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1.
Nat Immunol ; 25(2): 226-239, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38191855

RESUMO

Sepsis is a systemic response to infection with life-threatening consequences. Our understanding of the molecular and cellular impact of sepsis across organs remains rudimentary. Here, we characterize the pathogenesis of sepsis by measuring dynamic changes in gene expression across organs. To pinpoint molecules controlling organ states in sepsis, we compare the effects of sepsis on organ gene expression to those of 6 singles and 15 pairs of recombinant cytokines. Strikingly, we find that the pairwise effects of tumor necrosis factor plus interleukin (IL)-18, interferon-gamma or IL-1ß suffice to mirror the impact of sepsis across tissues. Mechanistically, we map the cellular effects of sepsis and cytokines by computing changes in the abundance of 195 cell types across 9 organs, which we validate by whole-mouse spatial profiling. Our work decodes the cytokine cacophony in sepsis into a pairwise cytokine message capturing the gene, cell and tissue responses of the host to the disease.


Assuntos
Citocinas , Sepse , Camundongos , Animais , Interleucina-6/genética , Fator de Necrose Tumoral alfa/metabolismo , Interferon gama , Sepse/genética
2.
Cell ; 148(3): 447-57, 2012 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-22304915

RESUMO

Although it is believed that neural activation can affect immune responses, very little is known about the neuroimmune interactions involved, especially the regulators of immune traffic across the blood-brain barrier which occurs in neuroimmune diseases such as multiple sclerosis (MS). Using a mouse model of MS, experimental autoimmune encephalomyelitis, we show that autoreactive T cells access the central nervous system via the fifth lumbar spinal cord. This location is defined by IL-6 amplifier-dependent upregulation of the chemokine CCL20 in associated dorsal blood vessels, which in turn depends on gravity-induced activation of sensory neurons by the soleus muscle in the leg. Impairing soleus muscle contraction by tail suspension is sufficient to reduce localized chemokine expression and block entry of pathogenic T cells at the fifth lumbar cord, suggesting that regional neuroimmune interactions may offer therapeutic targets for a variety of neurological diseases.


Assuntos
Barreira Hematoencefálica , Linfócitos T CD4-Positivos/citologia , Encefalomielite Autoimune Experimental/imunologia , Animais , Movimento Celular , Quimiocina CCL20/imunologia , Encefalomielite Autoimune Experimental/patologia , Gravitação , Interleucina-6/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/imunologia , Músculo Esquelético/inervação , Neuroimunomodulação , Medula Espinal/irrigação sanguínea
3.
J Immunol ; 211(1): 34-42, 2023 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-37212607

RESUMO

We recently discovered a (to our knowledge) new neuroimmune interaction named the gateway reflex, in which the activation of specific neural circuits establishes immune cell gateways at specific vessel sites in organs, leading to the development of tissue-specific autoimmune diseases, including a multiple sclerosis (MS) mouse model, experimental autoimmune encephalomyelitis (EAE). We have reported that peripheral-derived myeloid cells, which are CD11b+MHC class II+ and accumulate in the fifth lumbar (L5) cord during the onset of a transfer model of EAE (tEAE), play a role in the pain-mediated relapse via the pain-gateway reflex. In this study, we investigated how these cells survive during the remission phase to cause the relapse. We show that peripheral-derived myeloid cells accumulated in the L5 cord after tEAE induction and survive more than other immune cells. These myeloid cells, which highly expressed GM-CSFRα with common ß chain molecules, grew in number and expressed more Bcl-xL after GM-CSF treatment but decreased in number by blockade of the GM-CSF pathway, which suppressed pain-mediated relapse of neuroinflammation. Therefore, GM-CSF is a survival factor for these cells. Moreover, these cells were colocalized with blood endothelial cells (BECs) around the L5 cord, and BECs expressed a high level of GM-CSF. Thus, GM-CSF from BECs may have an important role in the pain-mediated tEAE relapse caused by peripheral-derived myeloid cells in the CNS. Finally, we found that blockade of the GM-CSF pathway after pain induction suppressed EAE development. Therefore, GM-CSF suppression is a possible therapeutic approach in inflammatory CNS diseases with relapse, such as MS.


Assuntos
Encefalomielite Autoimune Experimental , Esclerose Múltipla , Camundongos , Animais , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Doenças Neuroinflamatórias , Células Endoteliais/metabolismo , Sistema Nervoso Central , Dor/metabolismo , Células Mieloides , Recidiva
4.
Proc Natl Acad Sci U S A ; 116(15): 7425-7430, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30910977

RESUMO

Serum IgG, which is mainly generated from IgG-secreting plasma cells in the bone marrow (BM), protects our body against various pathogens. We show here that the protein SiiE of Salmonella is both required and sufficient to prevent an efficient humoral immune memory against the pathogen by selectively reducing the number of IgG-secreting plasma cells in the BM. Attenuated SiiE-deficient Salmonella induces high and lasting titers of specific and protective Salmonella-specific IgG and qualifies as an efficient vaccine against Salmonella A SiiE-derived peptide with homology to laminin ß1 is sufficient to ablate IgG-secreting plasma cells from the BM, identifying laminin ß1 as a component of niches for IgG-secreting plasma cells in the BM, and furthermore, qualifies it as a unique therapeutic option to selectively ablate IgG-secreting plasma cells in autoimmune diseases and multiple myeloma.


Assuntos
Células da Medula Óssea/imunologia , Imunidade Humoral , Imunoglobulina G/imunologia , Memória Imunológica , Plasmócitos/imunologia , Salmonella/imunologia , Animais , Células da Medula Óssea/citologia , Imunoglobulina G/genética , Laminina/genética , Laminina/imunologia , Camundongos , Camundongos Knockout , Plasmócitos/citologia , Salmonella/genética
5.
Plant Cell Physiol ; 60(3): 503-515, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30690508

RESUMO

Grain size is a key determiner of grain weight, one of the yield components in rice (Oryza sativa). Therefore, to increase grain yield, it is important to elucidate the detailed mechanisms regulating grain size. The Large grain (Lgg) mutant, found in the nonautonomous DNA-based active rice transposon1 (nDart1)-tagged lines of Koshihikari, is caused by a truncated nDart1-3 and 355 bp deletion in the 5' untranslated region of LGG, which encodes a putative RNA-binding protein, through transposon display and cosegregation analysis between grain length and LGG genotype in F2 and F3. Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9-mediated knockout and overexpression of LGG led to longer and shorter grains than wild type, respectively, showing that LGG regulates spikelet hull length. Expression of LGG was highest in the 0.6-mm-long young panicle and gradually decreased as the panicle elongated. LGG was also expressed in roots and leaves. These results show that LGG functions at the very early stage of panicle development. Longitudinal cell numbers of spikelet hulls of Lgg, knockout and overexpressed plants were significantly different from those of the wild type, suggesting that LGG might regulate longitudinal cell proliferation in the spikelet hull. RNA-Seq analysis of 1-mm-long young panicles from LGG knockout and overexpressing plants revealed that the expressions of many cell cycle-related genes were reduced in knockout plants relative to LGG-overexpressing plants and wild type, whereas some genes for cell proliferation were highly expressed in knockout plants. Taken together, these results suggest that LGG might be a regulator of cell cycle and cell division in the rice spikelet hull.


Assuntos
Oryza/metabolismo , Proteínas de Plantas/metabolismo , Elementos de DNA Transponíveis/genética , Grão Comestível/genética , Grão Comestível/metabolismo , Regulação da Expressão Gênica de Plantas , Oryza/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
6.
Anal Chem ; 91(14): 8979-8986, 2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31251568

RESUMO

The matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) technique is a promising approach for detecting the distribution of small molecules in a section of biological tissue. However, when a cryosection is created from fragile, hard, or whole-body samples, obtaining a high-quality section that maintains the distribution of the various components has been difficult. Since adhesive films have the potential to obtain high-quality cryosections, we attempted to utilize a conductive adhesive film for MALDI-MSI. To this end, cryosections of the whole body of a 9-day-old mouse were directly prepared on indium tin oxide (ITO) glass slides, nonconductive adhesive films, or conductive adhesive films, and the signal intensities from each section were measured by MALDI-MSI. We measured the differences in the ion intensity among these three slides/films by means of multivariate analyses and found that both the nonconductive and conductive adhesive films gave rise to high-quality sections in comparison with the ITO glass slide. The conductive adhesive film gave higher signals that were comparable to those of the ITO glass slide in comparison with the nonconductive adhesive film. We divided the frozen sections into two groups, a freeze-dried group and a thawed group, to examine the freeze-thaw effect on the signals of representative compounds of amino acids, cholesterol, and phosphatidylcholines. The freeze-dried samples were found to be useful for the analysis. These results indicate that the sections made with the conductive adhesive film under a freeze-dried condition can expand the utility of the MALDI-MSI analysis.

7.
Development ; 142(4): 787-96, 2015 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-25670797

RESUMO

The periodontal ligament (PDL) is a mechanosensitive noncalcified fibrous tissue connecting the cementum of the tooth and the alveolar bone. Here, we report that scleraxis (Scx) and osterix (Osx) antagonistically regulate tensile force-responsive PDL fibrogenesis and osteogenesis. In the developing PDL, Scx was induced during tooth eruption and co-expressed with Osx. Scx was highly expressed in elongated fibroblastic cells aligned along collagen fibers, whereas Osx was highly expressed in the perialveolar/apical osteogenic cells. In an experimental model of tooth movement, Scx and Osx expression was significantly upregulated in parallel with the activation of bone morphogenetic protein (BMP) signaling on the tension side, in which bone formation compensates for the widened PDL space away from the bone under tensile force by tooth movement. Scx was strongly expressed in Scx(+)/Osx(+) and Scx(+)/Osx(-) fibroblastic cells of the PDL that does not calcify; however, Scx(-)/Osx(+) osteogenic cells were dominant in the perialveolar osteogenic region. Upon BMP6-driven osteoinduction, osteocalcin, a marker for bone formation was downregulated and upregulated by Scx overexpression and knockdown of endogenous Scx in PDL cells, respectively. In addition, mineralization by osteoinduction was significantly inhibited by Scx overexpression in PDL cells without affecting Osx upregulation, suggesting that Scx counteracts the osteogenic activity regulated by Osx in the PDL. Thus, Scx(+)/Osx(-), Scx(+)/Osx(+) and Scx(-)/Osx(+) cell populations participate in the regulation of tensile force-induced remodeling of periodontal tissues in a position-specific manner.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ligamento Periodontal/metabolismo , Resistência à Tração/fisiologia , Fatores de Transcrição/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Células Cultivadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Interferência de RNA , Ratos , Ratos Wistar , Fator de Transcrição Sp7 , Fatores de Transcrição/genética
8.
Breed Sci ; 68(3): 381-384, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30100806

RESUMO

To obtain a clear intact section of a ripened rice grain, which is suitable for biochemical and histological analysis, the Kawamoto method using a specific adhesive film was applied using a cryomicrotome. The longitudinal and sagittal sections were easily obtained together with the cross-section, and cell characteristics were clearly discerned in the ripened grain. It was demonstrated that the Kawamoto method is readily applicable for intact sectioning of hard tissue, including ripened grain. Intact section sampling may be useful for enzymatic analysis and transcriptomic analysis of plant tissue.

9.
Mass Spectrom (Tokyo) ; 12(1): A0137, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38107656

RESUMO

The matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) technique was used to obtain the molecular images of cryosections without labeling. Although MALDI-MSI has been widely used to detect small molecules from biological tissues, issues remain due to the technical process of cryosectioning and limited mass spectrometry parameters. The use of a conductive adhesive film is a unique method to obtain high-quality sections from cutting tissue, such as bone, muscle, adipose tissue, and whole body of mice or fish, and we have reported the utilization of the film for MALDI-MSI in previous. However, some signal of the small molecules using the conductive adhesive films was still lower than on the indium tin oxide (ITO) glass slide. Here, the sample preparation and analytical conditions for MALDI-MSI using an advanced conductive adhesive film were optimized to obtain strong signals from whole mice heads. The effects of tissue thickness and laser ionization power on signal intensity were verified using MALDI-MSI. The phospholipid signal intensity was measured for samples with three tissue thicknesses (5, 10, and 20 µm); compared to the signals from the samples on the ITO glass slides, the signals with conductive adhesive films exhibited significantly higher intensities when a laser with a higher range of power was used to ionize the small molecules. Thus, the technique using the advanced conductive adhesive film showed an improvement in MALDI-MSI analysis.

10.
J Exp Med ; 219(6)2022 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-35579694

RESUMO

Neural circuits between lesions are one mechanism through which local inflammation spreads to remote positions. Here, we show the inflammatory signal on one side of the joint is spread to the other side via sensory neuron-interneuron crosstalk, with ATP at the core. Surgical ablation or pharmacological inhibition of this neural pathway prevented inflammation development on the other side. Mechanistic analysis showed that ATP serves as both a neurotransmitter and an inflammation enhancer, thus acting as an intermediary between the local inflammation and neural pathway that induces inflammation on the other side. These results suggest blockade of this neural pathway, which is named the remote inflammation gateway reflex, may have therapeutic value for inflammatory diseases, particularly those, such as rheumatoid arthritis, in which inflammation spreads to remote positions.


Assuntos
Interneurônios , Células Receptoras Sensoriais , Trifosfato de Adenosina , Humanos , Inflamação , Reflexo/fisiologia
11.
Methods Mol Biol ; 2230: 259-281, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33197019

RESUMO

A method for preparing frozen sections with an adhesive film is described. In order to observe fine structures and weak fluorescence of samples, new types of adhesive films [Cryofilm type 3C(16UF) and 4D(16UF)] are used. The adhesive film is made with very clear and very low autofluorescence. For gene analysis, a very thin adhesive film (LMD film) is used to cut by means of the laser microdissection (LMD). For MALDI mass spectrometry imaging (MALDI-MSI), a conductive adhesive film (Cryofilm type MS) is used to avoid electric charge of the sample. A biological sample is frozen quickly and freeze-embedded. The frozen sample is cut with a very sharp disposable blade made from fine tungsten carbide. The combination of the adhesive films and the blade can generate 3 micrometer thick sections from samples including bone, while it is also possible to generate 1 µm thick sections. The morphology of bone and soft tissues are preserved using this method. Cells such as osteoblasts, fibroblasts, and osteoclasts are clearly observed with an oil immersion lens at high magnification. Sections generated using the Cryofilm type 3C(16UF) shows weak fluorescent signals more clearly than sections generated with the previously reported adhesive films [Cryofilm type 2C(9) and 2C(10)]. Furthermore fluorescence of the fine structures in cells is clearly shown using a super-high-resolution microscope. Several staining and experimental methods such as histology, histochemistry, enzyme histochemistry, immunohistochemistry, and in situ hybridization can be performed on these sections. This method is also useful for preparing frozen sections of large sample such as a whole-body mouse and rat. In gene analysis, gene quality of sample collected from the section made with the LMD film is superior to that of sample made by a conventional method. The Cryofilm type MS makes almost complete section from tissues including hard tissues and large samples. The satisfactory signals are detected from the section with MALDI-MSI.


Assuntos
Osso e Ossos/ultraestrutura , Secções Congeladas/métodos , Histocitoquímica/métodos , Microtomia/métodos , Animais , Criopreservação/métodos , Fibroblastos/ultraestrutura , Imuno-Histoquímica/métodos , Hibridização In Situ/métodos , Camundongos , Microscopia/métodos , Osteoblastos/ultraestrutura , Ratos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
12.
Bone ; 149: 115969, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33892176

RESUMO

During tooth movement in orthodontic treatment, bone formation and resorption occur on the tension and compression sides of the alveolar bone, respectively. Although the bone formation activity increases in the periodontal ligament (PDL) on the tension side, the PDL itself is not ossified and maintains its homeostasis, indicating that there are negative regulators of bone formation in the PDL. Our previous report suggested that scleraxis (Scx) has an inhibitory effect on ossification of the PDL on the tension side through the suppression of calcified extracellular matrix formation. However, the molecular biological mechanisms of Scx-modulated inhibition of ossification in the tensioned PDL are not fully understood. The aim of the present study is to clarify the inhibitory role of Scx in osteoblast differentiation of PDL cells and its underlying mechanism. Our in vivo experiment using a mouse experimental tooth movement model showed that Scx expression was increased during early response of the PDL to tensile force. Scx knockdown upregulated expression of alkaline phosphatase, an early osteoblast differentiation marker, in the tensile force-loaded PDL cells in vitro. Transforming growth factor (TGF)-ß1-Smad3 signaling in the PDL was activated by tensile force and inhibitors of TGF-ß receptor and Smad3 suppressed the tensile force-induced Scx expression in PDL cells. Tensile force induced ephrin A2 (Efna2) expression in the PDL and Efna2 knockdown upregulated alkaline phosphatase expression in PDL cells under tensile force loading. Scx knockdown eliminated the tensile force-induced Efna2 expression in PDL cells. These findings suggest that the TGF-ß1-Scx-Efna2 axis is a novel molecular mechanism that negatively regulates the tensile force-induced osteoblast differentiation of PDL cells.


Assuntos
Efrina-A2 , Fator de Crescimento Transformador beta1 , Diferenciação Celular , Células Cultivadas , Ligamentos , Osteoblastos , Osteogênese , Ligamento Periodontal , Técnicas de Movimentação Dentária
13.
Histochem Cell Biol ; 133(5): 577-84, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20372918

RESUMO

Melatonin is known to regulate a variety of physiological processes including control of circadian rhythms, regulation of seasonal reproductive function, regulation of body temperature, free radical scavenging, and so forth. Accumulating evidence from in vitro and in vivo experiments has also suggested that melatonin may have an influence on skeletal growth and bone formation. However, little is known about the effects of melatonin on tooth development and growth, which thus remain to be elucidated. This study was performed to examine the possibility that melatonin might exert its influence on tooth development as well as skeletal growth. Immunohistochemical analysis revealed that melatonin 1a receptor (Mel1aR) was expressed in secretory ameloblasts, the cells of the stratum intermedium and stellate reticulum, external dental epithelial cells, odontoblasts, and dental sac cells. Reverse transcription-polymerase chain reaction and Western blot analysis showed that HAT-7, a rat dental epithelial cell line, expressed Mel1aR and its expression levels increased after the cells reached confluence. These results strongly suggest that melatonin may play a physiological role in tooth development/growth by regulating the cellular function of odontogenic cells in tooth germs.


Assuntos
Melatonina/metabolismo , Odontogênese/fisiologia , Receptor MT1 de Melatonina/metabolismo , Germe de Dente/metabolismo , Dente/crescimento & desenvolvimento , Ameloblastos/citologia , Ameloblastos/metabolismo , Animais , Animais Recém-Nascidos , Linhagem Celular , Criança , Esmalte Dentário/metabolismo , Papila Dentária/citologia , Papila Dentária/metabolismo , Saco Dentário/citologia , Saco Dentário/metabolismo , Dentina/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Expressão Gênica/genética , Humanos , Masculino , Mandíbula/citologia , Mandíbula/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Odontoblastos/citologia , Odontoblastos/metabolismo , Ratos , Receptor MT1 de Melatonina/genética , Germe de Dente/citologia
14.
Med Mol Morphol ; 43(2): 102-6, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20683698

RESUMO

Tight junctions (TJs) function primarily as a barrier against paracellular transport between epithelial cells and are composed mainly of occludin (OLD) and claudins (CLDs). The CLD family consists of 24 members that show tissue- or cell-specific expression. Ameloblasts, which originate from the oral epithelium, form enamel, and enamel proteins and minerals are transported across the ameloblastic layer during amelogenesis. We immunohistochemically examined the distribution patterns of TJs in ameloblasts by observing the expression patterns of OLD and CLDs (CLD-1 to CLD-10). Secretory ameloblasts contained OLD and CLD-1, -8, and -9 at the distal end of the cell. In mature ameloblasts, OLD and CLD-1, -6, -7, -8, -9, and -10 were present mainly at both the distal and proximal ends of the cell, regardless of whether the ameloblasts were ruffle-ended or smooth-ended. Mature ameloblasts in which only the proximal ends were stained for OLD and CLDs were also found. These results indicate that the expression patterns of CLDs and the distribution patterns of TJs change drastically between the secretory and mature ameloblast stages, suggesting that these patterns reflect the different functions of these cells, specifically in the transport of proteins and ions for enamel formation.


Assuntos
Ameloblastos/metabolismo , Diferenciação Celular , Claudinas/metabolismo , Perfilação da Expressão Gênica , Incisivo/metabolismo , Proteínas de Membrana/metabolismo , Junções Íntimas/metabolismo , Ameloblastos/citologia , Animais , Incisivo/citologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Contraste de Fase , Ocludina , Transporte Proteico
15.
Res Pract Thromb Haemost ; 4(1): 86-91, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31989088

RESUMO

BACKGROUND: Super-resolution microscopy has enabled high-resolution imaging of the actin cytoskeleton in megakaryocytes and platelets. These technologies have extended our knowledge of thrombopoiesis and platelet spreading using megakaryocytes and platelets cultured in vitro on matrix proteins. However, for better understanding of megakaryocytopoiesis and platelet production, high-resolution imaging of cells in an in vivo bone marrow microenvironment is required. Development of Kawamoto's film method greatly advanced the techniques of thin cryosectioning of hard tissues such as undecalcified bones. One obstacle that remains is the spherical aberration that occurs due to the difference in the refractive index for the light path, limiting the usage of Kawamoto's film method to lower magnification observation. OBJECTIVES: To overcome the weakness of the conventional Kawamoto's film method for higher magnification observation of undecalcified bone marrow. METHODS: We have modified the original method with a very simple method: flipping the film at the step of mounting the sections on the glass. RESULTS AND CONCLUSIONS: This new method successfully led to the adjustment of the refractive index and enabled super-resolution imaging of megakaryocytes in undecalcified mouse femurs. Our modified method will expand the application of Kawamoto's film method and enable precise analysis of megakaryocytopoiesis and platelet production in the bone marrow microenvironment under pathophysiological conditions.

16.
Acta Histochem Cytochem ; 50(4): 119-125, 2017 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-28928541

RESUMO

Tight junction (TJ) is one of the cell-cell junctions and known to have the barrier and fence functions between adjacent cells in both simple and stratified epithelia. We examined the distribution pattern, constitutive proteins, and permeability of TJ in the stratified squamous epithelium of the palatal mucosa of mice. Ultrastructural observations based on the ultrathin section and freeze-fracture methods revealed that poorly developed TJs are located at the upper layer of the stratum granulosum. The positive immunofluorescence of occludin (OCD), claudin (CLD)-1 and -4 were localized among the upper layer of the stratum granulosum showing a dot-like distribution pattern. And CLD-1 and -4 were localized among the stratum spinosum and the lower part of stratum granulosum additionally showed a positive reaction along the cell profiles. Western blotting of TJ constitutive proteins showed OCD, CLD-1, -2, -4, and -5 bands. The permeability test using biotin as a tracer revealed both the areas where biotin passed through beyond OCD positive points and the areas where biotin stopped at OCD positive points. These results show that poor TJs localize at the upper layer of the stratum granulosum of the palatal epithelium, and the TJs are leaky and include at least CLD-1 and -4.

17.
Elife ; 62017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28809157

RESUMO

Impact of stress on diseases including gastrointestinal failure is well-known, but molecular mechanism is not understood. Here we show underlying molecular mechanism using EAE mice. Under stress conditions, EAE caused severe gastrointestinal failure with high-mortality. Mechanistically, autoreactive-pathogenic CD4+ T cells accumulated at specific vessels of boundary area of third-ventricle, thalamus, and dentate-gyrus to establish brain micro-inflammation via stress-gateway reflex. Importantly, induction of brain micro-inflammation at specific vessels by cytokine injection was sufficient to establish fatal gastrointestinal failure. Resulting micro-inflammation activated new neural pathway including neurons in paraventricular-nucleus, dorsomedial-nucleus-of-hypothalamus, and also vagal neurons to cause fatal gastrointestinal failure. Suppression of the brain micro-inflammation or blockage of these neural pathways inhibited the gastrointestinal failure. These results demonstrate direct link between brain micro-inflammation and fatal gastrointestinal disease via establishment of a new neural pathway under stress. They further suggest that brain micro-inflammation around specific vessels could be switch to activate new neural pathway(s) to regulate organ homeostasis.


Assuntos
Encéfalo/fisiologia , Encefalomielite Autoimune Experimental/complicações , Gastroenteropatias/fisiopatologia , Hipotálamo/patologia , Vias Neurais/fisiologia , Estresse Fisiológico , Animais , Citocinas/metabolismo , Encefalomielite Autoimune Experimental/patologia , Camundongos , Linfócitos T/imunologia
18.
Nucl Med Commun ; 27(1): 81-9, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16340727

RESUMO

BACKGROUND: As apoptosis occurs in tumors within a short time after irradiation, the detection of the frequency of apoptosis may be useful as an indicator of the effect of treatment. For the evaluation of apoptosis under these conditions, tissue extraction from patients is indispensable. AIM: To develop a noninvasive imaging technique to measure and monitor apoptosis in tumor cells caused by X-irradiation using (125)I-radiolabeled annexin V. METHODS: The tumors used were human ependymoblastomas, which were transplanted into nude mice. The tumors were irradiated at 2, 5 or 10 Gy. (125)I-annexin V was administered intravenously 6 h after irradiation. In the 5 Gy irradiation group, the isotope was injected at various time intervals (3, 6 and 12 h) after irradiation. Three hours after the injection, the mice were sacrificed, the tumors were quickly removed and frozen sections were prepared at 6 and 40 microm thickness using a cryomicrotome. In autoradiographic imaging, the tumor-to-muscle ratios were compared in the respective irradiated groups. In addition, apoptosis detection by the in-situ end-labeling (Klenow) assay was conducted on the same sections. The number of Klenow-positive cells was counted in 100 x fields for each section. RESULTS: Both autoradiography and immunohistochemical staining showed a significantly higher frequency of apoptosis in the neoplasms in all irradiated groups than in the control group (P<0.05). Although immunohistochemical staining revealed a peak apoptosis frequency in the 5 Gy irradiated group, autoradiography revealed a peak in the group receiving a lower dose than 5 Gy. When the time from irradiation to annexin injection was varied, both imaging methods showed a peak apoptosis frequency in the group receiving the injection 6 h after irradiation. CONCLUSION: It is possible to predict the effect of treatment in cancer in a noninvasive manner by apoptosis imaging in vivo after radiotherapy.


Assuntos
Anexina A5 , Apoptose/efeitos da radiação , Radioisótopos do Iodo , Tumores Neuroectodérmicos Primitivos/diagnóstico por imagem , Tumores Neuroectodérmicos Primitivos/radioterapia , Animais , Relação Dose-Resposta à Radiação , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Prognóstico , Doses de Radiação , Cintilografia , Compostos Radiofarmacêuticos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Resultado do Tratamento , Células Tumorais Cultivadas
19.
Acta Histochem Cytochem ; 48(2): 37-45, 2015 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-26175546

RESUMO

To confirm the possible involvement of planar cell polarity proteins in odontogenesis, one group of core proteins, PRICKLE1, PRICKLE2, PRICKLE3, and PRICKLE4, was examined in enamel epithelial cells and ameloblasts by immunofluorescence microscopy. PRICKLE1 and PRICKLE2 showed similar localization in the proliferation and secretory zones of the incisor. Immunoreactive dots and short rods in ameloblasts and stratum intermedium cells were evident in the proliferation to differentiation zone, but in the secretion zone, cytoplasmic dots decreased and the distal terminal web was positive for PRICKLE1 and PRICKLE2. PRICKLE3 and PRICKLE4 showed cytoplasmic labeling in ameloblasts and other enamel epithelial cells. Double labeling of PRICKLE2 with VANGL1, which is another planar cell polarity protein, showed partial co-localization. To examine the transport route of PRICKLE proteins, PRICKLE1 localization was examined after injection of a microtubule-disrupting reagent, colchicine, and was compared with CX43, which is a membrane protein transported as vesicles via microtubules. The results confirmed the retention of immunoreactive dots for PRICKLE1 in the cytoplasm of secretory ameloblasts of colchicine-injected animals, but fewer dots were observed in control animals. These results suggest that PRICKLE1 and PRICKLE2 are transported as vesicles to the junctional area, and are involved in pattern formation of distal junctional complexes and terminal webs of ameloblasts, further implying a role in the formed enamel rod arrangement.

20.
PLoS One ; 10(12): e0144194, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26713442

RESUMO

Calcium-dependent transglutaminases (TGs) are a family of enzymes that catalyze protein cross-linking and/or attachment of primary amines in a variety of organisms. Mammalian TGs are implicated in multiple biological events such as skin formation, blood coagulation, and extracellular matrix stabilization. Medaka (Oryzias latipes) has been used as a model fish to investigate the physiological functions of mammalian proteins. By analysis of the medaka genome, we found seven TGs orthologues, some of which apparently corresponded to the mammalian TG isozymes, TG1, TG2, and Factor XIII. All orthologues had preserved amino acid residues essential for enzymatic activity in their deduced primary structures. In this study, we analyzed biochemical properties of two orthologues (OlTGK1 and OlTGK2) of mammalian epithelium-specific TG (TG1) that are significantly expressed at the transcriptional level. Using purified recombinant proteins for OlTGK1 and OlTGK2, we characterized their catalytic reactions. Furthermore, immunohistochemical analyses of fish sections revealed higher expression in the pancreas (OTGK1), intervertebral disk (OlTGK2) and pharyngeal teeth (OlTGK2) as well as in the skin epidermis.


Assuntos
Epiderme/enzimologia , Proteínas de Peixes/química , Transglutaminases/química , Sequência de Aminoácidos , Animais , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Humanos , Cinética , Dados de Sequência Molecular , Oryzias/metabolismo , Filogenia , Transglutaminases/genética , Transglutaminases/metabolismo
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