vital for different inferential models used in marine ecosystem management activities. In spite that various validation techniques are used for marking carbon calcium structures, the calcein marking technique for oysters had never been used for age validation in Pinctada mazatlanica. Thus the objectives of this study included: the evaluation of calcein to mark a shell growing-edge, and the efficacy of Coomassie Blue staining on posterior shell growth, to produce visible micro growth-bands that would enable age validation of juvenile mother-of- pearl oysters. Oysters were collected and cultivated at The Perlas del Cortez S. de R. L. MI. pearl-farming opera tion, in Pichilingue, La Paz Bay, Baja California Sur, Mexico; a total of 36 oysters (shell height 11.5-36.4mm) were injected with calcein (0.125g/L), and another 50 oysters (shell height 14.8-42.7mm) were submersed in calcein (0.4 and 0.7g/L). Shell slices of calcein-marked oysters were posteriourly stained with Coomassie Blue R-25 for micro growth-band recognition. Our results showed that Calcein marking only worked by submersion and produced a concise bright lime-green florescent band along the growing-edge with clear boundaries for both concentrations. However, marks resulted better at the lower calcein concentration (0.4g/L) with more “perfect” and “good” marks on the growing-edge (p=0.0012). Commassie Blue staining technique was successful, and allowed to conclude that one micro growth-band was laid down per day, similar to other oyster species. Mean 15-d increment of shell growth height was slightly greater at the lower calcein concentration ( =0.735mm) than at the higher one ( =0.577mm) (not significant difference, p=0.198). Calcein marking of shell growing- edges and Commassie Blue staining of posterior shell growth, as a method for age validation is recommended for shellfish shell growth-band counts. This will allow back-dating for estimation of very precise colonization dates, both spatially and temporally in future work.
La validación de la edad es el primer paso para determinar las edades de las especies de moluscos, esta información es de vital importancia para los diferentes modelos de inferencia utilizados en actividades de gestión de los ecosistemas marinos. Diversas técnicas de valida- ción se utilizan para marcar estructuras de carbonato de calcio, aunque la técnica de marcado de calceína en ostras nunca se había utilizado para la validación de la edad de P. mazatlanica. Los objetivos de este estudio fueron: evaluar la calceína como marcador interno de la concha y la eficiencia del azul de Coomassie en la tinción de la matriz proteica de la concha, para facilitar la observación y conteo de micro bandas de crecimiento que permiten validar la edad de las ostras juveniles de madre perla. Las ostras fue- ron recolectadas en la costa de la empresa Perlas del Cortez S. de RL MI., en Pichilingue en Bahía de La Paz, Baja California Sur, México. Se inyectaron 36 ostras (altura de concha 11.5-36.4mm) (0.125g/L de calceína) y otras 50 ostras (altura de la concha 14.8-42.7mm) se sumergieron (0.4 y 0.7g/L de calceína). Secciones de la concha marcadas con calceína fueron teñidos con azul de Coomassie R-25 para el reconocimiento de las micro bandas de crecimiento. El marcado con calceína fue exitoso por inmersión y produjo una banda fluorescente de color verde lima brillante con- cisa a lo largo del crecimiento interno de la concha. Sin embargo, las marcas fueron mejores a una concentración de calceína inferior (0.4g/L), con mayor cantidad de marcas “buenas” y “perfectas” (p=0.0012). La técnica de tinción con azul de Commassie también fue exitosa. Se detectó un crecimiento diario por micro banda, similar a lo encontrado en otras especies de ostras. La diferencia del crecimiento medio en relación a la altura de la concha en un lapso de 15 días, fue ligeramente mayor con una concentración de calceína inferior ( =0.735mm) que con la de mayor concentración ( =0.577mm), pero no significativamente (p=0.198). El marcado de conchas con calceína y tinción de matrices proteicas con azul de Coomassie posterior a su crecimiento, es recomendando como un método para la validación de la edad facilitando el conteo de micro bandas de crecimiento internas de la concha. Además, permitirá estimar edades con el fin de predecir fechas de colonización y ubicación de bancos naturales.
Subject(s)
Animals , Animal Identification Systems/methods , Indicators and Reagents/administration & dosage , Pinctada/growth & development , Rosaniline Dyes/administration & dosage , Aquaculture , Mexico , Pinctada/classification , Reproducibility of ResultsABSTRACT
Objective:To develop and optimize a novel assay for determination of cytotoxicity based Calcein -AM release.Methods:The target cells stained by Calcein-AM dye,then effectors and targets were incubated at E/T ratios from 30∶1-1∶1 for 4 h at 37℃,and the supernatant of reactions were detected by Fluorescence-Measurement to analyze specific cytotoxity.Results:The optimal excitation and emission wave lengths of Calcein were 485 nm and 515 nm.Dilutions of target cells stained by Calcein-AM had a linear relationship with measured fluorescence values.The Calcein-AM dye used to stain the living cells was shown to have a low spontaneous leakage rate-less than 15% in 4 hours at 37℃.Cytotoxicity activity of CIK showed a significant and positive correlation with E/T ratio when incubated at 4 h.Conclusion:The developed cytotoxicity test by Calcein-AM release is accurate and can avoid the application of radioactive reagents.
ABSTRACT
OBJECTIVE: To optimize the prescription of the novel temperature-sensitive liposomes(NTSL). METHODS: Calcein-loaded temperature-sensitive liposomes with different phospholipid composition (DPPC-MSPC-DSPE-PEG200=86:10:4 and N (20% HSPC + 80% DPPC) -MSPC-DSPE-PEG200=92:4:4) were prepared by the thin film -hydration method. Based on the fluorescence self-quenching off characteristics of calcein, the release behavior of the above liposomes was compared when exposed to 37°C (normal temperature) as well as their respective phase transition temperature (Tm). RESULTS: Compared with the traditional temperature-sensitive liposomes (TTSL), the NTSL showed more favorable temperature-sensitive release performance and more stable encapsulation ability to calcein, even with the amount of lysophospholipids was reduced by 60 percent. CONCLUSION: The application of NTSL helps to enhance the effect of temperature-sensitive release.
ABSTRACT
OBJECTIVE: To investigate the cellular delivery of calcein-loaded liposomes and the endocytosis mechanism.
ABSTRACT
The amidated analog of Plantaricin149, an antimicrobial peptide from Lactobacillus plantarum NRIC 149, directly interacts with negatively charged liposomes and bacterial membranes, leading to their lysis. In this study, four Pln149-analogs were synthesized with different hydrophobic groups at their N-terminus with the goal of evaluating the effect of the modifications at this region in the peptide's antimicrobial properties. The interaction of these peptides with membrane models, surface activity, their hemolytic effect on red blood cells, and antibacterial activity against microorganisms were evaluated. The analogs presented similar action of Plantaricin149a; three of them with no hemolytic effect (< 5%) until 0.5 mM, in addition to the induction of a helical element when binding to negative liposomes. The N-terminus difference between the analogs and Plantaricin149a retained the antibacterial effect on S. aureus and P. aeruginosa for all peptides (MIC50 of 19 µM and 155 µM to Plantaricin149a, respectively) but resulted in a different mechanism of action against the microorganisms, that was bactericidal for Plantaricin149a and bacteriostatic for the analogs. This difference was confirmed by a reduction in leakage action for the analogs. The lytic activity of Plantaricin149a is suggested to be a result of the peptide-lipid interactions from the amphipathic helix and the hydrophobic residues at the N-terminus of the antimicrobial peptide.
Subject(s)
Antimicrobial Cationic Peptides/metabolism , Bacteria/drug effects , Bacteriocins/metabolism , Cell Membrane/drug effects , Lipid Bilayers/metabolism , Antimicrobial Cationic Peptides/genetics , Bacteriocins/genetics , Lactobacillus plantarum/metabolism , Microbial Sensitivity Tests , Pseudomonas aeruginosa/drug effects , Staphylococcus aureus/drug effectsABSTRACT
Evaluation of apoptosis by flow cytometry is generally accomplished by methods that use annexin V-FITC as vital dye, which access phosphatidylserine exposed on the external membrane at the beginning of this process. In addition, the concomitant use of propidium iodide makes possible to verify the characteristic nuclear alterations in the late stages of apoptosis, as a consequence of the increase in membrane permeability. On the other hand, the use of calcein-AM in association with ethidium homodimer (EthD-1) allows the evaluation of cell apoptosis through detection of esterase activity and cellular membrane physical and chemical alterations. The aim of this study was to compare the sensibility of calcein-AM and EthD-1 with annexin V-FITC and propidium iodide for early apoptosis evaluation in peripheral blood mononuclear cell culture, obtained from HIV-infected patients. Apoptosis and cellular viability were detected and quantified by flow cytometry after 24 and 48 hours incubation times. Our results showed that calcein-AM/EthD-1 was more sensitive for apoptotic cell quantification in both incubation times than annexin V-FITC/propidium iodide (mean of 46.95 percent ± 3.56, p < 0.0001, for 24 hours and mean of 37.67 percent ± 2.47, p < 0.0014 for 48 hours), besides allowing to clearly define viable, apoptotic and dead cell populations.
Subject(s)
Humans , Apoptosis , /metabolism , Flow Cytometry/methods , Fluoresceins/metabolism , Fluorescent Dyes/metabolism , HIV Infections/pathology , Lymphocytes/physiology , Biomarkers/metabolism , Cell Membrane Permeability , Ethidium/analogs & derivatives , Ethidium/metabolism , Propidium/metabolism , Sensitivity and Specificity , Time FactorsABSTRACT
Amyloid beta (Abeta) neurotoxicity is believed to play a critical role in the pathogenesis of Alzheimer's disease (AD) mainly because of its deposition in AD brain and its neuronal toxicity. However, there have been discrepancies in Abeta-induced cytotoxicity studies, depending on the assay methods. Comparative analysis of Abeta42-induced in vitro cytotoxicity might be useful to elucidate the etiological role of Abeta in the pathogenesis of AD. In this study, MTT, CCK-8, calcein-AM/EthD-1 assays as well as thorough microscopic examinations were comparatively performed after Abeta42 treatment in a neuronal precursor cells (NT2) and a somatic cells (EcR293). Extensive formation of vacuoles was observed at the very early stage of Abeta42 treatment in both cells. Early observation of Abeta42 toxicity as seen in vacuole formation was also shown in MTT assay, but not in CCK-8 and calcein-AM/EthD-1 assays. In addition, Abeta42 treatment dramatically accelerated MTT formazan exocytosis, implying its effect on the extensive formation of cytoplasmic vacuoles. Abeta42 seems to cause indirect inhibition on the intracellular MTT reduction as well as vacuole formation and exocytosis enhancement. Following the acute cellular dysfunction induced by Abeta42, the prolonged treatment of micromolar concentration of Abeta42 resulted in slight inhibition on redox and esterase activity. The early Abeta42-induced vacuolated morphology and later chronic cytotoxic effect in neuronal cell might be linked to the chronic neurodegeneration caused by the accumulation of Abeta42 in AD patients' brain.