RESUMO
Sialic acids are unique sugars with negative charge and exert various biological functions such as regulation of immune systems, maintenance of nerve tissues and expression of malignant properties of cancers. Alpha 2,6 sialylated N-glycans, one of representative sialylation forms, are synthesized by St6gal1 or St6gal2 gene products in humans and mice. Previously, it has been reported that St6gal1 gene is ubiquitously expressed in almost all tissues. On the other hand, St6gal2 gene is expressed mainly in the embryonic and perinatal stages of brain tissues. However, roles of St6gal2 gene have not been clarified. Expression profiles of N-glycans with terminal α2,6 sialic acid generated by St6gal gene products in the brain have never been directly studied. Using conventional lectin blotting and novel sialic acid linkage-specific alkylamidationmass spectrometry method (SALSA-MS), we investigated the function and expression of St6gal genes and profiles of their products in the adult mouse brain by establishing KO mice lacking St6gal1 gene, St6gal2 gene, or both of them (double knockout). Consequently, α2,6-sialylated N-glycans were scarcely detected in adult mouse brain tissues, and a majority of α2,6-sialylated glycans found in the mouse brain were O-linked glycans. The majority of these α2,6-sialylated O-glycans were shown to be disialyl-T antigen and sialyl-(6)T antigen by mass spectrometry analysis. Moreover, it was revealed that a few α2,6-sialylated N-glycans were produced by the action of St6gal1 gene, despite both St6gal1 and St6gal2 genes being expressed in the adult mouse brain. In the future, where and how sialylated O-linked glycoproteins function in the brain tissue remains to be clarified.
Assuntos
Encéfalo/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/metabolismo , Sialiltransferases/genética , Animais , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Sialiltransferases/deficiência , Sialiltransferases/metabolismo , beta-D-Galactosídeo alfa 2-6-SialiltransferaseRESUMO
Gangliosides have been considered to modulate cell signals in the microdomain of the cell membrane, lipid/rafts, or glycolipid-enriched microdomain/rafts (GEM/rafts). In particular, cancer-associated gangliosides were reported to enhance the malignant properties of cancer cells. In fact, GD2-positive (GD2+) cells showed increased proliferation, invasion, and adhesion, compared with GD2-negative (GD2-) cells. However, the precise mechanisms by which gangliosides regulate cell signaling in GEM/rafts are not well understood. In order to analyze the roles of ganglioside GD2 in the malignant properties of melanoma cells, we searched for GD2-associating molecules on the cell membrane using the enzyme-mediated activation of radical sources combined with mass spectrometry, and integrin ß1 was identified as a representative GD2-associating molecule. Then, we showed the physical association of GD2 and integrin ß1 by immunoprecipitation/immunoblotting. Close localization was also shown by immuno-cytostaining and the proximity ligation assay. During cell adhesion, GD2+ cells showed multiple phospho-tyrosine bands, i.e., the epithelial growth factor receptor and focal adhesion kinase. The knockdown of integrin ß1 revealed that the increased malignant phenotypes in GD2+ cells were clearly cancelled. Furthermore, the phosphor-tyrosine bands detected during the adhesion of GD2+ cells almost completely disappeared after the knockdown of integrin ß1. Finally, immunoblotting to examine the intracellular distribution of integrins during cell adhesion revealed that large amounts of integrin ß1 were localized in GEM/raft fractions in GD2+ cells before and just after cell adhesion, with the majority being localized in the non-raft fractions in GD2- cells. All these results suggest that GD2 and integrin ß1 cooperate in GEM/rafts, leading to enhanced malignant phenotypes of melanomas.
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Gangliosídeos/metabolismo , Integrinas/metabolismo , Melanoma/patologia , Animais , Anticorpos Monoclonais/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Gangliosídeos/imunologia , Humanos , Integrina beta1/metabolismo , Espectrometria de Massas , Microdomínios da Membrana/metabolismo , Camundongos , Fenótipo , Fosfotirosina/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
In biological systems, extracellular vesicles including exosomes have recently been revealed to play a significant role in the communication between various cells, and the number of papers on this subject has dramatically increased. In current conventional exosome studies, the standard research method is to use liquid biopsies to analyze extracts of various disease exosomes. However, exosomes are only one of many key players in natural cellular interactions. Reproducing the phenomena occurring in vivo and investigating the interactions are required in order to examine their role fully. For exosome research, an alternative to the liquid biopsy method for observing natural interactions is the co-culturing technique. It does not require an exosome extraction procedure, and while the technique has been used in many studies thus far, its application to exosome research has been limited. However, the use of co-culturing technologies is necessary to examine the essential interactions of exosomes. An overview of exosome research methodologies and co-culturing systems is thus provided here.
Assuntos
Exossomos , Animais , Pesquisa Biomédica , Técnicas de Cocultura , HumanosRESUMO
BACKGROUND: Medullary cystic kidney disease Type 1 is an autosomal dominant tubulointerstitial kidney disease (ADTKD). Recently, mucin 1 (MUC1) was identified as a causal gene of medullary cystic kidney disease (ADTKD-MUC1). However, the MUC1 mutation was found to be a single cytosine insertion in a single copy of the GC-rich variable number of tandem repeats (VNTRs), which are very difficult to analyze by next-generation sequencing. To date, other mutations have not been detected in ADTKD-MUC1, and the mutant MUC1 protein has not been analyzed because of the difficulty of genetically modifying the VNTR sequence. METHODS: We conducted whole-exome analyses of an ADTKD family by next-generation sequencing. We also performed histopathological analyses of a renal biopsy from a pedigree family member. We constructed a mutant protein expression vector based on the patient genome sequence and characterized the nature of the mutant protein. RESULTS: We found a novel frameshift mutation before the VNTR in the MUC1 gene. The resulting mutant MUC1 protein had a very similar amino acid sequence and predicted 3D structure to the previously reported mutant protein. Notably, the recombinant mutant MUC1 protein was trapped in the cytoplasm and appeared to self-aggregate. The patient native mutant protein was also found in urine exosomes. CONCLUSIONS: This novel frameshift mutation in the MUC1 gene and consequent mutant protein may contribute to the future discovery of the pathophysiology of ADTKD-MUC1. The mutant MUC1 protein in urine exosomes may be used for non-DNA-related diagnosis.
Assuntos
Mutação da Fase de Leitura , Mucina-1/genética , Proteínas Mutantes/genética , Rim Policístico Autossômico Dominante/genética , Adulto , Sequência de Aminoácidos , Sequência de Bases , Exoma , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Linhagem , Adulto JovemRESUMO
CUBN encodes cubilin, which plays a role in the reabsorption of glomerular-filtered albumin in the proximal tubule. CUBN-related proteinuria was recently established as a new disease concept and may be present in proteinuric cases that were previously undiagnosed either genetically or histologically. We herein report a case of triplets diagnosed with chronic benign proteinuria due to CUBN variants 20 years after its onset. The proband, the first child of triplets, tested positive for urinary protein several times during the neonatal period. A urine screening test at 3 years old was positive. Proteinuria persisted for years within a non-nephrotic range. Kidney biopsy at 8 years old revealed minor glomerular abnormalities. Renin-angiotensin system inhibitors were started for albumin-based proteinuria but were ineffective. Since the two other triplets had similar courses, analyses of the NPHS1/2 and WT1 genes were performed but revealed no abnormalities. The triplets transitioned to adult care at 15 years old. CUBN-related proteinuria was reported in 2020; therefore, we re-analyzed their DNA samples and identified compound heterozygous variants in CUBN in all three triplets. The molecular diagnosis of CUBN-related proteinuria will save patients from unnecessary treatments and concerns about renal prognosis.
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In the clinical diagnosis of breast cancer, immunohistochemistry panels with estrogen receptor (ER), progesterone receptor (PgR), human epidermal growth factor receptor 2 (HER2) and Ki-67 are routinely used, and they have been proposed for the classification of breast tumors into distinct subtypes. Gene expression analysis with formalin-fixed paraffin-embedded material have also become widely available recently, but the prognostic values of corresponding gene panels compared with these four immunohistochemical panels had never tested. We independently evaluated the 5-year relapse risk-estimation scores using semiquantitative data of four immunohistochemical panels (Ku-IHC4 score) and compared these with the results of four-gene expression profiling of formalin-fixed paraffin-embedded specimens (Ku-FFPE4 score) in a consecutive series of 235 primary invasive breast cancer patients. Ku-IHC4 score was revealed to be an independent predictor of recurrence other than Ku-FFPE4 score in a multivariate model analyzed by classical clinical parameters (Ku-IHC4 score vs Ku-FFPE4 score; χ(2): 14.2 vs 2.5, P: 0.0002 vs 0.11). When patients were trichotomized into high-, intermediate- and low-risk groups using the thresholds determined from the approximately calculated 5-year relapse rate, Kaplan-Meier analyses showed a significant difference among the three groups in Ku-IHC4 score (log-rank, P<0.0001), but not in Ku-FFPE4 score. The high-risk group according to Ku-FFPE4 score showed contradictory low recurrence rates (Ku-IHC4 score vs Ku-FFPE4 score, 53.1 vs 24.8%), which might be caused by risk-dependently extended error ranges. We show that the Ku-IHC4 score, consisted with semiquantitative measures of immunohistochemistry, provides better prognostic information than the corresponding quantitative RNA measurements. Prognostication tools such as the Ku-IHC4 score may be potentially useful in screening which patients had better be assessed by further testing using other genes rather than ER, PgR, HER2 and Ki-67 to determine critical aspects of therapeutic decision making.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/classificação , Imuno-Histoquímica , Transcriptoma , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Antígeno Ki-67/análise , Antígeno Ki-67/biossíntese , Antígeno Ki-67/genética , Pessoa de Meia-Idade , Prognóstico , Receptor ErbB-2/análise , Receptor ErbB-2/biossíntese , Receptor ErbB-2/genética , Receptores de Estrogênio/análise , Receptores de Estrogênio/biossíntese , Receptores de Estrogênio/genética , Receptores de Progesterona/análise , Receptores de Progesterona/biossíntese , Receptores de Progesterona/genéticaRESUMO
BACKGROUND: Aurora A kinase, a centrosomal serine/threonine kinase which plays an essential role in chromosome segregation during cell division, is commonly amplified and/or over expressed in human malignancies. Aurora A is suggested to be one of the proliferation parameters which is an independent prognostic factor for early invasive breast cancer patients; however the individual clinical or prognostic relevance of this gene has been a matter of debate. METHODS: A comprehensive analysis of Aurora A at the levels of gene expression, gene copy number and protein expression was performed for 278 primary invasive breast cancer patients; and the correlation with clinical outcomes were investigated. RESULTS: Aurora A gene expression level not only correlated with gene amplification, but was also significantly associated with several clinicopathological parameters and patient prognosis. Patients with higher nuclear grade, negative progesterone receptor status and higher Ki67 expressed higher levels of Aurora A mRNA, which was associated not only with poor relapse-free survival (RFS) but was also found to be a significant multivariate parameter for RFS. Aurora A protein expression was also significantly associated with clinicopathological characteristics; lymph node status, nuclear grade, estrogen receptor status and Ki67, but not with prognosis. By contrast, Aurora A gene amplification correlated with tumor size, nuclear grade and Ki67, and had no prognostic value. CONCLUSION: Our data indicate that Aurora A gene expression is an effective tool, which defines both tumor proliferation potency and patient prognosis.
Assuntos
Aurora Quinase A/biossíntese , Biomarcadores Tumorais/análise , Neoplasias da Mama/metabolismo , Proliferação de Células , Aurora Quinase A/análise , Aurora Quinase A/genética , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Intervalo Livre de Doença , Feminino , Dosagem de Genes , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Prognóstico , Modelos de Riscos Proporcionais , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TranscriptomaRESUMO
BACKGROUND: Breast elastography (EG), which can objectively evaluate tumor stiffness, has been useful for differentiation of benign and malignant breast lesions. However, the value of EG for prediction of response to systemic therapy is poorly understood. METHODS: The baseline evaluations of EG in 55 patients who received neoadjuvant chemotherapy were reviewed. We investigated the correlation between tumor stiffness and response to neoadjuvant chemotherapy. Tumor stiffness was evaluated by the Tsukuba elasticity scoring system. RESULTS: The mean EG scores were significant lower for the clinical and pathologic complete response (pCR) groups than for the others. When we categorized patients into two groups according to tumor stiffness, 26 patients were assigned to the low EG group (soft, scores from 1 to 3) and 29 patients were assigned to the high EG group (hard, score 4 and 5). The low EG group had significantly higher clinical complete response and pCR rates than the high EG group (clinical complete response, low EG group 38 % vs. high EG group 10 %, P = 0.024; pCR, low EG group 50 % vs. high EG group 14 %, P = 0.003, respectively). Furthermore, multivariate analysis indicated that estrogen receptor, human epidermal growth factor receptor 2, and low EG (odds ratio 13.04, 95 % confidence interval 1.19-458.28, P = 0.035) were independent predictive factors of pCR. CONCLUSIONS: Tumor stiffness evaluated by EG bears predictive potential for response to neoadjuvant chemotherapy. Stiffness evaluated by EG may be recognized as a clinically significant tumor characteristic, comparable to other data obtained by functional imaging techniques.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/tratamento farmacológico , Carcinoma Ductal de Mama/diagnóstico por imagem , Carcinoma Ductal de Mama/tratamento farmacológico , Técnicas de Imagem por Elasticidade , Adulto , Idoso , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Quimioterapia Adjuvante , Distribuição de Qui-Quadrado , Feminino , Humanos , Antígeno Ki-67/metabolismo , Modelos Logísticos , Pessoa de Meia-Idade , Terapia Neoadjuvante , Valor Preditivo dos Testes , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Estudos Retrospectivos , Resultado do TratamentoRESUMO
Immune check point inhibitors (ICIs) are now increasingly used for cancer therapy. At the same time, by activating the immune system, ICIs induce unique side effects, termed immune-related adverse events (irAEs). Renal irAEs, although uncommon, result in acute tubulointerstitial nephritis. Recently, because of an increase in ICI administration, renal irAEs, including glomerulonephritis, are being increasingly reported. A 69-year-old man presented with nephrotic syndrome after use of the ICI nivolumab. He underwent renal biopsy and was diagnosed with membranous nephropathy (MN) without acute tubulointerstitial nephritis. Immunofluorescence staining was negative for IgG4 and phospholipase A2 receptor (PLA2R), suggesting a malignancy-associated pattern. Oral glucocorticoid therapy was started as the standard treatment for irAEs, which resulted in complete remission of the nephrotic syndrome in 20 months. We suggest his MN was induced or accelerated by immune activation due to nivolumab. It means that ICIs possibly induce not only acute tubulointerstitial nephritis but also nephrotic syndrome due to MN as renal irAEs which is treatable with glucocorticoid.
Assuntos
Adenocarcinoma de Pulmão , Glomerulonefrite Membranosa , Neoplasias Pulmonares , Síndrome Nefrótica , Adenocarcinoma de Pulmão/tratamento farmacológico , Idoso , Feminino , Glomerulonefrite Membranosa/diagnóstico , Glomerulonefrite Membranosa/tratamento farmacológico , Glucocorticoides/uso terapêutico , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Nefrite Intersticial , Síndrome Nefrótica/induzido quimicamente , Síndrome Nefrótica/diagnóstico , Síndrome Nefrótica/tratamento farmacológico , Nivolumabe/efeitos adversosRESUMO
Ganglioside GD3/GD2 are over-expressed in various neuroectoderm-derived tumors. Previous studies indicated that GD3 is involved in the enhancement of cancer properties such as rapid growth and increased invasiveness. However, little is known about the functions of GD3/GD2 in glioma cells and glioma microenvironments. To clarify the functions of GD3/GD2 in gliomas, we used a mouse glioma model based on the RCAS/Gtv-a system. At first, we compared the gliomas size between wild-type (WT) and GD3 synthase (GD3S) knockout (KO) mice, showing a less malignant histology and slower tumor growth in GD3S-KO mice than in WT mice. Immunohistochemistry of glioma sections from WT and GD3S-KO mice revealed that reactive microglia/macrophages showed different localization patterns between the two genetic types of mice. CD68+ cells were more frequently stained inside glioma tissues of GD3S-KO mice, while they were stained mainly around glioma tissues in WT mice. The number of CD68+ cells markedly increased in tumor tissues of GD3S-KO mice at 2 weeks after injection of transfectant DF-1 cells. Furthermore, CD68+ cells in GD3S(-/-) glioma tissues expressed higher levels of inducible nitric oxide synthase. We observed higher expression levels of pro-inflammatory cytokine genes in primary-cultured glioma cells of WT mice than in GD3S-KO mice. DNA microarray data also revealed differential expression levels of various cytokines and chemokines in glioma tissues between WT and GD3S-KO mice. These results suggest that expression of GD3S allows glioma cells to promote polarization of microglia/macrophages towards M2-like phenotypes by modulating the expression levels of chemokines and cytokines.
Assuntos
Glioma , Animais , Citocinas , Glioma/genética , Camundongos , Camundongos Knockout , Índice de Gravidade de Doença , Microambiente TumoralRESUMO
Microfluidics is applied in biotechnology research via the creation of microfluidic channels and reaction vessels. Filters are considered to be able to simulate microfluidics. A typical example is the cell culture insert, which comprises two vessels connected by a filter. Cell culture inserts have been used for years to study cell-to-cell communication. These systems generally have a bucket-in-bucket structure and are hereafter referred to as a vertical-type co-culture plate (VTCP). However, VTCPs have several disadvantages, such as the inability to simultaneously observe samples in both containers and the inability of cell-to-cell communication through the filters at high cell densities. In this study, we developed a novel horizontal-type co-culture plate (HTCP) to overcome these disadvantages and confirm its performance. In addition, we clarified the migration characteristics of substances secreted from cells in horizontal co-culture vessels. It is generally assumed that less material is exchanged between the horizontal vessels. However, the extracellular vesicle (EV) transfer was found to be twice as high when using HTCP. Other merits include control of the degree of co-culture via the placement of cells. We believe that this novel HTCP container will facilitate research on cell-to-cell communication in various fields.
RESUMO
A 74-year-old man presented with nephrotic syndrome and kidney insufficiency. Laboratory tests revealed monoclonal gammopathy of immunoglobulin A-lambda. Renal biopsy revealed diffuse mesangial proliferation and double-contoured basement membranes. Immunofluorescent analyses showed granular deposition of immunoglobulin A and C3 at the capillary walls and mesangial regions. Immunohistochemistry suggested monoclonal deposition of immunoglobulin A1-lambda. Electron microscopic analyses showed finely granular electron-dense deposits at mesangial and subendothelial areas. These findings suggested immunoglobulin A-type proliferative glomerulonephritis with monoclonal immunoglobulin deposits. Based on the results of bone marrow aspiration, multiple myeloma was diagnosed. Because the renal manifestation was considered to be affected by monoclonal gammopathy, chemotherapy was initiated rather than immunomodulatory therapy. Although bortezomib and dexamethasone proved ineffective, second chemotherapy with elotuzumab, lenalidomide, and dexamethasone was successful, and kidney function recovered. Effective treatments for proliferative glomerulonephritis with monoclonal immunoglobulin deposits have not been established. This represents the first description of a patient successfully treated for proliferative glomerulonephritis with monoclonal immunoglobulin deposits by chemotherapy using elotuzumab.
Assuntos
Glomerulonefrite Membranoproliferativa/diagnóstico , Imunoglobulina A/imunologia , Cadeias lambda de Imunoglobulina/imunologia , Mieloma Múltiplo/complicações , Mieloma Múltiplo/tratamento farmacológico , Idoso , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos Hormonais/administração & dosagem , Antineoplásicos Hormonais/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biópsia por Agulha/métodos , Medula Óssea/patologia , Dexametasona/administração & dosagem , Dexametasona/uso terapêutico , Glomerulonefrite Membranoproliferativa/etiologia , Glomerulonefrite Membranoproliferativa/imunologia , Glomerulonefrite Membranoproliferativa/patologia , Humanos , Imunoglobulina A/efeitos dos fármacos , Imunoglobulina A/metabolismo , Imuno-Histoquímica/métodos , Fatores Imunológicos/administração & dosagem , Fatores Imunológicos/uso terapêutico , Lenalidomida/administração & dosagem , Lenalidomida/uso terapêutico , Masculino , Microscopia Eletrônica/métodos , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/patologia , Síndrome Nefrótica/diagnóstico , Síndrome Nefrótica/etiologia , Paraproteinemias/etiologia , Paraproteinemias/imunologia , Insuficiência Renal/diagnóstico , Insuficiência Renal/etiologia , Resultado do TratamentoRESUMO
Renal fibrosis (RF) is an indicator for progression of chronic kidney disease (CKD). Although diabetic nephropathy (DN) is the leading cause of CKD and end-stage renal disease in Western populations, the ability of MRI to evaluate RF in DN patients has not been determined. As a first step to identify possible MRI methods for RF evaluation, we examined the use of diffusion tensor imaging (DTI) MRI to evaluate RF in a rat model of DN (SHR/NDmcr-cp(cp/cp): SHR/ND). The signal-to-noise ratio in DTI MRI was enhanced using a spin-echo sequence, and a special kidney attachment was developed for long-term stabilization. The changes in renal temperature and blood flow during measurement were minimal, suggesting the feasibility of this method. At 38 weeks of age, RF had aggressively accumulated in the outer stripe (OS) of the outer medulla. FA maps showed that this method was successful in visualizing and evaluating fibrosis in the OS of the SHR/ND rat kidney (r = 0.7697, P = 0.0126). Interestingly, in the FA color maps, the directions of water molecule diffusion in RF were random, but distinct from conventional water diffusion in brain neuron fibers. These findings indicate that DTI MRI may be able to evaluate RF in CKD by DN.
Assuntos
Nefropatias Diabéticas/diagnóstico por imagem , Imagem de Tensor de Difusão/métodos , Fibrose/diagnóstico por imagem , Rim/diagnóstico por imagem , Animais , Nefropatias Diabéticas/patologia , Modelos Animais de Doenças , Fibrose/patologia , Rim/patologia , RatosRESUMO
Autosomal recessive polycystic kidney disease (ARPKD) is an important childhood nephropathy, occurring 1 in 20,000 live births. The major clinical phenotypes are expressed in the kidney with dilatation of the collecting ducts, systemic hypertension, and progressive renal insufficiency, and in the liver with biliary dysgenesis, portal tract fibrosis, and portal hypertension. The systemic hypertension has been attributed to enhanced distal sodium reabsorption in the kidney, the structural defects have been ascribed to altered cellular morphology, and fibrosis to increased TGF-ß signaling in the kidney and biliary tract, respectively. The pathogenic mechanisms underlying these abnormalities have not been determined. In the current report, we find that disrupting PKHD1 results in altered sub-cellular localization and function of the C2-WWW-HECT domain E3 family of ligases regulating these processes. We also demonstrate altered activity of RhoA and increased TGF-ß signaling and ENaC activity. Linking these phenomena, we found that vesicles containing the PKHD1/Pkhd1 gene product, FPC, also contain the NEDD4 ubiquitin ligase interacting protein, NDFIP2, which interacts with multiple members of the C2-WWW-HECT domain E3 family of ligases. Our results provide a mechanistic explanation for both the cellular effects and in vivo phenotypic abnormalities in mice and humans that result from Pkhd1/PKHD1 mutation.
Assuntos
Ubiquitina-Proteína Ligases Nedd4/metabolismo , Rim Policístico Autossômico Recessivo/genética , Rim Policístico Autossômico Recessivo/metabolismo , Receptores de Superfície Celular/deficiência , Animais , Biomarcadores , Linhagem Celular , Modelos Animais de Doenças , Ativação Enzimática , Expressão Gênica , Humanos , Espaço Intracelular/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Biológicos , Mutação , Rim Policístico Autossômico Recessivo/patologia , Transporte Proteico , Ratos , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismoAssuntos
Colite/etiologia , Colo Sigmoide , Síndrome Nefrótica/etiologia , Adulto , Humanos , Masculino , Sífilis/complicaçõesRESUMO
A potent angiotensin II type-1 receptor blocker, azilsartan, has been reported to reduce blood pressure more effectively than candesartan. Interestingly, azilsartan can also restore the circadian rhythm of blood pressure. We hypothesized that azilsartan could also improve salt sensitivity; thus, we examined the effect of azilsartan on sodium handling in renal tubules. Subtotal nephrectomized C57BL/6 mice received azilsartan (1.0 mg/kg/day), candesartan (0.3 mg/kg/day), or vehicle via the oral route in conjunction with a normal- (0.3%) or high-salt (8.0%) diet. Two weeks later, the azilsartan group showed significantly lower blood pressure during the light period than the candesartan and vehicle groups (azilsartan: 103.1 ± 1.0; candesartan: 111.7 ± 2.7; vehicle: 125.5 ± 2.5 mmHg; P < 0.05; azilsartan or candesartan vs. vehicle). The azilsartan group also showed higher urinary fractional excretion of sodium during the dark period than the candesartan and vehicle groups (azilsartan: 21.37 ± 3.69%; candesartan: 14.17 ± 1.42%; vehicle: 13.85 ± 5.30%; P < 0.05 azilsartan vs. candesartan or vehicle). A pressure-natriuresis curve demonstrated that azilsartan treatment restored salt sensitivity. Immunofluorescence and western blotting showed lower levels of Na+-H+ exchanger-3 (NHE3) protein (the major sodium transporter in renal proximal tubules) in the azilsartan group, but not in the candesartan or vehicle groups. However, azilsartan did not affect NHE3 transcription levels. Interestingly, we did not observe increased expression of downstream sodium transporters, which would have compensated for the increased flow of sodium and water due to non-absorption by NHE3. We also confirmed the mechanism stated above using cultured opossum kidney proximal tubular cells. Results revealed that a proteasomal inhibitor (but not a lysosomal inhibitor) blocked the azilsartan-induced decrease in NHE3 protein expression, suggesting that azilsartan increases NHE3 ubiquitination. In conclusion, azilsartan (but not candesartan) improved salt sensitivity possibly by decreasing NHE3 expression via ubiquitin-proteasomal degradation.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Benzimidazóis/farmacologia , Túbulos Renais Proximais/efeitos dos fármacos , Oxidiazóis/farmacologia , Tolerância ao Sal/efeitos dos fármacos , Sódio na Dieta/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Compostos de Bifenilo , Pressão Sanguínea/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nefrectomia , Trocador 3 de Sódio-Hidrogênio , Tetrazóis/farmacologiaRESUMO
Histone acetylation is generally associated with gene activation and chromatin decondensation. Recent mass spectrometry analysis has revealed that histone H4 lysine 20, a major methylation site, can also be acetylated. To understand the function of H4 lysine 20 acetylation (H4K20ac), we have developed a specific monoclonal antibody and performed ChIP-seq analysis using HeLa-S3 cells. H4K20ac was enriched around the transcription start sites (TSSs) of minimally expressed genes and in the gene body of expressed genes, in contrast to most histone acetylation being enriched around the TSSs of expressed genes. The distribution of H4K20ac showed little correlation with known histone modifications, including histone H3 methylations. A motif search in H4K20ac-enriched sequences, together with transcription factor binding profiles based on ENCODE ChIP-seq data, revealed that most transcription activators are excluded from H4K20ac-enriched genes and a transcription repressor NRSF/REST co-localized with H4K20ac. These results suggest that H4K20ac is a unique acetylation mark associated with gene repression.
Assuntos
Histonas/metabolismo , Acetilação , Animais , Anticorpos Monoclonais/imunologia , Sítios de Ligação , Imunoprecipitação da Cromatina , Cromatografia Líquida de Alta Pressão , Células HeLa , Histonas/imunologia , Humanos , Lisina/metabolismo , Metilação , Camundongos , Microscopia de Fluorescência , Peptídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de Transcrição/metabolismo , Sítio de Iniciação de TranscriçãoRESUMO
Collagenofibrotic glomerulopathy is a rare glomerular disease characterized by extensive accumulation of atypical type III collagen fibers within the mesangial matrix and subendothelial space. Laboratory evaluation of this disease shows a marked increase in serum procollagen III peptide (P III P) levels. Here, we report the case of two brothers with collagenofibrotic glomerulopathy confirmed by histology. Patient 1 presented with proteinuria and hypertension and patient 2 presented with nephrotic-range proteinuria. Immunohistochemistry revealed strong staining for antibodies to type III collagen in the widened subendothelial spaces in both patients. Electron microscopy revealed numerous collagenous fibers in the mesangium and subendothelial space. P III P levels were elevated in both patients. Most reported cases of collagenofibrotic glomerulopathy, including the adult-onset type, have been sporadic. Within the limits of our literature search, this is only the third report of adult siblings with collagenofibrotic glomerulopathy confirmed by histology. This report indicates that it may be beneficial to measure serum P III P levels in the siblings of patients diagnosed with adult-onset collagenofibrotic glomerulopathy.
RESUMO
The newly identified gene, metastasisassociated in colon cancer 1 (MACC1), is suggested to be a transcriptional regulator of cMet, leading to cancer progression in colorectal cancer. To date however, little is known of the role of MACC1 in breast cancer. In a series of 300 breast cancer patients, we analyzed the association of MACC1 mRNA and protein expression with breast cancer survival using Cox proportional hazard models. In an in vitro study, we evaluated activities of cMet protein after transfection with a MACC1harboring plasmid as well as the binding ability of MACC1 to the cMet promoter using a chromatin immunoprecipitation (ChIP) assay. In survival analyses, reduced MACC1 expression was associated with patient mortality. MACC1 expression was an independent prognostic factor in multivariate analysis. In the cell lines tested, MACC1 expression was much higher in colorectal than in breast cancer cells. After cells were transfected with MACC1, cMet expression was not induced in MCF7 cells, whereas corresponding cMet expression was upregulated in SW480 cells. Further, SW480 cells transfected with MACC1 showed enhanced migratory ability, whereas in MDAMB231 cells, transfection of MACC1 had no impact on this ability. In ChIP assay, the binding of MACC1 to the cMet promoter was suggested in SW480 cells, but not in MCF7 cells. In conclusion, our findings provide some novel insights into the role of MACC1 in breast cancer, indicating that it plays different roles in breast and several other cancers. There is a possibility that MACC1 does not modulate the transcriptional role of cMet signaling in breast cancer.