RESUMEN
Musculocontractural Ehlers-Danlos syndrome caused by dermatan sulfate epimerase deficiency (mcEDS-DSE) is a rare connective tissue disorder. This is the first report describing the detailed and comprehensive clinical and pathophysiological features of mcEDS-DSE. The patient, with a novel homozygous nonsense variant (NM_013352.4:c.2601C>A:p.(Tyr867*)), exhibited mild skin hyperextensibility without fragility and small joint hypermobility, but developed recurrent large subcutaneous hematomas. Dermatan sulfate (DS) moieties on chondroitin sulfate/DS proteoglycans were significantly decreased, but remained present, in skin fibroblasts. Electron microscopy examination of skin specimens, including cupromeronic blue-staining to visualize glycosaminoglycan (GAG) chains, revealed coexistence of normally assembled collagen fibrils with attached curved GAG chains and dispersed collagen fibrils with linear GAG chains from attached collagen fibrils across interfibrillar spaces to adjacent fibrils. Residual activity of DS-epi1, encoded by DSE, and/or compensation by DS-epi2, a minor homolog of DS-epi1, may contribute to the mild skin involvement through this "mosaic" pattern of collagen fibril assembly.
Asunto(s)
Dermatán Sulfato , Síndrome de Ehlers-Danlos , Humanos , Colágeno/genética , Síndrome de Ehlers-Danlos/diagnóstico , Síndrome de Ehlers-Danlos/genética , Racemasas y Epimerasas , SulfotransferasasRESUMEN
Stem cell-based products have clinical and industrial applications. Thus, there is a need to develop quality control methods to standardize stem cell manufacturing. Here, we report a deep learning-based automated cell tracking (DeepACT) technology for noninvasive quality control and identification of cultured human stem cells. The combination of deep learning-based cascading cell detection and Kalman filter algorithm-based tracking successfully tracked the individual cells within the densely packed human epidermal keratinocyte colonies in the phase-contrast images of the culture. DeepACT rapidly analyzed the motion of individual keratinocytes, which enabled the quantitative evaluation of keratinocyte dynamics in response to changes in culture conditions. Furthermore, DeepACT can distinguish keratinocyte stem cell colonies from non-stem cell-derived colonies by analyzing the spatial and velocity information of cells. This system can be widely applied to stem cell cultures used in regenerative medicine and provides a platform for developing reliable and noninvasive quality control technology.
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Aprendizaje Profundo , Células Epidérmicas , Diferenciación Celular , Rastreo Celular , Células Cultivadas , Humanos , Queratinocitos , Control de Calidad , Células MadreRESUMEN
Loss-of-function variants in CHST14 cause a dermatan 4-O-sulfotransferase deficiency named musculocontractural Ehlers-Danlos syndrome-CHST14 (mcEDS-CHST14), resulting in complete depletion of the dermatan sulfate moiety of decorin glycosaminoglycan (GAG) chains, which is replaced by chondroitin sulfate. Recently, we uncovered structural alteration of GAG chains in the skin of patients with mcEDS-CHST14. Here, we conducted the first systematic investigation of Chst14 gene-deleted homozygote (Chst14-/-) mice. We used skin samples of wild-type (Chst14+/+) and Chst14-/- mice. Mechanical fragility of the skin was measured with a tensile test. Pathology was observed using light microscopy, decorin immunohistochemistry and electron microscopy (EM) including cupromeronic blue (CB) staining. Quantification of chondroitin sulfate and dermatan sulfate was performed using enzymatic digestion followed by anion-exchange HPLC. In Chst14-/- mice, skin tensile strength was significantly decreased compared with that in Chst14+/+ mice. EM showed that collagen fibrils were oriented in various directions to form disorganized collagen fibers in the reticular layer. Through EM-based CB staining, rod-shaped linear GAG chains were found to be attached at one end to collagen fibrils and protruded outside of the fibrils, in contrast to them being round and wrapping the collagen fibrils in Chst14+/+ mice. A very low level of dermatan sulfate disaccharides was detected in the skin of Chst14-/- mice by anion-exchange chromatography. Chst14-/- mice, exhibiting similar abnormalities in the GAG structure of decorin and collagen networks in the skin, could be a reasonable model for skin fragility of patients with mcEDS-CHST14, shedding light on the role of dermatan sulfate in maintaining skin strength.
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Síndrome de Ehlers-Danlos/genética , Piel/metabolismo , Sulfotransferasas/genética , Animales , Síndrome de Ehlers-Danlos/patología , Ratones , Ratones Noqueados , Sulfotransferasas/deficiencia , Sulfotransferasas/metabolismoRESUMEN
BACKGROUND: Classification of optical coherence tomography (OCT) images can be achieved with high accuracy using classical convolution neural networks (CNN), a commonly used deep learning network for computer-aided diagnosis. Classical CNN has often been criticized for suppressing positional relations in a pooling layer. Therefore, because capsule networks can learn positional information from images, we attempted application of a capsule network to OCT images to overcome that shortcoming. This study is our attempt to improve classification accuracy by replacing CNN with a capsule network. METHODS: From an OCT dataset, we produced a training dataset of 83,484 images and a test dataset of 1000 images. For training, the dataset comprises 37,205 images with choroidal neovascularization (CNV), 11,348 with diabetic macular edema (DME), 8616 with drusen, and 26,315 normal images. The test dataset has 250 images from each category. The proposed model was constructed based on a capsule network for improving classification accuracy. It was trained using the training dataset. Subsequently, the test dataset was used to evaluate the trained model. RESULTS: Classification of OCT images using our method achieved accuracy of 99.6%, which is 3.2 percentage points higher than that of other methods described in the literature. CONCLUSION: The proposed method achieved classification accuracy results equivalent to those reported for other methods for CNV, DME, drusen, and normal images.
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Algoritmos , Retinopatía Diabética/clasificación , Edema Macular/clasificación , Redes Neurales de la Computación , Tomografía de Coherencia Óptica/métodos , Retinopatía Diabética/complicaciones , Retinopatía Diabética/diagnóstico , Humanos , Edema Macular/diagnóstico , Edema Macular/etiología , Estudios RetrospectivosRESUMEN
Ehlers-Danlos syndrome (EDS) is a collection of inheritable diseases involving the musculoskeletal, integumentary and visual systems. Spondylodysplastic EDS-ZIP13 (spEDS-ZIP13: OMIM 612350) was recently defined as a new form of EDS. Although vasculitis has been found in many spEDS-ZIP13 patients, vascular pathology has not been included as a pathognomonic lesion of this type of EDS. We investigate the morphometry of the thoracic aorta in wild-type and Zip13-knockout (Zip13-KO) mice. Our assessment found abnormalities in the number and morphology of elastic and cellular components in the aortic wall, especially the tunica media, of Zip13-KO mice, indicating aortic fragility. Accordingly, our major findings (vascular smooth muscle cells with small nuclei, small percentage of elastic membrane area per tunica media, many large elastic flaps) should be considered vulnerable characteristics indicating fragility of the aorta in patients with spEDS-ZIP13.
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Aorta Torácica/anomalías , Síndrome de Ehlers-Danlos/patología , Músculo Liso Vascular/anomalías , Osteocondrodisplasias/patología , Animales , Aorta Torácica/patología , Proteínas de Transporte de Catión/genética , Elasticidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/ultraestructuraRESUMEN
Preclinical Research This article describes the properties of a novel topical NSAID (Nonsteroidal anti-inflammatory drug) patch, SFPP (S(+)-flurbiprofen plaster), containing the potent cyclooxygenase (COX) inhibitor, S(+)-flurbiprofen (SFP). The present studies were conducted to confirm human COX inhibition and absorption of SFP and to evaluate the analgesic efficacy of SFPP in a rat adjuvant-induced arthritis (AIA) model. COX inhibition by SFP, ketoprofen and loxoprofen was evaluated using human recombinant COX proteins. Absorption of SFPP, ketoprofen and loxoprofen from patches through rat skin was assessed 24 h after application. The AIA model was induced by injecting Mycobacterium tuberculosis followed 20 days later by the evaluation of the prostaglandin PGE2 content of the inflamed paw and the pain threshold. SFP exhibited more potent inhibitory activity against COX-1 (IC50 = 8.97 nM) and COX-2 (IC50 = 2.94 nM) than the other NSAIDs evaluated. Absorption of SFP was 92.9%, greater than that of ketoprofen and loxoprofen from their respective patches. Application of SFPP decreased PGE2 content from 15 min to 6 h and reduced paw hyperalgesia compared with the control, ketoprofen and loxoprofen patches. SFPP showed analgesic efficacy, and was superior to the ketoprofen and loxoprofen patches, which could be through the potent COX inhibitory activity of SFP and greater skin absorption. The results suggested SFPP can be expected to exert analgesic effect clinically.
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Analgésicos/administración & dosificación , Antiinflamatorios no Esteroideos/administración & dosificación , Artritis Experimental/tratamiento farmacológico , Inhibidores de la Ciclooxigenasa/administración & dosificación , Flurbiprofeno/administración & dosificación , Administración Tópica , Analgésicos/farmacocinética , Analgésicos/farmacología , Animales , Antiinflamatorios no Esteroideos/farmacocinética , Antiinflamatorios no Esteroideos/farmacología , Artritis Experimental/inmunología , Ciclooxigenasa 1/metabolismo , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa/farmacocinética , Inhibidores de la Ciclooxigenasa/farmacología , Dinoprostona/metabolismo , Modelos Animales de Enfermedad , Flurbiprofeno/farmacocinética , Flurbiprofeno/farmacología , Humanos , Masculino , Umbral del Dolor/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
In tracing the biological processes using fluorescent probes, it is desirable to shift the excitation/emission energy to a far-red/near-infrared (FR/NIR) region. In this study, we successfully synthesize FR fluorescent organic nanoparticles via ion-association between the malachite green (MG) cations and tetrakis(4-fluorophenyl)borate (TFPB) anions in the presence of a neutral stabilizing polymer. Binding of MG with TFPB results in the prominent appearance of an absorption band that can be assigned to an H-aggregate of MG. The fluorescence intensity as well as the fluorescence lifetime shows a significant increase with a decrease in the nanoparticle size. Since the MG dye is known as a local viscosity or environmental rigidity probe showing a rotational friction dependence of the excited state lifetime, we find that the rigidity of the organic nanoparticle is strongly size-dependent; that is, the smaller the size of the nanoparticle, the greater the rigidity of the nanoparticle. We also reveal that surface regions of the ion-based organic nanoparticles are more rigid than inner regions. The presence of H-aggregates that are almost non-fluorescent is the major origin of aggregation-caused quenching (ACQ) and still avoids the enhancement of the fluorescence quantum yield of the MG nanoparticles, so we develop a new approach to prevent H-aggregation inside the nanoparticle by incorporating photochemically inert, bulky phosphonium cations, which results in a 430-fold enhancement of its fluorescence yield. We believe that such a methodology will open up an avenue in the development of new types of fluorescent nanomaterials for many applications.
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Color , Nanopartículas/química , Tamaño de la Partícula , Colorantes de Rosanilina/química , Modelos Moleculares , Conformación Molecular , Povidona/química , Espectrometría de Fluorescencia , Tetrafenilborato/análogos & derivados , Tetrafenilborato/químicaRESUMEN
Loss-of-function mutations in carbohydrate sulfotransferase 14 (CHST14) cause musculocontractural Ehlers-Danlos syndrome-CHST14 (mcEDS-CHST14), characterized by multiple congenital malformations and progressive connective tissue fragility-related manifestations in the cutaneous, skeletal, cardiovascular, visceral and ocular system. The replacement of dermatan sulfate chains on decorin proteoglycan with chondroitin sulfate chains is proposed to lead to the disorganization of collagen networks in the skin. However, the pathogenic mechanisms of mcEDS-CHST14 are not fully understood, partly due to the lack of in vitro models of this disease. In the present study, we established in vitro models of fibroblast-mediated collagen network formation that recapacitate mcEDS-CHST14 pathology. Electron microscopy analysis of mcEDS-CHST14-mimicking collagen gels revealed an impaired fibrillar organization that resulted in weaker mechanical strength of the gels. The addition of decorin isolated from patients with mcEDS-CHST14 and Chst14-/- mice disturbed the assembly of collagen fibrils in vitro compared to control decorin. Our study may provide useful in vitro models of mcEDS-CHST14 to elucidate the pathomechanism of this disease.
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Síndrome de Ehlers-Danlos , Sulfotransferasas , Animales , Ratones , Decorina , Sulfotransferasas/genética , Síndrome de Ehlers-Danlos/genética , Matriz Extracelular/patología , ColágenoRESUMEN
Cytochrome P450(BSß), a H(2)O(2)-dependent cytochrome P450 catalyzing the hydroxylation of long-alkyl-chain fatty acids, lacks the general acid-base residue around the heme, which is indispensable for the efficient generation of the active species using H(2)O(2). On the basis of the crystal structure of the palmitic acid bound form of cytochrome P450(BSß), it was suggested that the role of the general acid-base function was provided by the carboxylate group of fatty acids. The participation of the carboxylate group of the substrate was supported by the fact that cytochrome P450(BSß) can catalyze oxidations of nonnatural substrates such as styrene and ethylbenzene in the presence of a series of short-alkyl-chain carboxylic acids as a dummy molecule of fatty acid. We refer to a series of short-alkyl-chain carboxylic acids as a "decoy molecule". As shown here, we have clarified the crystal structure of the decoy-molecule-bound form and elucidated that the location of its carboxylate group is virtually the same as that of palmitic acid in the heme cavity, indicating that the carboxylate group of the decoy molecule serves as the general acid-base catalyst. This result further confirms that the role of the acid-base function is satisfied by the carboxylate group of the substrates. In addition, the structure analysis of the substrate-free form has clarified that no remarkable structural change is induced by the binding of the decoy molecule as well as fatty acid. Consequently, whether the carboxylate group is positioned in the active site provides the switching mechanism of the catalytic cycle of cytochrome P450(BSß).
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Bacillus subtilis/enzimología , Sistema Enzimático del Citocromo P-450/química , Peróxido de Hidrógeno/química , Bacillus subtilis/metabolismo , Biocatálisis , Ácidos Carboxílicos/química , Dominio Catalítico , Cristalografía por Rayos X , Sistema Enzimático del Citocromo P-450/metabolismo , Modelos Moleculares , Conformación Molecular , Oxidación-ReducciónRESUMEN
We previously showed that cationic liposomes composed of cholesteryl-3ß-carboxyamidoethylene-N-hydroxyethylamine (OH-Chol) and 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) inhibited mast cell degranulation mediated by the cross-linking of high-affinity IgE receptors (FcεRI). In this study, we prepared three kinds of cationic liposomes composed of OH-Chol and DOPE in different ratios (0.28, 0.60, and 0.86 of OH-Chol in mol ratio, named as L-liposome, M-liposome, and H-liposome, respectively) and investigated their effects on mast cell activation. We found that mast cell degranulation evoked with antigen was inhibited by pretreatment with cationic liposomes in the composite ratio-dependent manner of OH-Chol and that the H-liposome showed the highest inhibitory effect on degranulation among three kinds of liposomes. Store-operated Ca2+ entry, phosphorylation of PI3K and Akt, and IL-4 secretion after antigen stimulation were reduced in dose-dependent manner of each liposome, but there were no differences between H-liposome and M-liposome. Meanwhile, microtubule acetylation, which is involved in the secretory granule transport, was significantly suppressed by H-liposome compared with M-liposome. These data suggested that the lipid composition in cationic liposomes themselves largely influenced the inhibition of mast cell activation as well as the efficiency of gene transfection.
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Lípidos/farmacología , Mastocitos/efectos de los fármacos , Animales , Calcio/metabolismo , Cationes/química , Células Cultivadas , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Lípidos/química , Liposomas/química , Mastocitos/metabolismo , RatasRESUMEN
Adult T-cell leukemia (ATL) is a mature T-cell malignancy caused by human T-cell leukemia virus type I infection, and 10%-25% of patients show central nervous system (CNS) involvement. CNS involvement significantly reduces survival and there are no effective treatments for CNS involvement. Therefore, an appropriate animal model is required to evaluate the inhibitory effects of novel drugs on the progression of ATL with CNS involvement. Here, we established a mouse model of ATL with CNS involvement using NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice inoculated with ATL cells intramuscularly in the postauricular region, and these mice showed paraparesis. Of the 10 mice inoculated with ATL cells intramuscularly (I.M.) at 5 weeks of age, 8 (80%) showed paraparesis, whereas none of the 10 mice inoculated with ATL cells subcutaneously (S.C.) showed paraparesis. In the I.M. group, PCR detected HTLV-1-specific genes in the thoracic and lumbar vertebrae; however, in the S.C. group, the vertebrae were negative for HTLV-1 genes. Histological analysis revealed a particularly high incidence of tumors, characterized by accumulation of the injected cells, in the thoracic vertebrae of mice in the I.M. group. Tumor cell infiltration was relatively high in the bone marrow. Spinal cord compression caused by invasion of the tumor mass outside the pia mater was observed in the thoracic vertebrae of the spinal cord. In conclusion, we have reported a mouse model of tumor growth with paraparesis that may be used to assess novel therapeutic agents for ATL with CNS involvement.
RESUMEN
Musculocontractural Ehlers-Danlos syndrome (mcEDS) due to CHST14/D4ST1 deficiency (mcEDS-CHST14) is a recently delineated type of EDS caused by biallelic loss-of-function mutations in CHST14, which results in the depletion of dermatan sulfate (DS). Clinical characteristics of mcEDS-CHST14 consist of multiple malformations and progressive fragility-related manifestations, including skin hyperextensibility and fragility. Skin fragility is suspected to result from the impaired assembly of collagen fibrils caused by alteration of the glycosaminoglycan (GAG) chain of decorin-proteoglycan (PG) from DS to chondroitin sulfate (CS). This systematic investigation of the skin pathology of patients with mcEDS-CHST14 comprised both immunostaining of decorin and transmission electron microscopy-based cupromeronic blue staining to visualize GAG chains. Collagen fibrils were dispersed in the affected papillary to reticular dermis; in contrast, they were regularly and tightly assembled in controls. Moreover, the fibrils exhibited a perpendicular arrangement to the affected epidermis, whereas fibrils were parallel to control epidermis. Affected GAG chains were linear, stretching from the outer surface of collagen fibrils to adjacent fibrils; in contrast, those of controls were curved, maintaining close contact with attached collagen fibrils. This is the first observation of compositional alteration, from DS to CS, of GAG side chains, which caused structural alteration of GAG side chains and resulted in spatial disorganization of collagen networks; this presumably disrupted the ring-mesh structure of GAG side chains surrounding collagen fibrils. McEDS-CHST14 provides a critical example of the importance of DS in GAG side chains of decorin-PG during assembly of collagen fibrils in maintenance of connective tissues.
Asunto(s)
Colágeno/metabolismo , Síndrome de Ehlers-Danlos , Glicosaminoglicanos/metabolismo , Piel/metabolismo , Piel/ultraestructura , Sulfotransferasas/genética , Adolescente , Adulto , Secuencia de Carbohidratos , Estudios de Casos y Controles , Niño , Preescolar , Colágeno/química , Colágeno/ultraestructura , Decorina/metabolismo , Dermatán Sulfato/metabolismo , Síndrome de Ehlers-Danlos/genética , Síndrome de Ehlers-Danlos/metabolismo , Síndrome de Ehlers-Danlos/patología , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Femenino , Glicosaminoglicanos/química , Glicosaminoglicanos/ultraestructura , Humanos , Masculino , Conformación Molecular , Mutación , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Piel/patología , Relación Estructura-Actividad , Sulfotransferasas/metabolismo , Adulto JovenRESUMEN
Superficial digital flexor tendon (SDFT) of the bovine hindlimb originates from the caudolateral aspect of the distal femur and finally inserts onto the plantar aspect of the middle phalanges. In the present study, morphology and morphometry of the bovine SDFT at the muscle-tendon junction (MTJ), middle metatarsus (mM) and tendon-bone interface (TBI) were investigated. Cross-sectional morphology at the three regions of SDFT were oval, semioval and ring-formed, respectively. Significant difference in cross-sectional area was found only between MTJ-mM and mM-TBI (P<0.05). Functional compression and friction from the adjacent structures could be the most potential interactions affecting such appearances. Morphometric data of tenocyte number, water content, and glycosaminoglycan (GAG) length and angle were found increasing in the proximodistal direction, except the fibril diameter and collagen fibril index (CFI). Statistical analyzes could reveal significant differences in average number of tenocytes (P<0.0001), CFI (between MTJ-mM and MTJ-TBI, P<0.05), water content (between MTJ-TBI, P<0.05), length of GAG chains (between MTJ-TBI, P<0.05), and angle of GAG chains (P<0.0001), respectively. The fibrillar characteristics at the three different areas, including fibril diameter distribution and interfibrillar distance, existed in conforming to the tensional axes in situ. In addition, length and angle of GAG chains were relevant to moving directions of the collagen fibrils.
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Bovinos/anatomía & histología , Colágenos Fibrilares/metabolismo , Tendones/anatomía & histología , Animales , Fémur/anatomía & histología , Colágenos Fibrilares/ultraestructura , Glicosaminoglicanos/metabolismo , Metatarso/anatomía & histología , Microscopía Electrónica de Transmisión/veterinaria , Músculo Esquelético/anatomía & histología , Tendones/ultraestructura , Tenocitos/metabolismo , Falanges de los Dedos del Pie/anatomía & histologíaRESUMEN
Ehlers-Danlos syndrome (EDS) is a group of hereditary diseases caused by mutation of extracellular matrix-related genes. Recently, spondylodysplastic EDS-Zip13 (spEDS-Zip13: OMIM 612350) was recognized as a new EDS type. This current study could reveal various morphometric differences of collagenous population in the proper substance of cornea between the wild type and spEDS-Zip13-knockout (Zip13-KO) mice. Blockade of Smad-signaling pathway might initiate these alterations. Predilected dissimilarity in level of transcriptional activity probably dictated morphology of keratocyte and shape and electron density of its nucleus. In addition, the imbalance of proteoglycans and glycosaminoglycans would also affect the diameter and arrangement of collagen fibrils. These findings would be considered as vulnerable characteristics of corneal stroma of the Zip13-KO mice.
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Córnea/patología , Enfermedades de la Córnea/genética , Síndrome de Ehlers-Danlos/genética , Animales , Proteínas de Transporte de Catión/genética , Enfermedades de la Córnea/patología , Queratocitos de la Córnea/patología , Síndrome de Ehlers-Danlos/patología , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Ehlers-Danlos syndrome (EDS) is a group of disorders caused by abnormalities in the extracellular matrix (ECM). Transforming growth factor-ß (TGF-ß) plays a crucial role in formation of the ECM by the SMAD (Sma-and Mad-related protein, mothers against decapentaplegic homolog) pathway. It has been reported that loss of function of zinc transporter ZRT/IRT-like protein 13 (ZIP13) is the cause of the spondylocheiro dysplastic form of EDS (SCD-EDS: OMIM 612350). Our previous study suggested that TGF-ß1 has a relationship with the skin pathological condition in the Zip13-Knockout (KO) mouse, which is a model of SCD-EDS. Thus far, effective treatment based on modern medicine for this syndrome has not yet been established. According to an approach of traditional Chinese medicine, the present study investigates the medicinal effects of Makomo (Zizania latifolia) on certain aspects of SCD-EDS, such as skin morphology and plasma TGF-ß1, in Zip13-KO mice. Increases in densities of collagen fibers and fibrils without a significant change in thickness of the dermal layer were observed in the group of mice fed a Makomo-containing diet. No change in the amount of collagen suggests that Makomo feed does not elevate collagen synthesis, but changes the length of glycosaminoglycan chains and decreases the distance between collagen fibrils. In conclusion, the changes of the skin structure suggest that Makomo can increase the mechanical strength of skin.
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Alimentación Animal , Proteínas de Transporte de Catión/metabolismo , Dieta , Poaceae , Piel/patología , Animales , Proteínas de Transporte de Catión/genética , Colágeno/metabolismo , Síndrome de Ehlers-Danlos/genética , Regulación de la Expresión Génica/efectos de los fármacos , Ratones , Ratones NoqueadosRESUMEN
The aim of this study was to determine the effects of prolyl-hydroxyproline (Pro-Hyp) on the proliferation and differentiation of rat stromal-vascular cells (SVCs) being cultured in a medium with (Pro-Hyp group) or without Pro-Hyp (control group). The results showed that there was no significant difference in proliferation rate of SVCs, lipid droplet (LD) diameter or intracellular concentration of triglycerides between two groups. However, the diameter range of LDs in the Pro-Hyp group tended to be smaller than that in the control group. Transmission electron microscopy showed a tendency for increase in the area of mitochondria and decrease in the number of mitochondria in the Pro-Hyp-treated SVCs. The mRNA expression levels of white adipose tissue differentiation markers (Cbp, Fabp and Serpina3k) were significantly lower, but those of the brown adipose tissue differentiation markers (Dio2, Ucp1 and Ucp3) were significantly higher in the Pro-Hyp group than in the control group. Our results suggested that Pro-Hyp can facilitate SVCs to differentiate into "brite/beige" adipocytes.
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Adipocitos Beige/efectos de los fármacos , Dipéptidos/farmacología , Adipocitos Beige/citología , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Regulación de la Expresión Génica , Gotas Lipídicas/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Ratas Sprague-Dawley , Células del Estroma/citología , Células del Estroma/efectos de los fármacos , Triglicéridos/metabolismoRESUMEN
The fine structures of different tendons in various animals at different ages have been studied extensively to reveal their arrangement and growth patterns. However, knowledge of the microstructures of the growing tenocytes in the tendons of piglets is still lacking. Thus, we performed the first morphometric analysis to describe the characteristics of tenocytes in the metacarpal superficial digital flexor tendon of 0-, 10- and 20-day-old piglets. In the present study, hydrochloric acid/collagenase digestion was applied to remove the interstitial connective tissue to obtain clear visualization of intact tenocytes and their cytoplasmic processes (Cp). Then, the morphometry of the tenocytes was investigated by optical and electron microscopy. The mean ± SE values of the fascicle area, number of tenocytes/fascicle, cell density, number of Cp/tenocyte, length of Cp, and thickness of Cp were compared among the three age groups. Significant differences (judged at P<0.05) were found in almost all morphometric aspects among the age groups, except for the number of Cp/cell (P=0.545) and thickness of the Cp (P=0.105). A decrease of cell density corresponded with an increase in the length of the Cp, which were extended to connect either with the Cp of the other tenocytes or the surrounding endotendineum. Moreover, an increase of the fascicle area reflected the increase in tendon diameter. The revealed morphometric characteristics are thus the outcome of tendon growth.
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Porcinos/crecimiento & desarrollo , Tendones/crecimiento & desarrollo , Tenocitos/ultraestructura , Animales , Recuento de Células/veterinaria , Microscopía/veterinaria , Microscopía Electrónica de Rastreo/veterinaria , Microscopía Electrónica de Transmisión/veterinaria , Porcinos/anatomía & histología , Tendones/citología , Tendones/ultraestructuraRESUMEN
Ehlers-Danlos syndrome (EDS) is a group of disorders caused by abnormalities that are identified in the extracellular matrix. Transforming growth factor-ß1 (TGF-ß1) plays a crucial role in formation of the extracellular matrix. It has been reported that the loss of function of zinc transporter ZRT/IRT-like protein 13 (ZIP13) causes the spondylocheiro dysplastic form of EDS (SCD-EDS: OMIM 612350), in which dysregulation of the TGF-ß1 signaling pathway is observed, although the relationship between the dermis abnormalities and peripheral TGF-ß1 level has been unclear. We investigated the characteristics of the dermis of the Zip13-knockout (KO) mouse, an animal model for SCD-EDS. Both the ratio of dermatan sulfate (DS) in glycosaminoglycan (GAG) components and the amount of collagen were decreased, and there were very few collagen fibrils with diameters of more than 150 nm in Zip13-KO mice dermis. We also found that the TGF-ß1 level was significantly higher in Zip13-KO mice serum. These results suggest that collagen synthesis and collagen fibril fusion might be impaired in Zip13-KO mice and that the possible decrease of decorin level by reduction of the DS ratio probably caused an increase of free TGF-ß1 in Zip13-KO mice. In conclusion, skin fragility due to defective ZIP13 protein may be attributable to impaired extracellular matrix synthesis accompanied by abnormal peripheral TGF-ß homeostasis.