RESUMO
This article reports on an effort to use the learning organization to change the existing mental model of a nursing team in order to transform from a nursing-oriented group to a patient-oriented group and to build shared vision, self-affirmation, and self-transcendence in order to change perspectives using team-learning spirit. The nursing group built partnerships with the patient group. The nursing group learning process fostered positive thinking logic and, under the inheritance of knowledge and technology, the patient-oriented group was successfully established. This model will be expanded to more patient-oriented groups. Through the experience sharing and joint supervision, this model may change lifestyles by implementing the concept of "My health, I care" and, ultimately, help patients achieve self-healthcare.
Assuntos
Aprendizagem , Equipe de Enfermagem/organização & administração , Assistência Centrada no Paciente/organização & administração , Humanos , Modelos de EnfermagemRESUMO
The hepatopancreas of oyster, Crassostrea virginica, was found to contain two unique glycosphingolipid (GSL) cleaving enzymes, ceramide glycanase (CGase) and ceramidase. These two enzymes were found to be tightly associated together through the consecutive purification steps including gel filtration, hydrophobic interaction and cation-exchange chromatographies. They were separated only by preparatory SDS-PAGE. The purified CGase was found to have a molecular mass of 52 kDa and pH optimum of 3.2-3.3. This enzyme prefers to hydrolyze the acidic GSLs, II3SO3LacCer and gangliosides over the neutral GSLs. Oyster ceramidase was found to have a molecular mass of 88 kDa and pH optimum of 4-4.5. Since oyster ceramidase greatly prefers ceramides with C6 to C8 fatty acids, C6-ceramide (N-hexanoyl-D-sphingosine) was used as the substrate for its purification and characterization. The oyster acid ceramidase also catalyzed the synthesis of ceramide from a sphingosine and a fatty acid. For the synthesis, C16 and C18 fatty acids were the best precursors. The amino acid sequences of the two cyanogenbromide peptides derived from the purified ceramidase were found to have similarities to those of several neutral and alkaline ceramidases reported. The tight association of CGase and ceramidase may indicate that CGase in oyster hepatopancreas acts as a vehicle to release ceramide from GSLs for subsequent generation of sphingosines and fatty acids by ceramidase to serve as signaling factors and energy source.
Assuntos
Ceramidases/metabolismo , Crassostrea/enzimologia , Glicosídeo Hidrolases/metabolismo , Glicoesfingolipídeos/metabolismo , Hepatopâncreas/enzimologia , Animais , Ceramidas/metabolismo , Crassostrea/metabolismo , Ácidos Graxos/metabolismo , Hepatopâncreas/metabolismoRESUMO
OBJECTIVE: This paper evaluates the effectiveness of motivational enhancement therapy plus cognitive behavioural therapy on depressive symptoms, glycosylated haemoglobin, fasting glucose, body mass index (BMI), and health-related quality of life in type II diabetes patients. METHODS: A controlled trial was conducted to compare patients who received the behavioural intervention with untreated controls on measures of health outcomes. A total of 31 intervention group participants and 30 controls were selected from patients that met the inclusion criteria from a hospital-based endocrinology outpatient department. The outcome measures including depressive symptoms, glycosylated haemoglobin, fasting glucose, BMI, and both physical and mental quality of life were collected before (T1), after (T2), and after 90 days (T3) following the intervention. RESULTS: The experimental group showed a significant reduction in glycosylated haemoglobin, fasting glucose, and depressive symptoms and a significant increase in physical quality of life and mental quality of life at T2 and T3, while patients in the control group with usual care showed no changes over time. CONCLUSION: The behavioural intervention facilitated a significant improvement in psychological adjustment and glycemic control, thus strengthening diabetes control skills and leading to healthy outcomes. It is feasible that nurses and psychiatrists can deliver the behavioural intervention for diabetes patients to decrease their depressive symptoms. Sharing discussion and problem-solving experiences is particularly helpful method for self-control, and these will be beneficially influential on further research.
Assuntos
Glicemia/análise , Terapia Cognitivo-Comportamental/métodos , Depressão/terapia , Diabetes Mellitus Tipo 2/psicologia , Hemoglobinas Glicadas/análise , Nível de Saúde , Qualidade de Vida/psicologia , Adulto , Idoso , Índice de Massa Corporal , Depressão/psicologia , Diabetes Mellitus Tipo 2/terapia , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
We have previously reported that oyster hepatopancreas contained three unusual α-ketoside hydrolases: (i) a 3-deoxy-d-manno-oct-2-ulosonic acid α-ketoside hydrolase (α-Kdo-ase), (ii) a 3-deoxy-D-glycero-D-galacto-non-2-ulosonic acid α-ketoside hydrolase and (iii) a bifunctional ketoside hydrolase capable of cleaving both the α-ketosides of Kdn and Neu5Ac (Kdn-sialidase). After completing the purification of Kdn-sialidase, we proceeded to clone the gene encoding this enzyme. Unexpectedly, we found that instead of expressing Kdn-sialidase, our cloned gene expressed α-Kdo-ase activity. The full-length gene, consisting of 1176-bp (392 amino acids, Mr 44,604), expressed an active recombinant α-Kdo-ase (R-α-Kdo-ase) in yeast and CHO-S cells, but not in various Escherichia coli strains. The deduced amino acid sequence contains two Asp boxes (S(277)PDDGKTW and S(328)TDQGKTW) commonly found in sialidases, but is devoid of the signature FRIP-motif of sialidase. The R-α-Kdo-ase effectively hydrolyzed the Kdo in the core-oligosaccharide of the structurally defined lipopolysaccharide (LPS), Re-LPS (Kdo(2)-Lipid A) from Salmonella minnesota R595 and E. coli D31m4. However, Rd-LPS from S. minnesota R7 that contained an extra outer core phosphorylated heptose was only slowly hydrolyzed. The complex type LPS from Neisseria meningitides A1 and M992 that contained extra 5-6 sugar units at the outer core were refractory to R-α-Kdo-ase. This R-α-Kdo-ase should become useful for studying the structure and function of Kdo-containing glycans.
Assuntos
Glicosídeo Hidrolases/metabolismo , Ostreidae/enzimologia , Açúcares Ácidos/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Cricetinae , Cricetulus , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Fígado/metabolismo , Dados de Sequência Molecular , Ostreidae/genética , Pâncreas/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
Globoid cell leukodystrophy (GLD) is a common neurodegenerative lysosomal storage disorder caused by a deficiency in galactocerebrosidase (GALC), an enzyme that cleaves galactocerebroside during myelination. Bone marrow transplantation has shown promise when administered to late-onset GLD patients. However, the side effects (e.g., graft vs. host disease), harsh conditioning regimens (e.g., myelosuppression), and variable therapeutic effects make this an unsuitable option for infantile GLD patients. We previously reported modest improvements in the twitcher mouse model of GLD after intracerebroventricular (ICV) injections of a low-dose of multipotent stromal cells (MSCs). Goals of this study were to improve bone marrow-derived MSC (BMSC) therapy for GLD by increasing the cell dosage and comparing cell type (e.g., transduced vs. native), treatment timing (e.g., single vs. weekly), and administration route (e.g., ICV vs. intraperitoneal [IP]). Neonatal twitcher mice received (a) 2 × 10(5) BMSCs by ICV injection, (b) 1 × 10(6) BMSCs by IP injection, (c) weekly IP injections of 1 × 10(6) BMSCs, or (d) 1 × 10(6) lentiviral-transduced BMSCs overexpressing GALC (GALC-BMSC) by IP injection. All treated mice lived longer than untreated mice. However, the mice receiving peripheral MSC therapy had improved motor function (e.g., hind limb strength and rearing ability), twitching symptoms, and weight compared to both the untreated and ICV-treated mice. Inflammatory cell, globoid cell, and apoptotic cell levels in the sciatic nerves were significantly decreased as a result of the GALC-BMSC or weekly IP injections. The results of this study indicate a promising future for peripheral MSC therapy as a noninvasive, adjunct therapy for patients affected with GLD.
Assuntos
Encéfalo/metabolismo , Leucodistrofia de Células Globoides/terapia , Células-Tronco Multipotentes/fisiologia , Células-Tronco Multipotentes/transplante , Animais , Encéfalo/patologia , Modelos Animais de Doenças , Feminino , Terapia Genética , Inflamação/terapia , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/metabolismo , Leucodistrofia de Células Globoides/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Multipotentes/metabolismo , Transplante de Células-Tronco/métodos , Células Estromais/metabolismo , Células Estromais/fisiologia , Células Estromais/transplante , Análise de SobrevidaRESUMO
Cancer stem cells are distinguished from normal adult stem cells by their stemness without tissue homeostasis control. Glycosphingolipids (GSLs), particularly globo-series GSLs, are important markers of undifferentiated embryonic stem cells, but little is known about whether or not ceramide glycosylation, which controls glycosphingolipid synthesis, plays a role in modulating stem cells. Here, we report that ceramide glycosylation catalyzed by glucosylceramide synthase, which is enhanced in breast cancer stem cells (BCSCs) but not in normal mammary epithelial stem cells, maintains tumorous pluripotency of BCSCs. Enhanced ceramide glycosylation and globotriosylceramide (Gb3) correlate well with the numbers of BCSCs in breast cancer cell lines. In BCSCs sorted with CD44(+)/ESA(+)/CD24(-) markers, Gb3 activates c-Src/ß-catenin signaling and up-regulates the expression of FGF-2, CD44, and Oct-4 enriching tumorigenesis. Conversely, silencing glucosylceramide synthase expression disrupts Gb3 synthesis and selectively kills BCSCs through deactivation of c-Src/ß-catenin signaling. These findings highlight the unexploited role of ceramide glycosylation in selectively maintaining the tumorous pluripotency of cancer stem cells. It speculates that disruption of ceramide glycosylation or globo-series GSL is a useful approach to specifically target BCSCs specifically.
Assuntos
Neoplasias da Mama/enzimologia , Ceramidas/metabolismo , Glucosiltransferases/metabolismo , Células-Tronco Neoplásicas/enzimologia , Animais , Antibióticos Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Antígeno CD24/metabolismo , Separação Celular , Sobrevivência Celular/efeitos dos fármacos , Transformação Celular Neoplásica , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Feminino , Glicosilação , Humanos , Receptores de Hialuronatos/metabolismo , Separação Imunomagnética , Células MCF-7 , Camundongos , Camundongos Nus , Transplante de Neoplasias , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Esferoides Celulares/efeitos dos fármacos , beta Catenina/metabolismoRESUMO
Well-differentiated small intestinal neuroendocrine tumors are rare malignancies. They arise from enterochromaffin cells and very little is known about differential microRNA (miRNA) expression. The aim of this study was to identify the miRNA profile of well-differentiated small intestinal neuroendocrine tumors, which may have a critical role in tumor development, progression and potentially develop miRNAs as novel clinical biomarkers. Specimens from two test groups, 24 small intestinal neuroendocrine tumor specimens at different stages of malignancy, are included in this study. Total RNA from the first test group, five primary tumors, five mesentery metastases and five liver metastases was hybridized onto the Affymetrix Genechip miRNA arrays to perform a genome-wide profile. The results were validated by using quantitative real-time PCR (QRT-PCR) and northern blot analyses. We then expanded the investigation to laser capture microdissected small intestinal neuroendocrine tumor cells and immuno-laser capture microdissected normal enterochromaffin cells of the first test group. Furthermore, a second test group, three primary tumors, three mesentery metastases and three liver metastases, was included in the study. Thus, two independent test groups validated the data by QRT-PCR. Moreover, we characterized nine miRNAs, five (miR-96, -182, -183, -196a and -200a), which are upregulated during tumor progression, whereas four (miR-31, -129-5p, -133a and -215) are downregulated. Several online software programs were used to predict potential miRNA target genes to map a number of putative target genes for the aberrantly regulated miRNAs, through an advanced and novel bioinformatics analysis. Our findings provide information about pivotal miRNAs, which may lead to further insights into tumorigenesis, progression mechanisms and novel therapeutic targets recognition.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Intestinais/genética , MicroRNAs/análise , Tumores Neuroendócrinos/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Northern Blotting , Feminino , Perfilação da Expressão Gênica , Humanos , Neoplasias Intestinais/patologia , Microdissecção e Captura a Laser , Masculino , Pessoa de Meia-Idade , Tumores Neuroendócrinos/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND AND PURPOSE: We sought to determine the prognostic value of a pre-treatment peripheral blood signature and the peripheral blood signature-based nomogram for patients with non-metastatic nasopharyngeal carcinoma (NPC). MATERIALS AND METHODS: We retrospectively collected 21 peripheral blood indicators from patients with NPC between 2004 and 2015. Data were randomly divided into a training and a validation set (ratio: 6:4). The peripheral blood signature was constructed based on candidate biomarkers using the least absolute shrinkage and selection operator Cox regression model. Multivariable logistic regression was applied to identify the independent risk factors of overall survival to build the nomogram. The predictive value of the peripheral blood nomogram was evaluated using time-dependent area under the curve, decision curve analysis, and calibration curve. RESULTS: In total, 6668 patients were enrolled with 4000 and 2668 in the training and validation cohorts, respectively. Four peripheral blood indicators, (white blood cell count, lymphocyte percentage, haemoglobin, and mean platelet volume), were included to construct the peripheral blood signature. Patients were divided into low- and high-risk groups using an optimal cut-off value of - 1.71142. Patients in the high-risk group had significantly lower overall, distant metastasis-free, and progression-free survival than patients in the low-risk group in both cohorts (P < 0.05). We constructed and validated a peripheral blood signature-based nomogram in combination with five vital clinical characteristics, (age, sex, tumour stage, nodal stage, and pre-treatment Epstein-Barr virus DNA), which showed favourable performance. CONCLUSION: Patients with NPC with different outcomes could be distinguished based on their peripheral blood signature score; the proposed peripheral blood signature-based nomogram offers individualised risk estimation.
Assuntos
Infecções por Vírus Epstein-Barr , Neoplasias Nasofaríngeas , Humanos , Carcinoma Nasofaríngeo/patologia , Estudos Retrospectivos , Herpesvirus Humano 4 , Prognóstico , Nomogramas , Fatores de Risco , Testes Hematológicos , Neoplasias Nasofaríngeas/patologiaRESUMO
OBJECTIVES: About 17.7-34.0 % of patients with recurrent or metastatic nasopharyngeal carcinoma (RM-NPC) responded well to anti-PD-1 monotherapy. We sought to establish a nomogram to estimate the progression-free survival (PFS) of RM-NPC patients receiving subsequent-line anti-PD-1 monotherapy. MATERIALS AND METHODS: This cohort study investigated consecutive RM-NPC patients undergoing anti-PD-1 monotherapy. A nomogram was developed in the training cohort (n = 161), using a Cox multivariate model with backward stepwise inclusion, and was validated in the validation cohort (n = 69). Its predictive accuracy was assessed using a concordance index (C-index) and calibration curve. The primary endpoint was PFS. Secondary endpoints included the objective response rate (ORR), disease control rate (DCR), and overall survival (OS). RESULTS: Liver metastasis, albumin, lactate dehydrogenase, monocyte-to-lymphocyte ratio, and plasma Epstein-Barr virus DNA were used to develop a nomogram that could separate patients into favourable- and unfavourable-prognosis groups. The C-index in the training and validation cohort were 0.70 and 0.68, respectively, which was confirmed by calibration curves. Median PFS (mPFS) was lower for the unfavourable-prognosis than for the favourable-prognosis group (1.80 vs 4.93; hazard ratio 2.49 [95 % confidence interval: 1.78-3.49]; p < 0.001), across all subgroups. OS exhibited the same pattern. The ORR and DCR were markedly lower in the unfavourable-prognosis than in the favourable-prognosis group. All results were confirmed in the validation cohort. CONCLUSION: Our model is a reliable prognostic indicator of PFS in RM-NPC patients undergoing anti-PD-1 monotherapy, allowing robust estimation of the immunotherapy benefit an individual might derive.
Assuntos
Infecções por Vírus Epstein-Barr , Neoplasias Nasofaríngeas , Humanos , Prognóstico , Carcinoma Nasofaríngeo/patologia , Estudos de Coortes , Estadiamento de Neoplasias , Recidiva Local de Neoplasia/patologia , Herpesvirus Humano 4 , Neoplasias Nasofaríngeas/patologiaRESUMO
Immunotherapy combined with antiangiogenic targeted therapy has improved the treatment of certain solid tumors, but effective regimens remain elusive for refractory recurrent/metastatic nasopharyngeal carcinoma (RM-NPC). We conducted a phase 2 trial to evaluate the safety and activity of camrelizumab plus apatinib in platinum-resistant (cohort 1, NCT04547088) and PD-1 inhibitor resistant NPC (cohort 2, NCT04548271). Here we report on the primary outcome of objective response rate (ORR) and secondary endpoints of safety, duration of response, disease control rate, progression-free survival, and overall survival. The primary endpoint of ORR was met for cohort 1 (65%, 95% CI, 49.6-80.4, n = 40) and cohort 2 (34.3%; 95% CI, 17.0-51.8, n = 32). Grade ≥ 3 treatment-related adverse events (TRAE) were reported in 47 (65.3%) of 72 patients. Results of our predefined exploratory investigation of predictive biomarkers show: B cell markers are the most differentially expressed genes in the tumors of responders versus non-responders in cohort 1 and that tertiary lymphoid structure is associated with higher ORR; Angiogenesis gene expression signatures are strongly associated with ORR in cohort 2. Camrelizumab plus apatinib combination effectiveness is associated with high expression of PD-L1, VEGF Receptor 2 and B-cell-related genes signatures. Camrelizumab plus apatinib shows promising efficacy with a measurable safety profile in RM-NPC patients.
Assuntos
Inibidores de Checkpoint Imunológico , Neoplasias Nasofaríngeas , Humanos , Carcinoma Nasofaríngeo/tratamento farmacológico , Platina , Neoplasias Nasofaríngeas/tratamento farmacológico , Neoplasias Nasofaríngeas/genética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêuticoRESUMO
Suppression therapy utilizes compounds that suppress translation termination at in-frame premature termination codons (PTCs) to restore full-length, functional protein. This approach may provide a treatment for diseases caused by nonsense mutations such as mucopolysaccharidosis type I-Hurler (MPS I-H). MPS I-H is a lysosomal storage disease caused by severe α-L-iduronidase deficiency and subsequent lysosomal glycosaminoglycan (GAG) accumulation. MPS I-H represents a good target for suppression therapy because the majority of MPS I-H patients carry nonsense mutations, and restoration of even a small amount of functional α-L-iduronidase may attenuate the MPS I-H phenotype. In this study, we investigated the efficiency of suppression therapy agents to suppress the Idua-W392X nonsense mutation in an MPS I-H mouse model. The drugs tested included the conventional aminoglycosides gentamicin, G418, amikacin, and paromomycin. In addition, the designer aminoglycosides NB54 and NB84, two compounds previously designed to mediate efficient PTC suppression with reduced toxicity, were also examined. Overall, NB84 suppressed the Idua-W392X nonsense mutation much more efficiently than any of the other compounds tested. NB84 treatment restored enough functional α-L-iduronidase activity to partially reverse abnormal GAG accumulation and lysosomal abundance in mouse embryonic fibroblasts derived from the Idua-W392X mouse. Finally, in vivo administration of NB84 to Idua-W392X mice significantly reduced urine GAG excretion and tissue GAG storage. Together, these results suggest that NB84-mediated suppression therapy has the potential to attenuate the MPS I-H disease phenotype.
Assuntos
Aminoglicosídeos/uso terapêutico , Drogas Desenhadas/uso terapêutico , Glicosaminoglicanos/metabolismo , Mucopolissacaridose I/tratamento farmacológico , Mucopolissacaridose I/metabolismo , Trissacarídeos/uso terapêutico , Aminoglicosídeos/química , Aminoglicosídeos/farmacologia , Animais , Sequência de Bases , Bioensaio , Códon sem Sentido/genética , Drogas Desenhadas/química , Drogas Desenhadas/farmacologia , Modelos Animais de Doenças , Embrião de Mamíferos/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Genes Reporter , Glicosaminoglicanos/urina , Iduronidase/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Trissacarídeos/química , Trissacarídeos/farmacologiaRESUMO
The twitcher mouse is an animal model of Krabbe's disease (KD), which is a neurodegenerative lysosomal storage disorder resulting from the absence of functional lysosomal enzyme galactocerebrosidase (GALC). This disease affects the central and peripheral nervous systems and in its most severe form results in death before the age of 2 in humans and approximately 30-40 days in mice. This study evaluates the effect of intracerebroventricular administration of mesenchymal stem cells derived from adipose tissue (ASCs) and bone marrow (BMSCs) on the pathology of KD. Subsequent to the intracerebroventricular injection of ASCs or BMSCs on postnatal day (PND) 3-4, body weight, lifespan, and neuromotor function were evaluated longitudinally beginning on PND15. At sacrifice, tissues were harvested for analysis of GALC activity, presence of myelin, infiltration of macrophages, microglial activation, inflammatory markers, and cellular persistence. Survival analysis curves indicate a statistically significant increase in lifespan in stem cell-treated twitcher mice as compared with control twitcher mice. Body weight and motor function were also improved compared with controls. The stem cells may mediate some of these benefits through an anti-inflammatory mechanism because the expression of numerous proinflammatory markers was downregulated at both transcriptional and translational levels. A marked decrease in the levels of macrophage infiltration and microglial activation was also noted. These data indicate that mesenchymal lineage stem cells are potent inhibitors of inflammation associated with KD progression and offer potential benefits as a component of a combination approach for in vivo treatment by reducing the levels of inflammation.
Assuntos
Tecido Adiposo/fisiologia , Medula Óssea/fisiologia , Leucodistrofia de Células Globoides/cirurgia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/fisiologia , Animais , Linhagem da Célula , Modelos Animais de Doenças , Galactosilceramidase/análise , Galactosilceramidase/metabolismo , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Humanos , Inflamação/cirurgia , Lisossomos/enzimologia , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/antagonistas & inibidoresRESUMO
The World Health Organization (WHO) developed the Caregiver Skills Training for Families of Children with Developmental Delays and Disabilities (CST) with support from Autism Speaks to address the resource gaps and worldwide needs for interventions for children with developmental disorders or delays, especially those with autism spectrum disorder (ASD), and their families. Evidence has indicated that parent-mediated interventions benefit both caregivers and children by strengthening caregivers' knowledge and confidence and children's social communication skills and behavioral regulation. The CST-Taiwan team began the prepilot field trial in 2017 and developed the project to serve families in various locations. This study (1) delineated the adaptations and promotion of CST-Taiwan; (2) determined the program's effectiveness in the promotional stage, in terms of caregiver and child outcomes, and (3) examined the maintenance of its effects. The materials, delivery, and facilitator training procedure of the original CST were adapted to Taiwan. The quantitative data indicated that CST-Taiwan is a promising program, it positively affected caregiver knowledge and confidence and reduced the severity of the children's autistic symptoms. The 3-month follow-up results suggested that the effects persisted. Thus, CST-Taiwan, and its promotional strategies are feasible and effective.
RESUMO
To facilitate the study of the chemical pathology of galactosylsphingosine (psychosine, GalSph) in Krabbe disease and glucosylsphingosine (GlcSph) in Gaucher disease, we have devised a facile method for the effective separation of these two glycosylsphingosines from other glycosphingolipids (GSLs) in Krabbe brain and Gaucher spleen samples. The procedure involves the use of acetone to selectively extract GalSph and GlcSph, respectively, from Krabbe brain and Gaucher spleen samples. Since acetone does not extract other GSLs except modest amounts of galactosylceramide, sulfatide, and glucosylceramide, the positively charged GalSph or GlcSph in the acetone extract can be readily separated from other GSLs by batchwise cation-exchange chromatography using a Waters Accell Plus CM Cartridge. GalSph or GlcSph enriched by this simple procedure can be readily analyzed by thin-layer chromatography or high-performance liquid chromatography.
Assuntos
Doença de Gaucher/patologia , Glicoesfingolipídeos/química , Leucodistrofia de Células Globoides/patologia , Psicosina/análogos & derivados , Psicosina/isolamento & purificação , Adolescente , Adulto , Animais , Química Encefálica , Criança , Pré-Escolar , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia em Camada Fina/métodos , Cães , Humanos , Lactente , Macaca mulatta , Camundongos , Camundongos Mutantes , Psicosina/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Baço/químicaRESUMO
Adrenocortical carcinoma is a rare aggressive disease commonly recurring regardless of radical surgery. Although data on genomic alterations in malignant tumors are accumulating, knowledge of molecular events of importance for initiation of adrenocortical transformation is scarce. In an attempt to recognize early molecular alterations, we used adrenals from young multiple endocrine neoplasia type 1 conventional knock-out mice (Men1+/-) closely mimicking the human MEN1 trait (i.e. transformation of pituitary, parathyroid, endocrine pancreatic, and adrenocortical cells). MicroRNA array and hierarchical clustering showed a distinct pattern. Twenty miRNAs were significantly upregulated and eleven were downregulated in Men1+/- compared to wild type littermates. The latter included the known suppressor miRNA miR-486-3p, which was chosen for transfection in human adrenocortical carcinoma cell lines H295R and SW13. Cell growth decreased in miR-486-3p overexpressing clones and levels of the predicted target gene fatty acid synthase (FASN) and its downstream product, palmitic acid, were lowered. In conclusion, heterozygous inactivation of Men1 in adrenals results in distinct miRNA profile regulating expression of genes with impact on tumorigenesis, e.g. transcription, nucleic acid and lipid metabolism. Low levels of miR-486-3p in the early stages of transformation may contribute to proliferation by increasing FASN and thus fatty acid production. FASN as a potentially druggable target for treatment of the devastating disease adrenocortical carcinoma warrants further studies.
Assuntos
Neoplasias do Córtex Suprarrenal/genética , Carcinoma Adrenocortical/genética , Regulação para Baixo , Ácido Graxo Sintase Tipo I/genética , MicroRNAs/genética , Proteínas Proto-Oncogênicas/genética , Neoplasias do Córtex Suprarrenal/metabolismo , Carcinoma Adrenocortical/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Aprendizado Profundo , Ácido Graxo Sintase Tipo I/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Among patients with the rare diagnosis of pancreatic neuroendocrine tumor (P-NET), a substantial proportion suffer from the inherited cancer syndrome multiple endocrine neoplasia type 1 (MEN1), which is caused by germline mutations of the MEN1 suppressor gene. Somatic mutations and loss of the MEN1 protein (menin) are frequently also found in sporadic P-NETs. Thus, a human neuroendocrine pancreatic cell line with biallelic inactivation of MEN1 might be of value for studying tumorigenesis. We used the polyclonal human P-NET cell line BON1, which expresses menin, serotonin, chromogranin A and neurotensin, to generate a monoclonal stable MEN1 knockout BON1 cell line (MEN1-KO-BON1) by CRISPR/Cas9 editing. Changes in morphology, hormone secretion, and proliferation were analyzed, and proteomics were assessed using nanoLC-MS/MS and Ingenuity Pathway Analysis (IPA). The menin-lacking MEN1-KO-BON1 cells had increased chromogranin A production and were smaller, more homogenous, rounder and grew faster than their control counterparts. Proteomic analysis revealed 457 significantly altered proteins, and IPA identified biological functions related to cancer, e.g., posttranslational modification and cell death/survival. Among 39 proteins with at least a two-fold difference in expression, twelve are relevant in glucose homeostasis and insulin resistance. The stable monoclonal MEN1-KO-BON1 cell line was found to have preserved neuroendocrine differentiation, increased proliferation, and an altered protein profile.
Assuntos
Biomarcadores Tumorais/metabolismo , Sistemas CRISPR-Cas , Edição de Genes , Tumores Neuroendócrinos/patologia , Neoplasias Pancreáticas/patologia , Proteoma/análise , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica , Humanos , Tumores Neuroendócrinos/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas/genética , Células Tumorais CultivadasRESUMO
The effect of inter-molecular carbohydrate-to-carbohydrate interaction on basic cell biological processes has been well documented and appreciated. In contrast, very little is known about the intra-molecular carbohydrate-to-carbohydrate interaction. The presence of an interaction between the GalNAc and the Neu5Ac in GM2 detected by NMR spectroscopy represents a well-defined intra-molecular carbohydrate-to-carbohydrate interaction. This intriguing interaction is responsible for the GM2-epitope, GalNAcbeta1-->4(Neu5Acalpha2-->3)Gal-, to exhibit a rigid and compact conformation. We hypothesized that this compact conformation may be the cause for both the GalNAc and the Neu5Ac in GM2 to be refractory to enzymatic hydrolysis and the GM2 activator protein is able to interact with the compact trisaccharide GM2-epitope, rendering the GalNAc and the Neu5Ac accessible to beta-hexosaminidase A and sialidase. We have used a series of structurally modified GM2 to study the effect of modifications of sugar chains on the conformation and enzymatic susceptibility of this ganglioside. Our hypothesis was borne out by the fact that when the GalNAcbeta1-->4Gal linkage in GM2 was converted to the GalNAcbeta1-->6Gal, both the GalNAc and the Neu5Ac became susceptible to beta-hexosaminidase A and sialidase, respectively, without GM2 activator protein. We hope our work will engender interest in identifying other intra-molecular carbohydrate-to-carbohydrate interactions in glycoconjugates.
Assuntos
Gangliosídeo G(M2)/química , Gangliosídeo G(M2)/metabolismo , Neuraminidase/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo , Acetilgalactosamina/química , Acetilgalactosamina/metabolismo , Configuração de Carboidratos , Cromatografia em Camada Fina , Epitopos/química , Gangliosídeo G(M2)/análogos & derivados , Galactose/metabolismo , Humanos , Hidrólise , Espectroscopia de Ressonância Magnética , Mutação/genética , PrótonsRESUMO
After the discovery of glycosphingolipid (GSL) glycan detaching enzymes, Rhodococcal endoglycoceramidase (EGCase) and leech ceramide glycanase (CGase), the method for enzymatically releasing glycans from GSLs has become the method of choice for preparing intact ceramide-free oligosaccharide chains from GSLs. This paper describes (1) the preparation of the intact oligosaccharides from GM1 (II(3)NeuAcGgOse(4)Cer) and GbOse(4)Cer as examples to show the use of CGase to prepare intact glycan chains from GSLs, and (2) the specificity and detergent requirements of Rhodococcal EGCases for the release of glycan chains from different GSLs.
Assuntos
Glicoesfingolipídeos/metabolismo , Oligossacarídeos/análise , Animais , Sequência de Carboidratos , Cromatografia em Camada Fina , Detergentes/farmacologia , Gangliosídeo G(M1)/análise , Globosídeos/análise , Globosídeos/química , Glicosídeo Hidrolases/metabolismo , Glicoesfingolipídeos/química , Hidrólise/efeitos dos fármacos , Sanguessugas , Dados de Sequência Molecular , Oligossacarídeos/química , Rhodococcus/enzimologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por Substrato/efeitos dos fármacosRESUMO
A number of studies have shown that a short peptide, the protein transduction domain (PTD) derived from the HIV-1 Tat protein (Tat-PTD) improved cellular uptake in vitro and distribution in vivo of recombinant proteins bearing such PTDs when administered systemically. To investigate the effects of Tat-PTD addition on the subcellular localization of the lysosomal enzyme galactocerebrosidase (GALC, EC 3.2.2.46) and with a view towards designing improved therapeutic strategies for Krabbe disease (globoid cell leukodystrophy), mouse GALC was tagged C-terminally with the Tat-PTD. Compared with unmodified GALC, GALC bearing a Tat-PTD, a myc epitope and 6 consecutive His residues [GALC-TMH (Tat-PTD, a myc epitope and 6 consecutive His residues)] was found to be secreted more efficiently. Also, GALC-TMH was found to be taken up by cells both via mannose-6-phosphate receptor (M6PR)-mediated endocytosis as well as by M6PR-independent mechanisms. GALC-TMH displayed increased M6PR-independent uptake in fibroblasts derived from twitcher mice (a murine model of globoid cell leukodystrophy) and in neurons derived from the mouse brain cortex compared with GALC lacking a Tat-PTD. Immunocytochemical analyses revealed that Tat-modified GALC protein co-localized in part with the lysosome-associated membrane protein-1. Complete correction of galactosylceramide accumulation was achieved in twitcher mouse fibroblasts lacking GALC activity following addition of GALC-TMH. Therefore, GALC-TMH not only maintained the features of the native GALC protein including enzymatic function, intracellular transport and location, but also displayed more efficient cellular uptake.
Assuntos
Galactosilceramidase/genética , Lisossomos/enzimologia , Lisossomos/genética , Transdução Genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Sequência de Aminoácidos , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Fibroblastos/metabolismo , Galactosilceramidase/metabolismo , Células HeLa , Humanos , Camundongos , Camundongos Mutantes Neurológicos , Dados de Sequência Molecular , Estrutura Terciária de Proteína/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
We previously observed that gangliosides GM2, GM1, and GM3 inhibit Ca2+-uptake via the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) in neurons and in brain microsomes. We now systematically examine the effect of various gangliosides and their analogs on Ca2+-uptake via SERCA and demonstrate that an exposed carboxyl group on the ganglioside sialic acid residue is required for inhibition. Thus, asialo-GM2 and asialo-GM1 have no inhibitory effect, and modifications of the carboxyl group of GM1 and GM2 into a hydroxymethyl residue (CH2OH), a methyl ester (COOCH3) or a taurine-conjugated amide (CONHCH2CH2SO3H) drastically diminish their inhibitory activities. We also demonstrate that the saccharides must be attached to a ceramide backbone in order to inhibit SERCA as the ceramide-free ganglioside saccharides only inhibit SERCA to a minimal extent. Finally, we attempted to use the ceramide-free ganglioside saccharides to antagonize the effects of the gangliosides on SERCA; although some reversal was observed, the inhibitory effects of the gangliosides were not completely abolished.