ABSTRACT
YAP/TAZ are wild over-activated in head and neck squamous cell carcinoma (HNSCC) with high potential as a direct therapy target for HNSCC treatments. However, the efforts on the directly targeting-YAP/TAZ therapies over the past decade, have very limited impacts, mainly caused by: 1. There is still none effective and specific YAP/TAZ inhibitor with clinical potential; 2. YAP/TAZ might not be directly targeted, because of their multiple important biological functions, such as: regulation of cell proliferation and survival, stem cell maintain, regulation of organ development, organ size control, and tissue regeneration. Interestingly, the over-activation of YAP/TAZ in HNSCC mainly be regulated by upstream abnormal molecular or biological events, instead of genes alteration of YAP/TAZ. Therefore, exploring the alternative molecular events regulating YAP/TAZ activation and molecular mechanism in HNSCC might help to uncover novel indirect targets of YAP/TAZ therapies for HNSCC prevention and treatment.
Subject(s)
Humans , Adaptor Proteins, Signal Transducing/metabolism , Head and Neck Neoplasms , Phosphoproteins/metabolism , Trans-Activators/metabolism , Transcription FactorsABSTRACT
BACKGROUND@#Arteriosclerosis obliterans (ASO) is a major cause of adult limb loss worldwide. Autophagy of vascular endothelial cell (VEC) contributes to the ASO progression. However, the molecular mechanism that controls VEC autophagy remains unclear. In this study, we aimed to explore the role of the GRB2 associated binding protein 1 (GAB1) in regulating VEC autophagy.@*METHODS@#In vivo and in vitro studies were applied to determine the loss of adapt protein GAB1 in association with ASO progression. Histological GAB1 expression was measured in sclerotic vascular intima and normal vascular intima. Gain- and loss-of-function of GAB1 were applied in VEC to determine the effect and potential downstream signaling of GAB1.@*RESULTS@#The autophagy repressor p62 was significantly downregulated in ASO intima as compared to that in healthy donor (0.80 vs. 0.20, t = 6.43, P < 0.05). The expression level of GAB1 mRNA (1.00 vs. 0.24, t = 7.41, P < 0.05) and protein (0.72 vs. 0.21, t = 5.97, P < 0.05) was significantly decreased in ASO group as compared with the control group. Loss of GAB1 led to a remarkable decrease in LC3II (1.19 vs. 0.68, t = 5.99, P < 0.05), whereas overexpression of GAB1 significantly led to a decrease in LC3II level (0.41 vs. 0.93, t = 7.12, P < 0.05). Phosphorylation levels of JNK and p38 were significantly associated with gain- and loss-of-function of GAB1 protein.@*CONCLUSION@#Loss of GAB1 promotes VEC autophagy which is associated with ASO. GAB1 and its downstream signaling might be potential therapeutic targets for ASO treatment.
Subject(s)
Adult , Humans , Adaptor Proteins, Signal Transducing , Arteriosclerosis Obliterans/genetics , Autophagy , GRB2 Adaptor Protein , Phosphoproteins/metabolism , Phosphorylation , Protein Binding , Signal TransductionABSTRACT
Acute effect of purified mimosine (MiMo) extracted from Leucaena leucocephala on testicular histopathology has been documented with seminal vesicle (SV) atrophy. Since protein phosphorylation and seminal secretions play important roles in sperm physiology, this study aimed to study the alteration of substances including tyrosine phosphorylated (TyrPho) proteins in seminal vesicle treated with MiMo. Male mice were divided into a control and experimental groups treated with purified MiMo at 3 doses of 15, 30, and 60 mg/KgBW, respectively for 35 consecutive days. The morphology and weights of SV were compared among groups. The levels of magnesium and fructosamine in SV fluid were assayed. The profiles of equally SV total proteins were compared using SDS-PAGE. The expression of seminal TyrPho proteins was detected by western blotting. Recent results showed the decreased weights of SV in MiMo treated mice compared to control. However MiMo in all doses did not affect the levels of magnesium and fructosamine in SV fluid. The SV protein expression of 130 and 55 kDas was obviously decreased in a high dose MiMo. In dose-dependent response, the expressions of 72 and 55 kDas TyrPho proteins of SV were increased. In conclusion, MiMo could affect SV morphological size and protein secretions especially TyrPho proteins.
El efecto agudo de la mimosina purificada (MiMo) extraída de Leucaena leucocephala en la histopatología testicular se ha documentado con atrofia de vesícula seminal (VS). Debido a que la fosforilación de proteínas y las secreciones seminales tienen un papel importante en la fisiología de los espermatozoides, este estudio tuvo como objetivo estudiar la alteración de sustancias como la proteína tirosina fosforilada (TyrPho) en vesículas seminales tratadas con MiMo. Los ratones se dividieron en un grupo control y un grupo experimental y se trataron con MiMo purificado en 3 dosis de 15, 30 y 60 mg / KgBW, respectivamente, durante 35 días seguidos. La morfología y los pesos de VS se compararon entre los grupos. Fueron analizados los niveles de magnesio y fructosamina en el fluido VS. Los perfiles de las proteínas totales de VS se compararon utilizando SDS-PAGE. La expresión de la proteína TyrPho en las vesículas seminales se detectó mediante transferencia de Western blot. Los resultados recientes muestran la disminución del peso de las VS en ratones tratados con MiMo, en comparación con el grupo control. Sin embargo, en ninguna de las dosis se vieron afectados por mimosina purificada los niveles de magnesio y fructosamina en el líquido de las VS. La expresión de la proteína en VS de 130 y 55 kDas disminuyó notablemente en una dosis alta de MiMo. En la respuesta dependiente de la dosis, aumentaron las expresiones de 72 y 55 kDas de las proteínas TyrPho en las VS. En conclusión, la mimosina purificada podría afectar el tamaño morfológico de las VS y la expresión de proteínas, especialmente las proteínas TyrPho.
Subject(s)
Animals , Male , Mice , Phosphoproteins/drug effects , Seminal Vesicles/drug effects , Mimosine/administration & dosage , Organ Size , Phosphoproteins/metabolism , Phosphorylation , Seminal Vesicles/pathology , Tyrosine/analogs & derivatives , Blotting, Western , Phosphotyrosine , Electrophoresis, Polyacrylamide Gel , Mice, Inbred ICR , Mimosine/pharmacologyABSTRACT
Tyrosine phosphorylated proteins have been localized and identified in male reproductive tissues such as testis and capacitated/ acrosome reacted sperm except epididymis. The changes of such proteins are associated with decreased sperm quality of valproic acid treatment. This study aimed to investigate the presence and alterations of protein phosphorylation in epididymal epithelium and fluid of rats treated VPA. Sixteen adult male rats were divided into control and VPA-treated groups (n=8/ each). Treated rats were injected with VPA (500 mg/ kgBW, intraperitoneally) for 10 consecutive days. At the end of experiment, the monoclonal antiphosphotyrosine (clone 4G10) was used for immunohistochemistry to probe tyrosine phosphorylated proteins and also to examine the expression of such proteins using immuno-Western blotting in epididymal tissue and fluid. The result showed that positive reactivity of phosphorylated proteins was clearly observed in cytoplasmic principle cells, nuclei of apical & basal cells and sperm mass surrounded with epididymal fluids. The profiles of phosphorylated proteins in epididymal fluid were 182, 127, 80, 70, 57, 45, 34, and 31 kDas, respectively. Interestingly, VPA affected the changes of phosphorylated proteins and β actin in head, body, and tail epididymal fluids. We conclude that tyrosine phosphorylated proteins were detected in epididymal epithelium and fluid. The expressions of those proteins and actin were altered under VPA treating.
Las proteínas tirosina fosforiladas han sido localizadas e identificadas en tejidos reproductores masculinos tales como testículos y espermatozoides, capacitados a nivel acrosómico, excepto en el epidídimo. Los cambios de estas proteínas están asociadas con una disminución de la calidad del esperma en el tratamiento con ácido valproico (AVP). Este estudio tuvo como objetivo investigar la presencia y las alteraciones de la fosforilación de proteínas en el epitelio epididimal y en el fluido espermático de ratas tratadas con AVP. Dieciséis ratas macho adultas se dividieron en dos grupos: control y tratadas con AVP (n = 8 / cada uno). A las ratas tratadas se les inyectó AVP por vía intraperitoneal (500 mg / kg de peso corporal) durante 10 días consecutivos. Al final del experimento, se realizó inmunohistoquímica con la anti-fosfotirosina monoclonal (clon 4G10) para sondear las proteínas tirosina fosforiladas y también para examinar la expresión de tales proteínas usando inmunotransferencia Western, en tejido y fluido epididimarios. El resultado mostró reactividad positiva de proteínas fosforiladas en células citoplásmicas principales, en los núcleos de las células apicales y basales y en la masa de esperma rodeada por fluidos epididimarios. Los perfiles de proteínas fosforiladas en el fluido epididimal fueron 182, 127, 80, 70, 57, 45, 34 y 31 kDas, respectivamente. El AVP provocó cambios en las proteínas fosforiladas y en la β actina de los fluidos epididimarios de cabeza, cuerpo y cola del epidídimo. Concluimos que las proteínas tirosina fosforiladas se detectaron en el epitelio y el fluido epididimarios. Las expresiones de esas proteínas y de la β actina se alteraron bajo tratamiento con AVP.
Subject(s)
Animals , Male , Rats , Phosphoproteins/drug effects , Tyrosine/drug effects , Valproic Acid/administration & dosage , Actins/drug effects , Anticonvulsants/administration & dosage , Phosphoproteins/metabolism , Phosphorylation , Tyrosine/metabolism , Immunohistochemistry , Blotting, Western , Actins/metabolism , Rats, Sprague-Dawley , Phosphotyrosine , EpididymisABSTRACT
SUMMARY: Spermatogenesis is a major process in testis occurring from puberty through life span of males. The tyrosine phosphorylation is assumed to play roles in spermatogenesis because this process is important for cell proliferations, divisions, and differentiations. However, the localizations and identifications of phosphorylated proteins in testicular tissue of adult male rats are still unclear. Therefore, this study attempted to immuno-localize and identify such proteins in testicular tissues of Sprague-Dawley rats. The monoclonal anti-phosphotyrosine (clone 4G10) was used to probe tyrosine phosphorylated proteins and also to examine the expression of such proteins using immuno-Western blotting in rat testis. The result showed that positive reactivity of tyrosine phosphorylated proteins was clearly observed in interstitial endocrine cells (Leydig cells), sustentocytes (Sertoli cells), spermatogonia, spermatocytes, and spermatids (round and elongated), respectively. The expressions of testicular tyrosine phosphorylated proteins were 200, 131, 93, 70, 60, and 48 kDas, respectively. In conclusion, testicular tyrosine phosphorylated proteins were localized in both germinal epithelium and interstitial endocrine cells of adult Sprague-Dawley rats.
RESUMEN: La espermatogénesis es un proceso importante en los testículos que ocurre desde la pubertad a lo largo de la vida de los machos. Se supone que la fosforilación de la tirosina desempeña papeles en la espermatogénesis, debido a que este proceso es importante para las proliferaciones, divisiones y diferenciaciones celulares. Sin embargo, las localizaciones e identificaciones de proteínas fosforiladas en el tejido testicular de ratas macho adultas todavía no están claras. Por lo tanto, este estudio intentó inmuno-localizar e identificar dichas proteínas en tejidos testiculares de ratas Sprague-Dawley. La anti-fosfotirosina monoclonal (clon 4G10) se usó para sondar proteínas tirosina fosforiladas y también para examinar la expresión de tales proteínas usando inmunotransferencia Western en testículo de rata. El resultado mostró que la actividad positiva de las proteínas tirosina fosforiladas se observó claramente en endocrinocitos intersticiales (células de Leydig), sustentocitos (células de Sertoli), espermatogonias, espermatocitos y espermátidas (redondas y alargadas), respectivamente. Las expresiones de las proteínas tirosina fosforiladas testiculares fueron de 200, 131, 93, 70, 60 y 48 kDas, respectivamente. En conclusión, las proteínas tirosina fosforiladas fueron localizadas en ambos epitelios germinales y endocrinocitos intersticiales de ratas adultas Sprague-Dawley.
Subject(s)
Animals , Male , Rats , Phosphoproteins/analysis , Phosphoproteins/metabolism , Testis/chemistry , Tyrosine/analysis , Tyrosine/metabolism , Immunohistochemistry , Blotting, Western , Rats, Sprague-DawleyABSTRACT
Abstract The aim of this study was to assess the in vitro and in vivo effects of short-interfering RNAs (siRNAs) against rabies virus phosphoprotein (P) mRNA in a post-infection treatment for rabies as an extension of a previous report (Braz J Microbiol. 2013 Nov 15;44(3):879-82). To this end, rabies virus strain RABV-4005 (related to the Desmodus rotundus vampire bat) were used to inoculate BHK-21 cells and mice, and the transfection with each of the siRNAs was made with Lipofectamine-2000™. In vitro results showed that siRNA 360 was able to inhibit the replication of strain RABV-4005 with a 1 log decrease in virus titter and 5.16-fold reduction in P mRNA, 24 h post-inoculation when compared to non-treated cells. In vivo, siRNA 360 was able to induce partial protection, but with no significant difference when compared to non-treated mice. These results indicate that, despite the need for improvement for in vivo applications, P mRNA might be a target for an RNAi-based treatment for rabies.
Subject(s)
Animals , Phosphoproteins/genetics , Rabies/veterinary , Rabies virus/genetics , Viral Proteins/genetics , Chiroptera/virology , RNA, Small Interfering/genetics , RNA Interference , Phosphoproteins/metabolism , Rabies/virology , Rabies virus/physiology , Viral Proteins/metabolism , Virus Replication , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/metabolismABSTRACT
During gonad and adrenal development, the POD-1/capsulin/TCF21transcription factor negatively regulates SF-1/NR5A1expression, with higher SF-1 levels being associated with increased adrenal cell proliferation and tumorigenesis. In adrenocortical tumor cells, POD-1 binds to the SF-1 E-box promoter region, decreasing SF-1 expression. However, the modulation of SF-1 expression by POD-1 has not previously been described in normal adrenal cells. Here, we analyzed the basal expression of Pod-1 and Sf-1 in primary cultures of glomerulosa (G) and fasciculata/reticularis (F/R) cells isolated from male Sprague-Dawley rats, and investigated whether POD-1 overexpression modulates the expression of endogenous Sf-1 and its target genes in these cells. POD-1 overexpression, following the transfection of pCMVMycPod-1, significantly decreased the endogenous levels of Sf-1 mRNA and protein in F/R cells, but not in G cells, and also decreased the expression of the SF-1 target StAR in F/R cells. In G cells overexpressing POD-1, no modulation of the expression of SF-1 targets, StAR and CYP11B2, was observed. Our data showing that G and F/R cells respond differently to ectopic POD-1 expression emphasize the functional differences between the outer and inner zones of the adrenal cortex, and support the hypothesis that SF-1 is regulated by POD-1/Tcf21 in normal adrenocortical cells lacking the alterations in cellular physiology found in tumor cells.
Subject(s)
Animals , Male , Adrenal Cortex/metabolism , Basic Helix-Loop-Helix Transcription Factors/metabolism , Phosphoproteins/metabolism , Steroidogenic Factor 1/metabolism , Adrenal Cortex/cytology , Basic Helix-Loop-Helix Transcription Factors/genetics , Electrophoresis, Polyacrylamide Gel , Gene Expression , Immunoblotting , Primary Cell Culture , Phosphoproteins/analysis , Rats, Sprague-Dawley , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , RNA, Messenger/analysis , Steroidogenic Factor 1/analysis , Zona Fasciculata/cytology , Zona Fasciculata/metabolism , Zona Glomerulosa/cytology , Zona Glomerulosa/metabolism , Zona Reticularis/cytology , Zona Reticularis/metabolismSubject(s)
Animals , Mice , /metabolism , Adipocytes/metabolism , Carrier Proteins/metabolism , Lipolysis/physiology , Membrane Proteins/metabolism , Phosphoproteins/metabolism , /genetics , Adipocytes/cytology , Carrier Proteins/genetics , Membrane Proteins/genetics , Protein Structure, Tertiary , Proteolysis , Phosphoproteins/genetics , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/metabolismSubject(s)
Animals , Rats , Apoptosis , Insulin-Secreting Cells/drug effects , Signal Transduction , /metabolism , Cell Line, Tumor , /metabolism , DNA Fragmentation , Enzyme Activation , I-kappa B Proteins/metabolism , Immunoprecipitation , Insulin-Secreting Cells/metabolism , JNK Mitogen-Activated Protein Kinases/metabolism , Lipopeptides/pharmacology , /metabolism , Palmitates , Protein Binding , Phosphoproteins/metabolism , Poly(ADP-ribose) Polymerases/metabolism , RNA Interference , /genetics , /agonists , /genetics , /metabolismABSTRACT
Neointimal proliferation after vascular injury is a key mechanism of restenosis, a major cause of percutaneous transluminal angioplasty failure and artery bypass occlusion. Emodin, an anthraquinone with multiple physiological activities, has been reported to inhibit proliferation of vascular smooth muscle cells (VSMCs) that might cause intimal arterial thickening. Thus, in this study, we established a rat model of balloon-injured carotid artery and investigated the therapeutic effect of emodin and its underlying mechanism. Intimal thickness was analyzed by hematoxylin and eosin staining. Expression of Wnt4, dvl-1, beta-catenin and collagen was determined by immunohistochemistry and/or western blotting. The proliferation of VSMC was evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and electron microscopy. MicroRNA levels were quantified by real-time quantitative PCR. Emodin relieved injury-induced artery intimal thickness. Results of western blots and immunohistochemistry showed that emodin suppressed expression of signaling molecules Wnt4/Dvl-1/beta-catenin as well as collagen protein in the injured artery. In addition, emodin enhanced expression of an artery injury-related microRNA, miR-126. In vitro, MTT assay showed that emodin suppressed angiotensin II (AngII)-induced proliferation of VSMCs. Emodin reversed AngII-induced activation of Wnt4/Dvl-1/beta-catenin signaling by increasing expression of miR-126 that was strongly supported by transfection of mimic or inhibitor for miR-126. Emodin prevents intimal thickening via Wnt4/Dvl-1/beta-catenin signaling pathway mediated by miR-126 in balloon-injured carotid artery of rats.
Subject(s)
Animals , Male , Rats , Adaptor Proteins, Signal Transducing/metabolism , Carotid Arteries/drug effects , Carotid Artery Injuries/drug therapy , Cell Proliferation/drug effects , Emodin/therapeutic use , MicroRNAs/metabolism , Phosphoproteins/metabolism , Rats, Sprague-Dawley , Signal Transduction/drug effects , Tunica Intima/drug effects , Wnt4 Protein/metabolism , beta Catenin/metabolismABSTRACT
PURPOSE: Foxo3 in female reproduction has been reported to regulate proliferation of granulose cells that form follicles. There are no reports so far that discuss on the role of Foxo3 in males. This study was designed to outline the role of Foxo3 in the testes. MATERIALS AND METHODS: Testes from mice at birth to postpartum week (PPW) 5 were isolated and examined for the expression of Foxo3 using immunostaining. To elucidate role of Foxo3 in Leydig cells, R2C cells were treated with luteinizing hormone (LH) and the phosphorylation of Foxo3. Testosterone and steroidogenic acute regulatory (StAR) protein levels were measured after constitutive active [triple mutant (TM)] human FOXO3 adenovirus was transduced and StAR promoter assay was performed. RESULTS: Foxo3 expression in the testicles started from birth and lasted until PPW 3. After PPW 3, most Foxo3 expression occurred in the nuclei of Leydig cells; however, at PPW 5, Foxo3 was expressed in both the nucleus and cytoplasm. When R2C cells were treated with luteinizing hormone, Foxo3 phosphorylation levels by AKT increased. After blocking the PI3K pathway, LH-induced phosphorylated Foxo3 levels decreased, indicating that LH signaling regulates Foxo3 localization. When active FOXO3-TM adenovirus was introduced into a Leydig tumor cell line, the concentrations of testosterone and StAR protein decreased. When FOXO3 and a StAR promoter vector were co-transfected into HEK293 cells for a reporter assay, FOXO3 inhibited the StAR promoter. CONCLUSION: FOXO3 affects testosterone synthesis by inhibiting the formation of StAR protein. LH hormone, meanwhile, influences Foxo3 localization, mediating its function.
Subject(s)
Animals , Humans , Male , Mice , Cellular Senescence/physiology , Cell Nucleus/metabolism , Cytoplasm/metabolism , Forkhead Transcription Factors/metabolism , HEK293 Cells , Leydig Cells/drug effects , Luteinizing Hormone/blood , Phosphatidylinositol 3-Kinases , Phosphoproteins/metabolism , Phosphorylation , Signal Transduction/drug effects , Testosterone/bloodABSTRACT
Viral gene oncotherapy is emerging as a biotherapeutic cancer treatment modality based on targeted killing of cancer cells by viral genes. Newcastle disease virus (NDV) has the property to cause selective oncolysis of tumor cells sparing normal cells. NDV has a single stranded negative sense RNA genome, which is 15,186 nucleotide long and consists of six genes, which codes for eight proteins. NDV like other paramyxoviruses has the ability to generate multiple proteins from the P gene. P protein is encoded by an unedited transcript of the P gene, whereas the V and W protein are the results of RNA editing event in which one and two G residues are inserted at a conserved editing site within the P gene mRNA resulting in V and W transcripts, respectively. Although NDV is known to cause oncolysis by triggering apoptosis, the role of different viral proteins in selective oncolysis is still unclear. P gene edited products are known for its anti-apoptotic property in homologous host. In the present study, NDV P gene and its RNA edited products were amplified, cloned, sequenced and in vitro expression was done in HeLa cells. Further constructs were assayed for their apoptosis inducing ability in HeLa cells. Preliminary study suggested that P, V and W proteins are not apoptotic to HeLa cells.
Subject(s)
Amino Acid Sequence , Animals , Annexin A5/metabolism , Base Sequence , Chickens , Cloning, Molecular , Gene Expression Regulation, Viral , Genes, Viral/genetics , HeLa Cells , Humans , Molecular Sequence Data , Newcastle disease virus/genetics , Open Reading Frames/genetics , Phosphoproteins/chemistry , Phosphoproteins/genetics , Phosphoproteins/metabolism , Reproducibility of Results , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
OBJECTIVE@#To discuss the myocardial expression of Spry1 and MAPK proteins of viral myocarditis (VMC), to reveal its mechanism of sudden death, and to provide guides for forensic identification of sudden cardiac death.@*METHODS@#Thirty Balb/c male mice were randomly divided into VMC group and control group, inoculated intraperitoneally with Coxsackievirus B3 and Eagel's solution, respectively. After the mice were sacrificed, the cardiac tissues of the mice were taken to proceed regular pathological examination. The changes of Spry1 protein, Spry1 mRNA and MAPK protein were detected by immunohistochemistry, Western blotting and real-time PCR.@*RESULTS@#Under light microscope, the pathologic changes included myocardial interstitial edema, inflammatory cells infiltration, myocardial necrosis, and focal and patchy necrosis of myocardial fiber in VMC group. The expression of Spry1 protein in VMC group was lower than that in control group (P < 0.05). There was slightly decreased expression of Spry1 of the mRNA level in VMC group (P > 0.05). But the MAPK protein expression in VMC group was higher than that in control group (P < 0.05).@*CONCLUSION@#The pathway of MAPK/ERK involving Spry1 protein accelerates the expression of collagen, which may contribute to arrhythmia, heart failure and even sudden cardiac death.
Subject(s)
Animals , Male , Mice , Adaptor Proteins, Signal Transducing/metabolism , Coxsackievirus Infections/pathology , Death, Sudden, Cardiac/pathology , Disease Models, Animal , Immunohistochemistry , Membrane Proteins/metabolism , Mice, Inbred BALB C , Mitogen-Activated Protein Kinases/metabolism , Myocarditis/virology , Myocardium/pathology , Phosphoproteins/metabolism , RNA, Messenger/metabolism , Random Allocation , Real-Time Polymerase Chain ReactionABSTRACT
Early diabetic nephropathy is characterized by glomerular hyperpermeability as a result of impaired glomerular filtration structure caused by hyperglycemia, glycated proteins or irreversible advanced glycosylation endproducts (AGE). To investigate the effect of ginseng total saponin (GTS) on the pathologic changes of podocyte ZO (zonula occludens)-1 protein and podocyte permeability induced by diabetic conditions, we cultured mouse podocytes under: 1) normal glucose (5 mM, = control); 2) high glucose (HG, 30 mM); 3) AGE-added; or 4) HG plus AGE-added conditions and treated with GTS. HG and AGE increased the dextran filtration of monolayered podocytes at early stage (2-8 hr) in permeability assay. In confocal imaging, ZO-1 colocalized with actin filaments and beta-catenin at cell contact areas, forming intercellular filtration gaps. However, these diabetic conditions suppressed ZO-1 immunostainings and disrupted the linearity of ZO-1. In Western blotting, diabetic conditions also decreased cellular ZO-1 protein levels at 6 hr and 24 hr. GTS improved such quantitative and qualitative changes. These findings imply that HG and AGE have an influence on the redistribution and amount of ZO-1 protein of podocytes thereby causing hyperpermeability at early stage, which can be reversed by GTS.
Subject(s)
Animals , Mice , Actin Cytoskeleton/metabolism , Cell Line , Diabetic Nephropathies/physiopathology , Glomerular Filtration Rate , Glucose/pharmacology , /pharmacology , Hyperglycemia/physiopathology , Membrane Proteins/metabolism , Panax , Permeability/drug effects , Phosphoproteins/metabolism , Plant Preparations/pharmacology , Podocytes/drug effects , Saponins/pharmacology , beta Catenin/metabolismABSTRACT
We sought to determine the effects of activation of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) on multilocularization of adipocytes in adult white adipose tissue (WAT). Male C57BL/6 normal, db/db, and ob/ob mice were treated with agonists of PPAR-gamma, PPAR-alpha, or beta3-adrenoceptor for 3 weeks. To distinguish multilocular adipocytes from unilocular adipocytes, whole-mounted adipose tissues were co-immunostained for perilipin and collagen IV. PPAR-gamma activation with rosiglitazone or pioglitazone induced a profound change of unilocular adipocytes into smaller, multilocular adipocytes in adult WAT in a time-dependent, dose-dependent, and reversible manner. PPAR-alpha activation with fenofibrate did not affect the number of locules or remodeling. db/db and ob/ob obese mice exhibited less multilocularization in response to PPAR-gamma activation compared to normal mice. Nevertheless, all adipocytes activated by PPAR-gamma contained a single nucleus regardless of locule number. Multilocular adipocytes induced by PPAR-gamma activation contained substantially increased mitochondrial content and enhanced expression of uncoupling protein-1, PPAR-gamma coactivator-1-alpha , and perilipin. Taken together, PPAR-gamma activation induces profound multilocularization and enhanced mitochondrial biogenesis in the adipocytes of adult WAT. These changes may affect the overall function of WAT.
Subject(s)
Animals , Male , Mice , Adipocytes/cytology , Adipose Tissue, White/cytology , Cell Nucleus Division , Hypoglycemic Agents/pharmacology , Ion Channels/metabolism , Mice, Inbred C57BL , Mice, Obese , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , PPAR alpha/agonists , PPAR gamma/agonists , Phosphoproteins/metabolism , Receptors, Adrenergic, beta-3/agonists , Thiazolidinediones/pharmacology , Trans-Activators/metabolismABSTRACT
Inorganic phosphate (Pi) plays a critical role in diverse cellular functions, and regulating the Pi balance is accomplished by sodium-dependent Pi co-transporter (NPT). Pulmonary NPT has recently been identified in mammalian lungs. However, to date, many of the studies that have involved Pi have mainly focused on its effect on bone and kidney. Therefore, current study was performed to discover the potential effects of low Pi on the lung of developing transgenic mice expressing the renilla/firefly luciferase dual reporter gene. Two-weeks old male mice divided into 2 groups and these groups were fed either a low PI diet or a normal control diet (normal: 0.5% Pi, low: 0.1% Pi) for 4 weeks. After 4 weeks of the diet, all the mice were sacrificed. Their lungs were harvested and analyzed by performing luciferase assay, Western blotting, kinase assay and immunohistochemistry. Our results demonstrate that low Pi affects the lungs of developing mice by disturbing protein translation, the cell cycle and the expression of fibroblast growth factor-2. These results suggest that optimally regulating Pi consumption may be important to maintain health.
Subject(s)
Animals , Male , Mice , Blotting, Western , Carrier Proteins/metabolism , Immunohistochemistry , Lung/drug effects , Mice, Transgenic , Phosphoproteins/metabolism , Phosphorus, Dietary/administration & dosage , Phosphorylation , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Sodium-Phosphate Cotransporter Proteins, Type IIa/metabolismABSTRACT
DNA damage activates several mechanisms such as DNA repair and cell cycle checkpoints. The Saccharomyces cerevisiae heterotrimeric checkpoint clamp consisting of the Rad17, Mec3 and Ddc1 subunits is an early response factor to DNA damage and activates checkpoints. This complex is structurally similar to the proliferating cell nuclear antigen (PCNA), which serves as a sliding clamp platform for DNA replication. Growing evidence suggests that PCNA-like complexes play a major role in DNA repair as they have been shown to interact with and stimulate several proteins, including specialized DNA polymerases. With the aim of extending our knowledge concerning the link between checkpoint activation and DNA repair, we tested the possibility of a functional interaction between the Rad17/Mec3/Ddc1 complex and the replicative DNA polymerases alpha, delta and epsilon. The analysis of sensitivity response of single and double mutants to UVC and 8-MOP + UVA-induced DNA damage suggests that the PCNA-like component Mec3p of S. cerevisiae neither relies on nor competes with the third subunit of DNA polymerase delta, Pol32p, for lesion removal. No enhanced sensitivity was observed when inactivating components of DNA polymerases alpha and epsilon in the absence of Mec3p. The hypersensitivity of pol32delta to photoactivated 8-MOP suggests that the replicative DNA polymerase delta also participates in the repair of mono- and bi-functional DNA adducts. Repair of UVC and 8-MOP + UVA-induced DNA damage via polymerase delta thus occurs independent of the Rad17/Mec3/Ddc1 checkpoint clamp.
Subject(s)
Cell Cycle Proteins , DNA-Directed DNA Polymerase/metabolism , DNA Repair , Phosphoproteins/metabolism , Nuclear Proteins/metabolism , DNA-Binding Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Proliferating Cell Nuclear Antigen/metabolism , DNA-Directed DNA Polymerase/classification , DNA, Fungal , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/geneticsABSTRACT
To compare the stem niche in different culture conditions of limbal epithelial cells, the suspended human limbal epithelial cells (HLECs) were seeded on the 3T3-pretreated plates and the other suspended cells were plated on amniotic membranes (AMs) which were either cryo-preserved or freeze-dried. All were cultured for 10 to 12 days. Reverse transcription-polymerase chain reaction (RT-PCR) for ATP-binding casette, subfamily G, member 2 (ABCG2), p63, cytokeratin 12, and connexin 43 were performed in cultivated HLECs and their expression levels were compared. The mRNA expression of all markers examined showed no statistically significant differences between the cells on cryo-preserved and on freeze-dried AM. The expression of p63 and cytokeratin 12 in cultivated cells on AMs were significantly lower than those in 3T3-cocultured cells on RT-PCR and immunofluorescent staining. Cultivated HLECs on AMs showed reduced proliferation and differentiation while maintaining stem-property regardless of the preservative method of AM.
Subject(s)
Animals , Humans , Mice , 3T3 Cells , Cell Culture Techniques/instrumentation , Cells, Cultured , Cytological Techniques , DNA Primers/chemistry , Epithelial Cells/metabolism , Immunohistochemistry/methods , Keratin-12/metabolism , Models, Biological , Phosphoproteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Stem Cells/cytology , Trans-Activators/metabolismABSTRACT
Endothelins (ETs), which were originally found to be potent vasoactive transmitters, were known to be implicated in nervous system, but the mode of mechanism remains unclear. ETs (ET-1, ET-2, and ET-3) were added to HN33 (mouse hippocampal neuron chi neuroblastoma) cells. Among the three types of ET, only ET-1 increased the intracellular calcium levels in a PLC dependent manner with the induction of ERK 1/2 activation. As the result of ET-1 exposure, the survival rate of HN33 cells and the PKCalpha translocation into the plasma membrane were increased. We suggest that ET-1 participated in the neuroprotective effect involving the calcium-PKCalpha-ERK1/2 pathway.
Subject(s)
Animals , Mice , Apoptosis/drug effects , Calcium/metabolism , Cell Line , Cell Survival/drug effects , Cytosol/drug effects , Endothelin-1/pharmacology , Endothelin-2/pharmacology , Endothelin-3/pharmacology , Estrenes/pharmacology , Extracellular Signal-Regulated MAP Kinases/metabolism , Immunoblotting , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Neurons/cytology , Neuroprotective Agents/pharmacology , Phosphoproteins/metabolism , Protein Kinase C-alpha/metabolism , Protein Transport/drug effects , Pyrrolidinones/pharmacology , SerumABSTRACT
Glycogen synthase kinase 3 (GSK3) was recently suggested to be a potential target of psychotropics used in psychiatric illnesses such as schizophrenia and bipolar disorder. Relevant studies have found that antipsychotic drugs regulate GSK3 activity via an increase in either inhibitory serine phosphorylation or amount of GSK3 after acute or subchronic treatment. Recent evidence shows that GSK3 is regulated by dopaminergic or serotonergic systems implicated in the pathophysiology and treatment mechanisms of schizophrenia and bipolar disorder. Therefore, antipsychotics may regulate GSK3 via antagonizing dopaminergic or serotonergic activity. However, the signaling pathway that is involved in GSK3 regulation by dopaminergic or serotonergic systems has not been well established. Haloperidol is a typical antipsychotic with potent dopamine D(2) receptor antagonism. Clozapine is an atypical antipsychotic with potent serotonin 5HT(2) receptor antagonism. We injected rats with haloperidol or clozapine and examined the phosphorylation and amount of GSK3alpha/beta and its well-known upstream regulators Akt and Dvl in the rat frontal cortex by Western blotting. Both haloperidol and clozapine induced Ser21/9 phosphorylation of GSK3GSK3alpha/beta. Haloperidol increased the Ser473 phosphorylation of Akt transiently, whereas clozapine maintained the increase for 1 h. Haloperidol did not affect the phosphorylation and amount of Dvl, whereas clozapine increased both phosphorylation and the amount of Dvl. Our results suggest that GSK3 activity may be regulated by both typical and atypical antipsychotics and that Akt or Dvl, depending on the D(2)- or 5HT(2)- receptor antagonism properties of typical and atypical antipsychotics, mediate the regulation differently.