RESUMEN
Polysorbate 80 (PS80), a nonionic surfactant used in pharmaceutical formulation, is known to be incompatible with m-cresol, an antimicrobial agent for multi-dose injectable formulations. This incompatibility results in increased turbidity caused by micelle aggregation progressing over weeks or longer, where storage temperature, ionic strength, and component concentration influence the aggregation kinetics. Small-angle neutron scattering (SANS) analysis of PS80/m-cresol solutions over a pharmaceutically relevant concentration range of each component reveals the cause of aggregation, the coalescence mechanism, and aggregate structure. PS80 solutions containing m-cresol concentrations below ≈2.0 mg/mL and above ≈4.5 mg/mL are kinetically stable and do not aggregate over a 50 h period. At 5 mg/mL of m-cresol, the mixture forms a kinetically stable microemulsion phase, despite being well below the aqueous solubility limit of m-cresol. Solutions containing intermediate m-cresol concentrations (2.0-4.5 mg/mL) are unstable, resulting in aggregation, coalescence, and eventual phase separation. In unstable solutions, two stages of aggregate growth (nucleation and power-law growth) are observed at m-cresol concentrations at or below ≈3.6 mg/mL. At higher m-cresol concentrations, aggregates experience a third stage of exponential growth. A single kinetic model is developed to explain the stages of aggregate growth observed in both kinetic mechanisms. This work establishes the phase diagram of PS80/m-cresol solution stability and identifies component concentrations necessary for producing stable formulations.
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Polisorbatos , Tensoactivos , Cresoles , Cinética , Polisorbatos/química , Dispersión del Ángulo Pequeño , Tensoactivos/químicaRESUMEN
Breast cancer, the most common malignancy among women, is closely associated with mutations in the tumor suppressor gene BRCA. DSS1, a component of the TRanscription-EXport-2 (TREX-2) complex involved in transcription and mRNA nuclear export, stabilizes BRCA2 expression. DSS1 is also related to poor prognosis in patients with breast cancer owing to the induction of chemoresistance. Recently, BRCA2 was shown to be associated with the TREX-2 component PCID2, which prevents DNA:RNA hybrid R-loop formation and transcription-coupled DNA damage. This study aimed to elucidate the involvement of these TREX-2 components and BRCA2 in the chemosensitivity of breast carcinomas. Our results showed that compared with that in normal breast tissues, DSS1 expression was upregulated in human breast carcinoma, whereas PCID2 expression was comparable between normal and malignant tissues. We then compared patient survival time among groups divided by high or low expressions of DSS1, BRCA2, and PCID2. Increased DSS1 expression was significantly correlated with poor prognosis in recurrence-free survival time, whereas no differences were detected in the high and low BRCA2 and PCID2 expression groups. We performed in vitro analyses, including propidium iodide nuclear staining, single-cell gel electrophoresis, and clonogenic survival assays, using breast carcinoma cell lines. The results confirmed that DSS1 depletion significantly increased chemosensitivity, whereas overexpression conferred chemoresistance to breast cancer cell lines; however, BRCA2 expression did not affect chemosensitivity. Similar to DSS1, PCID2 expression was also inversely correlated with chemosensitivity. These results strongly suggest that DSS1 and PCID2 depletion is closely associated with increased chemosensitivity via BRCA2-independent DNA damage. Together with the finding that DSS1 is not highly expressed in normal breast tissues, these results demonstrate that DSS1 depletion confers a druggable trait and may contribute to the development of novel chemotherapeutic strategies to treat DSS1-depleted breast carcinomas independent of BRCA2 mutations.
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Proteína BRCA2/genética , Neoplasias de la Mama/genética , Daño del ADN/genética , Resistencia a Antineoplásicos/genética , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismoRESUMEN
Interfacial stresses can destabilize therapeutic formulations containing monoclonal antibodies (mAbs), which is proposed to be a result of adsorption and aggregation at the air-water interface. To increase protein stability, pharmaceutical industries add surfactants, such as Polysorbate 20 (PS20), into protein formulations to minimize mAb adsorption at the interface but rarely quantify this process. We determine that mAb adsorption in surfactant-free solutions creates a monolayer with significant viscoelasticity, which can influence measurements of bulk mAb solution viscosity. In contrast, PS20 absorption leads to an interface with negligible interfacial viscosity that protects the air-water interface from mAb adsorption. These studies were performed through a combined study of surface tensiometry, interfacial rheology, capillary viscometry, and neutron reflectometry to determine the surface activity of a model surfactant, PS20, and mAb system, which can be useful for the successful formulation developments of biotherapeutics.
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Anticuerpos Monoclonales , Agua , Adsorción , Neutrones , Reología , Propiedades de Superficie , TensoactivosRESUMEN
As a typical representative of transformation thermodynamics, which is the counterpart of transformation optics, the thermal cloak has been explored extensively while most current research focuses on the structural design instead of adaptability and practicability in a dynamic environment. The evaluation of energy processes involved in the thermal cloak under dynamic conditions are also lacking, which is essential to the engineering application of this functional structure. In this paper, based on the dynamic environment of a sinusoidal form with ambient amplitude, distribution density, phase, and temperature difference as variables, we evaluated the cloaking performance and environmental response of a 2D thermal cloak. Considering the heat dissipation and energy loss in the whole procedure, local entropy production rate and response entropy were introduced to analyze the different influences of each environmental parameter on the cloaking system. Moreover, we constructed a series of comprehensive schemes to obtain the fitting equation as well as an appropriate scope to apply the thermal cloak. The results are beneficial to the novel use of the concept of entropy and valuable for further improving the working efficiency and potential engineering applications of the thermal cloak.
RESUMEN
BACKGROUND.: Viral load (VL) monitoring for patients receiving antiretroviral therapy (ART) is recommended worldwide. However, the costs of frequent monitoring are a barrier to implementation in resource-limited settings. The extent to which personalized monitoring frequencies may be cost-effective is unknown. METHODS.: We created a simulation model parameterized using person-level longitudinal data to assess the benefits of flexible monitoring frequencies. Our data-driven model tracked human immunodeficiency virus (HIV)-infected individuals for 10 years following ART initiation. We optimized the interval between viral load tests as a function of patients' age, gender, education, duration since ART initiation, adherence behavior, and the cost-effectiveness threshold. We compared the cost-effectiveness of the personalized monitoring strategies to fixed monitoring intervals every 1, 3, 6, 12, and 24 months. RESULTS.: Shorter fixed VL monitoring intervals yielded increasing benefits (6.034 to 6.221 discounted quality-adjusted life-years [QALYs] per patient with monitoring every 24 to 1 month over 10 years, respectively, standard error = 0.005 QALY), at increasing average costs: US$3445 (annual monitoring) to US$5393 (monthly monitoring) per patient, respectively (standard error = US$3.7). The adaptive policy optimized for low-income contexts achieved 6.142 average QALYs at a cost of US$3524, similar to the fixed 12-month policy (6.135 QALYs, US$3518). The adaptive policy optimized for middle-income resource settings yields 0.008 fewer QALYs per person, but saves US$204 compared to monitoring every 3 months. CONCLUSIONS.: The benefits from implementing adaptive vs fixed VL monitoring policies increase with the availability of resources. In low- and middle-income countries, adaptive policies achieve similar outcomes to simpler, fixed-interval policies.
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Terapia Antirretroviral Altamente Activa/economía , Infecciones por VIH/tratamiento farmacológico , Recursos en Salud , ARN Viral/sangre , Carga Viral , Adolescente , Adulto , Terapia Antirretroviral Altamente Activa/métodos , Terapia Antirretroviral Altamente Activa/estadística & datos numéricos , Recuento de Linfocito CD4 , Ensayos Clínicos como Asunto , Simulación por Computador , Análisis Costo-Beneficio , Femenino , Infecciones por VIH/economía , Humanos , Masculino , Carga Viral/economía , Carga Viral/métodos , Adulto JovenRESUMEN
Human chromosome 21 is known to be associated with the high risk of hematological malignancy but with resistance to breast cancer in the study of Down syndrome. In human cancers, we previously observed the significant alterations of the protein expression encoded by the ganp/MCM3AP gene on human chromosome 21q22.3. Here, we investigated GANP protein alterations in human breast cancer samples (416 cases) at various stages by immunohistochemical analysis. This cohort study clearly showed that expression of GANP is significantly decreased in human breast cancer cases with poor prognosis as an independent risk factor (relapse-free survival, hazard ratio = 2.37, 95% confidence interval, 1.27-4.42, P = 0.007 [univariate analysis]; hazard ratio = 2.70, 95% confidence interval, 1.42-5.13, P = 0.002 [multivariate analysis]). To investigate whether the altered GANP expression is associated with mammary tumorigenesis, we created mutant mice that were conditionally deficient in the ganp/MCM3AP gene using wap-cre recombinase transgenic mice. Mammary gland tumors occurred at a very high incidence in female mammary gland-specific GANP-deficient mice after severe impairment of mammary gland development during pregnancy. Moreover, tumor development also occurred in female post parous GANP-heterodeficient mice. GANP has a significant role in the suppression of DNA damage caused by estrogen in human breast cancer cell lines. These results indicated that the GANP protein is associated with breast cancer resistance.
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Acetiltransferasas/genética , Neoplasias de la Mama/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Neoplasias Mamarias Animales/genética , Recurrencia Local de Neoplasia/genética , Acetiltransferasas/biosíntesis , Adulto , Anciano , Animales , Neoplasias de la Mama/patología , Carcinogénesis/genética , Línea Celular Tumoral , Cromosomas Humanos Par 10/genética , Daño del ADN/genética , Estrógenos/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/biosíntesis , Neoplasias Mamarias Animales/patología , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Recurrencia Local de Neoplasia/patología , Estadificación de Neoplasias , EmbarazoRESUMEN
The development of biofilms at air/water or oil/water interfaces has important ramifications on several applications, but it has received less attention than biofilm formation on solid surfaces. A key difference between the growth of biofilms on solid surfaces versus liquid interfaces is the range of complicated boundary conditions the liquid interface can create that may affect bacteria, as they adsorb onto and grow on the interface. This situation is exacerbated by the existence of complex interfaces in which interfacially adsorbed components can even more greatly affect interfacial boundary conditions. In this work, we present evidence as to how particle-laden interfaces impact biofilm growth at an air/water interface. We find that particles can enhance the rate of growth and final strength of biofilms at liquid interfaces by providing sites of increased adhesive strength for bacteria. The increased adhesion stems from creating localized areas of hydrophobicity that protrude in the water phase and provide sites where bacteria preferentially adhere. This mechanism is found to be primarily controlled by particle composition, with particle size providing a secondary effect. This increased adhesion through interfacial conditions creates biofilms with properties similar to those observed when adhesion is increased through biological means. Because of the generally understood ubiquity of increased bacteria attachment to hydrophobic surfaces, this result has general applicability to pellicle formation for many pellicle-forming bacteria.
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Biopelículas , Nanopartículas del Metal/química , Poliestirenos/química , Aire , Adhesión Bacteriana , Dimetilsulfóxido , Elasticidad , Escherichia coli/fisiología , Tamaño de la Partícula , Reología , Plata/química , Propiedades de Superficie , Viscosidad , AguaRESUMEN
In an effort to develop new drug candidates with enhanced anticancer activity, our team synthesized and assessed the cytotoxicity of a series of novel xanthone derivatives with two longer 3,6-disubstituted amine carbonyl methoxy side chains on either benzene ring in selected human cancer cell lines. An MTT assay revealed that a set of compounds with lower IC50 values than the positive control, 5-FU, exhibited greater anticancer effects. The most potent derivative (XD8) exhibited anticancer activity in MDA-MB-231, PC-3, A549, AsPC-1, and HCT116 cells lines with IC50 values of 8.06, 6.18, 4.59, 4.76, and 6.09µM, respectively. Cell cycle analysis and apoptosis activation suggested that the mechanism of action of these derivatives includes cell cycle regulation and apoptosis induction.
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Antineoplásicos/farmacología , Xantonas/farmacología , Antineoplásicos/síntesis química , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Humanos , Relación Estructura-Actividad , Xantonas/síntesis químicaRESUMEN
Hyaluronic acid solutions have been widely studied due to their relevance to the rheological behavior of synovial fluid and joint lubrication. Ambulatory joint motion is typically large oscillatory deflections; therefore, large amplitude oscillatory shear strain experiments are used to examine the relevant non-linear viscoelastic properties of these solutions. Using the sequence of physical processes method to analyze data provides time dependent viscoelastic moduli, which exhibit a clear physiologically relevant behavior to hyaluronic acids non-linear viscoelasticity. In particular, it is seen that during peak strain/acceleration, the time dependent elastic modulus peaks and the loss modulus is at a minimum. The hyaluronic acid can provide an immediate elastic response to sudden forces, acting like a shock absorber during sudden changes in direction of motion or maximum deflection. However, during peak rate, the elastic modulus is at a minimum and the loss modulus is at a maximum, which provides greater efficacy to hydrodynamic shear lubrication.
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Elasticidad , Ácido Hialurónico/metabolismo , Dinámicas no Lineales , Líquido Sinovial/metabolismo , Humanos , Rodilla/fisiología , Modelos Biológicos , Movimiento , Reología , Estrés Mecánico , Factores de Tiempo , ViscosidadRESUMEN
Numerous cofactors modulate the gene regulatory activity of glucocorticoid receptors (GRs) by affecting one or more of the following three major transcriptional properties: the maximal activity of agonists (A(max)), the potency of agonists (EC(50)), and the partial agonist activity of antisteroids (PAA). Here, we report that the recently described nuclear protein, Pax2 transactivation domain interaction protein (PTIP)-associated protein 1 (PA1), is a new inhibitor of GR transactivation. PA1 suppresses A(max), increases the EC(50), and reduces the PAA of an exogenous reporter gene in a manner that is independent of associated PTIP. PA1 is fully active with, and strongly binds to, the C-terminal half of GR. PA1 reverses the effects of the coactivator TIF2 on GR-mediated gene induction but is unable to augment the actions of the corepressor SMRT. Analysis of competition assays between PA1 and TIF2 with an exogenous reporter indicates that the kinetic definition of PA1 action is a competitive decelerator at two sites upstream from where TIF2 acts. With the endogenous genes IGFBP1 and IP6K3, PA1 also represses GR induction, increases the EC(50), and decreases the PAA. ChIP and re-ChIP experiments indicate that PA1 accomplishes this inhibition of the two genes via different mechanisms as follows: PA1 appears to increase GR dissociation from and reduce GR transactivation at the IGFBP1 promoter regions but blocks GR binding to the IP6K3 promoter. We conclude that PA1 is a new competitive decelerator of GR transactivation and can act at more than one molecularly defined step in a manner that depends upon the specific gene.
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Proteínas Portadoras/química , Proteínas Portadoras/fisiología , Proteínas Nucleares/química , Proteínas Nucleares/fisiología , Receptores de Glucocorticoides/química , Sitios de Unión , Unión Competitiva , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Línea Celular , Línea Celular Tumoral , Proteínas de Unión al ADN , Genes Reporteros , Glucocorticoides/metabolismo , Humanos , Cinética , Proteínas Nucleares/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , ARN Interferente Pequeño/metabolismo , Activación Transcripcional , Técnicas del Sistema de Dos HíbridosRESUMEN
NELF-B is a BRCA1-interacting protein and subunit (with NELF-A, -C/D, and -E) of the human negative elongation factor (NELF) complex, which participates in RNA polymerase II pausing shortly after transcription initiation, especially for synchronized gene expression. We now report new activities of NELF-B and other NELF complex subunits, which are to attenuate glucocorticoid receptor (GR)-mediated gene induction, reduce the partial agonist activity of an antagonist, and increase the EC50 of an agonist during nonsynchronized expression of exogenous and endogenous reporters. Stable knockdown of endogenous NELF-B has the opposite effects on an exogenous gene. The GR ligand-binding domain suffices for these biological responses. ChIP assays reveal that NELF-B diminishes GR recruitment to promoter regions of two endogenous genes. Using a new competition assay, NELF-A and NELF-B are each shown to act independently as competitive decelerators at two steps after the site of GR action and before or at the site of reporter gene activity. A common motif in each NELF was identified that is required for full activity of both NELF-A and NELF-B. These studies allow us to position the actions of two new modulators of GR-regulated transactivation, NELF-A and NELF-B, relative to other factors in the overall gene induction sequence.
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Regulación de la Expresión Génica/fisiología , Receptores de Glucocorticoides/metabolismo , Elementos de Respuesta/fisiología , Factores de Transcripción/metabolismo , Transcripción Genética/fisiología , Factores de Elongación Transcripcional/metabolismo , Animales , Células COS , Chlorocebus aethiops , Técnicas de Silenciamiento del Gen , Humanos , Estructura Terciaria de Proteína , Ratas , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/genética , Factores de Transcripción/genética , Factores de Elongación Transcripcional/genéticaRESUMEN
TTLL5/STAMP (tubulin tyrosine ligase-like family member 5) has multiple activities in cells. TTLL5 is one of 13 TTLLs, has polyglutamylation activity, augments the activity of p160 coactivators (SRC-1 and TIF2) in glucocorticoid receptor-regulated gene induction and repression, and displays steroid-independent growth activity with several cell types. To examine TTLL5/STAMP functions in whole animals, mice were prepared with an internal deletion that eliminated several activities of the Stamp gene. This mutation causes both reduced levels of STAMP mRNA and C-terminal truncation of STAMP protein. Homozygous targeted mutant (Stamp(tm/tm)) mice appear normal except for marked decreases in male fertility associated with defects in progressive sperm motility. Abnormal axonemal structures with loss of tubulin doublets occur in most Stamp(tm/tm) sperm tails in conjunction with substantial reduction in α-tubulin polyglutamylation, which closely correlates with the reduction in mutant STAMP mRNA. The axonemes in other structures appear unaffected. There is no obvious change in the organs for sperm development of WT versus Stamp(tm/tm) males despite the levels of WT STAMP mRNA in testes being 20-fold higher than in any other organ examined. This defect in male fertility is unrelated to other Ttll genes or 24 genes previously identified as important for sperm function. Thus, STAMP appears to participate in a unique, tissue-selective TTLL-mediated pathway for α-tubulin polyglutamylation that is required for sperm maturation and motility and may be relevant for male fertility.
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Proteínas Portadoras/metabolismo , Eliminación de Gen , Infertilidad Masculina/metabolismo , Motilidad Espermática , Espermatozoides/metabolismo , Testículo/metabolismo , Animales , Proteínas Portadoras/genética , Regulación de la Expresión Génica/genética , Infertilidad Masculina/genética , Infertilidad Masculina/patología , Masculino , Ratones , Ratones Mutantes , Coactivador 1 de Receptor Nuclear/genética , Coactivador 1 de Receptor Nuclear/metabolismo , Coactivador 2 del Receptor Nuclear/genética , Coactivador 2 del Receptor Nuclear/metabolismo , Procesamiento Proteico-Postraduccional/genética , Espermatozoides/patología , Testículo/patología , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMEN
The tumor vascular system, which is critical to the survival and growth of solid tumors, has been an attractive target for anticancer research. Building on studies that show that some flavonoids have anticancer vascular effects, we developed and analyzed the flavonoid derivative R24 [3, 6-bis (2-oxiranylmethoxy)-9H-xanthen-9-one]. A CAM assay revealed that R24 disrupted neovascular formation; fewer dendrites were detected and overall dendritic length was shorter in the R24-treated chicken embryos. The antiproliferative effect of R24 was measured by MTT assay in A549 (lung cancer), AsPC-1 (pancreatic cancer), HCT-116 (colorectal cancer), and PC-3 (prostate cancer) cell lines. R24 reduced proliferation with an IC50 of 3.44, 3.59, 1.22, and 11.83 µM, respectively. Cell-cycle analysis and Annexin-V/propidium iodide staining showed that R24 induced apoptosis. In addition, R24 regulated intracellular ROS production in a dose-dependent manner. CM-H2DCFDA staining indicated that intracellular ROS production increased with the R24 dose. In summary, we found that R24 exhibits potent antiangiogenic and antiproliferative effects, induces apoptosis, and promotes ROS production.
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Flavonoides/farmacología , Neoplasias/irrigación sanguínea , Neovascularización Patológica/prevención & control , Especies Reactivas de Oxígeno/metabolismo , Línea Celular Tumoral , HumanosRESUMEN
Optimal triage biodosimetry would include risk stratification within minutes, and it would provide useful triage despite heterogeneous dosimetry, cytokine therapy, mixed radiation quality, race, and age. For regulatory approval, the U.S. Food and Drug Administration (FDA) Biodosimetry Guidance requires suitability for purpose and a validated species-independent mechanism. Circulating cell-free DNA (cfDNA) concentration assays may provide such triage information. To test this hypothesis, cfDNA concentrations were measured in unprocessed monkey plasma using a branched DNA (bDNA) technique with a laboratory developed test. The cfDNA levels, along with hematopoietic parameters, were measured over a 7-day period in Rhesus macaques receiving total body radiation doses ranging from 1 to 6.5 Gy. Low-dose irradiation (0-2 Gy) was easily distinguished from high-dose whole-body exposures (5.5 and 6.5 Gy). Fold changes in cfDNA in the monkey model were comparable to those measured in a bone marrow transplant patient receiving a supralethal radiation dose, suggesting that the lethal threshold of cfDNA concentrations may be similar across species. Average cfDNA levels were 50 ± 40 ng/mL [±1 standard deviation (SD)] pre-irradiation, 120 ± 13 ng/mL at 1 Gy; 242 ± 71 ng/mL at 2 Gy; 607 ± 54 at 5.5 Gy; and 1585 ± 351 at 6.5 Gy (±1 SD). There was an exponential increase in cfDNA concentration with radiation dose. Comparison of the monkey model with the mouse model and the Guskova model, developed using Chernobyl responder data, further demonstrated correlation across species, supporting a similar mechanism of action. The test is available commercially in a Clinical Laboratory Improvement Amendments (CLIA) ready form in the U.S. and the European Union. The remaining challenges include developing methods for further simplification of specimen processing and assay evaluation, as well as more accurate calibration of the triage category with cfDNA concentration cutoffs.
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Ácidos Nucleicos Libres de Células , Macaca mulatta , Triaje , Animales , Ácidos Nucleicos Libres de Células/sangre , Triaje/métodos , Humanos , Masculino , Ratones , Relación Dosis-Respuesta en la Radiación , Radiometría/métodos , Irradiación Corporal TotalRESUMEN
Amifostine is a first-line cytoprotective drug used to prevent radiotherapy-induced or chemotherapy-induced injuries. However, its mechanism of action is not well understood. In this study, freshly harvested bone marrow cells were treated with amifostine and analyzed with a series of mitochondrial indices. In vitro results showed that bone marrow cells treated with amifostine 0.5 h before irradiation (0.5 Gy) experienced several benefits, as compared to vehicle controls, including (1) reduced reactive oxygen species levels, which reduced the production of free radicals; (2) better preservation of mitochondria, as indicated by MitoTracker-positive staining and the increased intensity of staining; (3) reduced apoptosis, as demonstrated by Annexin V staining; and (4) a better proliferation rate, as illustrated by MTT assay. Our in vitro studies showed that amifostine-treated mice exhibited (1) higher ATP production; (2) reduced plasma IL-2 levels, suppressing the immune response triggered by radiotoxicity; and (3) enhanced radiation-induced production of granulocyte colony-stimulating factor. All of these processes benefit recovery from radiation-induced damage.
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Amifostina/farmacología , Células de la Médula Ósea/efectos de los fármacos , Médula Ósea/efectos de los fármacos , Citocinas/metabolismo , Mitocondrias/efectos de los fármacos , Protectores contra Radiación/farmacología , Adaptación Fisiológica/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Animales , Médula Ósea/crecimiento & desarrollo , Médula Ósea/metabolismo , Células de la Médula Ósea/metabolismo , Masculino , Ratones , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
In this study, we compared two in vitro collagen production assays ([(3)H]-proline incorporation and Sirius Red) for their ability to determine the pattern shift from soluble to deposited collagen. The effect of the antifibrotic agent, triptolide (TPL), on collagen production was also studied. The results showed that: (1) 48 h after NIH 3T3 (murine embryo fibroblast) and HFL-1(human fetal lung fibroblast) were exposed to transforming growth factor-beta 1 (TGF-ß), there was an increase in soluble collagen in the culture medium; (2) on day 4, soluble collagen declined, whereas deposited collagen increased; (3) Sirius Red was easier to use than [(3)H]-proline incorporation and more consistently reflected the collagen pattern shift from soluble to deposited; (4) the in vitro Sirius Red assay took less time than the in vivo assay to determine the effect of TPL. Our results suggest that: (a) the newly synthesized soluble collagen can sensitively evaluate an agent's capacity for collagen production and (b) Sirius Red is more useful than [(3)H]-proline because it is easier to use, more convenient, less time consuming, and does not require radioactive material.
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Compuestos Azo , Bioensayo , Colágeno/metabolismo , Embrión de Mamíferos/metabolismo , Feto/metabolismo , Fibroblastos/metabolismo , Pulmón/metabolismo , Animales , Antineoplásicos Alquilantes/farmacología , Células Cultivadas , Colorantes , Diterpenos/farmacología , Embrión de Mamíferos/citología , Embrión de Mamíferos/efectos de los fármacos , Compuestos Epoxi/farmacología , Feto/citología , Feto/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Humanos , Pulmón/citología , Pulmón/efectos de los fármacos , Ratones , Células 3T3 NIH , Fenantrenos/farmacología , Prolina/metabolismo , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Although glycoproteins possess a variety of functional and structural roles in intracellular and intercellular activities, the effect of ionizing radiation (IR) on glycosylation is largely unknown. To explore this effect, we established a sandwich assay in which PHA-L, a phytohaemagglutinin that agglutinates leukocytes, was used as a coating layer to capture glycoproteins containing complex oligosaccharides; the bound glycoproteins were then measured. C57BL/6 mice were exposed to 0, 3, 6, or 10 Gy, and the plasma was collected at 6, 12, 18, 24, 48, 72, or 168 h and then analyzed for galactose/N-acetylgalactosamine (Gal/GalNAc) containing proteins. We found that (1) the sandwich assay accurately measured the level of glycoproteins, (2) 6-12 h after IR, the amount of glycoproteins containing GalNAc increased, and (3) at 72 and 168 h, 10 Gy was associated with a decrease in Gal/GalNAc. These IR-induced alterations might relate to the release of glycoproteins into the blood and the damage of the proteins and genes that are related to the glycosylation process.
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Acetilgalactosamina/sangre , Galactosa/sangre , Glicoproteínas/sangre , Glicosilación/efectos de la radiación , Manosa/sangre , Irradiación Corporal Total , Acetilgalactosamina/análogos & derivados , Animales , Ensayo de Inmunoadsorción Enzimática , Masculino , Ratones , Ratones Endogámicos C57BL , Fitohemaglutininas/metabolismoRESUMEN
Various members of the fibroblast growth factor (FGF) family mitigate radiation-induced damage. We designed and synthesized the binding domain peptide of FGF-2 (FGF-P) with a dimer form resistant to peptidase and examined its mitigatory effect on murine bone marrow cells. NIH Swiss mice were exposed to different doses of total body irradiation (TBI) and treated with ten doses of 5 mg/kg FGF-P. We achieved the following results: (1) FGF-P stimulated the growth of bone marrow cells harvested from mice exposed to 3 Gy; (2) on day 25 after 6 Gy TBI, the number of leukocytes and granulocytes was higher in the FGF-P group than in the vehicle-alone group; (3) FGF-P significantly increased the number of pro-B and pre-B cells; and (4) FGF-P treatment in vivo increased the long-term hematopoietic stem cells (LT-HSC) in bone marrow. These data reveal the underlying mechanism by which FGF-P rescued a significant percentage of the exposed mice. The increase of LT-HSC in bone marrow leads to a concomitant increase of pro-B and pre-B cells followed by leukocytes and granulocytes, which in turn enhance immunity against infection.
Asunto(s)
Linfocitos B/efectos de los fármacos , Médula Ósea/efectos de los fármacos , Médula Ósea/efectos de la radiación , Factores de Crecimiento de Fibroblastos/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Irradiación Corporal Total , Animales , Linfocitos B/patología , Linfocitos B/efectos de la radiación , Médula Ósea/patología , Células Cultivadas , Células Madre Hematopoyéticas/patología , Células Madre Hematopoyéticas/efectos de la radiación , Masculino , RatonesRESUMEN
Interleukin 11 (IL-11) is a multifunctional cytokine isolated from bone marrow (BM)-derived stromal cells that promotes hematopoiesis and prolongs the life span of lethally irradiated animals. However, the underlying mechanism for the protective effect of IL-11 on BM is unclear. In this study, we explored the effect of IL-11 on irradiated BM cells. Freshly harvested BM cells were pretreated with 20 ng/ml of recombinant IL-11 for 30 min, irradiated with a dose of 0.5 Gy, cultured for 24 h, and then subjected to several assays. In vitro data showed that, as compared to the vehicle controls, IL-11: (1) reduced the production of reactive oxygen species; (2) reduced the alteration of mitochondrial membrane potential; (3) increased MitoTracker staining, suggesting that the number of mitochondria and their functions were better maintained; and (4) reduced apoptosis of BM cells and enhanced BM cell proliferation. In vivo studies of mice pretreated with saline or 100 µg/kg of IL-11 at 12 and 2 h before 10-Gy total body irradiation (TBI) demonstrated that G-CSF and IL-6 were significantly upregulated, whereas IL-2 and IL-4 were reduced. We found that IL-11 protects mitochondrial functions, acts with G-CSF and IL-6 to stimulate the growth of radiation-damaged BM, and reduces the immune response to radiation injury.
Asunto(s)
Médula Ósea/efectos de los fármacos , Médula Ósea/efectos de la radiación , Interleucina-11/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/efectos de la radiación , Traumatismos Experimentales por Radiación/metabolismo , Traumatismos Experimentales por Radiación/prevención & control , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Médula Ósea/metabolismo , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/efectos de la radiación , Procesos de Crecimiento Celular/efectos de los fármacos , Procesos de Crecimiento Celular/efectos de la radiación , Factor Estimulante de Colonias de Granulocitos/metabolismo , Interleucina-11/metabolismo , Interleucina-6/metabolismo , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de la radiación , Ratones , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Irradiación Corporal Total/métodosRESUMEN
In this study, we investigated the response of irradiated bone marrow cells to granulocyte colony-stimulating factor (G-CSF). Freshly harvested bone marrow cells were treated with either saline (vehicle control) or 20 ng/ml of G-CSF. Thereafter, cells were separated into nonirradiated (no-IR) and irradiated (IR, 0.5 Gy) groups. IR cells exhibited a higher proliferation rate in response to G-CSF, as compared to the no-IR cells. Reduced levels of reactive oxygen species indicated that G-CSF-treated IR cells produced fewer free radicals, as compared to the no-IR cells. The G-CSF-treated IR cells also had a lower apoptotic rate than their no-IR counterparts. Furthermore, G-CSF-treated IR cells exhibited less alteration of mitochondrial membrane potential, as compared to the no-IR cells. Finally, the mitochondrial number increased in the G-CSF-treated IR cells. The radiation-induced increase in plasma IL-6 in vivo could be enhanced by the administration of G-CSF. The data suggest that radiation potentiates the response of bone marrow cells to G-CSF treatment.