RESUMEN
OBJECTIVE: To establish a quick analytical method using quantitative PCR for marker gene analysis to identify the functions of iTreg cells and subsequently curtail the harvest time for iTreg cells. RESULTS: The data from the marker gene analysis indicated that varying proportions of iTreg cells could reveal the various expression levels of these genes. FoxP3 expression increased to a considerable degree. By using the same iTreg population, the mixed lymphocyte reaction assay was conducted for 5 days. The suppression percentage of T-cells was dependent on the proportion of iTreg cells, indicating that gene expression levels can represent the biological functions of iTreg cells. By using human peripheral blood mononuclear cells for Treg cell induction, the marker gene expression analysis showed a difference between iTreg cells and uninduced T cells. CONCLUSION: Marker gene analysis requires only 1 day to identify the functions of human iTreg cells can save time in clinical application and might prevent graft-versus-host disease occurrence effectively.
Asunto(s)
Linfocitos T CD4-Positivos , Factores de Transcripción Forkhead/genética , Perfilación de la Expresión Génica/métodos , Marcadores Genéticos/genética , Linfocitos T Reguladores , Animales , Linfocitos T CD4-Positivos/química , Linfocitos T CD4-Positivos/clasificación , Linfocitos T CD4-Positivos/metabolismo , Técnicas de Cocultivo , Citometría de Flujo , Factores de Transcripción Forkhead/metabolismo , Humanos , Leucocitos Mononucleares , Prueba de Cultivo Mixto de Linfocitos , Ratones , Ratones Endogámicos BALB C , Linfocitos T Reguladores/química , Linfocitos T Reguladores/clasificación , Linfocitos T Reguladores/metabolismoRESUMEN
BACKGROUND: The polymorphic CAG repeats of the androgen receptor (AR) gene have been suggested to affect the risk of breast cancer, but the results are controversial. In addition, the relationship between patients' CAG genotype and the prognosis has not been investigated. OBJECTIVE: The purpose of this study is to access the association between the polymorphic CAG repeats and the incidence and prognosis of breast cancer. METHODS: One hundred and fifty-six breast cancer cases and 108 healthy controls from Taipei Veteran General Hospital were enrolled. The length of CAG repeats was analyzed among by means of PCR amplification. The logistic regression model was used for cross-sectional analyses of prevalent breast cancer.Furthermore, we categorized the cases according to the average length of both CAG alleles (CAGn ≥ 23 versus < 23). Outcomes were disease-free survival and mortality. The Cox proportional hazards model and Kaplan-Meier estimate were used for survival analysis. RESULTS: The median age was 56 (51-64) and 46 (37-52) in breast cancer patients and healthy controls, respectively. The median of CAGn was 22.5 (21.5-24) in study group and 23 (21.5-24) in controls. Our study showed the length of CAG repeats did not contribute to breast cancer or benign breast tumors (HR 1.01; 95% CI, 0.90-1.13). In the median follow-up of 6.59 years, we found the CAGn ≥ 23 (n = 75) could be a poor prognosis (adjust HR, 3.08; 95% CI, 1.42-6.67, p = 0.004). CONCLUSION: The CAG polymorphism is not associated with development of breast cancer, but patients with more CAG repeats of the AR gene are prone to poor prognoses.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/mortalidad , Polimorfismo Genético/genética , Receptores Androgénicos/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Adulto , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/patología , Estudios de Casos y Controles , Femenino , Estudios de Seguimiento , Predisposición Genética a la Enfermedad , Humanos , Técnicas para Inmunoenzimas , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Receptor ErbB-2/metabolismo , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Tasa de SupervivenciaRESUMEN
Fluorescent carbon nanodots (C-dots; 4.3 ± 0.8 nm) from fresh tender ginger juice provide high suppression of the growth of human hepatocellular carcinoma cells (HepG2), with low toxicity to normal mammary epithelial cells (MCF-10A) and normal liver cells (FL83B). The inhibition is selective to HepG2 over other tested cancer cells, including human lung cancer cell line (A549), human breast cancer cell line (MDA-MB-231), and human cervical cancer cell line (HeLa). Western blot results reveal that the C-dots up-regulate the expression of p53 protein only in the HepG2 cell line. The 50% inhibiting concentration (IC50) value of the C-dots on HepG2 cells is 0.35 mg mL-1. Image cytometry results show significant uptake of C-dots by HepG2 cells that induce intracellular production of reactive oxygen species (ROS, 18.2-fold increased), while other cells remain almost the same in ROS levels after treatment with C-dots (1.11 mg mL-1). The C-dots trigger the pro-apoptotic factor to promote HepG2 cell apoptosis. The C-dots effectively inhibit the growth of tumors in nude mice (104 ± 14 vs. 3.7 ± 0.2 mg with and without treatment within 14 days).
RESUMEN
The expression of neutrophil gelatinase-associated lipocalin (NGAL) is up-regulated in some cancers; therefore NGAL has potential as a tumor biomarker. Although the regulation mechanism for this is unknown, one study has shown that it is likely to involve a microRNA (miRNA). Here, we investigate the relation between miRNA expression and NGAL expression, and the role of NGAL in tumorigenesis. Using miRNA target-detecting software, we analyze the mRNA sequence of NGAL and identify a target site for microRNA-138 (miR-138) in nucleotides 25-53 of the 3' UTR. We then analyze NGAL and miR-138 expression in three cancer cell lines originating from breast, endometrial and pancreatic carcinomas (the MCF-7, RL95-2 and AsPC-1 cell lines), respectively, using quantitative (real-time) PCR and western blot analysis. Metastasis is a critical event in cancer progression, in which malignant cell proliferation, migration and invasion increase. To determine whether miR-138-regulated NGAL expression is associated with metastasis, the proliferation and migration of the cell line are examined after miR-138 transfection. Using nude mice, we examine both the tumorigenicity of these cell lines and of miR-138-transfected cancer cells in vivo, as well as the effect of treating tumors with an antibody against NGAL. Our results show that these cancer cell lines down-regulate NGAL when miR-138 is highly expressed. Ectopic transfection of miR-138 suppresses NGAL expression and cell migration in RL95-2 and AsPC-1 cells, demonstrating that miR-138-regulated NGAL expression is associated with cell migration. Additionally, injection of the NGAL antibody diminishes NGAL-mediated tumorigenesis in nude mice, and miR-138 transfection of cancer cells reduces tumor formation. As the cell proliferation data showed that the tumor size should be regulated by NGAL-related cell growth. Taken together, our results indicate that NGAL may be a good target for cancer therapy and suggest that miR-138 acts as a tumor suppressor and may prevent metastasis.
Asunto(s)
Transformación Celular Neoplásica/genética , Gelatinasas/genética , Lipocalinas/genética , MicroARNs/genética , Neutrófilos/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Transformación Celular Neoplásica/metabolismo , Regulación hacia Abajo , Gelatinasas/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Lipocalinas/metabolismo , Ratones , Ratones Desnudos , MicroARNs/metabolismo , TransfecciónRESUMEN
AIM: The present study was aimed at investigating the effect of trans-6-(4-chlorobutyl)-5-hydroxy-4-(phenylthio)-1-tosyl-5,6-dihydropyridine-2(1H)-one (HTDP-2), a novel synthetic compound, on the release of endogenous glutamate in rat cerebrocortical nerve terminals (synaptosomes) and exploring the possible mechanism. METHODS: The release of glutamate was evoked by the K⺠channel blocker 4-aminopyridine (4-AP) and measured by an on-line enzyme-coupled fluorimetric assay. We also used a membrane potential-sensitive dye to assay nerve terminal excitability and depolarization, and a Ca²âº indicator, Fura-2-acetoxymethyl ester, to monitor cytosolic Ca²âº concentrations ([Ca²âº](c)). RESULTS: HTDP-2 inhibited the release of glutamate evoked by 4-AP in a concentration-dependent manner. Inhibition of glutamate release by HTDP-2 was prevented by the chelating intraterminal Ca²âº ions, and by the vesicular transporter inhibitor bafilomycin A1, but was insensitive to the glutamate transporter inhibitor DL-threo-ß-benzyloxyaspartate. HTDP-2 did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization whereas it decreased the 4-AP-induced increase in [Ca²âº](c). Furthermore, the inhibitory effect of HTDP-2 on the evoked glutamate release was abolished by the N-, and P/Q-type Ca²âº channel blocker ω-conotoxin MVIIC, but not by the ryanodine receptor blocker dantrolene, or the mitochondrial Naâº/Ca²âº exchanger blocker CGP37157. CONCLUSION: Based on these results, we suggest that, in rat cerebrocortical nerve terminals, HTDP-2 decreases voltage-dependent Ca²âº channel activity and, in so doing, inhibits the evoked glutamate release.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/fisiología , Corteza Cerebral/fisiología , Ácido Glutámico/fisiología , Fármacos Neuroprotectores/farmacología , Piridonas/farmacología , Compuestos de Tosilo/farmacología , 4-Aminopiridina/farmacología , Sistema de Transporte de Aminoácidos X-AG/metabolismo , Animales , Calcio/análisis , Calcio/fisiología , Bloqueadores de los Canales de Calcio/síntesis química , Bloqueadores de los Canales de Calcio/toxicidad , Canales de Calcio/metabolismo , Clonazepam/análogos & derivados , Clonazepam/farmacología , Citosol/fisiología , Dantroleno/farmacología , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Interacciones Farmacológicas , Ácido Glutámico/análisis , Ácido Glutámico/toxicidad , Masculino , Potenciales de la Membrana , Terminaciones Nerviosas/fisiología , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/toxicidad , Bloqueadores de los Canales de Potasio/farmacología , Piridonas/síntesis química , Piridonas/toxicidad , Ratas , Ratas Sprague-Dawley , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/farmacología , Sinaptosomas/fisiología , Tiazepinas/farmacología , Compuestos de Tosilo/síntesis química , Compuestos de Tosilo/toxicidadRESUMEN
Folic acid plays an important role in neuronal development. A series of newly synthesized bioactive compounds (NSCs) was reported to exhibit immunoactive and neuroprotective functions. The isolated and combined effects of folic acid and NSCs against beta-amyloid (Abeta)-induced cytotoxicity are poorly understood. These effects were tested using human microglia cells (C13NJ) subjected to Abeta(25-35) challenge. According to an MTT assay, treatment of C13NJ cells with Abeta(25-35) at 10-100 microM for 48 h induced 18%-43% cellular death in a dose-dependent manner (p < 0.05). Abeta(25-35) treatment at 25 microM induced nitrite oxide (NO) release, elevated superoxide production, and reduced the distribution of cells in the S phase. Preincubation of C13NJ with 100 microM folic acid protected against Abeta(25-35)-induced cell death, which coincided with a reduction in NO release by folic acid supplements. NSC47 at a level of 50 microM protected against Abeta(25-35)-induced cell death and reduced Abeta-promoted superoxide production (p < 0.05). Folic acid in combination with NSC47 at their cytoprotective doses did not synergistically ameliorate Abeta(25-35)-associated NO release, superoxide production, or cell cycle arrest. Taken together, folic acid or NSC treatment alone, but not the combined regimen, protected against Abeta(25-35)-induced cell death, which may partially, if not completely, be mediated by free radical-scavenging effects.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Productos Biológicos/farmacología , Ácido Fólico/farmacología , Microglía/efectos de los fármacos , Fragmentos de Péptidos/toxicidad , Ciclo Celular , Relación Dosis-Respuesta a Droga , Humanos , Superóxidos/metabolismoRESUMEN
The aim of this study is to investigate cancer patients' response and side effects associated with transdermal therapeutic fentanyl (TTS-F), whose pain was hardly controlled by nonweak/weak opioids in Taiwan. From 2005 to 2006, 822 outpatients received TTS-F to collect pain assessment forms and diaries for 4 weeks. Most (78.7%) patients were initially prescribed 25 microg/h TTS-F. Doses were adjusted weekly at clinicians' discretion, according to pain assessment and side effects. Patients receiving 50 microg/h, 75 microg/h, and > 75 microg/h TTS-F had increased from 17.5% to 32.1%, 1.8% to 3.4%, and 1.9% to 2.2%, respectively, by week 2; further small increases were found in weeks 3 and 4. Pain palliation improved from 60.6% during week 1 to 78.6% at week 4. The common adverse effects were nausea/vomiting. Patient's compliance was >90%. This study found that the TTS-F is effective and well tolerated.
Asunto(s)
Analgésicos Opioides/administración & dosificación , Fentanilo/administración & dosificación , Neoplasias/complicaciones , Dolor/tratamiento farmacológico , Cooperación del Paciente/estadística & datos numéricos , Administración Cutánea , Adulto , Anciano , Anciano de 80 o más Años , Atención Ambulatoria/estadística & datos numéricos , Estudios de Cohortes , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Masculino , Persona de Mediana Edad , Dolor/etiología , Taiwán , Resultado del Tratamiento , Adulto JovenRESUMEN
A new synthetic compound, 6-hydroxy-2-tosylisoquinolin-1(2H)-one (2-OH), was selected for immunopharmacological activity tests. The effects of 2-OH on human peripheral blood mononuclear cell (PBMC) proliferation were determined by tritiated thymidine uptake. Compared to phytohemagglutinin (PHA; 5 microg/mL) stimulation, 2-OH significantly enhanced PBMC proliferation in a dose-dependent manner. The 50% enhancement activity (EC(50)) for 2-OH was 4.4+/-0.1 microM. In addition, effects of 2-OH on interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) production in PBMC were determined by enzyme immunoassay. Results demonstrated that 2-OH stimulated IL-2 and IFN-gamma production in PBMC. Data from reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR indicated that IL-2 and IFN-gamma mRNA expression in PBMC could be induced by 2-OH. Therefore, 2-OH enhanced IL-2 and IFN-gamma production in PBMC by modulation their gene expression. We suggest that 2-OH may be an immunomodulatory agent.
Asunto(s)
Expresión Génica/efectos de los fármacos , Factores Inmunológicos/farmacología , Interferón gamma/genética , Interleucina-2/genética , Leucocitos Mononucleares/efectos de los fármacos , Quinolonas/farmacología , Proliferación Celular , Humanos , Factores Inmunológicos/síntesis química , Factores Inmunológicos/química , Leucocitos Mononucleares/inmunología , Quinolonas/síntesis química , Quinolonas/químicaRESUMEN
AIM: Excessive glutamate release has been proposed to be involved in the pathogenesis of several neurological diseases. In this study, we investigated the effect of HDT-1 (3, 4, 4a, 5, 8, 8a-hexahydro-6,7-dimethyl-4a-(phenylsulfonyl)- 2-tosylisoquinolin-1(2H)-one), a novel synthetic compound, on glutamate release in rat cerebrocortical nerve terminals and explored the possible mechanism. METHODS: The release of glutamate was evoked by the K+ channel blocker 4-aminopyridine (4-AP) or the high external [K+] and measured by one-line enzyme-coupled fluorometric assay. We also determined the loci at which HDT-1 impinges on cerebrocortical nerve terminals by using membrane potentialsensitive dye to assay nerve terminal excitability and depolarization, and Ca2+ indicator Fura-2 to monitor Ca2+ influx. RESULTS: HDT-1 inhibited the release of glutamate evoked by 4-AP and KCl in a concentration-dependent manner. HDT-1 did not alter the resting synaptosomal membrane potential or 4-APevoked depolarization. Examination of the effect of HDT-1 on cytosolic [Ca2+] revealed that the diminution of glutamate release could be attributed to reduction in voltage-dependent Ca2+ influx. Consistent with this, the HDT-1-mediated inhibition of glutamate release was significantly prevented in synaptosomes pretreated with the N- and P/Q-type Ca2+ channel blocker omega-conotoxin MVIIC. CONCLUSION: In rat cerebrocortical nerve terminals, HDT-1 inhibits glutamate release through a reduction of voltage-dependent Ca2+ channel activity and subsequent decrease of Ca2+ influx into nerve terminals, rather than any upstream effect on nerve terminal excitability.
Asunto(s)
Corteza Cerebral/metabolismo , Antagonistas de Aminoácidos Excitadores , Ácido Glutámico/metabolismo , Isoquinolinas/farmacología , Sinaptosomas/metabolismo , 4-Aminopiridina/farmacología , Animales , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Citosol/efectos de los fármacos , Citosol/metabolismo , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Bloqueadores de los Canales de Potasio/farmacología , Cloruro de Potasio/farmacología , Ratas , Ratas Sprague-Dawley , Sinaptosomas/efectos de los fármacosRESUMEN
Arsenic trioxide (As2O3) has recently received a great deal of attention because of its capacity to cause complete remission of acute promyelocytic leukemia (APL). To evaluate possible toxicity on the male reproductive system during arsenic therapy, male mice were used as a model. Outbred mice (ICR/CD1 and S-W, 6 weeks old) were subcutaneously administered As2O3 continuously for 5 days, with a 2-day interval, for a period of 3 weeks. As2O3 doses were 0, 0.15, 0.3, 1.5, and 3.0 mg/kg of body weight, respectively. No mice died in any dosage group. Our data showed no significant changes in food consumption or in the weight of the body, liver, testis, or epididymis after As2O3 treatment. Using histological observation to identify the stages of seminiferous tubules, we showed that As2O3 treatment resulted in the inhibition of spermatogenesis. The frequency of mature seminiferous tubules (stages VII and VIII) was markedly decreased after As2O3 treatment. A significant decrease in sperm motility and viability also was found with computer-assisted sperm analysis (CASA) and a SYBR14/PI staining assay. Using an enzyme-linked immunosorbent assay (ELISA), we found a significant decrease in levels of plasma luteinizing hormone (LH) at a dose of 3.0 mg/kg body weight. No significant difference was found in plasma follicle-stimulating hormone (FSH) in all dosages. A significant decrease was found in plasma testosterone in all dosages, but no difference in intratesticular testosterone, with the exception of As2O3 at a dose of 3.0 mg/kg body weight. Moreover, there was a significant decrease in the levels of mRNA involved in testicular testosterone synthesis, cytochrome P450 side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), and cytochrome P450 17-alpha hydroxylase/C17-20 lyase (Cyp17). The use of immunohistological observation showed no obvious difference in the testosterone level of Leydig cells of mice treated with As2O3 at doses of 0.3 and 1.5 mg/kg body weight. We concluded that As2O3 treatment caused damage to sperm mobility and viability. As2O3 treatment disturbed spermatogenesis via reducing gene expression of the key enzymes in testosterone synthesis.
Asunto(s)
Expresión Génica/efectos de los fármacos , Inhibidores de Crecimiento/toxicidad , Óxidos/toxicidad , Espermatogénesis/efectos de los fármacos , Testosterona/metabolismo , Animales , Animales no Consanguíneos , Trióxido de Arsénico , Arsenicales , Supervivencia Celular/efectos de los fármacos , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Inyecciones Subcutáneas , Células Intersticiales del Testículo/efectos de los fármacos , Células Intersticiales del Testículo/metabolismo , Hormona Luteinizante/sangre , Masculino , Ratones , Ratones Endogámicos ICR , ARN Mensajero/metabolismo , Túbulos Seminíferos/efectos de los fármacos , Túbulos Seminíferos/metabolismo , Motilidad Espermática/efectos de los fármacos , Espermatogénesis/fisiología , Espermatozoides/efectos de los fármacos , Espermatozoides/patología , Espermatozoides/fisiología , Testículo/química , Testículo/metabolismo , Testosterona/análisis , Testosterona/genéticaRESUMEN
Stromal cell monolayers have been an important means of studying the regulation of hematopoiesis, because they produce cytokines. Cytosine arabinoside, vincristine, daunorubicin, and doxorubicin are common drugs for hematological cancer therapy, and they may have some effects on bone marrow stroma during chemotherapy. The aim of this study was to elucidate interactions between the bone marrow stromal microenvironment and leukemic cells after drug treatment. We tested the hypothesis that human HS-5 stromal cells, pretreated with anticancer drugs, affected the growth of leukemic K562 cells by changing the cytokines in the culture microenvironment. Thereafter, proliferation of K562 cells increased nearly 2.5-fold compared the co-cultivation with drugs-pretreated HS-5 stromal cells and drugs-untreated HS-5 stromal cells. The results indicated that co-cultivation with HS-5 stromal cells pretreated with drugs caused significant K562 cell proliferation. Cytokines in the microenvironment were detected via the RayBio((R))Human Cytokine Antibody Array Membrane. The levels of the cytokines CKbeta, IL-12, IL-13, IGFBP-2, MCP-1, MCP-3, MCP-4, MDC, MIP-1beta and MIP-1delta were decreased, with a particularly marked decrease in MIP-3alpha. In co-culture medium, there was a 20-fold decrease in MIP-3alpha in daunorubicin-pretreated HS-5 cells and at least a 3-fold decrease in Ara-C-pretreated cells. This indicated a significant effect of anticancer drugs on the stromal cell line. Using phosphorylated Erk and pRb proteins as cell proliferation markers, we found that phosphorylation of these markers in K562 cells was inhibited during co-cultivation with drug-pretreated stromal cells in MIP-3alpha-supplemented medium and restored by MIP-3alpha antibody supplement. In conclusion, anticancer drug pretreatment suppresses the negative control exerted by HS-5 cells on leukemic cell proliferation, via modulation of cytokines in the microenvironment, especially at the level of MIP-3alpha.
Asunto(s)
Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Quimiocina CCL20/farmacología , Células del Estroma/efectos de los fármacos , Western Blotting , Supervivencia Celular , Técnicas de Cocultivo , Medios de Cultivo Condicionados , Citocinas/biosíntesis , Citocinas/genética , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Humanos , Células K562 , Sales de Tetrazolio , TiazolesRESUMEN
This study is focused on the toxicological effect of cyclophosphamide on male mice reproductive system. In the present study, cyclophosphamide was injected intraperitoneally (ip) at the level of 50-200mg/kg body weight into 6-weeks old ICR male mice once in a week for a period of 5 weeks. The animals were sacrificed after 1st and 5th week of last injection. Reduction in weight of testis and epididymis were observed both in 1st and 5th week group mice after administration with increasing concentration of cyclophosphamide. The weight of the body significantly decreased in both 1st and 5th week group in mice treated with 200mg/kg cyclophosphamide. The weight of the testis significantly decreased with all doses of cyclophosphamide in 1st week group, whereas, in 5th week group significant reduction was observed only in 200mg/kg dose of cyclophosphamide. The sperm motility was analyzed with Computer-Assisted Sperm Analysis (CASA). The motility of caudal sperm decreased with increasing concentration of cyclophosphamide in the 1st week group, whereas, it revived after 5th week. The total sperm counts in the epididymis of 1st week group mice declined significantly while significant restoration of the same was observed with mice treated with 50,100 and 150 mg/kg doses in the 5th week group. The intact acrosome was lower with 150 and 200mg/kg doses in both 1st and 5th week group. The live sperm was reduced to 29% in mice treated with 200mg/kg in the 5th week group. The decrease in the pregnancy rate of female mice was 17, 50, 58 and 100% when mated with male mice injected with 50, 100, 150 and 200mg/kg dose, respectively. Seminiferous tubules of mouse testis were severely damaged in the 1st week group. However, reinstate of sperm within the seminiferous tubules was observed in the 5th week group mice. Significant decrease in serum luteinizing hormone (LH) was observed in the 1st week group treated with 50, 100, 150 and 200mg/kg dose of cyclophosphamide. However, no significant difference was observed in the serum follicle-stimulating hormone (FSH), whereas, a decrease of about 98% in serum testosterone level was observed in cyclophosphamide treated mice. The decrease in the mean testosterone levels of cyclophosphamide treated mice served as proof for the damage of testis. These results demonstrate that cyclophosphamide caused temporary interference of normal male reproductive system with low dose treatment, but might be permanent dysfunction in high dose treatment.
Asunto(s)
Antineoplásicos Alquilantes/toxicidad , Ciclofosfamida/toxicidad , Fertilización/efectos de los fármacos , Espermatogénesis/efectos de los fármacos , 3-Hidroxiesteroide Deshidrogenasas/genética , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Reacción Acrosómica/efectos de los fármacos , Animales , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Hormona Folículo Estimulante/sangre , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hormona Luteinizante/sangre , Masculino , Ratones , Ratones Endogámicos ICR , ARN Mensajero/metabolismo , Recuento de Espermatozoides , Motilidad Espermática/efectos de los fármacos , Testículo/efectos de los fármacos , Testículo/enzimología , Testículo/patología , Testosterona/sangreRESUMEN
Under in vitro conditions, incubation with 0.3% bovine serum albumin (BSA) and 1.8 mM CaCl(2) induces mouse sperm capacitation and increases the consequential acrosome-reaction. The effect of mouse uterine 24p3 protein on such stimulated sperm has been investigated to understand the biological function of the 24p3 protein. Variations in the intracellular pH (pHi), calcium concentration, cAMP levels and tyrosine phosphorylation in cytosol were determined and on in vitro mouse fertilization was evaluated. The presence of 24p3 protein reduced the response of sperm to BSA and calcium by suppressing the elevation of intracellular pH, calcium uptake, cAMP accumulation and protein tyrosine phosphorylation of BSA/calcium-stimulated sperm and showed inhibitory effect on mouse in vitro fertilization. The results indicated the inhibition of the BSA-stimulated sperm acrosome reaction by 24p3 protein then suppressed sperm fertilization. We suggested that the 24p3 protein acts as an in vitro inhibitor of the acrosome reaction in BSA stimulated sperm and this might be an anti-fertilization factor in vitro.
Asunto(s)
Reacción Acrosómica/fisiología , Proteínas de Fase Aguda/metabolismo , Fertilización/fisiología , Proteínas Oncogénicas/metabolismo , Proteínas de Fase Aguda/fisiología , Análisis de Varianza , Animales , Cloruro de Calcio , AMP Cíclico/metabolismo , Femenino , Fertilización In Vitro , Citometría de Flujo , Concentración de Iones de Hidrógeno , Lipocalina 2 , Lipocalinas , Masculino , Ratones , Proteínas Oncogénicas/fisiología , Fosforilación , Albúmina Sérica Bovina , Espermatozoides/metabolismo , Tirosina/metabolismo , Útero/químicaRESUMEN
The aim of this study was to illustrate the further process of 24p3 protein after association with epididymal spermatozoa. We have previously identified a caput-initiated 24p3 protein, which interacts with the spermatozoa surface in vitro. In the present study, we investigate another role of the 24p3 protein with spermatozoa. Mouse epididymal spermatozoa exhibit the ability to bind spontaneously with exogenous 24p3 protein, a part of which is further internalized into the spermatozoa in epididymal caput. We have now focused on this issue using freshly prepared spermatozoa from caudal region of epididymis. First, the cytosolic fractionation of spermatozoa has revealed that biotinylated 24p3 protein signal could be detected by supplying biotinylated protein under 37 degrees C incubation after 30 min at this experiment. Further, flow cytometric analysis of FITC-protein containing spermatozoa has revealed two distinct types of fluorescent spermatozoa, and microscopical experimentation with fluorescent FITC-24p3 protein has shown that the 24p3 protein did accumulate in the cytosolic portion of spermatozoa. All of these events, which showed protein uptake into the cell, demonstrated time- and temperature-dependence of endocytotic characteristics, these constituting the critical points in the process of endocytosis for spermatozoa as for other cells. Using a fluorometric method, the binding affinities of ferrous ion and ferric ion to 24p3 protein were shown to be (1.5+/-0.2)x10(6) and (3.0+/-0.4)x10(7)M(-1), respectively. We have also determined the internalization of this protein in the transition of iron into spermatozoa. We report here that spermatozoa, from the caudal epididymis, demonstrate the ability to bind with 24p3 protein and further internalize it and deliver the ferric ion to the spermatozoa via protein internalization. We suggest that the 24p3 protein plays a physiological role in spermatozoa in the context of protein-ligand complex internalization.
Asunto(s)
Proteínas de Fase Aguda/metabolismo , Endocitosis/fisiología , Iones/metabolismo , Hierro/química , Hierro/metabolismo , Proteínas Oncogénicas/metabolismo , Espermatozoides/metabolismo , Animales , Proteínas Portadoras/metabolismo , Citoplasma/química , Epidídimo/citología , Femenino , Citometría de Flujo , Ligandos , Lipocalina 2 , Lipocalinas , Masculino , Ratones , Unión Proteica , Espermatozoides/citología , Temperatura , Factores de TiempoRESUMEN
Menadione is a commonly used compound that causes oxidative stress. We investigated the influence of lipid peroxidation on the apoptotic response of mouse myogenic C2C12 cells following menadione-induced oxidative stress. The presence of hypodiploid cells and phosphatidylserine translocation were assayed to detect apoptotic cells. Menadione at 10-40 micro M induced cell apoptosis. Menadione at dose of 80 micro M induced both apoptosis and necrosis. At a 160 micro M dosage, menadione induced cell necrosis. Caspase 3 activation is required for menadione-induced apoptosis. Incubation of cells with 40 micro M menadione resulted in the depletion of cellular glutathione and increased lipid peroxidation. Pre-treatment of cells with cysteine suppressed the menadione-induced apoptosis and prevented changes in reactive oxygen species levels, glutathione levels and lipid peroxidation. Pre-treatment of cells with deferoxamine mesylate, an iron chelator, also reduced both menadione-induced apoptosis and lipid peroxidation. However, this did not prevent menadione-induced glutathione depletion. Thus, the inhibition of lipid peroxidation by deferoxamine mesylate prevented apoptosis even though cellular glutathione remained depleted. Our data suggest that menadione-induced apoptosis is directly linked to iron-dependent lipid peroxidation.
Asunto(s)
Apoptosis/efectos de los fármacos , Peroxidación de Lípido/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Estrés Oxidativo/fisiología , Vitamina K 3/antagonistas & inhibidores , Vitamina K 3/toxicidad , Animales , Apoptosis/fisiología , Caspasa 3 , Caspasas/metabolismo , Cisteína/farmacología , Deferoxamina/farmacología , Interacciones Farmacológicas , Citometría de Flujo , Glutatión/antagonistas & inhibidores , Glutatión/metabolismo , Quelantes del Hierro/farmacología , Peróxidos Lipídicos/metabolismo , Ratones , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Mioblastos/citología , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Necrosis , Ploidias , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The 24p3 protein is a 25 kDa glycoprotein that is secreted into the uterine fluid during the proestrous phase of mice. We assessed the effects on spermatozoa motility and on the functions of mouse spermatozoa using the computer-assisted sperm analysis method, cytochemical staining and detection of the protein tyrosine phosphorylation pattern. Compared with the control cells, sperm motility was stimulated by the addition of 24p3 protein into the medium. Introducing 24p3 protein enhanced progressive motility but did not promote the appearance of hyperactivated movement. The presence of 24p3 protein in the medium did not allow the cells to undergo the capacitated protein tyrosine phosphorylation pattern and acrosome reaction. The tyrosine phosphorylation pattern shows phosphoproteins in the range of Mr 50,000-106,000 correlated with the sperm progressive motility after the addition of 24p3 protein into the medium. Using flow cytometry, we assessed the changes in the intracellular pH and measured the intracellular cAMP concentration with an immunodetection kit. The results indicated that the elevation in intracellular pH from 6.67 to 6.89, increase of intracellular cAMP accumulation, and protein tyrosine phosphorylation might be the factors in enhancement of sperm motility as the 24p3 protein bound to the spermatozoa. The 24p3 protein may have a role in regulating flagellar motility.
Asunto(s)
Proteínas de Fase Aguda/farmacología , Proteínas Oncogénicas/farmacología , Motilidad Espermática/efectos de los fármacos , Espermatozoides/citología , Espermatozoides/efectos de los fármacos , Reacción Acrosómica , Animales , AMP Cíclico/metabolismo , Femenino , Concentración de Iones de Hidrógeno , Lipocalina 2 , Lipocalinas , Masculino , Ratones , Ratones Endogámicos ICR , Fosforilación , Fosfotirosina/metabolismo , Espermatozoides/metabolismo , Factores de TiempoRESUMEN
Pulmonary function in 42 patients with chronic myelogenous leukemia (CML) was tested before and after HLA-matched (39 related, 3 unrelated) allogeneic bone marrow transplantation (BMT) between 1985 and 1999. Pulmonary function tests (PFTs) including ventilatory capacity, lung volumes, and diffusion capacity for carbon monoxide (DLCO) were performed before and 3, 6, 12, and 24 months after BMT, and every 12 months thereafter. Possible pre- and post-BMT risk factors were evaluated for their influence on pulmonary function. Patients were divided into two groups according to their survival duration for more than 12 months or not. Pretransplant PFTs were essentially normal except for mild reduction in DLCO values in the short-term survival group. Overall pulmonary function changes revealed persistent and significant decrease of forced vital capacity (FVC) and DLCO values after BMT. The DLCO values reached abnormal levels (< 80%) and showed a trend of incomplete recovery. Decrease of forced expiratory volume in the first second (FEV1) and vital capacity were also noted but the FEV1/FVC ratio remained within normal limits after BMT. Transient fall of total lung capacity after BMT was noted. However, its values did not reach abnormal levels such as to cause restrictive ventilatory impairment. Possible risk factors including gender, smoking, bronchiolitis obliterans, acute and chronic graft-versus-host disease (GVHD) were found to have significant influences on posttransplant pulmonary function changes by multiple regression analysis. Most patients except those who developed bronchiolitis obliterans were clinically asymptomatic. Development of bronchiolitis obliterans was the most important factor to cause both clinical symptoms and impaired pulmonary function. In summary, pulmonary function changes before and after HLA-matched allogeneic BMT in long-term survivors of CML only showed modest dysfunction. The primary negative presentation with the development of oxygenation defect had no clinical significance in most patients. The influences on the impairment of pulmonary function were multifactorial.