RESUMEN
The human immunodeficiency virus (HIV) destroys CD4+ lymphocytes and monitoring these cells is one of the best techniques for studying HIV infection. In the present study a novel bioluminescent probe, super RLuc8-sFv, is developed in order to detect human CD4+ cells by fusion of an anti-human CD4 sFv to the C-terminus of super RLuc8. The results indicate that the probe can bind to CD4+ cells via its sFv domain; also it emits visible light through its signalling domain. Super RLuc8-sFv provides a new gateway for detection of human CD4+ cells using luminometric-based assays and may reduce the difficulties involved in, and the cost of, HIV-related diagnostic tests.
Asunto(s)
Linfocitos T CD4-Positivos , Luciferasas , Proteínas Luminiscentes , Sondas Moleculares , Anticuerpos de Cadena Única , Antígenos CD4/química , Antígenos CD4/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Humanos , Luciferasas/química , Mediciones Luminiscentes/métodos , Proteínas Luminiscentes/química , Modelos Moleculares , Sondas Moleculares/química , Unión Proteica , Conformación Proteica , Anticuerpos de Cadena Única/químicaRESUMEN
A novel and advanced Fc-binding probe FcUni-RLuc namely has been produced and functionally assayed for labelling IgGs. The Fc antibody binding sequence HWRGWV was fused to Renilla luciferase, and the purified probe was employed for bioluminescence enzyme-linked immunoabsorbance assay of Her2 positive cells.
Asunto(s)
Anticuerpos Monoclonales Humanizados/metabolismo , Neoplasias de la Mama/diagnóstico , Fragmentos Fc de Inmunoglobulinas/metabolismo , Luciferasas de Renilla/metabolismo , Sondas Moleculares , Ingeniería de Proteínas , Receptor ErbB-2/metabolismo , Anticuerpos Monoclonales Humanizados/genética , Bioensayo/métodos , Neoplasias de la Mama/metabolismo , Femenino , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Luciferasas de Renilla/genética , Mediciones Luminiscentes/métodos , Unión Proteica , Receptor ErbB-2/genética , Transducción de Señal , Trastuzumab , Células Tumorales CultivadasRESUMEN
The parent and nanosized starch, and lipid encapsulated K6[SiW11O39Co(H2O)]·nH2O (abbreviated as SEP, LEP and SiW11Co, respectively), as potent antitumor candidates, were synthesized and characterized by FT-IR spectroscopy, ICP, TG analysis, SEM and TEM images. The results show that the SiW11Co retains its parent structure after encapsulation by starch and lipid nanoparticles. Antitumor activity tests of SiW11Co and its encapsulated forms were carried out on two types of human cancer cells, MCF-7 and HEK-293 by MTT method. The encapsulated forms exhibited the higher antitumor activity compared to the parent SiW11Co. However, this observed enhancement for the lipid encapsulated form is more than the starch counterpart, which can be related to its smaller size. These results showed that these compounds can be novel antitumor candidates. The calf thymus DNA (abbreviated as ctDNA) binding ability of SiW11Co was also investigated, using UV-Vis absorption spectroscopy, fluorescence quenching and fluorescence Scatchard plots. Absorption spectra tracing reveal 10% hyperchromism for SiW11Co. The values of 1.8×10(4) and 1.2×10(4)M(-1) were obtained for association binding constant of SiW11Co to ctDNA at R⩾1 and R<1, respectively (R is defined as the mole ratio of SiW11Co to ctDNA). It was shown that the interaction of SiW11Co with ctDNA depended on the R values. The obtained results of absorption titration rejected the intercalating binding mode and suggest the groove or outside stacking binding for SiW11Co. These results were authenticated by fluorescence quenching experiments and scatchard plots.
Asunto(s)
Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Cobalto/química , ADN/metabolismo , Nanocápsulas/química , Compuestos de Tungsteno/metabolismo , Compuestos de Tungsteno/farmacología , Antineoplásicos/química , Células HEK293 , Humanos , Lípidos/química , Células MCF-7 , Almidón/química , Compuestos de Tungsteno/químicaRESUMEN
Cell lines derived from mammalian are dominant systems for the production of recombinant therapeutic proteins because of their capacity for correct protein folding, assembly and post-translational modification. In the search of an efficient method for the production of a recombinant protein using animal cell culture, we investigated the effects of different treatment including fetal calf serum concentration, glycerol and culture temperature on a Chinese hamster ovary (CHO) cell line on the production of recombinant human growth hormone (rhGH) and recombinant Chinese hamster ovary (rCHO) viability. The GH production was assessed using ELISA and western blotting methods and cell viability was determined by flow cytometry. The production of recombinant protein increased by 2-fold when stimulatory chemical such as glycerol was added in two stages, first cells were cultured without glycerol for a period of time in order to obtain enough cell density and then glycerol was added to achieve high specific productivity.. Moreover, glycerol addition increased cell viability. Low culture temperature (below 37°C) led to enhanced cellular productivity of the rhGH by 3-fold but decreased cell viability. These findings indicate that quite simple factors such as culture temperature and addition of simple chemicals may lead to the improvement of industrial process for the production of recombinant proteins such as rhGH.
RESUMEN
The ctDNA-binding properties and in vitro antitumor activity of three water soluble Keggin type polyoxometalates (POMs): K6H[CoW11O39CpZr]·nH2O, K6H[CoW11O39CpTi·nH2O and K7H2[CoW11O39CpFe]·nH2O (abbreviated as CoWCpZr, CoWCpTi and CoWCpFe, respectively) were investigated using UV-Vis absorption spectroscopy, fluorescence spectrophotometry, cyclic voltammetry and MTT assay. The results of UV-Vis, fluorescence and cyclic voltammetry rule out intercalating binding mode and propose the groove or outside stacking binding of these POMs with ctDNA. The values of 1.30×10(4) M(-1), 1.15×10(4) M(-1) and 3.10×10(3) M(-1) were obtained for binding constant of CoWCpZr, CoWCpTi and CoWCpFe to ctDNA, respectively. The redox potential of POMs shift to more negative values in the presence of ctDNA which can be related to domination of electrostatic interaction in this system. The antitumor activity tests of these polyoxometalates (POMs) were carried out on two types of human cancer cells, MCF-7 and HEK-293 by MTT method. The results show the higher antitumor activity of CoWCpFe respect to two other that is related to its highest penetrating effectiveness for MCF-7 cells. Therefore, the antitumor activity of these POMs depends not only on their affinity to ctDNA but also strongly on their penetration ability to the cell membrane.
Asunto(s)
Antineoplásicos/química , ADN/química , Compuestos Organometálicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , ADN/metabolismo , Células HEK293 , Humanos , Células MCF-7 , Modelos Moleculares , Compuestos Organometálicos/farmacología , Espectrometría de Fluorescencia , Espectroscopía de Absorción de Rayos XRESUMEN
Evidence shows that probiotic bacteria can undergo substantial structural and morphological changes in response to environmental stresses, including antibiotics. Therefore, this study investigated the effects of penicillin G (0.015, 0.03, and 0.06 mg/l) on the morphology and adhesion of Lactobacillus acidophilus ATCC 4356, including the colony morphotype, biofilm production, hydrophobicity, H2O2 formation, S-layer structure, and slpA gene expression. Whereas only smooth colonies grew in the presence of penicillin, rough and smooth colony types were observed in the control group. L. acidophilus ATCC 4356 was found to be hydrophobic under normal conditions, yet its hydrophobicity decreased in the presence of the antibiotic. No biofilm was produced by the bacterium, despite testing a variety of different culture conditions; however, treatment with penicillin G (0.015-0.06 mg/l) significantly decreased its production of H2O2 formation and altered the S-layer protein structure and slpA gene expression. The S-protein expression decreased with 0.015 mg/l penicillin G, yet increased with 0.03 and 0.06 mg/l penicillin G. In addition, the slpA gene expression decreased in the presence of 0.015 mg/l of the antibiotic. In conclusion, penicillin G was able to alter the S-layer protein production, slpA gene expression, and certain physicochemical properties of Lactobacillus acidophilus ATCC 4356.
Asunto(s)
Antibacterianos/farmacología , Lactobacillus acidophilus/efectos de los fármacos , Lactobacillus acidophilus/crecimiento & desarrollo , Penicilina G/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Interacciones Hidrofóbicas e Hidrofílicas/efectos de los fármacos , Lactobacillus acidophilus/química , Lactobacillus acidophilus/fisiología , Propiedades de Superficie/efectos de los fármacosRESUMEN
In many conditions, bacterial surface properties are changed as a result of variation in growth medium and conditions. This study examined the influence of bile salt concentrations (0-0.1%) on colony morphotype, hydrophobicity, H2O2 concentration, S-layer protein production and slpA gene expression in Lactobacillus acidophilus ATCC 4356. It was observed that two types of colonies (R and S) were in the control group and the stress condition. When the bile level increased in the medium, the amount of S type was more than the R. A stepwise increment in the bile concentration resulted in a stepwise decline in the maximum growth rate. The results showed that hydrophobicity was increased in 0.01%-0.02% bile but it was decreased in 0.1% bile. Treatment by bile (0.01%- 0.1%) profoundly decreased H2O2 formation. S-layer protein and slpA gene expression was also altered by stress condition. S-protein expression was increased in stress condition. slpA gene expression increased in 0.01%-0.05% bile and it decreased in 0.1% bile. However, we found that different of bile salt concentrations influence on morphology and some surface properties of L. acidophilus ATCC 4356. These changes were very different in the 0.1% bile. It appears that the bacteria respond abruptly to 0.1% bile.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Ácidos y Sales Biliares/farmacología , Adhesión Celular/fisiología , Lactobacillus acidophilus/fisiología , Proteínas Bacterianas/genética , Recuento de Colonia Microbiana , Regulación de la Expresión Génica/efectos de los fármacos , Interacciones Hidrofóbicas e Hidrofílicas , Lactobacillus acidophilus/química , Lactobacillus acidophilus/efectos de los fármacos , ARN Bacteriano/química , ARN Bacteriano/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
GH treatment during critical illness and sepsis may increase mortality. A family of negative regulators of cytokine signalling, the suppressors of cytokine signalling (SOCS), have been characterised. SOCS provide a mechanism for cross-talk between the cytokine receptors, including GH. Here, we have investigated the impact of nutrition and GH treatment on GH receptor, SOCS1, SOCS-2, SOCS-3 and cytokine-inducible SH2-containing protein (CIS) hepatic mRNA expression in a rat model of sepsis, caecal ligation and puncture (CLP). Four groups of rats were studied: control (food given ad libitum, n=7), CLP only (n=8), CLP and total parenteral nutrition (TPN) (n=9), and CLP, TPN and GH (n=10). CLP rats underwent surgery and 18 h later received saline or TPN or TPN+GH for 6 h before they were killed. Serum IGF-I levels were lower in all CLP groups (P<0.001). The combination of TPN and GH treatment increased IGF-I levels compared with the saline-treated CLP rats (P<0.01), but IGF-I levels remained lower than control animals (P<0.001). GH receptor and GH-binding protein expression in liver was reduced in animals subjected to CLP and was unaffected by nutrition or GH treatment. Hepatic SOCS-1 was detectable in normal rats, induced in all CLP animals but was unaffected by nutrition and GH. Hepatic SOCS-2 expression was difficult to detect in normal and CLP rats but was greatly induced in CLP rats treated with GH. Hepatic SOCS-3 expression was only just detectable in the control group but was elevated in all CLP groups and unaffected by nutrition and GH. Hepatic CIS expression was difficult to detect in normal rats, was not induced by CLP but was induced by both nutrition and GH. In conclusion, CLP induced low IGF-I levels associated with increased expression of SOCS-1 and SOCS-3, both of which are known to inhibit GH receptor signalling. GH induced SOCS-2 and CIS in the CLP rat despite resistance with respect to IGF-I generation, and parenteral feeding induced CIS in the CLP rat. Thus, there is potential for a complex interaction between GH and cytokine signalling at the level of SOCS expression whereby the inflammatory response may alter GH signalling and GH may influence the inflammatory response.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Citocinas/metabolismo , Proteínas de Unión al ADN , Hormona del Crecimiento/farmacología , Hígado/metabolismo , Nutrición Parenteral Total , Proteínas Represoras , Sepsis/metabolismo , Transducción de Señal/efectos de los fármacos , Transactivadores , Factores de Transcripción , Análisis de Varianza , Animales , Northern Blotting , Proteínas Portadoras/análisis , Proteínas Portadoras/genética , Hormona del Crecimiento/metabolismo , Factor I del Crecimiento Similar a la Insulina/análisis , Hígado/química , Masculino , Proteínas/análisis , Proteínas/genética , ARN Mensajero/análisis , Ratas , Ratas Wistar , Receptores de Somatotropina/genética , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Proteína 1 Supresora de la Señalización de Citocinas , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de CitocinasRESUMEN
High-dose GH therapy, with GH doses 10-20 times the normal replacement dose for GH-deficient adults, has been used as an anti-catabolic agent in a number of different patient groups. A recent study, however, has shown an increase in mortality in critically ill patients treated with high-dose GH. The increased mortality was associated with multiorgan failure, septic shock, and uncontrolled infection, suggesting that GH may have altered the immune response. The GH receptor and GH are both expressed in peripheral blood mononuclear cells (PBMCs); thus, GH could act as either an endocrine or an autocrine modulator of the immune response. We have examined the hypothesis that high-dose GH therapy may induce proinflammatory cytokines, which are implicated in septic shock. To do this we measured cytokine production by PBMCs incubated in conditions that simulated high-dose GH therapy, and we measured cytokine levels in patients undergoing laparoscopic cholecystectomy who were randomized to receive either high-dose GH therapy (13 IU/m2 x day) or placebo. To confirm the biological activity of GH in our cell culture system we used a Stat5 functional assay. In this assay GH induced a bell-shaped curve, with a maximal response at GH levels between 100-1,000 ng/mL. PBMCs from healthy volunteers were incubated with GH in doses from 1-1,000 ng/mL for 6-72 h under resting conditions and after activation with endotoxin and the mixed lymphocyte reaction. Studies were repeated with PBMCs from six individuals using a GH dose of 100 ng/mL (the level of GH found after high-dose GH therapy) and an endotoxin dose that gave a submaximal response (0.01 ng/mL). GH had no effect on cell proliferation or the production of tumor necrosis factor-alpha (TNFalpha), interleukin-6 (IL-6), or interferon-gamma (IFNgamma). In patients undergoing laparoscopic cholecystectomy there was a time-related effect of surgery on cytokine levels. There was a rise in IL-6 and a fall in TNFalpha at 24 h after surgery; however, high-dose GH therapy had no effect on the cytokine response. We considered the possibility that endogenous GH production by PBMCs could influence the cytokine response in activated PBMCs; however, incubation of PBMCs in the presence of the GH receptor antagonist, B2036, had no effect on TNFalpha, IL-6, or IFNgamma production by PBMCs in either the mixed lymphocyte reaction or when activated by endotoxin. These results suggest that high-dose GH therapy does not alter the proinflammatory cytokine response to surgery or endotoxin. The results do not exclude an effect of GH on the immune response, but they suggest that the mortality seen in critically ill patients may be due to factors other than immune modulation.