RESUMEN

OBJECTIVE: To establish the absorption and elution test for relatively quantitive obtaining anti-A and anti-B blood group IgG antibodies in the plasma of O-type RhD-positive pregnant women. METHODS: 95 cases of the O-type RhD-positive pregnant women plasma samples were randomly selected for obtaining the IgG antibodies of anti-A and anti-B blood group, with absorption test under 37 ℃ and elution test under 56 ℃, and the IgG anti-A and anti-B antibody titers of plasma and elution were determined by the microcolumn gel anti-human globulin test. The differences and correlation between the titers of IgG antibodies in the eluent and plasma were compared and analyzed. RESULTS: After a logarithmic transformation (Log2), there was no statistically difference between IgG antibody anti-A difference value and anti-B difference value in the eluent and plasma (P >0.05). The titer of IgG antibody in the eluent was positively correlated with the titer of IgG antibody in the plasma (r =0.914). The linear equation for IgG antibody titers fitted by a scatter plot between the eluent and plasma was Y=-3.55+0.96X. CONCLUSION: The absorption and elution test can be used to obtain the anti-A and anti-B IgG antibodies in the plasma of O-type RhD-positive pregnant women, whose plasma origin IgG titer is greater than 8. Meanwhile, the acquisition of anti-A antibodies was as effective as anti-B antibodies at the same time, and the antibodies obtained are positive proportional to their respective concentrations in the plasma.

Asunto(s)

Sistema del Grupo Sanguíneo ABO , Inmunoglobulina G , Humanos , Inmunoglobulina G/sangre , Femenino , Embarazo , Sistema del Grupo Sanguíneo ABO/inmunología , Sistema del Grupo Sanguíneo Rh-Hr/inmunología

RESUMEN

PURPOSE: Circular RNAs (circRNAs) are increasingly recognized for their important roles in various cancers, including papillary thyroid cancer (PTC). The specific mechanisms by which the circLIF receptor subunit alpha (circLIFR, hsa_circ_0072309) influences PTC progression remain largely unknown. METHODS: In our study, CircLIFR, miR-429, and TIMP2 levels were assessed using reverse transcription-quantitative PCR. The roles of circLIFR and miR-429 in PTC cells were determined using Cell Counting Kit-8, colony formation, wound healing, and Transwell assays. Western blotting was utilized to examine the levels of TIMP2. The direct interaction between circLIFR, TIMP2, and miR-429 was confirmed using dual-luciferase reporter, RNA immunoprecipitation, and fluorescence in situ hybridization assays. RESULTS: In PTC tissues and cells, a decrease in circLIFR and TIMP2 levels, accompanied by an increase in miR-429 levels, was observed. Overexpression of circLIFR or downregulation of miR-429 effectively suppressed the proliferation and migration of PTC cells. Conversely, the knockdown of circLIFR or overexpression of miR-429 had the opposite effect. Furthermore, circLIFR overexpression suppressed tumor growth in vivo. Mechanistically, circLIFR modulated TIMP2 expression by serving as a sponge for miR-429. Rescue experiments indicated that the antitumor effect of circLIFR could be reversed by miR-429. CONCLUSION: This study confirmed circLIFR as a novel tumor suppressor delayed PTC progression through the miR-429/TIMP2 axis. These findings suggested that circLIFR held promise as a potential therapeutic target for PTC.

Asunto(s)

Proliferación Celular , Progresión de la Enfermedad , MicroARNs , ARN Circular , Cáncer Papilar Tiroideo , Neoplasias de la Tiroides , Inhibidor Tisular de Metaloproteinasa-2 , Animales , Femenino , Humanos , Masculino , Ratones , Línea Celular Tumoral , Movimiento Celular/genética , Regulación Neoplásica de la Expresión Génica , Ratones Endogámicos BALB C , Ratones Desnudos , MicroARNs/genética , ARN Circular/genética , Cáncer Papilar Tiroideo/genética , Cáncer Papilar Tiroideo/patología , Cáncer Papilar Tiroideo/metabolismo , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patología , Neoplasias de la Tiroides/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/genética , Inhibidor Tisular de Metaloproteinasa-2/metabolismo

RESUMEN

Long noncoding RNAs (lncRNAs), which are more than 200 nucleotides in length and do not encode proteins, play crucial roles in governing gene expression at both the transcriptional and posttranscriptional levels. These molecules demonstrate specific expression patterns in various tissues and developmental stages, suggesting their involvement in numerous developmental processes and diseases, notably cancer. Despite their widespread acknowledgment and the growing enthusiasm surrounding their potential as diagnostic and prognostic biomarkers, the precise mechanisms through which lncRNAs function remain inadequately understood. A few lncRNAs have been studied in depth, providing valuable insights into their biological activities and suggesting emerging functional themes and mechanistic models. However, the extent to which the mammalian genome is transcribed into functional noncoding transcripts is still a matter of debate. This review synthesizes our current understanding of lncRNA biogenesis, their genomic contexts, and their multifaceted roles in tumorigenesis, highlighting their potential in cancer-targeted therapy. By exploring historical perspectives alongside recent breakthroughs, we aim to illuminate the diverse roles of lncRNA and reflect on the broader implications of their study for understanding genome evolution and function, as well as for advancing clinical applications.

RESUMEN

Objective To investigate the effect of long intergenic non-coding RNA COX2 (lincRNA-COX2) on apoptosis and polarization of Listeria monocytogenes (Lm)-infected RAW264.7 cells. Methods RAW264.7 cells were cultured and divided into control group (uninfected cells), Lm infection group, negative control of small interfering RNA (si-NC) group, si-NC and Lm infection group, small interfering RNA of lincRNA-COX2 (si-lincRNA-COX2) group, si-lincRNA-COX2 and Lm infection group. RAW264.7 cells were infected with MOI=10 Lm for 6 hours, and then the inhibition efficiency of siRNA transfection was detected by fluorescence microscope and quantitative real-time PCR (qRT-PCR). The expression levels of cleaved-caspase-3(c-caspase-3), caspase-3, B-cell lymphoma-2 (Bcl2), Bcl2 associated X protein (BAX), arginase 1 (Arg1), inducible nitric oxide synthase (iNOS) were detected by Western blot analysis. Results c-caspase-3/caspase-3, BAX/Bcl2 and iNOS were significantly up-regulated, while the level of Arg1 was down-regulated in Lm-infected RAW264.7 cells compared with control group. LincRNA-COX2 knockdown inhibited the increase of protein levels for BAX/Bcl2, c-caspase-3/caspase-3 and iNOS in Lm-infected RAW264.7 cells, while the level of Arg1 in Lm-infected RAW264.7 cells was up-regulated. Conclusion Knockdown of lincRNA-COX2 can inhibit cell apoptosis and suppress the macrophage polarization into M1 type in Lm-infected RAW264.7 cells.

Asunto(s)

Ciclooxigenasa 2 , Listeria monocytogenes , Macrófagos , ARN Largo no Codificante , Apoptosis/genética , Proteína X Asociada a bcl-2/metabolismo , Caspasa 3/genética , Caspasa 3/metabolismo , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Listeria monocytogenes/metabolismo , Listeria monocytogenes/patogenicidad , Macrófagos/metabolismo , Macrófagos/microbiología , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , ARN Interferente Pequeño/genética , Animales , Ratones

RESUMEN

Currently, the pathogenesis of prostate diseases is still under investigation, but it is generally clinically recognized to be related to the imbalance of prostate cell viability. Trichomonas vaginalis macrophage migration inhibitory factor (TvMIF) has been reported to induce the proliferation and invasion of prostate cancer cells, but for normal PECs, the relationship between them has not been reliably confirmed. Therefore, this research aims to determine the influence of macrophage TvMIF on prostate epithelial cells (PECs) and its preliminary mechanism. The activity of RWPE-1 human normal prostate epithelial cells, the inflammatory response state, the expression of miR-451, and the effect of miR-451 on RWPE-1 were detected after TvMIF intervention. We found that TvMIF can enhance RWPE-1 cell proliferation and activate inflammatory factors by suppressing miR-451, thus taking part in the development and proliferation of diseases such as prostatic hyperplasia and prostatitis.

Asunto(s)

Factores Inhibidores de la Migración de Macrófagos , MicroARNs , Neoplasias de la Próstata , Tricomoniasis , Trichomonas vaginalis , Proliferación Celular , Células Epiteliales/metabolismo , Humanos , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Masculino , MicroARNs/metabolismo , Próstata/patología , Neoplasias de la Próstata/patología , Tricomoniasis/metabolismo , Tricomoniasis/patología , Trichomonas vaginalis/genética , Trichomonas vaginalis/metabolismo

RESUMEN

Listeria monocytogenes crossing the blood-brain barrier in the form of "Trojan Horse" is of great significance for the establishment of bacterial encephalitis and meningitis. Induction of cell migration and crossing the blood-brain barrier is very important to understand the Listeria pathogenesis. The Rho GTPases family is considered a key factor in regulating cell migration. This study was designed to investigate the expression of Rho GTPases and their effect on the behavior of cell migration and the stimulation of immune factors. Selective Rho GTPases were investigated by real-time PCR and Western blot. Among these, the expression of RhoA was significantly increased following the infection of Listeria monocytogenes in macrophages. Further, we found that RhoA improves the migration of macrophages and expression of IL-1ß, IL-6, and TNF-α. The expression of IL-1ß, IL-6 and TNF-α possibly facilitates the migration and adhesion of macrophages to cross the blood-brain barrier. This study provides preliminary ground to investigate the detailed mechanism of Listeria monocytogenes crossing the blood-brain barrier.

Asunto(s)

Listeria monocytogenes , Listeriosis , Barrera Hematoencefálica/metabolismo , Citocinas/metabolismo , Humanos , Listeria monocytogenes/metabolismo , Macrófagos/metabolismo , Proteínas de Unión al GTP rho/genética

RESUMEN

Listeria monocytogenes (Lm) can lead to high mortality rates relative to other foodborne pathogens. Lm-induced inflammation is partly characterized by macrophage activation. Long non-coding RNAs (lncRNAs) have important roles in various biological processes. However, it is unknown how lncRNAs regulate the host response to Lm infection. To identify the role of lncRNA in Lm infection, we used in vitro and in vivo models. We found that lincRNA-Cox2 was highly expressed in Lm-infected RAW264.7 cells. LincRNA-Cox2 knockdown resulted in reduced proinflammatory cytokines, apoptosis, migration ability and enhanced phagocytosis of Lm. LincRNA-Cox2 knockdown also reduced the phosphorylation of Janus kinase 3 (JAK3) and signal transducer and activator of transcription (STAT3) and the nuclear translocation of nuclear factor (NF)-κB P65, which are known to be involved in inflammatory responses. Experimentally inhibiting the protein and phosphorylation levels of STAT3 resulted in reduced proinflammatory cytokines and enhanced phagocytosis of Lm by the RAW264.7 cells. Our research suggests that lincRNA-Cox2 plays important roles in inflammation, the phagocytic function and cell migration ability of RAW264.7 cells by activating interleukin (IL)-6/JAK3/STAT3 signaling, and lincRNA-Cox2 also regulates NF-κB P65 nuclear translocation. Our research provides new insights into the regulatory role of lincRNA-Cox2 in Lm infection.

Asunto(s)

Listeria monocytogenes , ARN Largo no Codificante , Interleucina-6/genética , Janus Quinasa 3 , FN-kappa B , ARN Largo no Codificante/genética

RESUMEN

Listeria monocytogenes is a food-borne pathogen responsible for neurolisteriosis, which is potentially lethal in immunocompromised individuals. Microglia are the main target cells for L. monocytogenes in central nervous system (CNS). However, the precise mechanisms by which they trigger neuroinflammatory processes remain unknown. The BV2 microglial cell line and a murine model of L. monocytogenes infection were used for experiments in this study. Listeria monocytogenes induced pyroptosis and nucleotide binding and oligomerization, leucine-rich repeat, pyrin domain-containing 3 (NLRP3) inflammasome activation in BV2. Pharmacological inhibition of the NLRP3 inflammasome attenuated L. monocytogenes-induced pyroptosis. Moreover, inhibition of nuclear factor kappa-B (NF-κB) and extracellular regulated protein kinases (ERK) pathways induced a decrease in caspase1 activation and mature IL-1ß-17 secretion. Our collective findings support critical involvement of the NLRP3 inflammasome in L. monocytogenes-induced neuroinflammation and, to an extent, ROS production. In addition, ERK and NF-κB signaling play an important role in activation of the NLRP3 inflammasome, both in vitro and in vivo.

Asunto(s)

Inflamasomas/inmunología , Listeria monocytogenes/fisiología , Listeriosis/inmunología , Microglía/microbiología , FN-kappa B/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Especies Reactivas de Oxígeno/inmunología , Animales , Humanos , Inflamasomas/genética , Listeria monocytogenes/genética , Listeriosis/genética , Listeriosis/microbiología , Listeriosis/fisiopatología , Sistema de Señalización de MAP Quinasas , Ratones , Microglía/citología , Microglía/inmunología , FN-kappa B/genética , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Piroptosis , Transducción de Señal

RESUMEN

Objective To investigate the role of Ras homolog gene (Rho) A/Rho-associated coiled-coil containing protein kinase (ROCK) signaling pathway in tumor necrosis factor α (TNF-α) promoting hyper-permeability of vascular endothelial cells infected by Listeria monocytogenes (Lm) . Methods The cultured human umbilical vein endothelial cells (HUVECs) were divided into a control group (uninfected cells), TNF-α treatment group (100 ng/mL TNF-α, for 2 hours), Lm infection group (infected with MOI=10 Lm for 2 hours, then added gentamicin for 0.5 hour), Lm infection and TNF-α treatment group (infected with Lm and then treated with 100 ng/mL TNF-α for 2 hours), and Y-27632 inhibitor group combined with Lm infection and TNF-α treatment (treated with 50 µmol/L ROCK inhibitor Y-27632 for 30 minutes, and then Lm infection and TNF-α treatment as above). The protein levels of RhoA, zonula occluden-1 (ZO-1), occludin and ROCK in HUVECs were detected by Western blot analysis; the permeability of HUVECs was analyzed by the horseradish peroxidase (HRP) leakage; and the distribution of F-actin in HUVECs was detected by fluorescein isothiocyanate (FITC)-labeled phalloidine staining. Results TNF-α reduced the expression of tight junction protein ZO-1 and occludin in Lm-infected HUVECs, promoted its hyper-permeability and cytoskeletal rearrangement, and up-regulated the expression of RhoA and ROCK. ROCK inhibitor Y-27632 obviously inhibited the cytoskeleton rearrangement and hyper-permeability of HUVECs induced by TNF-α. Conclusion TNF-α can enhance hyper-permeability of HUVECs infected by Lm, which may be regulated by RhoA/Rock signaling pathway.

Asunto(s)

Células Endoteliales de la Vena Umbilical Humana/microbiología , Listeria monocytogenes , Transducción de Señal , Factor de Necrosis Tumoral alfa/farmacología , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Células Cultivadas , Citoesqueleto/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Permeabilidad

RESUMEN

Toxoplasma Gondii is an obligate intracellular protozoan. T. gondii tachyzoites can invade nucleated host cells and inhibit their apoptosis. Therefore, the goal of the present study was to determine whether rhoptry protein 16 (ROP16) secreted by the invading T. gondii can reduce the apoptotic response of the host cell. For this purpose, a vector for in vitro overexpression of T. gondii ROP16 was constructed and used to transfect human 293T cells. Cells transfected with the vector robustly expressed ROP16, as evidenced by Western blotting. Apoptosis of 293T cells was induced by incubation with 0.5 µg/mL actinomycin D (ActD) for 24 h, and its magnitude was measured using Annexin V-FITC/PI and TUNEL assays. Cells transfected by ROP16-expressing vector were characterized by a significantly lower level of apoptosis measured by both techniques.Moreover, caspase-3 activity was also reduced. Thus, ROP16 inhibited ActD-induced apoptosis of human 293T cells, documenting the ability of this rhoptry protein to modulate apoptosis of host cells infected by T. gondii.

Asunto(s)

Toxoplasma , Apoptosis , Células HEK293 , Humanos , Toxoplasma/genética

RESUMEN

The exact mechanism of delayed death of Toxoplasma gondii is not known. FAS II synthesis in the apicoplast of T. gondii is essential for the survival of Toxoplasma gondii, while ß-hydroxyacylacyl carrier protein dehydratase (FabZ) is indispensable for fatty acid synthesis. The present study investigated the relationship between the delayed death of T. gondii by inducing metabolic disorders due to suppression the expression of FabZ. A tetracycline-induced knockout vector inserted with T. gondii fabZ gene was constructed, and transfected into T. gondii TATi strain by electroporation. The stable mutants with tetracycline-induced knockout were selected, expression of FabZ was suppressed by using anhydrotetracycline (ATc), and FAS II deficient tachyzoites were prepared. The Western blot and qPCR results revealed that proliferation of FAS II deficient tachyzoites was not significantly different compared to the normal tachyzoites at 24 h and 48 h; however, after 72 h, the number of T. gondii tachyzoites in the ATc treated group was significantly (p < 0.05) less than that of non-treated group, indicating the delayed death of T. gondii caused by the loss of apicoplast and decrease in the expression of FabZ, which inhibited the FAS II metabolism. The results of this study can be used for prevention of toxoplasmosis by inducing delayed death of T. gondii.

Asunto(s)

Enfermedades Metabólicas , Toxoplasma , Antibacterianos , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa , Tetraciclina , Toxoplasma/genética

RESUMEN

OBJECTIVE: To explore the apoptosis of HepG2 cells infected by Listeria monocytogenes EGD strain (Lm-EGD) as well as Rho family small GTPases RhoA expression. METHODS: HepG2 cells were infected with Lm-EGD (MOI=10 and MOI=100) and collected 1 hour and 20 hours after infection. After harvesting, the apoptosis of HepG2 cells was determined by flow cytometry combined with annexin V-FITC/PI assay. RhoA and caspase 3 mRNAs were analyzed by reverse-transcription PCR. The caspase 3 activity was detected by colorimetric assay. And Western blotting was used to detect RhoA expression in HepG2 cells. RESULTS: Lm invasion promoted HepG2 cell apoptosis and down-regulated RhoA mRNA and protein expression. Additionally, caspase 3 expression was up-regulated following Lm infection. CONCLUSION: Lm infection could promote host cell apoptosis and down-regulate RhoA expression.

Asunto(s)

Apoptosis , Listeria monocytogenes/patogenicidad , Proteína de Unión al GTP rhoA/fisiología , Caspasa 3/metabolismo , Regulación hacia Abajo , Células Hep G2 , Humanos , ARN Mensajero/análisis , Proteína de Unión al GTP rhoA/análisis , Proteína de Unión al GTP rhoA/genética

RESUMEN

Toxoplasma gondii is an obligate intracellular parasite that may manipulate host cell mitochondrial apoptosis pathways. In our experiment, 293T cells were transfected with the p3×FLAG-CMV-Myc-ROP18 vector and expressed the ROP18-Myc fusion protein. Cell apoptosis was induced by 0.5 µg/mL actinomycin D (ActD) and was detected by Annexin V-FITC/PI assay. The cell mitochondrial membrane potential was determined by JC-1. Cytochrome c (Cyto-c) from mitochondria and the cytoplasm was measured by Western blot. The Bcl-2 and Bax coding gene expression levels were detected by real-time PCR. We found, in vitro, that T. gondii ROP18 significantly suppressed 293T cell apoptosis induced by ActD and maintained mitochondrial membrane potential and integrity, thereby preventing the release of Cyto-c from mitochondria into the cytoplasm. The ratio of Bcl-2/Bax in ROP18-overexpressing cells was significantly higher than that of the negative control. Therefore, we speculate that ROP18 could suppress host cell apoptosis via the mitochondrial apoptosis pathway in vitro.

Asunto(s)

Apoptosis/fisiología , Potencial de la Membrana Mitocondrial/fisiología , Mitocondrias/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Toxoplasma/metabolismo , Anexina A5 , Western Blotting , Línea Celular , Citocromos c/metabolismo , Dactinomicina/farmacología , Fluoresceína-5-Isotiocianato/análogos & derivados , Células HEK293 , Humanos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Protozoarias , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Recombinantes de Fusión/metabolismo , Toxoplasma/genética , Transfección , Proteína X Asociada a bcl-2/metabolismo

RESUMEN

BACKGROUND/AIMS: IGF-1 can act as an endocrine hormone and its signaling server as essential roles in regulating tumorigenesis. Polymorphisms in IGF-1 have been reported associated bad prognosis of with human cancer, but their association with the risk of human gastric cancer (GC) has not been found so far. In this study rs6218 located in the 3'UTR of IGF-1 was selected to evaluate its relationship with the risk of GC among Chinese population. METHODS: Questionnaire, SNaPshot genotype assay, real time PCR assay, cell transfection and the dual luciferase reporter assay were used in our study. RESULTS: SNP rs6218 in IGF-1 3'-UTR was involved in the occurrence of GC by acting as a tumor promotion factor while rs6128 acting as a risk factor. SNP rs6128 was also could be regulated by miR-603 which caused an up-regulation of IGF-1 in patients with UC and CC genotype. Furthermore, the carriers of UC and CC genotype presented a big tumor size as well as the high probability of metastasis. CONCLUSION: In conclusion, our findings have shown that the SNP rs6218 in IGF-1 3'-UTR, through disrupting the regulatory role of miR-603 in IGF-1 expression, rs16128 in IGF-1 might act as a promotion factor in the pathogenesis of GC.

Asunto(s)

Regiones no Traducidas 3' , Regulación Neoplásica de la Expresión Génica , Factor I del Crecimiento Similar a la Insulina/genética , MicroARNs/genética , Polimorfismo de Nucleótido Simple , Neoplasias Gástricas/genética , Anciano , Pueblo Asiatico , Estudios de Casos y Controles , Femenino , Genes Reporteros , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Luciferasas/genética , Luciferasas/metabolismo , Metástasis Linfática , Masculino , MicroARNs/metabolismo , Persona de Mediana Edad , Transducción de Señal , Estómago , Neoplasias Gástricas/etnología , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Carga Tumoral

RESUMEN

OBJECTIVE: To construct a 293T mutant cell line over-expressing ROP18 of Toxoplasma gondii by Tet-on lentivirus expression system. METHODS: Rop18 gene of T. gondii was amplified by PCR, and inserted into a lentiviral vector pLVCT-tTR-KRAB. The recombinant plasmid pLVCT-tTR-KRAB-ROP18 (6 µg) and 293T human embryonic kidney cells were co-transfected with psPAX2 (4 µg) and pMD2.G (2 µg) for the packaging. The result of co-transfection was evaluated by fluorescence microscopy. At 48 h and 72 h after co-transfection, the supernatant of the packaging lentivirus was collected for the 293T cell infection. The doxycycline (DOX) was added into the medium to induce the ROP18 expression in 293T cells. The ROP18 fusion expression was observed under fluorescence microscope and detected by RT-PCR after induction. RESULTS: PCR product of the gene fragment encoding ROP18 was 960 bp. The recombinant plasmid pLVCT-tTR-KRAB-ROP18 was identified by PCR and restriction enzyme digestion. Green fluorescence was observed in 293T cells at 48 h post-transfection. Bright green fluorescence was observed in 293T cells at 24 h after DOX induction. RT-PCR results showed that a 960 bp specific band (ROP18 gene) was detectable in 293T cells. CONCLUSION: 293T cell line stably expressing ROP18 is established with Tet-on lentivirus expression system.

Asunto(s)

Vectores Genéticos , Lentivirus , Proteínas Serina-Treonina Quinasas/genética , Toxoplasma , Células HEK293 , Humanos , Reacción en Cadena de la Polimerasa , Proteínas Protozoarias , Transfección

RESUMEN

This study aimed to explore Toxoplasma gondii nucleus coding apicoplast protein acyl carrier protein (ACP) synthesis and trafficking in delayed death. The recombinant T. gondii ACP was expressed by prokaryotic expression method, and anti-ACP polyclonal antibody was obtained from rabbit immune. T. gondii "delayed death" was induced by clindamycin (CLDM), and ACP transcription was determined by real-time PCR assay. The expression of ACP with transit type (t-ACP) and mature type (m-ACP) was determined by Western blotting with anti-ACP polyclonal antibody. The mutant-expressed ACP fused with green fluorescent protein (GFP) tag was constructed by pHX-ACP-GFP. The distribution of ACP in "delayed death" was observed by ACP-GFP fusion protein with a confocal microscope. T. gondii ACP transcription and t-ACP expression had no significant decrease in the early 4 h of "delayed death," but there has been a significant decrease in 6 h. The expression of m-ACP had a significant decrease in 4 h which occurred earlier than the t-ACP expression. The number of brightly dot green fluorescence in ACP-GFP mutant decreased with prolonged time. There was very little brightly dot green fluorescence in ACP-GFP mutant when treated with CLDM for 6 h. CLDM could suppress apicoplast proliferation and induce T. gondii "delayed death"; however, it could not directly suppress nucleus coding ACP transcription and expression. T. gondii lacking of apicoplast had a barrier of transit peptide cleavage and t-ACP could not be transformed into m-ACP. The reason for the decrease in ACP expression could be due to excessive t-ACP synthesis in tachyzoites resulting in a negative feedback for the ACP coding gene transcription.

Asunto(s)

Proteína Transportadora de Acilo/metabolismo , Apicoplastos/metabolismo , Proteínas Protozoarias/metabolismo , Toxoplasma/fisiología , Proteína Transportadora de Acilo/genética , Animales , Anticuerpos Antiprotozoarios/inmunología , Núcleo Celular/metabolismo , Proteínas Fluorescentes Verdes , Mutación , Transporte de Proteínas , Proteínas Protozoarias/genética , Conejos , Proteínas Recombinantes de Fusión , Toxoplasma/genética , Toxoplasma/inmunología

RESUMEN

Slit2 is often overexpressed in cancers. Slit2 is a secreted protein that binds to Roundabout (Robo) receptors to regulate cell growth and migration. Here, we employed several complementary mouse models of intestinal cancers, including the Slit2 transgenic mice, the ApcMin/+ spontaneous intestinal adenoma mouse model, and the DMH/DSS-induced colorectal carcinoma model to clarify function of Slit2/Robo1 signaling in intestinal tumorigenesis. We showed that Slit2 and Robo1 are overexpressed in intestinal tumors and may contribute to tumor generation. The Slit2/Robo1 signaling can induce precancerous lesions of the intestine and tumor progression. Ectopic expression of Slit2 activated Slit2/Robo1 signaling and promoted tumorigenesis and tumor growth. This was mediated in part through activation of the Src signaling, which then down-regulated E-cadherin, thereby activating Wnt/ß-catenin signaling. Thus, Slit2/Robo1 signaling is oncogenic in intestinal tumorigenesis.

Asunto(s)

Adenoma/enzimología , Carcinoma/enzimología , Neoplasias Colorrectales/enzimología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Mucosa Intestinal/enzimología , Proteínas del Tejido Nervioso/metabolismo , Receptores Inmunológicos/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismo , Familia-src Quinasas/metabolismo , Adenoma/genética , Adenoma/patología , Animales , Carcinoma/genética , Carcinoma/patología , Proliferación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Genes APC , Células HCT116 , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Mucosa Intestinal/patología , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Persona de Mediana Edad , Proteínas del Tejido Nervioso/genética , Interferencia de ARN , Receptores Inmunológicos/genética , Transfección , Carga Tumoral , Proteínas Roundabout

RESUMEN

OBJECTIVE: To construct a beta-hydroxyacyl-acyl carrier protein dehydratase (FABZ) mutant of Toxoplasma gondii with tetracycline inducible expression system. METHODS: The fabz gene was amplified from T. gondii genomic DNA, and then used to construct the tetracycline inducible expression vector pTetO7-Sag1-FABZ-Ty-DHFR. The vector was transfected into TATi strain by electroporation. The FABZ defective mutant was selected by pyrimethamine and limitting dilution assay. The expression of Ty-tagged mutant was detected by Western blotting. 5 x 10(5) tachyzoites of FABZ defective mutant were cultured in HFF in the presence of anhydrotetracycline (ATc, 1 microg/ml) for 24 h and 48 h, respectively. The expression of Ty-tagged FABZ protein in the mutant was detected by Western blotting. RESULTS: The mutant could express the transit peptide (t-FABZ) and mature FABZ (m-FABZ) with the Ty-epitope tag. After ATc added in culture medium for 24 h and 48 h, the expression of t-FABZ in the mutant decreased significantly (P<0.05). CONCLUSION: The FABZ mutant is constructed with a tetracycline inducible expression system.

Asunto(s)

Hidroliasas/metabolismo , Toxoplasma/enzimología , Antibacterianos , ADN Bacteriano , Vectores Genéticos , Hidroliasas/genética , Mutación , Tetraciclina , Transfección

RESUMEN

Recently, several reports have revealed that some simian adenoviruses (AdVs) strains show a close relationship to human AdVs. In the present study, a simian AdV strain named SAdV-ch1 was detected in chimpanzees in China, and its complete genome was determined. Phylogenetic analysis revealed SAdV-ch1 clustering in a clade that was separate from all of the other simian AdVs but genetically close to a human AdV strain, HAdV-18 (GenBank no. GU191019), sharing 92.5 % sequence identity with it. Recombination analysis provided evidence that a recombination event had occurred between SAdV-ch1 and HAdV-61 (JF964962), where SAdV-ch1 exchanged partial of its hexon gene segment with HAdV-61, leading to the recombinant HAdV-31 (AM749299).

Asunto(s)

Adenovirus Humanos/genética , Adenovirus de los Simios/genética , Enfermedades del Simio Antropoideo/virología , Genoma Viral , Pan troglodytes , Adenovirus Humanos/clasificación , Adenovirus de los Simios/clasificación , Animales , Humanos , Filogenia

RESUMEN

In this study, we evaluated four methods to separate and purify Toxoplasma gondii tachyzoites from in vivo and in vitro culture systems, including trypsin digestion, purification with a 3-µm filter, CF-11 cellulose purification, and Percoll purification. Our results indicate that both purification with a 3-µm filter and CF11 cellulose purification methods remove leukocytes or HeLa cells, and can therefore be used as candidate methods for the purification of in vivo and in vitro culture products. Trypsin digestion had a high tachyzoite recovery rate, but 22.35% of leukocytes and 69.64% of HeLa cells remained in the purified products. Percoll solution [30% (v/v)] also had a high tachyzoite recovery rate, but 3.44% of leukocytes and 61.61% of HeLa cells remained in the purified products. The 40% Percoll solution was also a candidate method for purifying tachyzoites from in vivo culture products, with a 65.45% tachyzoite recovery rate and without leukocytes.

Asunto(s)

Toxoplasma/aislamiento & purificación , Animales , Celulosa , Centrifugación , Eritrocitos/parasitología , Filtración , Células HeLa , Humanos , Leucocitos/parasitología , Ratones , Povidona , Dióxido de Silicio , Tripsina/metabolismo