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1.
Leukemia ; 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31462733

RESUMO

Studies have shown that mutant calreticulin (CALR) constitutively activates the thrombopoietin (TPO) receptor MPL and thus plays a causal role in the development of myeloproliferative neoplasms (MPNs). To further elucidate the molecular mechanism by which mutant CALR promotes MPN development, we studied the subcellular localization of mutant CALR and its importance for the oncogenic properties of mutant CALR. Here, mutant CALR accumulated in the Golgi apparatus, and its entrance into the secretion pathway and capacity to interact with N-glycan were required for its oncogenic capacity via the constitutive activation of MPL. Mutant CALR-dependent MPL activation was resistant to blockade of intracellular protein trafficking, suggesting that MPL is activated before reaching the cell surface. However, removal of MPL from the cell surface with trypsin shut down downstream activation, implying that the surface localization of MPL is required for mutant CALR-dependent activation. Furthermore, we found that mutant CALR and MPL interact on the cell surface. Based on these findings, we propose a model in which mutant CALR induces MPL activation on the cell surface to promote MPN development.

2.
Pediatr Surg Int ; 35(8): 911-914, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31203385

RESUMO

PURPOSE: The cadmium (Cd) chick model has been described as a reliable model of omphalocele. Skeletal anomalies, including lumber lordosis, can be seen in the Cd chick model, as well as in the human omphalocele. Bone deformations, such as lordosis, are associated with high bone mineral density (BMD). Recently, three-dimensional microcomputed tomography (3DMCT) has been used to investigate skeletal development in small animal embryos. We used 3DMCT to test the hypothesis that the BMD is increased in the Cd-induced omphalocele chick model. METHODS: After a 60-h incubation, chicks were exposed to either chick saline or Cd in ovo. Chick embryos were harvested at embryonic day 16.5 (E16.5) and were divided into control (n = 8) and Cd (n = 9). Chicks were then scanned by 3DMCT. The body volume, bone volume, bone/body volume ratio, bone mineral quantity and BMD were analysed statistically (significance was accepted at p < 0.05). RESULTS: Bone mineral density (mg/cm3) was significantly increased in the Cd group compared to control group (235.3 ± 11.7 vs 223.4 ± 4.6, p < 0.05), whereas there was no significant difference in the bone/body volume ratio between the Cd group and the control group (0.7 ± 0.1 vs 0.6 ± 0.0). The body volume (cm3) (0.3 ± 0.2 vs 0.3 ± 0.1), bone volume (cm3) (0.2 ± 0.2 vs 0.2 ± 0.1), and bone mineral quantity (mg) (51.3 ± 41.6 vs 41.5 ± 16.5) were not significantly different between the two groups. CONCLUSIONS: Increased BMD may be associated with lordosis of the vertebral column in the Cd-induced omphalocele chick model, stimulating osteogenesis by activating the canonical Wnt signalling pathway.

3.
J Neurosurg Spine ; : 1-4, 2019 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-31100724

RESUMO

OBJECTIVELumbar surgery via a lateral approach is a minimally invasive and highly useful procedure. However, care must be taken to avoid its potentially fatal complications of intestinal and vascular injuries. The object of this study was to evaluate the usefulness of intraoperative ultrasound in improving the safety of lateral lumbar spine surgery.METHODSA transvaginal ultrasound probe was inserted into the operative field, and the intestinal tract, kidney, psoas muscle, and vertebral body were identified using B-mode ultrasound. The aorta, vena cava, common iliac vessels, and lumbar arteries and their associated branches were identified using the color Doppler mode.RESULTSThe study cohort comprised 100 patients who underwent lateral lumbar spine surgery, 92 via a left-sided approach. The intestinal tract and kidney lateral to the psoas muscle on the anatomical approach pathway were visualized in 36 and 26 patients, respectively. A detachment maneuver displaced the intestinal tract and kidneys in an anteroinferior direction, enabling confirmation of the absence of organ tissues above the psoas. In all patients, the major vessels anterior to the vertebral bodies and the lumbar arteries and associated branches in the psoas on the approach path were clearly visualized in the Doppler mode, and their orientation, location, and positional relationship with regard to the vertebral bodies, intervertebral discs, and psoas were determined.CONCLUSIONSWhen approaching the lateral side of the lumbar spine in the retroperitoneal space, intraoperative ultrasound allows real-time identification of the blood vessels surrounding the lumbar spine, intestinal tract, and kidney in the approach path and improves the safety of surgery without increasing invasiveness.

4.
J Cell Sci ; 132(11)2019 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-31076512

RESUMO

Peroxisomes cooperate with mitochondria in the performance of cellular metabolic functions, such as fatty acid oxidation and the maintenance of redox homeostasis. However, whether peroxisomes also regulate mitochondrial fission-fusion dynamics or mitochondrion-dependent apoptosis remained unclear. We now show that genetic ablation of the peroxins Pex3 or Pex5, which are essential for peroxisome biogenesis, results in mitochondrial fragmentation in mouse embryonic fibroblasts (MEFs) in a manner dependent on Drp1 (also known as DNM1L). Conversely, treatment with 4-PBA, which results in peroxisome proliferation, resulted in mitochondrial elongation in wild-type MEFs, but not in Pex3-knockout MEFs. We further found that peroxisome deficiency increased the levels of cytosolic cytochrome c and caspase activity under basal conditions without inducing apoptosis. It also greatly enhanced etoposide-induced caspase activation and apoptosis, which is indicative of an enhanced cellular sensitivity to death signals. Taken together, our data unveil a previously unrecognized role for peroxisomes in the regulation of mitochondrial dynamics and mitochondrion-dependent apoptosis. Effects of peroxin gene mutations on mitochondrion-dependent apoptosis may contribute to pathogenesis of peroxisome biogenesis disorders.This article has an associated First Person interview with the first author of the paper.

5.
eNeuro ; 6(3)2019 May/Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31118204

RESUMO

The Golgi apparatus plays an indispensable role in posttranslational modification and transport of proteins to their target destinations. Although it is well established that the Golgi apparatus requires an acidic luminal pH for optimal activity, morphological and functional abnormalities at the neuronal circuit level because of perturbations in Golgi pH are not fully understood. In addition, morphological alteration of the Golgi apparatus is associated with several neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis. Here, we used anatomical and electrophysiological approaches to characterize morphological and functional abnormalities of neuronal circuits in Golgi pH regulator (GPHR) conditional knock-out mice. Purkinje cells (PCs) from the mutant mice exhibited vesiculation and fragmentation of the Golgi apparatus, followed by axonal degeneration and progressive cell loss. Morphological analysis provided evidence for the disruption of basket cell (BC) terminals around PC soma, and electrophysiological recordings showed selective loss of large amplitude responses, suggesting BC terminal disassembly. In addition, the innervation of mutant PCs was altered such that climbing fiber (CF) terminals abnormally synapsed on the somatic spines of mutant PCs in the mature cerebellum. The combined results describe an essential role for luminal acidification of the Golgi apparatus in maintaining proper neuronal morphology and neuronal circuitry.

6.
iScience ; 15: 536-551, 2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31132747

RESUMO

A short form of cellular FLICE-inhibitory protein encoded by CFLARs promotes necroptosis. Although necroptosis is involved in various pathological conditions, the detailed mechanisms are not fully understood. Here we generated transgenic mice wherein CFLARs was integrated onto the X chromosome. All male CFLARs Tg mice died perinatally due to severe ileitis. Although necroptosis was observed in various tissues of CFLARs Tg mice, large numbers of intestinal epithelial cells (IECs) died by apoptosis. Deletion of Ripk3 or Mlkl, essential genes of necroptosis, prevented both necroptosis and apoptosis, and rescued lethality of CFLARs Tg mice. Type 3 innate lymphoid cells (ILC3s) were activated and recruited to the small intestine along with upregulation of interleukin-22 (Il22) in CFLARs Tg mice. Deletion of ILC3s or Il22 rescued lethality of CFLARs Tg mice by preventing apoptosis, but not necroptosis of IECs. Together, necroptosis-dependent activation of ILC3s induces lethal ileitis in an IL-22-dependent manner.

7.
Life Sci ; 221: 293-300, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30797017

RESUMO

AIM: Cathepsin L (Ctsl) plays a pivotal role in lysosomal and autophagic proteolysis. Previous investigations revealed that partial hepatectomy (PH) decreases biosynthesis of cathepsins in liver, followed by suppression of lysosomal and autophagic proteolysis during liver regeneration. Conversely, it was reported that autophagy-deficiency suppressed liver regeneration. Thus, the purpose of this study is to determine if Ctsl deficiency affects liver regeneration after PH. METHODS: 70% of PH was performed in male Ctsl-deficient mice (Ctsl-/-) and wild-type littermates (Ctsl +/+) after PH. Mice were sacrificed and wet weight of the whole remaining liver was measured. Bromodeoxyuridine (BrdU)-immunostaining of liver sections was performed. Expression of cyclin D1, p62, LC-3, Nrf2, cleaved-Notch1, Hes1 was evaluated by western blot analysis. NQO1 mRNA expression was measured by realtime-PCR. RESULTS: After a 70% of PH, the liver mass was significantly restored within 5 days in Ctsl-/- mice compared to wild-type. Ctsl-deficiency enhanced the increases in both the rate of BrdU-positive cells and cyclin D1 expression after PH more than wild-type mice. On the other hand, Ctsl-deficiency upregulated p62, cleaved-Notch1 and Hes1 expression after PH. Moreover, the protein level of Nrf2 in the nucleus and mRNA expression of NQO1 in the liver after PH was also up-regulated in Ctsl-/- mice. CONCLUSIONS: These findings suggest that accumulation of p62 due to loss of Ctsl plays an important role in liver regeneration through activation of Nrf2-Notch1 signaling. Taken together, Ctsl might be a new therapeutic target on disorder of liver regeneration.


Assuntos
Catepsina L/deficiência , Regeneração Hepática/fisiologia , Animais , Autofagia , Catepsina L/metabolismo , Catepsinas , Células Cultivadas , Hepatectomia , Fígado , Lisossomos , Masculino , Camundongos , Camundongos Knockout , Fator 2 Relacionado a NF-E2 , Proteólise , Receptores Notch , Transdução de Sinais , Fatores de Transcrição
8.
Hum Mol Genet ; 28(11): 1894-1904, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30689867

RESUMO

Parkinson's disease (PD) is characterized by dopaminergic (DA) cell loss and the accumulation of pathological alpha synuclein (asyn), but its precise pathomechanism remains unclear, and no appropriate animal model has yet been established. Recent studies have shown that a heterozygous mutation of glucocerebrosidase (gba) is one of the most important genetic risk factors in PD. To create mouse model for PD, we crossed asyn Bacterial Artificial Chromosome transgenic mice with gba heterozygous knockout mice. These double-mutant (dm) mice express human asyn in a physiological manner through its native promoter and showed an increase in phosphorylated asyn in the regions vulnerable to PD, such as the olfactory bulb and dorsal motor nucleus of the vagus nerve. Only dm mice showed a significant reduction in DA cells in the substantia nigra pars compacta, suggesting these animals were suitable for a prodromal model of PD. Next, we investigated the in vivo mechanism by which GBA insufficiency accelerates PD pathology, focusing on lipid metabolism. Dm mice showed an increased level of glucosylsphingosine without any noticeable accumulation of glucosylceramide, a direct substrate of GBA. In addition, the overexpression of asyn resulted in decreased GBA activity in mice, while dm mice tended to show an even further decreased level of GBA activity. In conclusion, we created a novel prodromal mouse model to study the disease pathogenesis and develop novel therapeutics for PD and also revealed the mechanism by which heterozygous gba deficiency contributes to PD through abnormal lipid metabolism under conditions of an altered asyn expression in vivo.

9.
PLoS One ; 13(9): e0203944, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30222787

RESUMO

Recent studies have shown that superoxide dismutase 1 (SOD1), SOD2, and SOD3 are significantly decreased in human osteoarthritic cartilage. SOD activity is a marker that can be used to comprehensively evaluate the enzymatic capacities of SOD1, SOD2, and SOD3; however, the trend of SOD activity in end-stage osteoarthritic tissues remains unknown. In the present study, we found that SOD activity in end-stage osteoarthritic synovium of the knee was significantly lower than that in control synovium without the influence of age. The SOD activity was significantly lower in the end-stage knee osteoarthritic cartilage than in the control, but a weak negative correlation was observed between aging and SOD activity. However, SOD activity in end-stage hip osteoarthritic cartilage was significantly lower than that in control cartilage without the influence of aging. The relationship between osteoarthritis and SOD activity was stronger than the relationship between aging and SOD activity. These results indicate that direct regulation of SOD activity in joint tissues may lead to suppression of osteoarthritis progression.

10.
Proc Natl Acad Sci U S A ; 115(39): E9115-E9124, 2018 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-30209220

RESUMO

Leucine-rich repeat kinase 2 (LRRK2) has been associated with a variety of human diseases, including Parkinson's disease and Crohn's disease, whereas LRRK2 deficiency leads to accumulation of abnormal lysosomes in aged animals. However, the cellular roles and mechanisms of LRRK2-mediated lysosomal regulation have remained elusive. Here, we reveal a mechanism of stress-induced lysosomal response by LRRK2 and its target Rab GTPases. Lysosomal overload stress induced the recruitment of endogenous LRRK2 onto lysosomal membranes and activated LRRK2. An upstream adaptor Rab7L1 (Rab29) promoted the lysosomal recruitment of LRRK2. Subsequent family-wide screening of Rab GTPases that may act downstream of LRRK2 translocation revealed that Rab8a and Rab10 were specifically accumulated on overloaded lysosomes dependent on their phosphorylation by LRRK2. Rab7L1-mediated lysosomal targeting of LRRK2 attenuated the stress-induced lysosomal enlargement and promoted lysosomal secretion, whereas Rab8 stabilized by LRRK2 on stressed lysosomes suppressed lysosomal enlargement and Rab10 promoted lysosomal secretion, respectively. These effects were mediated by the recruitment of Rab8/10 effectors EHBP1 and EHBP1L1. LRRK2 deficiency augmented the chloroquine-induced lysosomal vacuolation of renal tubules in vivo. These results implicate the stress-responsive machinery composed of Rab7L1, LRRK2, phosphorylated Rab8/10, and their downstream effectors in the maintenance of lysosomal homeostasis.


Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Lisossomos/enzimologia , Estresse Fisiológico , Proteínas rab de Ligação ao GTP/metabolismo , Células 3T3 , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células HEK293 , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Lisossomos/genética , Camundongos , Camundongos Knockout , Fosforilação , Células RAW 264.7 , Proteínas rab de Ligação ao GTP/genética
12.
Autophagy ; 14(11): 1991-2006, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30025494

RESUMO

Macroautophagy/autophagy is considered as an evolutionarily conserved cellular catabolic process. In this study, we aimed to elucidate the role of autophagy in vascular smooth muscle cells (SMCs) on atherosclerosis. SMCs cultured from mice with SMC-specific deletion of the essential autophagy gene atg7 (Atg7cKO) showed reduced serum-induced cell growth, increased cell death, and decreased cell proliferation rate. Furthermore, 7-ketocholestrerol enhanced apoptosis and the expression of CCL2 (chemokine [C-C motif] ligand 2) with the activation of TRP53, the mouse ortholog of human and rat TP53, in SMCs from Atg7cKO mice. In addition, Atg7cKO mice crossed with Apoe (apolipoprotein E)-deficient mice (apoeKO; Atg7cKO:apoeKO) showed reduced medial cellularity and increased TUNEL-positive cells in the descending aorta at 10 weeks of age. Intriguingly, Atg7cKO: apoeKO mice fed a Western diet containing 1.25% cholesterol for 14 weeks showed a reduced survival rate. Autopsy of the mice demonstrated the presence of aortic rupture. Analysis of the descending aorta in Atg7cKO:apoeKO mice showed increased plaque area, increased TUNEL-positive area, decreased SMC-positive area, accumulation of macrophages in the media, and adventitia and perivascular tissue, increased CCL2 expression in SMCs in the vascular wall, medial disruption, and aneurysm formation. In conclusion, our data suggest that defective autophagy in SMCs enhances atherosclerotic changes with outward arterial remodeling.

13.
Sci Rep ; 8(1): 7229, 2018 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-29739985

RESUMO

Apples are well known to have various benefits for the human body. Procyanidins are a class of polyphenols found in apples that have demonstrated effects on the circulatory system and skeletal organs. Osteoarthritis (OA) is a locomotive syndrome that is histologically characterized by cartilage degeneration associated with the impairment of proteoglycan homeostasis in chondrocytes. However, no useful therapy for cartilage degeneration has been developed to date. In the present study, we detected beneficial effects of apple polyphenols or their procyanidins on cartilage homeostasis. An in vitro assay revealed that apple polyphenols increased the activities of mitochondrial dehydrogenases associated with an increased copy number of mitochondrial DNA as well as the gene expression of peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α), suggesting the promotion of PGC-1α-mediated mitochondrial biogenesis. Apple  procyanidins also enhanced proteoglycan biosynthesis with aggrecan upregulation in primary chondrocytes. Of note, oral treatment with apple procyanidins prevented articular cartilage degradation in OA model mice induced by mitochondrial dysfunction in chondrocytes. Our findings suggest that apple procyanidins are promising food components that inhibit OA progression by promoting mitochondrial biogenesis and proteoglycan homeostasis in chondrocytes.

14.
PLoS One ; 13(5): e0198039, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29795667

RESUMO

Loss of integrity of the blood-brain barrier (BBB) in ischemic stroke victims initiates a devastating cascade of events causing brain damage. Maintaining the BBB is important to preserve brain function in ischemic stroke. Unfortunately, recombinant tissue plasminogen activator (tPA), the only effective fibrinolytic treatment at the acute stage of ischemic stroke, also injures the BBB and increases the risk of brain edema and secondary hemorrhagic transformation. Thus, it is important to identify compounds that maintain BBB integrity in the face of ischemic injury in patients with stroke. We previously demonstrated that intravenously injected phosphorylated recombinant heat shock protein 27 (prHSP27) protects the brains of mice with transient middle cerebral artery occlusion (tMCAO), an animal stroke-model. Here, we determined whether prHSP27, in addition to attenuating brain injury, also decreases BBB damage in hyperglycemic tMCAO mice that had received tPA. After induction of hyperglycemia and tMCAO, we examined 4 treatment groups: 1) bovine serum albumin (BSA), 2) prHSP27, 3) tPA, 4) tPA plus prHSP27. We examined the effects of prHSP27 by comparing the BSA and prHSP27 groups and the tPA and tPA plus prHSP27 groups. Twenty-four hours after injection, prHSP27 reduced infarct volume, brain swelling, neurological deficits, the loss of microvessel proteins and endothelial cell walls, and mortality. It also reduced the rates of hemorrhagic transformation, extravasation of endogenous IgG, and MMP-9 activity, signs of BBB damage. Therefore, prHSP27 injection attenuated brain damage and preserved the BBB in tPA-injected, hyperglycemic tMCAO experimental stroke-model mice, in which the BBB is even more severely damaged than in simple tMCAO mice. The attenuation of brain damage and BBB disruption in the presence of tPA suggests the effectiveness of prHSP27 and tPA as a combination therapy. prHSP27 may be a novel therapeutic agent for ischemic stroke patients whose BBBs are injured following tPA injections.

15.
Cell Death Differ ; 25(12): 2130-2146, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29568058

RESUMO

There is compelling evidence that glial-immune interactions contribute to the progression of neurodegenerative diseases. The adaptive immune response has been implicated in disease processes of amyotrophic lateral sclerosis (ALS), but it remains unknown if innate immune signaling also contributes to ALS progression. Here we report that deficiency of the innate immune adaptor TIR domain-containing adaptor inducing interferon-ß (TRIF), which is essential for certain Toll-like receptor (TLR) signaling cascades, significantly shortens survival time and accelerates disease progression of ALS mice. While myeloid differentiation factor 88 (MyD88) is also a crucial adaptor for most TLR signaling pathways, MyD88 deficiency had only a marginal impact on disease course. Moreover, TRIF deficiency reduced the number of natural killer (NK), NK-T-lymphocytes, and CD8-T cells infiltrating into the spinal cord of ALS mice, but experimental modulation of these populations did not substantially influence survival time. Instead, we found that aberrantly activated astrocytes expressing Mac2, p62, and apoptotic markers were accumulated in the lesions of TRIF-deficient ALS mice, and that the number of aberrantly activated astrocytes was negatively correlated with survival time. These findings suggest that TRIF pathway plays an important role in protecting a microenvironment surrounding motor neurons by eliminating aberrantly activated astrocytes.

16.
Cell Microbiol ; 20(8): e12846, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29582580

RESUMO

Streptococcus pneumoniae is the most common causative agent of community-acquired pneumonia and can penetrate epithelial barriers to enter the bloodstream and brain. We investigated intracellular fates of S. pneumoniae and found that the pathogen is entrapped by selective autophagy in pneumolysin- and ubiquitin-p62-LC3 cargo-dependent manners. Importantly, following induction of autophagy, Rab41 was relocated from the Golgi apparatus to S. pneumoniae-containing autophagic vesicles (PcAV), which were only formed in the presence of Rab41-positive intact Golgi apparatuses. Moreover, subsequent localization and regulation of K48- and K63-linked polyubiquitin chains in and on PcAV were clearly distinguishable from each other. Finally, we found that E3 ligase Nedd4-1 was recruited to PcAV and played a pivotal role in K63-linked polyubiquitin chain (K63Ub) generation on PcAV, promotion of PcAV formation, and elimination of intracellular S. pneumoniae. These findings suggest that Nedd4-1-mediated K63Ub deposition on PcAV acts as a scaffold for PcAV biogenesis and efficient elimination of host cell-invaded pneumococci.

17.
Artigo em Inglês | MEDLINE | ID: mdl-29596938

RESUMO

BACKGROUND: A delicate balance between cell death and keratinocyte proliferation is crucial for normal skin development. Previous studies have reported that cellular FLICE (FADD-like ICE)-inhibitory protein plays a crucial role in prevention of keratinocytes from TNF-α-dependent apoptosis and blocking of dermatitis. However, a role for cellular FLICE-inhibitory protein in TNF-α-independent cell death remains unclear. OBJECTIVE: We investigated contribution of TNF-α-dependent and TNF-α-independent signals to the development of dermatitis in epidermis-specific Cflar-deficient (CflarE-KO) mice. METHODS: We examined the histology and expression of epidermal differentiation markers and inflammatory cytokines in the skin of CflarE-KO;Tnfrsf1a+/- and CflarE-KO;Tnfrsf1a-/- mice. Mice were treated with neutralizing antibodies against Fas ligand and TNF-related apoptosis-inducing ligand to block TNF-α-independent cell death of CflarE-KO;Tnfrsf1a-/- mice. RESULTS: CflarE-KO;Tnfrsf1a-/- mice were born but experienced severe dermatitis and succumbed soon after birth. CflarE-KO;Tnfrsf1a+/- mice exhibited embryonic lethality caused by massive keratinocyte apoptosis. Although keratinocytes from CflarE-KO;Tnfrsf1a-/- mice still died of apoptosis, neutralizing antibodies against Fas ligand and TNF-related apoptosis-inducing ligand substantially prolonged survival of CflarE-KO;Tnfrsf1a-/- mice. Expression of inflammatory cytokines, such as Il6 and Il17a was increased; conversely, expression of epidermal differentiation markers was severely downregulated in the skin of CflarE-KO;Tnfrsf1a-/- mice. Treatment of primary keratinocytes with IL-6 and, to a lesser extent, IL-17A suppressed expression of epidermal differentiation markers. CONCLUSION: TNF receptor superfamily 1 (TNFR1)-dependent or TNFR1-independent apoptosis of keratinocytes promotes inflammatory cytokine production, which subsequently blocks epidermal differentiation. Thus blockade of both TNFR1-dependent and TNFR1-independent cell death might be an alternative strategy to treat skin diseases when treatment with anti-TNF-α antibody alone is not sufficient.

18.
PLoS One ; 13(1): e0191108, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29342181

RESUMO

Green fluorescent protein (GFP) is tremendously useful for investigating many cellular and intracellular events. The monomeric GFP mNeonGreen is about 3- to 5-times brighter than GFP and monomeric enhanced GFP and shows high photostability. The maturation half-time of mNeonGreen is about 3-fold faster than that of monomeric enhanced GFP. However, the cDNA sequence encoding mNeonGreen contains some codons that are rarely used in Homo sapiens. For better expression of mNeonGreen in human cells, we synthesized a human-optimized cDNA encoding mNeonGreen and generated an expression plasmid for humanized mNeonGreen under the control of the cytomegalovirus promoter. The resultant plasmid was introduced into HEK293 cells. The fluorescent intensity of humanized mNeonGreen was about 1.4-fold higher than that of the original mNeonGreen. The humanized mNeonGreen with a mitochondria-targeting signal showed mitochondrial distribution of mNeonGreen. We further generated an expression vector of humanized mNeonGreen with 3xFLAG tags at its carboxyl terminus as these tags are useful for immunological analyses. The 3xFLAG-tagged mNeonGreen was recognized well with an anti-FLAG-M2 antibody. These plasmids for the expression of humanized mNeonGreen and mNeonGreen-3xFLAG are useful tools for biological studies in mammalian cells using mNeonGreen.


Assuntos
Corantes/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Animais , Células COS , Cercopithecus aethiops , Códon , DNA/biossíntese , Células HEK293 , Humanos , Mitocôndrias/metabolismo , Plasmídeos
19.
Heliyon ; 3(11): e00462, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29264419

RESUMO

The genus Cordyceps and its specific ingredient, cordycepin, have attracted much attention for multiple health benefits and expectations for lifespan extension. We analyzed whether Cordyceps militaris (CM), which contains large amounts of cordycepin, can extend the survival of Dahl salt-sensitive rats, whose survival was reduced to ∼3 months via a high-salt diet. The survival of these life-shortened rats was extended significantly when supplemented with CM, possibly due to a minimization of the effects of stroke. Next, we analyzed the effect of CM on hypertension-sensitive organs, the central nervous systems (CNS), heart, kidney and liver of these rats. We attempted to ascertain how the organs were improved by CM, and we paid particular attention to mitochondria and autophagy functions. The following results were from CM-treated rats in comparison with control rats. Microscopically, CNS neurons, cardiomyocytes, glomerular podocytes, renal epithelial cells, and hepatocytes all were improved. However, immunoblot and immunohistochemical analysis showed that the expressions of mitochondria-related proteins, ATP synthase ß subunit, SIRT3 and SOD2, and autophagy-related proteins, LC3-II/LC3-I ratio and cathepsin D all were reduced significantly in the CNS neurons, but increased significantly in the cells of the other three organs, although p62 was decreased in its expression in all the organs tested. Activity of Akt and mTOR was enhanced but that of AMPK was reduced in the CNS, while such kinase activity was completely the opposite in the other organs. Together, the influence of CM may differ between mitochondria and autophagy functioned between the two organ groups, as mitochondria and autophagy seemed to be repressed and promoted, respectively, in the CNS, while both mitochondria and autophagy were activated in the others. This could possibly be related to the steady or improved cellular activity in both the organs, which might result in the life extension of these rats.

20.
Sci Rep ; 7(1): 14972, 2017 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-29097807

RESUMO

TDP-43 is an RNA-binding protein important for many aspects of RNA metabolism. Abnormal accumulation of TDP-43 in the cytoplasm of affected neurons is a pathological hallmark of the neurodegenerative diseases frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Several transgenic mouse models have been generated that recapitulate defects in TDP-43 accumulation, thus causing neurodegeneration and behavioural impairments. While aging is the key risk factor for neurodegenerative diseases, the specific effect of aging on phenotypes in TDP-43 transgenic mice has not been investigated. Here, we analyse age-dependent changes in TDP-43 transgenic mice that displayed impaired memory. We found the accumulation of abundant poly-ubiquitinated protein aggregates in the hippocampus of aged TDP-43 transgenic mice. Intriguingly, the aggregates contained some interneuron-specific proteins such as parvalbumin and calretinin, suggesting that GABAergic interneurons were degenerated in these mice. The abundance of aggregates significantly increased with age and with the overexpression of TDP-43. Gene array analyses in the hippocampus and other brain areas revealed dysregulation in genes linked to oxidative stress and neuronal function in TDP-43 transgenic mice. Our results indicate that the interneuron degeneration occurs upon aging, and TDP-43 accelerates age-dependent neuronal degeneration, which may be related to the impaired memory of TDP-43 transgenic mice.

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