RESUMO
BACKGROUND: The standard for sentinel lymph node biopsy (SLNB), the dual technique (radiolabelled tracer and blue dye), has several drawbacks. A novel magnetic technique without these drawbacks has been evaluated in a number of clinical trials. It uses a magnetic tracer and a handheld magnetometer to identify and excise sentinel lymph nodes. A systematic review and meta-analysis was performed to assess the performance and utility of the magnetic in comparison to the standard technique. METHODS: MEDLINE, PubMed, Embase and the Cochrane online literature databases were used to identify all original articles evaluating the magnetic technique for SLNB published up to April 2016. Studies were included if they were prospectively conducted clinical trials comparing the magnetic with the standard technique for SLNB in patients with breast cancer. RESULTS: Seven studies were included. The magnetic technique was non-inferior to the standard technique (z = 3·87, P < 0·001), at a 2 per cent non-inferiority margin. The mean identification rates for the standard and magnetic techniques were 96·8 (range 94·2-99·0) and 97·1 (94·4-98·0) per cent respectively (risk difference (RD) 0·00, 95 per cent c.i. -0·01 to 0·01; P = 0·690). The total lymph node retrieval was significantly higher with the magnetic compared with the standard technique: 2113 (1·9 per patient) versus 2000 (1·8 per patient) (RD 0·05, 0·03 to 0·06; P = 0·003). False-negative rates were 10·9 (range 6-22) per cent for the standard technique and 8·4 (2-22) per cent for the magnetic technique (RD 0·03, 0·00 to 0·06; P = 0·551). The mean discordance rate was 3·9 (range 1·7-6·9) per cent. CONCLUSION: The magnetic technique for SLNB is non-inferior to the standard technique, with a high identification rate but with a significantly higher lymph node retrieval rate.
Assuntos
Neoplasias da Mama/patologia , Imãs , Linfonodo Sentinela/patologia , Neoplasias da Mama/cirurgia , Ensaios Clínicos como Assunto , Estudos de Viabilidade , Feminino , Humanos , Metástase Linfática , Magnetometria , Sensibilidade e Especificidade , Biópsia de Linfonodo Sentinela/efeitos adversos , Biópsia de Linfonodo Sentinela/métodosRESUMO
BACKGROUND AND PURPOSE: Tenascin-C (TnC) is a multi-domain extracellular matrix glycoprotein that is expressed at a high level during embryogenesis but is almost absent during normal postnatal life. This multi-domain complex molecule is reported to associate with both pro-inflammatory and anti-inflammatory signalling cascades. In this study, we examined how TnC modulated intestinal inflammation. EXPERIMENTAL APPROACH: TnC pathophysiology was evaluated in cultures of rat intestinal subepithelial myofibroblasts (ISEMF) and intestinal epithelial cells. Wild-type and TnC(-/-) mice were treated with dextran sodium sulfate (DSS) to induce colitis. KEY RESULTS: DSS-induced colitis in mice markedly increased TnC in the damaged mucosal areas and up-regulated mRNA for TnC, pro-inflammatory cytokines and growth factors (PDGF-B and TGF-ß1). In addition, 2,4,6-trinitrobenzene sulfonic acid-induced colitis and SAMP1/Yit mice, a model of spontaneous Crohn's disease, also exhibited increased mucosal TnC in colon and ilea respectively. PDGF receptor-α (PDGFRα) positive ISEMF were the primary TnC-producing cells in colon tissues. Accordingly, ISEMF collected from the rat colon constitutively expressed both TnC and PDGFRα. PDGF-BB and TGF-ß1 up-regulated both TnC mRNA and protein levels in ISEMF. Knock-down of TnC gene increased susceptibility to DSS-induced colitis, compared with TnC(+/+) littermates. TnC(-/-) mice showed marked abrasion of intestinal mucosal barrier and increased inflammatory scores. Moreover, TnC accelerated both trans-well migration and wound healing in epithelial cells. CONCLUSIONS AND IMPLICATIONS: The pharmacological profiles of PDGF-BB and TGF-ß in colitis tissues and ISEMF suggest that increased TnC production during inflammation contributed to epithelial cell migration, remodelling and protection of intestinal barriers.
Assuntos
Antiulcerosos , Mucosa Intestinal/efeitos dos fármacos , Miofibroblastos/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Tenascina/biossíntese , Fator de Crescimento Transformador beta/farmacologia , Actinas/biossíntese , Actinas/genética , Animais , Western Blotting , Movimento Celular , Colite/metabolismo , Colite/patologia , Citocinas/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Imunofluorescência , Óperon Lac , Masculino , Camundongos , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Miofibroblastos/efeitos dos fármacos , RNA/biossíntese , RNA/genética , Ratos , Ratos Sprague-Dawley , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Cicatrização/efeitos dos fármacosRESUMO
Although similar patterns of phenotypic diversification are often observed in phylogenetically independent lineages, differences in the magnitude and direction of phenotypic divergence have been also observed among independent lineages, even when exposed to the same ecological gradients. The stickleback family is a good model with which to explore the ecological and genetic basis of parallel and nonparallel patterns of phenotypic evolution, because there are a variety of populations and species that are locally adapted to divergent environments. Although the patterns of phenotypic divergence as well as the genetic and ecological mechanisms have been well characterized in threespine sticklebacks, Gasterosteus aculeatus, we know little about the patterns of phenotypic diversification in other stickleback lineages. In eastern Hokkaido, Japan, there are three species of ninespine sticklebacks, Pungitius tymensis and the freshwater type and the brackish-water type of the P. pungitius-P. sinensis species complex. They utilize divergent habitats along coast-stream gradients of rivers. Here, we investigated genetic, ecological and phenotypic divergence among three species of Japanese ninespine sticklebacks. Divergence in trophic morphology and salinity tolerance occurred in the direction predicted by the patterns observed in threespine sticklebacks. However, the patterns of divergence in armour plate were different from those previously found in threespine sticklebacks. Furthermore, the genetic basis of plate variation may differ from that in threespine sticklebacks. Because threespine sticklebacks are well-established model for evolutionary research, the sympatric trio of ninespine sticklebacks will be an invaluable resource for ecological and genetic studies on both common and lineage-specific patterns of phenotypic diversification.
Assuntos
Especiação Genética , Smegmamorpha/anatomia & histologia , Smegmamorpha/fisiologia , Animais , Ecossistema , Japão , Fenótipo , Isolamento Reprodutivo , Smegmamorpha/genética , EstômagoRESUMO
Situs inversus totalis represents a complete mirror image anatomy of the normal arrangement of the thoracic and abdominal viscera. This rare condition may pose possible surgical problems due to anatomical abnormality. There were few reports of surgical treatment for lung cancer patient with situs inversus totalis. In this case report, we describe a 74-year-old patient with situs inversus totalis and primary lung cancer who underwent successful left upper lobectomy and systemic lymph node dissection. For this rare condition, detail preoperative evaluation of mirror image anatomy with computed tomography and bronchofiber optic examination was thought to be a key to carry out safe operative procedure.
Assuntos
Neoplasias Pulmonares/complicações , Situs Inversus/complicações , Carcinoma de Pequenas Células do Pulmão/complicações , Idoso , Humanos , MasculinoRESUMO
In vertebrates, sperm development and maturation are directly regulated by gonadal steroid hormone secretion. The relationships among the expression of genes encoding steroidogenic proteins and receptors for gonadotropins, and testicular steroid production have not yet been comprehensively determined in male teleosts. In this study, the changes in levels of mRNAs encoding follicle-stimulating hormone (FSH) receptor, luteinizing hormone (LH) receptor, steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage, 3beta-hydroxysteroid dehydrogenase/delta5-4-isomerase, cytochrome P450 17alpha-hydroxylase/17,20-lyase, cytochrome P450 11beta-hydroxylase, 11beta-hydroxysteroid dehydrogenase and 20beta-hydroxysteroid dehydrogenase were determined by real-time, quantitative PCR assays and related to changes in serum steroid levels throughout the reproductive cycle in male rainbow trout. Serum 11-ketotestosterone and 17alpha,20beta-dihydroxy-4-pregnen-3-one levels were measured by RIA. Although the pattern of change in the mRNA levels for the enzymes was variable, the increases in steroidogenic enzyme mRNAs started prior to a significant increase of serum steroid levels. The patterns of transcript levels of FSH and LH receptors suggest that changes in StAR and steroidogenic enzyme transcripts are largely mediated by the FSH receptor during early and mid-spermatogenesis and by the LH receptor during late spermatogenesis and spermiation. Levels of StAR (10-fold) and P450 17alpha-hydroxylase/17,20-lyase (sevenfold) transcripts changed with the greatest magnitude and were closely related to the changes in serum steroids, suggesting that changes in StAR and P450 17alpha-hydroxylase/17,20-lyase abundance are likely to be the major influences on overall steroidogenic output during the reproductive cycle in male rainbow trout.
Assuntos
Oncorhynchus mykiss/metabolismo , Fosfoproteínas/genética , Receptores Acoplados a Proteínas G/genética , Espermatogênese/fisiologia , Esteroide 17-alfa-Hidroxilase/genética , Testículo/metabolismo , 17-Hidroxiesteroide Desidrogenases/genética , 20-Hidroxiesteroide Desidrogenases/genética , 3-Hidroxiesteroide Desidrogenases/genética , Animais , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Sistema Enzimático do Citocromo P-450/genética , Expressão Gênica , Masculino , Oxigenases de Função Mista/genética , RNA Mensageiro/análise , RNA Ribossômico 18S/análise , Receptores do FSH/genética , Receptores do LH/genética , Proteínas Ribossômicas/genética , Esteroide 11-beta-Hidroxilase/genéticaRESUMO
PURPOSE: To investigate the feasibility of volumetric cine imaging in human cardiac studies by comparing in vivo and in vitro coronary angiography using a 256-detector row computed tomography (CT) without ECG gating. MATERIAL AND METHODS: The left and right coronary arteries of two domestic pigs were scanned in vivo and in vitro in cine mode using the 256-detector row CT. The device scanned approximately 100 mm in the cranio-caudal direction with one rotation, with a slice thickness of 0.5 mm. RESULTS: The coronary arteries could be observed to the third-degree branches in vitro, but could be visualized clearly only to the proximal portion (first-degree or second-degree branches) in vivo. CONCLUSION: Application of cardiac volumetric cine imaging with 256-detector row CT may be a promising means of obtaining diagnostic information and has potential for adoption to human studies.
Assuntos
Angiografia Coronária/métodos , Tomografia Computadorizada por Raios X/métodos , Animais , Estudos de Viabilidade , Processamento de Imagem Assistida por Computador , Técnicas In Vitro , SuínosRESUMO
Lowering of what we term a driver's Activation State (AS) during monotonous driving conditions may increase the risk of an accident. To develop an in-car environment that allows active driving - "Biofee dforward System" - we have investigated the effects of applying a stimulus of increased inspired oxygen fraction (F
RESUMO
Monotonous automobile operation in our daily life may cause the lowering of what might be termed an activation state of the human body, resulting in an increased risk of an accident. We therefore propose to create a more suitable environment in-car so as to allow active operation of the vehicle, hopefully thus avoiding potentially dangerous situations during driving. In order to develop such an activation method as a final goal, we have firstly focused on the acquisition of physiological variables, including cardiovascular parameters, during presentation to the driver of a monotonous screen image, simulating autonomous travel of constant-speed on a motorway. Subsequently, we investigated the derivation of a driver's activation index. During the screen image presentation, a momentary electrical stimulation of about 1 second duration was involuntarily applied to a subject's shoulder to obtain a physiological response. We have successfully monitored various physiological variables during the image presentation, and results suggest that a peculiar pattern in the beat-by-beat change of blood pressure in response to the involuntary stimulus may be an appropriate, and feasible, index relevant to activation state.
RESUMO
A 62-year-old female patient, who was diagnosed with sigmoid colon cancer with multiple liver metastases, was admitted to our hospital. She underwent sigmoidectomy with D3 lymph node dissection on January 31, 2000. In addition to that, she received hepatic intra-arterial infusion of levoforinate (l-LV) 250 mg and 5-fluorouracil (5-FU) 500 mg for combined multiple hepatic metastases starting on postoperative day 14, and these medications were administered over 48 hours once weekly by infuser pump. The tumor diminished by 59% 2 months after the start of administration and further diminished at 4 months. PR was achieved. Cancer metastasis to the cerebellum and metastasis to the lung were detected at month 9 and month 11, respectively, but the liver metastatic tumor continued to diminish in size, ultimately becoming undetectable by CT scan at month 10. Surgery and radiotherapy were performed for the cerebellar metastasis, and intravenous administration of a combination of l-LV and 5-FU was performed systemically for the pulmonary metastatic tumor. At present, the patient receives regular outpatient treatment continuously. To our knowledge, there has been no report on the combination therapy with l-LV and 5-FU through the hepatic artery. Since good antitumor efficacy was demonstrated in the present patient, this case is described in this report together with four other cases of hepatic metastasis from colorectal cancer.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/secundário , Neoplasias do Colo Sigmoide/patologia , Feminino , Fluoruracila/administração & dosagem , Artéria Hepática , Humanos , Infusões Intra-Arteriais , Leucovorina/administração & dosagem , Pessoa de Meia-IdadeRESUMO
Hepatoblastomas (HBs) were induced in B6C3F1 male mice by diethylnitrosamine (DEN) and sodium phenobarbital (PB). Six-week-old mice received a single intraperitoneal dose of DEN followed by a continuous treatment with PB in diet at a concentration of 0 (group 1) or 500 (group 2) ppm for 50 weeks. HBs were observed in 13 of 21 (62%) group 2 mice, with typical histologic features as reported previously, while no such tumors were observed in group 1. Seven of 13 (54%) HBs were found in and/or adjacent to hepatocellular adenomas (HCAs) or hepatocellular carcinomas (HCCs). Immunohistochemically, all HBs were positive for S-100 protein but negative for keratin, alpha-fetoprotein (AFP), albumin (ALB) and vimentin, while HCC cells occasionally reacted positively for AFP with a mosaic pattern. HCC and HCA cells were occasionally positive for ALB. Non-neoplastic hepatocytes and normal bile ducts were positively stained for ALB and keratin/S-100 protein, respectively. S-100 protein is known to be expressed in many mesenchymal tissues and neoplasms including neuroectodermal elements but negative in cells of the hepatic lineage. Thus, the present immunohistochemical results suggested that mesenchymal differentiation occurs in mouse HB cells as observed in human HBs, one of the most frequent infant liver tumors in humans. Although the susceptibility of mouse HBs to PB-promotion suggests a hepatocytic histogenesis, the present immunohistochemical results support the hypothesis that the mouse HB is derived from pluripotent endodermal stem-like cells.
Assuntos
Neoplasias Hepáticas Experimentais/patologia , Albuminas/análise , Alquilantes/toxicidade , Animais , Dietilnitrosamina/toxicidade , Imuno-Histoquímica , Queratinas/análise , Neoplasias Hepáticas Experimentais/induzido quimicamente , Masculino , Camundongos , Fenobarbital/toxicidade , Distribuição Aleatória , Proteínas S100/análise , Vimentina/análise , alfa-Fetoproteínas/análiseRESUMO
Transforming growth factor alpha (TGF-alpha) is a potent stimulator of normal hepatocyte proliferation, considered to have relationship to the liver regeneration or carcinogenesis. In this study, we investigated immunohistochemically the association between expression of TGF-alpha and cell proliferation activity in mouse hepatoblastomas (HBs) and hepatocellular carcinomas (HCCs) induced in B6C3F1 mice by diethylnitrosamine and sodium phenobarbital. The TGF-alpha-positive rate in HBs (29.2%) was significantly higher than that in HCCs (12.7%). Likewise, the proliferating cell nuclear antigen-positive rate (22.2%) was higher than the HCC value (14.5%). On the individual data for both TGF-alpha and PCNA, most of the HBs showed higher positive rates than HCCs. In HBs, TGF-alpha was localized only in the nuclei, whereas some HCC cells stained positive both in their nuclei and cytoplasm (0.6%). These results suggest expression of TGF-alpha and its localization might be linked to cell proliferation and play a role in malignant progression of mouse HBs.
Assuntos
Carcinoma Hepatocelular/metabolismo , Dietilnitrosamina/farmacologia , Hepatoblastoma/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Fenobarbital/farmacologia , Antígeno Nuclear de Célula em Proliferação/análise , Fator de Crescimento Transformador alfa/biossíntese , Administração Oral , Animais , Testes de Carcinogenicidade/métodos , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/patologia , Divisão Celular/fisiologia , Núcleo Celular/química , Núcleo Celular/efeitos dos fármacos , Citoplasma/química , Citoplasma/efeitos dos fármacos , Dietilnitrosamina/administração & dosagem , Hepatoblastoma/induzido quimicamente , Hepatoblastoma/patologia , Imuno-Histoquímica , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/patologia , Masculino , Camundongos , Camundongos Endogâmicos , Fenobarbital/administração & dosagem , Antígeno Nuclear de Célula em Proliferação/imunologia , Fatores de Tempo , Fator de Crescimento Transformador alfa/imunologiaRESUMO
FRS2 has been identified in mammalian cells as a protein that is tyrosine phosphorylated and binds to Grb2 and Shp2 in response to fibroblast growth factor (FGF) or nerve growth factor (NGF) stimulation. But neither its existence in other vertebrate classes or invertebrates nor its function during embryonic development has been defined. Here we have identified and characterized a Xenopus homolog of FRS2 (xFRS2). xFRS2 is tyrosine phosphorylated in early embryos, and overexpression of an unphosphorylatable form of xFRS2 interferes with FGF-dependent mesoderm formation. The Src family kinase Laloo, which was shown to function in FGF signaling during early Xenopus development, binds to xFRS2 and promotes tyrosine phosphorylation of xFRS2. Moreover, xFRS2 and Laloo are shown to bind to Xenopus FGF receptor 1. These results suggest that xFRS2 plays an important role in FGF signaling in cooperation with Laloo during embryonic development.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Desenvolvimento Embrionário , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Transdução de Sinais/fisiologia , Proteínas de Xenopus , Xenopus laevis/embriologia , Quinases da Família src/metabolismo , Sequência de Aminoácidos , Animais , Padronização Corporal/fisiologia , Clonagem Molecular , Embrião não Mamífero/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Humanos , Hibridização In Situ , Substâncias Macromoleculares , Proteínas de Membrana/química , Proteínas de Membrana/genética , Dados de Sequência Molecular , Fosfoproteínas/química , Fosfoproteínas/genética , Fosforilação , Ligação Proteica , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Alinhamento de Sequência , Xenopus laevis/metabolismo , Quinases da Família src/genéticaRESUMO
A transgenic mouse containing the first exon of the human Huntington's disease (HD) gene has revealed a variety of behavioral and pathophysiological anomalies reminiscent of certain aspects of human Huntington's disease (HD). The present study has found that expression of the extracellular matrix glycoprotein tenascin-C appears to be unaffected in astroglial cells in wild-type and R6/2 transgenic mice that express the mutant huntingtin protein but that it is conspicuously absent in two neuronal populations within the cerebral cortex and thalamus of the R6/2 mice. Loss of tenascin-C expression begins between the fourth and eighth postnatal weeks, coincidental with the onset of abnormal behavioral phenotype and the appearance of intranuclear inclusion bodies and neuropil aggregates. By 12 weeks, R6/2 mice exhibit a complete absence of tenascin-C neuronal immunolabeling, a disappearance of cRNA probe-positive neurons across discrete cytoarchitectonic regions of the dorsal thalamus (e.g., the ventromedial, parafascicular, lateral posterior, and posterior thalamic groups) and frontal cortex, and an accompanying thalamic astrogliosis. The loss of neuronal tenascin-C expression includes structures that are known to send converging excitatory axonal projections to the caudate-putamen, the structure that is most at risk for neurodegeneration in HD. Altered neuronal expression of tenascin-C in R6/2 mice implicates altered transcriptional activities of the mutant huntingtin protein. The abnormal biochemistry and possibly abnormal activity of thalamostriate and corticostriate projection neurons may also affect abnormal neuronal activities in their primary connectional target, the neostriatum, which is severely compromised in HD.
Assuntos
Córtex Cerebral/fisiologia , Doença de Huntington/fisiopatologia , Camundongos Knockout/fisiologia , Tenascina/genética , Tálamo/fisiologia , Animais , Química Encefálica/genética , Córtex Cerebral/citologia , Modelos Animais de Doenças , Éxons , Feminino , Expressão Gênica/fisiologia , Humanos , Imuno-Histoquímica , Hibridização In Situ , Óperon Lac , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuroglia/fisiologia , Neurônios/fisiologia , RNA Mensageiro/análise , Tenascina/análise , Tálamo/citologiaRESUMO
We have systematically characterized gene expression patterns in 49 adult and embryonic mouse tissues by using cDNA microarrays with 18,816 mouse cDNAs. Cluster analysis defined sets of genes that were expressed ubiquitously or in similar groups of tissues such as digestive organs and muscle. Clustering of expression profiles was observed in embryonic brain, postnatal cerebellum, and adult olfactory bulb, reflecting similarities in neurogenesis and remodeling. Finally, clustering genes coding for known enzymes into 78 metabolic pathways revealed a surprising coordination of expression within each pathway among different tissues. On the other hand, a more detailed examination of glycolysis revealed tissue-specific differences in profiles of key regulatory enzymes. Thus, by surveying global gene expression by using microarrays with a large number of elements, we provide insights into the commonality and diversity of pathways responsible for the development and maintenance of the mammalian body plan.
Assuntos
Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Animais , Sequência de Bases , Sistema Nervoso Central/metabolismo , Primers do DNA , DNA Complementar , Regulação da Expressão Gênica no Desenvolvimento , CamundongosRESUMO
Using the new transgenic mice produced by mating gpt delta with p53 knockout, mutation induction by heavy-ion irradiation and the effect of p53 background on such induction were studied. After the whole body irradiation with 10 Gy of 135 MeV/u carbon-ion beam, the genomic DNA was isolated from the different organs and the lambda DNA was rescued as a phage. Mutations in the transgene on the lambda DNA were determined by the spi(-) selection (deletion assay). The spi(-) mutation was induced by the above irradiation, but enhancement of the mutant frequency by the knockout of p53 gene was found not in the phages recovered from liver but in those from kidney. We are now making an effort to determine the nature of spi(-) mutation to confirm such p53 effect.
Assuntos
Raios gama , Genes p53/efeitos da radiação , Íons Pesados , Mutação , Raios X , Animais , Relação Dose-Resposta à Radiação , Rim/efeitos da radiação , Fígado/efeitos da radiação , Masculino , Camundongos , Camundongos Transgênicos , Baço/efeitos da radiação , Testículo/efeitos da radiação , Irradiação Corporal TotalAssuntos
Transplante de Células/métodos , Hepatócitos/transplante , Fígado/fisiologia , Animais , Animais Recém-Nascidos , Divisão Celular , Hepatectomia , Hepatócitos/citologia , Hepatócitos/fisiologia , Fígado/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Baço , Transplante HeterólogoRESUMO
Stem cells responsible for tissue maintenance and repair are found in a number of organs. However, hepatic stem cells assumed to play a key role in liver development and regeneration remain to be well characterized. To address this issue, we set up a culture system in which primitive hepatic progenitor cells formed colonies. By combining this culture system with fluorescence-activated cell sorting (FACS), cells forming colonies containing distinct hepatocytes and cholangiocytes were identified in the fetal mouse liver. These cells express both CD49f and CD29 (alpha6 and beta1 integrin subunits), but do not mark for hematopoietic antigens such as CD45, TER119, and c-Kit. When transplanted into the spleen, these cells migrated to the recipient liver and differentiated into liver parenchymal cells. Our data demonstrate that hepatic progenitor cells are enriched by FACS and suggest approaches to supplanting organ allografting and improving artificial-organ hepatic support.
Assuntos
Fígado/citologia , Fígado/embriologia , Células-Tronco/citologia , Animais , Antígenos CD/análise , Diferenciação Celular , Divisão Celular , Movimento Celular , Separação Celular , Citometria de Fluxo , Hepatócitos/citologia , Integrina alfa6 , Integrina beta1/análise , Camundongos , Camundongos Endogâmicos BALB C , Baço/fisiologia , Transplante de Células-Tronco , Células-Tronco/imunologia , Células-Tronco/fisiologiaRESUMO
Peptidylarginine deiminase catalyzes the post-translational modification of proteins through the conversion of arginine to citrulline in the presence of calcium ions. In rodents, peptidylarginine deiminase has been classified into four isoforms, types I, II, III, and IV, which are distinct in their molecular weights, substrate specificities, and tissue localization. Of these isoforms, only type III was detected in epidermis and hair follicles. Although the role of this enzyme in these tissues is not yet clear, indirect data have shown that several structural proteins such as filaggrin, trichohyalin, and keratin are substrates for peptidylarginine deiminase. In this study, we cloned the full-length cDNA of human peptidylarginine deiminase type III (3142 bp) from cultured human keratinocytes by reverse transcription-polymerase chain reaction and by rapid amplification of cDNA ends methods. This cDNA contained a 1995 bp open reading frame encoding 664 amino acids (Mr = 74 770). To explore the physicochemical and enzymatic properties of human peptidylarginine deiminase type III, we constructed a plasmid for producing a recombinant human peptidylarginine deiminase type III in bacteria. The enzymatic characteristics of the recombinant enzyme were very similar to those of the rodent peptidylarginine deiminase type III. The recombinant enzyme showed the catalytic activities toward structural proteins of epidermis and hair follicle, filaggrin and trichohyalin, in which the deiminations maxima of about 60% and 13% arginine residues were observed in filaggrin and trichohyalin, respectively. An immunohistochemical study of human scalp skin with a monospecific anti-peptidyl-arginine deiminase type III antibody revealed that the type III enzyme was localized to the inner root sheath and outer root sheath of hair follicles. Peptidylarginine deiminase type III in the inner root sheath was notable between supramatrix and keratogenous zone and was scarcely detected in cornified hair zone. The enzyme was also expressed in the cuticle layer of hair. On the other hand, expression of the enzyme in the epidermis was very low. These data imply that human peptidylarginine deiminase type III is the predominant isoform in hair follicles and may function as a modulator of hair structural proteins, including trichohyalin during hair and hair follicle formation.
Assuntos
Hidrolases , Isoenzimas , Sequência de Aminoácidos , Formação de Anticorpos , Sequência de Bases , Western Blotting , Clonagem Molecular , DNA Complementar/química , Feminino , Proteínas Filagrinas , Humanos , Hidrolases/genética , Hidrolases/imunologia , Imuno-Histoquímica , Proteínas de Filamentos Intermediários/genética , Isoenzimas/genética , Isoenzimas/imunologia , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas , Proteínas Recombinantes/química , Sequências Repetitivas de Ácido Nucleico , Pele/químicaRESUMO
Gene delivery has shown potential in a variety of applications, including basic research, therapies for inborn genetic defects, cancer, AIDS, tissue engineering, and vaccination. Most available systems have serious drawbacks, such as safety hazards, inefficiency under in vivo-like conditions, and expensive production. When using naked DNA, for instance, a large amount of ultrapure DNA has to be applied as a result of degradation by nucleases. Similarly, the use of eukaryotic histones, synthetic peptides, or peptide nucleic acids may be limited by high production costs. We have demonstrated a biotechnologically feasible and economical approach for gene delivery using the histone-like protein from the hyperthermostable eubacterium Thermotoga maritima, TmHU as an efficient gene transfer reagent. HU can be easily isolated from recombinant Escherichia coli, is extraordinarily stable, and protects dsDNA from thermal denaturation. This study demonstrates its use as an inexpensive tool for gene delivery.
Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/genética , Técnicas de Transferência de Genes , Transfecção/métodos , Células 3T3 , Animais , Linhagem Celular , Proteínas de Ligação a DNA/química , Relação Dose-Resposta a Droga , Células Eucarióticas , Feminino , Galactosídeos/metabolismo , Histonas/química , Humanos , Indóis/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/metabolismo , Temperatura , Thermotoga maritima/química , Fatores de TempoRESUMO
Germ line mutations of the RET proto-oncogene are responsible for the development of multiple endocrine neoplasia type 2A (MEN 2A), an inherited cancer syndrome characterized by medullary thyroid carcinoma, pheochromocytoma, and parathyroid hyperplasia. To study the mechanism of tissue-specific tumor development by RET with a MEN2A (cysteine 634-->arginine) mutation, we generated transgenic mice by introducing the RET-MEN2A gene fused to Moloney murine leukemia virus long terminal repeat. Expression of the transgene and its product was detected at variable levels in a variety of tissues including thyroid, heart, liver, colon, parotid gland, and brain. All of 29 mice analyzed developed thyroid C-cell hyperplasia or medullary carcinoma, accompanying high levels of serum calcitonin. In addition, development of mammary or parotid gland adenocarcinoma was observed in one-half of the transgenic mice. RET dimerization and its complex formation with Shc and Grb2 adaptor proteins were detected in tumor tissues. Unexpectedly, no tumor formation was found in other tissues despite RET-MEN2A expression where RET dimerization was undetectable. Because these tissues but not tumors expressed glial cell line-derived neurotrophic factor family receptor alpha (GFR alpha) at high levels, this suggested that GFR alpha expression may interfere in the dimerization of the RET-MEN2A mutant proteins, leading to tissue-specific tumor development in vivo.