RESUMEN
Protein kinases are the family of attractive enzyme targets for drug design with relevance to cancer biology. Serine arginine protein kinase 1 (SRPK1) is responsible for the phosphorylation of serine/arginine (SR)-rich proteins. Alternative Splicing Factor/Splicing Factor 2 (ASF/SF2) involved in mRNA editing. ASF/SF2 is over expressed in many cancers and plays crucial roles in the cell survival. Phosphorylation of ASF/SF2 is decisive for its functions in cancer. In search of potential anticancer therapeutic agents for attenuating phosphorylation of ASF/SF2, we have explored specific and potential inhibitors of SRPK1 from natural and drug like compounds databases using in-silico methods. Compound ZINC02154892 (C02) was found to be the most potent inhibitor for SRPK1. In-vitro molecular and cell biology studies have shown C02 as a potent and specific inhibitor of phosphorylation of ASF/SF2 and cell survival in leukemic cell line. Structural analysis of SRPK1 with compound C02 revealed a unique pattern of binding targeting ATP binding site along with inhibiting recruitment of ASF/SF2 by SRPK1. The possibilities of compound C02 to be used as a lead compound paving way for the development of potent and specific inhibitors of SRPK1 for designing of novel potential anticancer inhibitor is inferred from the current studies.
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Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Células A549 , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Células HeLa , Humanos , Concentración 50 Inhibidora , Células Jurkat , Células K562 , Simulación del Acoplamiento Molecular , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/metabolismo , Factores de Empalme Serina-Arginina/metabolismoRESUMEN
Oryza punctata Kotschy ex Steud. (BB, 2n = 24) is a wild species of rice that has many useful agronomic traits. An interspecific hybrid (AB, 2n = 24) was produced by crossing O. punctata and Oryza sativa variety Punjab Rice 122 (PR122, AA, 2n = 24) to broaden the narrow genetic base of cultivated rice. Cytological analysis of the pollen mother cells (PMCs) of the interspecific hybrids confirmed that they have 24 chromosomes. The F1 hybrids showed the presence of 19-20 univalents and 1-3 bivalents. The interspecific hybrid was treated with colchicine to produce a synthetic amphiploid (AABB, 2n = 48). Pollen fertility of the synthetic amphiploid was found to be greater than 50% and partial seed set was observed. Chromosome numbers in the PMCs of the synthetic amphiploid were 24II, showing normal pairing. Flow cytometric analysis also confirmed doubled genomic content in the synthetic amphiploid. Leaf morphological and anatomical studies of the synthetic amphiploid showed higher chlorophyll content and enlarged bundle sheath cells as compared with both of its parents. The synthetic amphiploid was backcrossed with PR122 to develop a series of addition and substitution lines for the transfer of useful genes from O. punctata with least linkage drag.
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Cruzamientos Genéticos , Hibridación Genética , Oryza/genética , Fitomejoramiento , Ploidias , Cromosomas de las Plantas , Estudios de Asociación Genética , Meiosis/genética , Oryza/anatomía & histología , Hojas de la PlantaRESUMEN
BACKGROUND: The immune response against tuberculosis in lions is still poorly defined and our understanding is hampered by the lack of lion specific reagents. The process for producing antibodies against a specific antigen is laborious and not available to many research laboratories. As the search for antibody cross-reactivity is an important strategy for immunological studies in veterinary medicine, we have investigated the use of commercially available antibodies to characterize T cell subsets in African lions (Panthera leo). RESULTS: Commercially available antibodies were screened and investigated the influence of two different sample processing methods, as well as the effect of time delay on cell surface marker expression on lion lymphocytes. Using commercially available antibodies, we were able to identify CD4+, CD5+, CD8+, CD14+, CD25+, CD44+ and CD45+ T lymphocytes in samples obtained by density gradient centrifugation as well as red cell lysis of lion whole blood. Two distinct lymphocyte populations, which differed in size and phenotype, were observed in the samples processed by density gradient centrifugation. CONCLUSION: Commercially available antibodies are able to differentiate between T lymphocyte subsets including immune effector cells in African lion whole blood, and possibly give insight into unique specie phenotypes.
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Anticuerpos/metabolismo , Citometría de Flujo/métodos , Linfocitos/citología , Panthera , Animales , Proyectos Piloto , Linfocitos T/citologíaRESUMEN
There is a lack of suitable correlates of immune protection against Mycobacterium tuberculosis (Mtb) infection. T cells and monocytes play key roles in host immunity against Mtb. Thus, a method that allows assessing their interaction would contribute to the understanding of immune regulation in tuberculosis (TB). We have established imaging flow cytometer (IFC) based in vitro assay for the analysis of early events in T cell-monocyte interaction, upstream of cytokine production and T cell proliferation. This was achieved through short term stimulation of peripheral blood mononuclear cells (PBMC) from healthy Norwegian blood donors with Mycobacterium bovis Bacille Calmette-Guérin (BCG). In our assay, we examined the kinetics of BCG uptake by monocytes using fluorescently labeled BCG and T cell-monocyte interaction based on synapse formation (CD3/TCR polarization). Our results showed that BCG stimulation induced a gradual increase in the proportion of conjugated T cells displaying NF-κB translocation to the nucleus in a time dependent manner, with the highest frequency observed at 6â¯h. We subsequently tested PBMC from a small cohort of active TB patients (nâ¯=â¯7) and observed a similar BCG induced NF-κB translocation in T cells conjugated with monocytes. The method allowed for simultaneous evaluation of T cell-monocyte conjugates and T cell activation as measured by NF-κB translocation, following short-term challenge of human PBMC with BCG. Whether this novel approach could serve as a diagnostic or prognostic marker needs to be investigated using a wide array of Mtb specific antigens in a larger cohort of patients with different TB infection status.
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Antígenos Bacterianos/inmunología , Citometría de Flujo/métodos , Monocitos/inmunología , Mycobacterium bovis/inmunología , Mycobacterium tuberculosis/inmunología , Linfocitos T/inmunología , Tuberculosis/inmunología , Proliferación Celular , Humanos , Activación de Linfocitos , Monocitos/patología , Linfocitos T/patología , Tuberculosis/diagnóstico , Tuberculosis/patologíaRESUMEN
BACKGROUND: The significance of FMS-like tyrosine kinase 3 (FLT3)-ITD mutation in acute myeloid leukemia (AML) prognosis has been well established. The aims of this study were to investigate the prognostic impact of the FLT3 protein (CD135) expression and its association with FLT3-ITD mutation, and to identify its role in minimal residual disease. PATIENTS AND METHODS: CD135 was measured by flow cytometry on leukemic blasts of 257 adults with de novo AML. High expression of CD135 ≥ 20% was correlated with clinical, laboratory, and other prognostic factors that influenced treatment outcome. FLT3-ITD mutation was tested by PCR. RESULTS: The frequency of CD135 expression was 138 (53.7%) of 257. FLT3-ITD was detected in (21.4%). Positive CD135 expression was associated with high total leukocyte count (P = .006), platelet count (P = .003), monocytic leukemia (P < .001), and CD34 (P = .008) and CD117 (P = .006) expression. CD135 expression ≥ 25% was a predictor of FLT3-ITD mutation (P = .03). CD135 overexpression was a negative predictor of complete remission and of postinduction minimal residual disease at days 14 and 28 (P < .001). CD135 had an adverse impact on overall and disease-free survival (68.5% vs. 15%, P = .002). Multivariate analysis indicated CD135 was the sole independent prognostic factor for overall survival (hazard ratio = 2.49; 95% confidence interval, 1.855-3.345; P < .001). CONCLUSION: CD135 is emerging as a prognostic factor, a new marker for minimal residual disease, and a potential novel therapeutic target of AML.
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Biomarcadores de Tumor/análisis , Citometría de Flujo , Leucemia Monocítica Aguda/inmunología , Células Progenitoras Linfoides/inmunología , Tirosina Quinasa 3 Similar a fms/análisis , Adolescente , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/genética , Supervivencia sin Enfermedad , Egipto , Femenino , Predisposición Genética a la Enfermedad , Humanos , Leucemia Monocítica Aguda/tratamiento farmacológico , Leucemia Monocítica Aguda/genética , Leucemia Monocítica Aguda/mortalidad , Células Progenitoras Linfoides/efectos de los fármacos , Masculino , Persona de Mediana Edad , Mutación , Neoplasia Residual , Fenotipo , Valor Predictivo de las Pruebas , Estudios Prospectivos , Estudios Retrospectivos , Factores de Tiempo , Adulto Joven , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
The number of white blood cell (WBC) in semen is an important indicator of genital tract inflammation in male infertility. The peroxidase assay is the recommended reference method for seminal WBC counting. However, it is time-consuming and may cause relatively heavy workload in daily routine. Meanwhile, the main component in the reagent of peroxidase test is harmful to human and the environment. In this study, we evaluated the analytical performance of the Sysmex UF-1000i that is a urine flow cytometer as a screening tool for genital tract infection in male infertility patients through the counting of seminal WBC. We examined 143 semen samples and compared the results of UF-1000i and manual microscopy. The intra-assay variability, stability and linearity studies were performed. The intravariability (CV %) of seminal WBC count by Sysmex UF-1000i was 2.34%-9.65%. The method of UF-1000i displayed a good agreement with the reference assay of manual microscopy, and the r value for correlation of seminal WBC count between UF-1000i and manual microscopy was over 0.999 (p < 0.001). The Sysmex UF-1000i is capable of producing reliable seminal WBC count consistent with that obtained by manual microscopy. It is a suitable alternative to the manual microscopy, thus reduces the workload.
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Enfermedades de los Genitales Masculinos/diagnóstico , Infertilidad Masculina/diagnóstico , Inflamación/diagnóstico , Tamizaje Masivo/instrumentación , Semen/citología , Adulto , Pruebas de Enzimas/métodos , Enfermedades de los Genitales Masculinos/complicaciones , Humanos , Infertilidad Masculina/etiología , Inflamación/complicaciones , Recuento de Leucocitos/instrumentación , Recuento de Leucocitos/métodos , Masculino , Tamizaje Masivo/métodos , Microscopía , Persona de Mediana Edad , Peroxidasa/metabolismo , Análisis de Semen/instrumentación , Análisis de Semen/métodos , Carga de Trabajo/estadística & datos numéricosRESUMEN
Objective To investigate the role of the activated rhuPAa-melittin in ovarian cancer cells and to study the inhibitory effect of rhuPAa-melittinon on ovarian cancer cells. Methods rhuPAa-melittin was used to treat the ovarian cancer cells at different concentrations for 48 hrs. Then flow cytometery was applied to detect the cell cycle and cell apoptosis. rhuPAa-melittin protein was delivered to the mouse mode to investigate the effect of rhuPAa-melittin on the growth of the xenotransplanted tumor. Results rhuPAa-melittin was used to treat ovarian cancer cells at the concentration of 0 ,4 ,8 and 16 μg/mL for 48 hrs ,respectively. The results of cell apoptosis assay was 1.16%,3.83%,6.51% and 10.2%,respectively. Moreover,different concentrations of rhuPAa-melittin had no effects on the cells at G0/G1 phase,rhuPAa-melittin inhibited S phase cells to process into G2/M phase, contributing to suppressing the growth of ovarian cancer cells. Conclusion The in vivo and in vitro studies revealed that rhuPAa-melittin inhibits the growth of ovarian cancer.
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Objective Establish detection method to measure Vibrio vulnificus rapidly and accurately.MethodsUsing flow cytometry(FCM)and a 5'-FITC fluorescent labeled aptamer with high binding affinity to detect Vibrio vulnificus rapidly.Measure a series of concentrations of Vibrio vulnificus to identify the Limit of Blank, Lower Limit of Detection, Linearity Range, etc.ResultsCombined application of FCM and the aptamer can detect Vibrio vulnificus rapidly with the duration less than 1 hour and lower limit of detection as low as 29 CFU/mL.Conclusion The aptamer targeting Vibrio vulnificus is an excellent detective element, while FCM can realize accurate quantitative detection.The detection method has great application potential.
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Objective To investigate the role of the activated rhuPAa-melittin in ovarian cancer cells and to study the inhibitory effect of rhuPAa-melittinon on ovarian cancer cells. Methods rhuPAa-melittin was used to treat the ovarian cancer cells at different concentrations for 48 hrs. Then flow cytometery was applied to detect the cell cycle and cell apoptosis. rhuPAa-melittin protein was delivered to the mouse mode to investigate the effect of rhuPAa-melittin on the growth of the xenotransplanted tumor. Results rhuPAa-melittin was used to treat ovarian cancer cells at the concentration of 0 ,4 ,8 and 16 μg/mL for 48 hrs ,respectively. The results of cell apoptosis assay was 1.16%,3.83%,6.51% and 10.2%,respectively. Moreover,different concentrations of rhuPAa-melittin had no effects on the cells at G0/G1 phase,rhuPAa-melittin inhibited S phase cells to process into G2/M phase, contributing to suppressing the growth of ovarian cancer cells. Conclusion The in vivo and in vitro studies revealed that rhuPAa-melittin inhibits the growth of ovarian cancer.
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The aim of the present study was to investigate the differences in biological characteristics between the rhodamine 123 (Rh123)high and Rh123low subpopulations of the renal cancer cell line 786-O and to identify evidence for the existence of cancer stem cells in renal cell carcinoma (RCC) cells. In vitro cultured RCC 786-O cells were stained with Rh123, analyzed and sorted with flow cytometry. The differences in proliferative activity, long-term differentiation and radiation sensitivity between the two subpopulations were measured and the oncogenicity of each subpopulation was evaluated according to their neoplastic growth ability in soft agar and tumor-forming ability in NOD/SCID immunodeficient mice. There were two subpopulations in the cultured 786-O cells, Rh123high and Rh123low cells. Rh123low cells were the majority among 786-O renal carcinoma cells and barely formed solid tumors in NOD/SCID mice and colonies in soft agar. By contrast, the Rh123high cells were the minority, exhibited high proliferative activity, differentiation ability and resistance to radiation and showed high tumorigenesis potential and colony forming efficiency. The Rh123high cells had stem-like characteristics in cultured RCC 786-O cells in vitro.
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Immunophenotyping of acute leukemia is one of the most important clinical application of Flow cytometery. The aim of this work is to review recent advances in flow cytometery methods, quality control, troubleshooting and its prevention and data analysis of acute leukemia. Multiparameter flow cytometery is a useful adjunct to morphology and cytochemistry and it is an invaluable tool in the diagnosis of acute leukemia.
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Objective To explore the effect of STI571 on the CD_~117 expression and differentiation of bone marrow mononuclear cells in patients with acute non-lymphocytic leukemia(ANLL). Methods CD_~117 expression before and after STI571 treatment in vitro was assayed by FACS, and the cell differentiation was observed by Giemsa and peroxidase staining. Results After different concentrations of STI571 treatment in vitro, there was different degrees of differentiation in granulocytes and monocytes from patients with ANLL. With the increase of STI571 concentration, the number of progranulocyte and promyelocyte gradually decreased, the number of myelocyte, metamyelocyte and monocyte increased, and the positive degree of peroxidase staining increased. CD_~117 expression significantly decreased after STI571 treatment compared with before it. Conclusion STI571 could induce leukemia cells of ANLL into terminal differentiation by inhibiting c-kit tyrosine kinase.