RESUMEN
Objective: To elucidate the roles of interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) in cell cycle regulation and proliferation of rheumatoid arthritis fibroblast-like synovial cells (RA-FLSs). Methods: Under stimulation with IL-6/soluble interleukin-6 receptor (sIL-6R) and TNF-α, we examined the expression of cell cycle regulators [p16INK4a, p21Cip1, p27Kip1, cyclin-dependent kinase-4 (CDK4), CDK6, Cyclin D, Cyclin E, and retinoblastoma protein (pRB)] by quantitative polymerase chain reaction, Western blotting, and immunofluorescence staining. The expression of pRB, with or without 10% foetal bovine serum, was examined by Western blotting. DNA synthesis and cell viability were examined by the BrdU assay and WST-8 assay, respectively. After transfection with siRNA/p16INK4a, siRNA/p21Cip1, siRNA/p27Kip1, siRNA/CDK4, or siRNA/CDK6, RA-FLSs were successively stimulated with or without IL-6/sIL-6R or TNF-α to determine cell viability. Results: IL-6/sIL-6R significantly decreased the expression of p16INK4a, and increased p21Cip1, Cyclin E1, CYCLIN D, and pRB. TNF-α decreased the expression of CDK4, and significantly increased p27Kip1, CDK6, Cyclin E1/E2, CYCLIN D, CYCLIN E, pRB, and phosphorylated pRB (phospho-pRB). By immunofluorescence staining, CYCLIN D and phospho-pRB were simultaneously stained in the single cell. In serum-free culture, the expression of pRB was apparently decreased. DNA synthesis and cell viability were significantly increased by IL-6/sIL-6R and TNF-α. Silencing of CDK6 attenuated the cell viability induced by IL-6 and TNF-α. Conclusion: The results indicate that IL-6 and TNF-α interact with each other in regulating the cell cycle and accelerate the proliferation of RA-FLSs.
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Artritis Reumatoide/genética , Regulación de la Expresión Génica , Interleucina-6/genética , Sinoviocitos/patología , Factor de Necrosis Tumoral alfa/genética , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Western Blotting , Ciclo Celular , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Interleucina-6/biosíntesis , ARN/genética , Sinoviocitos/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
AIMS: Primary central nervous system lymphoma (PCNSL) manifest aggressive clinical behaviour and have poor prognosis. Although constitutive activation of the nuclear factor-κB (NF-κB) pathway has been documented, knowledge about the genetic alterations leading to the impairment of the NF-κB pathway in PCNSLs is still limited. This study was aimed to unravel the underlying genetic profiles of PCNSL. METHODS: We conducted the systematic sequencing of 21 genes relevant to the NF-κB signalling network for 71 PCNSLs as well as the pyrosequencing of CD79B and MYD88 mutation hotspots in a further 35 PCNSLs and 46 glioblastomas (GBMs) for validation. RESULTS: The results showed that 68 out of 71 PCNSLs had mutations in the NF-κB gene network, most commonly affecting CD79B (83%), MYD88 (76%), TBL1XR1 (23%), PRDM1 (20%) and CREBBP1 (20%). These mutations, particularly CD79B and MYD88, frequently coincided within each tumour in various combinations, simultaneously affecting diverse pathways within the network. No GBMs had hotspot mutation of CD79B Y196 and MYD88 L265. CONCLUSIONS: The prevalence of CD79B and MYD88 mutations in PCNSLs was considerably higher than reported in systemic diffuse large B-cell lymphomas. This observation could reflect the paucity of antigen stimuli from the immune system in the central nervous system (CNS) and the necessity to substitute them by the constitutive activation of CD79B and MYD88 that would initiate the signalling cascades. These hotspot mutations may serve as a genetic hallmark for PCNSL serving as a genetic marker for diagnose and potential targets for molecular therapy.
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Antígenos CD79/genética , Neoplasias del Sistema Nervioso Central/genética , Linfoma de Células B Grandes Difuso/genética , Factor 88 de Diferenciación Mieloide/genética , Anciano , Análisis Mutacional de ADN , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación , Reacción en Cadena de la PolimerasaRESUMEN
BACKGROUND: Immune checkpoint inhibitors (ICIs) are indicated for various cancers and are the mainstay of cancer immunotherapy. They are often associated with ICI-related pneumonitis (CIP), however, hindering a favorable clinical course. Recently, non-oncology concomitant drugs have been reported to affect the efficacy and toxicity of ICIs; however, the association between these drugs and the risk for CIP is uncertain. The aim of this study was to assess the impact of baseline concomitant drugs on CIP incidence in ICI-treated advanced cancer patients. PATIENTS AND METHODS: This was a single-center retrospective study that included a cohort of 511 patients with advanced cancer (melanoma and non-small-cell lung, head and neck, genitourinary, and other types of cancer) treated with ICIs. Univariable analysis was conducted to identify baseline co-medications associated with CIP incidence. A propensity score matching analysis was used to adjust for potential CIP risk factors, and multivariable analysis was carried out to assess the impact of the identified co-medications on CIP risk. RESULTS: Forty-seven (9.2%) patients developed CIP. In these patients, the organizing pneumonia pattern was the dominant radiological phenotype, and 42.6% had grade ≥3 CIP, including one patient with grade 5. Of the investigated baseline co-medications, the proportion of antiplatelet drugs (n = 50, 9.8%) was higher in patients with CIP (23.4% versus 8.4%). After propensity score matching, the CIP incidence was higher in patients with baseline antiplatelet drugs (22% versus 6%). Finally, baseline antiplatelet drug use was demonstrated to increase the risk for CIP incidence regardless of cancer type (hazard ratio, 3.46; 95% confidence interval 1.21-9.86). CONCLUSIONS: An association between concomitant antiplatelet drug use at baseline and an increased risk for CIP was seen in our database. This implies the importance of assessing concomitant medications for CIP risk management.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Neumonía , Humanos , Inhibidores de Agregación Plaquetaria/efectos adversos , Estudios Retrospectivos , Neumonía/inducido químicamente , Neumonía/epidemiologíaRESUMEN
BACKGROUND: The renin-angiotensin system (RAS) is thought to have a role in carcinogenesis, and RAS inhibition may prevent tumour growth. METHODS: We retrospectively investigated the impact of angiotensin I-converting enzyme inhibitors (ACEIs) and angiotensin II type-1 receptor blockers (ARBs) in 155 patients with pancreatic cancer receiving gemcitabine monotherapy. Patients were divided into three groups: the ACEI/ARB group (27 patients receiving an ACEI or ARB for hypertension (HT)), the non-ACEI/ARB with HT group (25 patients receiving antihypertensive drugs other than ACEIs or ARBs), and the non-HT group (103 patients receiving no antihypertensive drugs). RESULTS: Patient characteristics were not different, except for age and HT medications. Progression-free survival (PFS) was 8.7 months in the ACEI/ARB group, 4.5 months in the non-ACEI/ARB with HT group, and 3.6 months in the non-HT group. Overall survival (OS) was 15.1 months in the ACEI/ARB group, 8.9 months in the non-ACEI/ARB with HT group, and 9.5 months in the non-HT group. The use of ACEIs/ARBs was a significant prognostic factor for both PFS (P=0.032) and OS (P=0.014) in the multivariate analysis. CONCLUSIONS: The ACEIs/ARBs in combination with gemcitabine might improve clinical outcomes in patients with advanced pancreatic cancer. Prospective trials are needed to test this hypothesis.
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Bloqueadores del Receptor Tipo 1 de Angiotensina II/administración & dosificación , Inhibidores de la Enzima Convertidora de Angiotensina/administración & dosificación , Antihipertensivos/administración & dosificación , Antimetabolitos Antineoplásicos/uso terapéutico , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/tratamiento farmacológico , Anciano , Anciano de 80 o más Años , Desoxicitidina/administración & dosificación , Desoxicitidina/uso terapéutico , Quimioterapia Combinada , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neoplasias Pancreáticas/mortalidad , Pronóstico , Estudios Retrospectivos , GemcitabinaRESUMEN
In dynamic nuclear polarization (DNP) experiments applied to organic solids for creating nonequilibrium, high (1)H spin polarization, an efficient buildup of (1)H polarization is attained by partially deuterating the material of interest with an appropriate (1)H concentration. In such a dilute (1)H spin system, it is shown that the (1)H spin diffusion rate and thereby the buildup efficiency of (1)H polarization can further be enhanced by continually applying radiofrequency irradiation for deuterium decoupling during the DNP process. As experimentally confirmed in this work, the electron spin polarization of the photoexcited triplet state is mainly transferred only to those (1)H spins, which are in the vicinity of the electron spins, and (1)H spin diffusion transports the localized (1)H polarization over the whole sample volume. The (1)H spin diffusion coefficients are estimated from DNP repetition interval dependence of the initial buildup rate of (1)H polarization, and the result indicates that the spin diffusion coefficient is enhanced by a factor of 2 compared to that without (2)H decoupling.
RESUMEN
NEDD8/Rub1 is a ubiquitin (Ub)-like molecule that covalently ligates to target proteins through an enzymatic cascade analogous to ubiquitylation. This modifier is known to target all cullin (Cul) family proteins. The latter are essential components of Skp1/Cul-1/F-box protein (SCF)-like Ub ligase complexes, which play critical roles in Ub-mediated proteolysis. To determine the role of the NEDD8 system in mammals, we generated mice deficient in Uba3 gene that encodes a catalytic subunit of NEDD8-activating enzyme. Uba3(-/-) mice died in utero at the periimplantation stage. Mutant embryos showed selective apoptosis of the inner cell mass but not of trophoblastic cells. However, the mutant trophoblastic cells could not enter the S phase of the endoreduplication cycle. This cell cycle arrest was accompanied with aberrant expression of cyclin E and p57(Kip2). These results suggested that the NEDD8 system is essential for both mitotic and the endoreduplicative cell cycle progression. beta-Catenin, a mediator of the Wnt/wingless signaling pathway, which degrades continuously in the cytoplasm through SCF Ub ligase, was also accumulated in the Uba3(-/-) cytoplasm and nucleus. Thus, the NEDD8 system is essential for the regulation of protein degradation pathways involved in cell cycle progression and morphogenesis, possibly through the function of the Cul family proteins.
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Ubiquitinas/fisiología , Animales , Apoptosis , Ciclo Celular , División Celular , Clonación Molecular , Marcación de Gen/métodos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Morfogénesis , Proteína NEDD8 , Fase S , Trofoblastos/citología , Ubiquitinas/genéticaRESUMEN
The aim of this study was to evaluate the effect of human serum (HS) on growth and differentiation capacity of human synovium-derived mesenchymal stem cells (MSC) in comparison to cells grown in fetal bovine serum (FBS). Human MSCs were isolated from the synovium of knee joints of three donors and the cells were cultured individually in varying concentrations of allogenic HS or FBS. Bovine MSCs were isolated from synovium and cultured in the same manner. Cell proliferation was assessed by the tetrazolium assay after passage 3. The capacity for chondrogenic and osteogenic differentiation was investigated in specific media followed by 1,9-dimethylmethylene blue assay and alcian blue staining, or by alizarin red staining, respectively. Human MSCs proliferated significantly more rapidly in the presence of HS than with equivalent levels of FBS. Chondrogenic or osteogenic differentiation occurred to nearly identical levels in HS or FBS. The results of this study indicate that HS is superior for the culture of human MSCs compared with FBS in terms of cellular expandability, without losing chondrogenic or osteogenic differentiation capacity. Coupled with the advantage in eliminating the potential risk accompanied with the use of xeno-derived materials, pooled, well-characterized HS could be a useful reagent to promote cellular expansion for clinical synovial stem cell-based therapy.
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Técnicas de Cultivo de Célula , Diferenciación Celular , Medios de Cultivo , Células Madre Mesenquimatosas/citología , Animales , Bovinos , Proliferación Celular , Condrogénesis , Humanos , Trasplante de Células Madre Mesenquimatosas , Osteogénesis , Suero , Ingeniería de TejidosRESUMEN
Hepatitis B virus X protein (HBx) has many cellular functions and is a major factor in hepatitis and hepatocellular carcinoma caused by HBV infection. A proteomic approach was used to search for HBx-interacting proteins in order to elucidate the molecular mechanism of hepatocarcinogenesis. HBx was attached to myc and flag tags (MEF tags) and expressed in 293T cells; the protein complex formed within the cells was purified and characterized by mass spectrometry. COP9 signalosome (CSN) subunits 3 and 4 were subsequently identified as HBx-interacting proteins. In addition, CSN subunit 5, Jun activation domain-binding protein 1 (Jab1), was shown to be a novel cellular target of HBx. In vivo and in vitro interactions between HBx and Jab1 were confirmed by standard immunoprecipitation and GST pull-down assays. An analysis of HBx deletion constructs showed that amino acids 30-125 of HBx were responsible for binding to Jab1. Confocal laser microscopy demonstrated that HBx was mainly localized in the cytoplasm, while Jab1 was found mainly in the nucleus and partially in the cytoplasm, and that the two proteins colocalized in the cytoplasm. The cotransfection of HBx and Jab1 resulted in substantial activator protein 1 (AP-1) activation and knockdown of endogenous Jab1 attenuated AP-1 activation caused by HBx. In addition, the coexpression of HBx and Jab1 potentiated phosphorylation of JNK, leading to the subsequent phosphorylation of c-Jun, whereas the level of c-Jun and JNK phosphorylation induced by HBx was decreased in Jab1 knockdown cells. These results suggest that the interaction between HBx and Jab1 enhances HBx-mediated AP-1 activation.
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Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptido Hidrolasas/metabolismo , Transactivadores/fisiología , Factor de Transcripción AP-1/metabolismo , Complejo del Señalosoma COP9 , Línea Celular , Citoplasma/química , Humanos , Péptidos y Proteínas de Señalización Intracelular/análisis , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Espectrometría de Masas , Complejos Multiproteicos/química , Péptido Hidrolasas/análisis , Péptido Hidrolasas/química , Fosforilación , Subunidades de Proteína , Proteínas Proto-Oncogénicas c-jun/metabolismo , Transactivadores/análisis , Transactivadores/química , Proteínas Reguladoras y Accesorias ViralesRESUMEN
A recent study revealed that the p110alpha (PIK3CA), catalytic subunit of phosphatidylinositol 3-kinase (PI3K), is somatically mutated in many types of cancer. For example, PIK3CA is mutated in an estimated 35.6% of hepatocellular carcinoma (HCC) cases. To measure the frequency of PIK3CA hotspot mutations in Japanese HCC patients, exons 9 and 20 of the PIK3CA gene were sequenced in 47 clinical HCC samples. Contrary to expectations, no hotspot mutations were found any of the HCC samples. In addition, we found abnormally migrating waves near the end of exon 9 in the PCR chromatograms from 13 of the 47 samples. PCR amplification and subsequent cloning and sequencing revealed that these chromatograms contained two distinct sequences, the wild-type p110alpha sequence and a different sequence found on human chromosome 22q11.2, the Cat Eye Syndrome region, which contains a putative pseudogene of PIK3CA. These abnormally migrating waves were also found in noncancerous liver tissue, indicating that this was not a result of HCC-associated mutations. Therefore, it is likely that the percentage of hotspot mutations in the PIK3CA gene of Japanese HCC patients is lower than was previously reported.
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Carcinoma Hepatocelular/genética , Exones/genética , Neoplasias Hepáticas/genética , Mutación/genética , Fosfatidilinositol 3-Quinasas/genética , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma Hepatocelular/epidemiología , Fosfatidilinositol 3-Quinasa Clase I , Femenino , Humanos , Japón/epidemiología , Neoplasias Hepáticas/epidemiología , Masculino , Persona de Mediana Edad , Reacción en Cadena de la PolimerasaRESUMEN
To investigate the mechanisms of peroxisome assembly and the molecular basis of peroxisome assembly disorders, we isolated and characterized a peroxisome-deficient CHO cell mutant, ZP139, which was found to belong to human complementation group II, the same group as that of our earlier mutant, ZP105. These mutants had a phenotypic deficiency in the import of peroxisomal targeting signal type 1 (PTS1) proteins. Amino-terminal extension signal (PTS2)-mediated transport, including that of 3-ketoacyl coenzyme A thiolase, was also defective in ZP105 but not in ZP139. PEX5 cDNA, encoding the PTS1 receptor (PTS1R), was isolated from wild-type CHO-K1 cells. PTS1R's deduced primary sequence comprised 595 amino acids, 7 amino acids less than the human homolog, and contained seven tetratricopeptide repeat (TPR) motifs at the C-terminal region. Chinese hamster PTS1R showed 94, 28, and 24% amino acid identity with PTS1Rs from humans, Pichia pastoris, and Saccharomyces cerevisiae, respectively. A PTS1R isoform (PTS1RL) with 632 amino acid residues was identified in CHO cells; for PTS1R, 37 amino acids were inserted between residues at positions 215 and 216 of a shorter isoform (PTS1RS). Southern blot analysis of CHO cell genomic DNA suggested that these two isoforms are derived from a single gene. Both types of PEX5 complemented impaired import of PTS1 in mutants ZP105 and ZP139. PTS2 import in ZP105 was rescued only by PTS1RL. This finding strongly suggests that PTS1RL is also involved in the transport of PTS2. Mutations in PEX5 were determined by reverse transcription-PCR: a G-to-A transition resulted in one amino acid substitution: Gly298Glu of PTS1RS (G335E of PTS1RL) in ZP105 and Gly485Glu of PTS1RS (G522E of PTS1RL) in ZP139. Both mutations were in the TPR domains (TPR1 and TPR6), suggesting the functional consequence of these domains in protein translocation. The implications of these mutations are discussed.
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Microcuerpos/genética , Receptores Citoplasmáticos y Nucleares/genética , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , Proteínas Fúngicas , Regulación de la Expresión Génica , Humanos , Microcuerpos/metabolismo , Datos de Secuencia Molecular , Mutación , Receptor de la Señal 2 de Direccionamiento al Peroxisoma , Receptor de la Señal 1 de Direccionamiento al Peroxisoma , Alineación de SecuenciaRESUMEN
Epigenetic gene regulation linked to oncogenic pathways is an important focus of cancer research. KDM3A, a histone H3 lysine 9 (H3K9) demethylase, is known to have a pro-tumorigenic function. Here, we showed that KDM3A contributes to liver tumor formation through the phosphatidylinositol 3-kinase (PI3K) pathway, which is often activated in hepatocellular carcinoma. Loss of Kdm3a attenuated tumor formation in Pik3ca transgenic (Tg) mouse livers. Transcriptome analysis of pre-cancerous liver tissues revealed that the expression of activator protein 1 (AP-1) target genes was induced by PI3K activation, but blunted upon Kdm3a ablation. Particularly, the expression of Cd44, a liver cancer stem marker, was regulated by AP-1 in a Kdm3a-dependent manner. We identified Cd44-positive hepatocytes with epithelial-mesenchymal transition-related expression profiles in the Pik3ca Tg liver and confirmed their in vivo tumorigenic capacity. Notably, the number and tumor-initiating capacity of Cd44-positive hepatocytes were governed by Kdm3a. As a mechanism in Kdm3a-dependent AP-1 transcription, Kdm3a recruited c-Jun to the AP-1 binding sites of Cd44, Mmp7 and Pdgfrb without affecting c-Jun expression. Moreover, Brg1, a component of the SWI/SNF chromatin remodeling complex, interacted with c-Jun in a Kdm3a-dependent manner and was bound to the AP-1 binding site of these genes. Finally, KDM3A and c-JUN were co-expressed in 33% of human premalignant lesions with PI3K activation. Our data suggest a critical role for KDM3A in the PI3K/AP-1 oncogenic axis and propose a novel strategy for inhibition of KDM3A against liver tumor development under PI3K pathway activation.
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Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Neoplasias Hepáticas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Factor de Transcripción AP-1/metabolismo , Animales , Carcinogénesis , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Epigénesis Genética , Humanos , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/patología , Neoplasias Hepáticas Experimentales/enzimología , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/patología , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Fosforilación , Transducción de SeñalRESUMEN
5-fluorouracil (5-FU), although a widely used chemotherapeutic agent, has a limited effect in the treatment of human solid tumors due to their resistance to the cytotoxic effects of 5-FU. Escherichia coli uracil phosphoribosyltransferase (UPRT) is a pyrimidine salvage enzyme that catalyzes the synthesis of UMP from uracil and 5-phosphoribosyl-alpha-1-diphosphate. The present study demonstrates that adenovirus-mediated transduction of E. coli UPRT gene results in marked sensitization of colon, gastric, liver, and pancreas cancer cell lines to low concentration of 5-FU in vitro. The in vitro bystander effect was observed when only 10% of the hepatoma Hep3B cells were infected with UPRT-expressing adenovirus. In addition, 5-FU treatment of human hepatoma or gastric cancer xenografts in nude mice transduced with UPRT was demonstrated to result in significant in vivo antitumor effects. The adenovirus vector transduction of the UPRT gene followed by 5-FU administration is representative of a new chemosensitization strategy for cancer gene therapy.
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Adenoviridae/genética , Escherichia coli/enzimología , Fluorouracilo/farmacología , Genes Bacterianos , Neoplasias Experimentales/enzimología , Pentosiltransferasa/genética , Transfección , Células Tumorales Cultivadas/enzimología , Animales , Northern Blotting , Células Cultivadas , Cartilla de ADN/química , Regulación Enzimológica de la Expresión Génica , Terapia Genética , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias Experimentales/tratamiento farmacológico , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
AIMS: The aim of this study was to determine whether chilled irrigation saline decreases the incidence of clinical upper limb palsy (ULP; a reduction of one grade or more on manual muscle testing; MMT), based on the idea that ULP results from thermal damage to the nerve roots by heat generated by friction during bone drilling. METHODS: Irrigation saline for drilling was used at room temperature (RT, 25.6°C) in open-door laminoplasty in 400 patients (RT group) and chilled to a mean temperature of 12.1°C during operations for 400 patients (low-temperature (LT) group). We assessed deltoid, biceps, and triceps brachii muscle strength by MMT. ULP occurring within two days post-operatively was categorised as early-onset palsy. RESULTS: The incidence of ULP (4.0% vs 9.5%, p = 0.003), especially early-onset palsy (1.0% vs 5.5%, p < 0.001), was significantly lower for the LT group than for the RT group. Multivariate analysis indicated that RT irrigation saline use, concomitant foraminotomy, and opened side were significant predictors for ULP. DISCUSSION: Using chilled irrigation saline during bone drilling significantly decreased the ULP incidence, particularly the early-onset type, and shortened the recovery period for ULP. Chilled irrigation saline can thus be recommended as a simple method for preventing ULP. TAKE HOME MESSAGE: Chilled irrigation during laminoplasty reduces C5 palsy.
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Vértebras Cervicales/cirugía , Crioterapia/métodos , Laminoplastia/métodos , Parálisis/prevención & control , Irrigación Terapéutica/métodos , Adulto , Anciano , Anciano de 80 o más Años , Brazo/inervación , Pérdida de Sangre Quirúrgica , Femenino , Humanos , Laminoplastia/efectos adversos , Masculino , Persona de Mediana Edad , Fuerza Muscular/fisiología , Músculo Esquelético/fisiología , Tempo Operativo , Complicaciones Posoperatorias/prevención & control , Estudios Prospectivos , Cloruro de Sodio/administración & dosificaciónRESUMEN
Sharpin (Shank-associated RH domain-interacting protein, also known as SIPL1) is a multifunctional molecule that participates in various biological settings, including nuclear factor-κB signaling activation and tumor suppressor gene inhibition. Sharpin is upregulated in various types of cancers, including hepatocellular carcinoma (HCC), and is implicated in tumor progression. However, the exact roles of Sharpin in tumorigenesis and tumor progression remain largely unknown. Here we report novel mechanisms of HCC progression through Sharpin overexpression. In our study, Sharpin was upregulated in human HCC tissues. Increased Sharpin expression enhanced hepatoma cell invasion, whereas decrease in Sharpin expression by RNA interference inhibited invasion. Microarray analysis identified that Versican, a chondroitin sulfate proteoglycan that plays crucial roles in tumor progression and invasion, was also upregulated in Sharpin-expressing stable cells. Versican expression increased in the majority of HCC tissues and knocking down of Versican greatly attenuated hepatoma cell invasion. Sharpin expression resulted in a significant induction of Versican transcription synergistically with Wnt/ß-catenin pathway activation. Furthermore, Sharpin-overexpressing cells had high tumorigenic properties in vivo. These results demonstrate that Sharpin promotes Versican expression synergistically with the Wnt/ß-catenin pathway, potentially contributing to HCC development. A Sharpin/Versican axis could be an attractive therapeutic target for this currently untreatable cancer.
RESUMEN
We isolated peroxisome biogenesis mutants from Chinese hamster ovary (CHO) cells, using the 9-(1'-pyrene)nonanol/ultraviolet (P9OH/ UV) method and wild-type CHO-K1 cells that had been stably transfected with cDNA encoding Pex2p (formerly peroxisome assembly factor-1, PAF-1). Three mutant cell clones, ZP110, ZP111, and ZP114, showed cytosolic localization of catalase, thereby indicating a defect in peroxisome biogenesis, whereas ZP112 and ZP113 contained fewer but larger catalase-positive particles. Mutant ZP115 displayed an aberrant, tubular structure immunoreactive to anti-catalase antibody. Mutants lacking morphologically recognizable peroxisomes also showed the typical peroxisome assembly-defective phenotype such as severe loss of catalase latency and resistance to 12-(1'-pyrene)dodecanoic acid (P12)/UV treatment. ZP110 and ZP111, and ZP114 were found to belong to two novel complementation groups, respectively, by complementation group analysis with cDNA transfection and cell fusion. Cell fusion with fibroblasts from patients with peroxisome biogenesis disorders such as Zellweger syndrome revealed that ZP110 and ZP114 could not be classified to any of human complementation groups. Thus, ZP110/ZP111 and ZP114 are the first, two peroxisome-deficient cell mutants of newly identified complementation groups distinct from the ten mammalian groups previously characterized.
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Microcuerpos/enzimología , Microcuerpos/ultraestructura , Mutación , Acetil-CoA C-Aciltransferasa/biosíntesis , Acil-CoA Oxidasa , Animales , Células CHO , Catalasa/análisis , Fusión Celular , Cricetinae , Citosol/enzimología , Digitonina/farmacología , Fibroblastos , Prueba de Complementación Genética , Humanos , Ácidos Láuricos/farmacología , Mamíferos , Proteínas de la Membrana/genética , Mutagénesis , Oxidorreductasas/biosíntesis , Factor 2 de la Biogénesis del Peroxisoma , Rayos Ultravioleta , Síndrome de Zellweger/metabolismoRESUMEN
Proliferation of seminal vesicle cells and the concentration of testicular and serum androgens (testosterone and 5 alpha-androgens) from birth to adulthood were investigated in mice. The weight of the seminal vesicles increased significantly from day 0 to day 10 after birth (approximately 0.1-1 mg), remained nearly constant in the next 10 days, and increased again (1-50 mg) thereafter. As an index of cell proliferation, two distinct peaks of 5-[125I]iodo-2'-deoxyuridine ([125I]IdUrd) uptake by the whole seminal vesicles were found at days 8 and 30. Types of proliferating cells examined by autoradiography after injection of [3H]thymidine were partially testis-dependent fibromuscular and epithelial cells in the first peak and largely testis-dependent epithelial cells in the second peak. The [125I]IdUrd uptake values found around day 20 and after day 40 were similar to those in neonatally castrated mice. Concentrations of testicular and serum androgens were relatively high on days 8, 30, 35, 40, and 60 (0.2-0.7 ng/mg tissue and 1.3-2.2 ng/ml, respectively) but were very low on day 18 (0.02 ng/mg tissue and 0.4 ng/ml). These findings lead to the hypothesis that androgens secreted from neonatal and prepubertal mouse testes play a major role in the proliferation of seminal vesicle cells and that the quiescent interval of androgen secretion and action occurs around the 20th day after birth in mice.
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Andrógenos/metabolismo , Vesículas Seminales/crecimiento & desarrollo , Testículo/crecimiento & desarrollo , Envejecimiento , Andrógenos/sangre , Animales , División Celular , Idoxuridina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Vesículas Seminales/citología , Testículo/metabolismo , Testosterona/metabolismoRESUMEN
Studies were made of pancreastatin (PST) secretion from a human PST-producing cell line (QGP-1N) in response to various secretagogues. Cells with immunoreactivity for PST were observed in monolayer cultures of QGP-1N cells. Carbachol stimulated PST secretion and the intracellular Ca2+ mobilization concentration dependently in the range of 10(-6)-10(-4) M. The PST secretion and Ca2+ mobilization induced by carbachol were inhibited by atropine. The calcium ionophore (A23187) stimulated PST secretion. However, cholecystokinin and gastrin-releasing peptide did not stimulate either PST secretion or Ca2+ mobilization. Secretin also did not stimulate PST secretion. The glucose concentration in the culture medium had no effect on PST secretion. These results suggest that PST secretion is mainly regulated by acetylcholine through a muscarinic receptor, and that an increase in intracellular Ca2+ plays an important role in stimulus-secretion coupling in QGP-1N cells.
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Acetilcolina/fisiología , Adenoma de Células de los Islotes Pancreáticos/metabolismo , Hormonas Pancreáticas/metabolismo , Atropina/farmacología , Calcimicina/farmacología , Calcio/metabolismo , Carbacol/farmacología , Cromogranina A , Péptido Liberador de Gastrina , Humanos , Neoplasias Pancreáticas/metabolismo , Parasimpatolíticos/farmacología , Péptidos/farmacología , Piperidinas/farmacología , Pirenzepina/análogos & derivados , Pirenzepina/farmacología , Receptores Muscarínicos/fisiología , Sincalida/farmacología , Células Tumorales CultivadasRESUMEN
The concentration and molecular form of pancreastatin-like immunoreactivity (PST-LI) in urine of normal subjects and patients with noninsulin-dependent diabetes mellitus or chronic renal failure were examined. PST-LI output (mean +/- SEM) in urine of normal subjects was 74.6 +/- 8.5 pmol/day and 87.1 +/- 11.7 pmol/g creatinine. That in patients with noninsulin-dependent diabetes mellitus was 78.1 +/- 9.0 (SEM) pmol/day and 85.6 +/- 9.0 pmol/g creatinine and was not significantly different from that in normal subjects. Gel filtration analysis showed that PST-LI molecules excreted in urine of these two groups were smaller than human pancreastatin (43-52) (hPST-10) of C-terminal fragment. The PST-LI molecular forms were deduced to be nonbioactive from the result that hPST-10 did not inhibit pancreatic exocrine secretion. PST-LI excretion in patients with chronic renal failure was 258.5 +/- 62.9 pmol/day and 713.2 +/- 219.6 pmol/g creatinine. A molecular form corresponding to hPST-52 and a larger form eluted in the high mol wt region (approximately mol wt 15 K) were detected by gel filtration of urine from these patients, indicating that PST-LI is excreted in urine without degradation in patients with chronic renal failure. These results support the suggestion that the kidney may play an important role in PST degradation or metabolism.
Asunto(s)
Diabetes Mellitus Tipo 2/orina , Fallo Renal Crónico/orina , Hormonas Pancreáticas/orina , Cromatografía en Gel , Cromogranina A , Humanos , Hormonas Pancreáticas/inmunología , Fragmentos de Péptidos/orina , RadioinmunoensayoRESUMEN
Plasma pancreastatin (PST)-like immunoreactivity in normal subjects and patients with various diseases was estimated by a RIA, using antiserum raised against a synthetic C-terminal peptide of human PST deduced from the sequence of human chromogranin-A. The mean level +/- SEM was 13.2 +/- 0.6 pmol/L in normal subjects, but was significantly higher in patients with chronic renal failure (526.7 +/- 48.5). An immunoreactive form corresponding to a human PST-like sequence [human chromogranin-A-(250-301)] and a larger form were detected by gel filtration of plasma from these patients, suggesting accumulation of the larger molecular form in these patients. A significant increase in PST-like immunoreactivity was also found in patients with liver cirrhosis (20.8 +/- 3.0 pmol/L), but not in patients with noninsulin-dependent diabetes mellitus, chronic pancreatitis, or pancreatic cancer. Elevated levels were found in 16 of the 21 patients with small cell lung carcinoma examined. High levels were also found in 3 of 11 patients with islet cell tumor.