RESUMO
Interspecific hybridization is an important evolutionary process, which has significant influence on the diversity within and between participating taxa. Although interspecific hybridization in terrestrial and freshwater organisms has been subjected to many detailed studies, studies in marine realm have been limited in terms of both numbers and detail. In this study, the potential for interspecific hybridization between two rockfishes, Sebastes vulpes and S. zonatus, occurring in the western North Pacific, was assessed on the basis of 177 specimens collected from three sampling localities within the main geographic distribution of both species, and analysed using a combination of amplified fragment length polymorphisms (AFLP), mitochondrial DNA (mtDNA) markers and morphometric characters. Bayesian-based individual genetic assignment based on 364 AFLP loci detected a total of 63 (35.6%) hybrid specimens in the data set, the presence of interspecific hybrids also being rigorously supported by mtDNA analysis using partial sequences from the control region and morphological analysis based on 31 morphometric characters. Hybrids from all three localities were found, showing a common pattern of biased introgression across the localities whereby hybrids were more closely related to S. zonatus than to S. vulpes. Apart from this common pattern, rates of hybridization varied considerably among the localities, being greater in the northern localities. Variations in the local rates of hybridization were associated with variations in habitat segregation and thermal regime, implying that vertical water temperature regimes determined the extent of habitat segregation of the two species and, accordingly, the opportunity for hybridization.
Assuntos
Peixes/genética , Hibridização Genética , Animais , Ecossistema , Feminino , Peixes/anatomia & histologia , Japão , Masculino , Oceano Pacífico , TemperaturaRESUMO
OX40 expressed on activated T cells is known to be an important costimulatory molecule on T cell activation in vitro. However, the in vivo functional significance of the interaction between OX40 and its ligand, OX40L, is still unclear. To investigate the role of OX40L during in vivo immune responses, we generated OX40L-deficient mice and a blocking anti-OX40L monoclonal antibody, MGP34. OX40L expression was demonstrated on splenic B cells after CD40 and anti-immunoglobulin (Ig)M stimulation, while only CD40 ligation was capable of inducing OX40L on dendritic cells. OX40L-deficient and MGP34-treated mice engendered apparent suppression of the recall reaction of T cells primed with both protein antigens and alloantigens and a significant reduction in keyhole limpet hemocyanin-specific IgG production. The impaired T cell priming was also accompanied by a concomitant reduction of both T helper type 1 (Th1) and Th2 cytokines. Furthermore, antigen-presenting cells (APCs) derived from the mutant mice revealed an impaired intrinsic APC function, demonstrating the importance of OX40L in both the priming and effector phases of T cell activation. Collectively, these results provide convincing evidence that OX40L, expressed on APCs, plays a critical role in antigen-specific T cell responses in vivo.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Glicoproteínas de Membrana , Receptores do Fator de Necrose Tumoral/imunologia , Animais , Anticorpos Monoclonais/imunologia , Linfócitos B/imunologia , Células Cultivadas , Meios de Cultura , Citocinas/biossíntese , Células Dendríticas/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofenotipagem , Isoantígenos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligante OX40 , Receptores do Fator de Necrose Tumoral/genética , Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Fatores de Necrose TumoralRESUMO
BACKGROUND: A striking efficiency of interferon (IFN)-based anticancer therapy for advanced hepatocellular carcinoma (HCC) has been reported. Because its clinical efficiency greatly depends on each patient's local response, prediction of local response is crucial. METHODS: Continuous exposure of IFN-alpha to parental PLC/PRF/5 cells (PLC-P) and a limiting dilution method resulted in the establishment of IFN-resistant cell clones (PLC-Rs). Microarray analyses of PLC-P and PLC-Rs identified insulin-like growth factor-binding protein 7 (IGFBP7) as one of the most significantly downregulated genes in PLC-Rs. Changes in anticancer effects of IFN-alpha were examined in HCC cells after genetic manipulation of IGFBP7 expression. The correlation between immunohistochemically determined IGFBP7 expression and the response to IFN-alpha/5-fluorouracil (5-FU) therapy was investigated in surgically resected HCC specimens. RESULTS: PLC-R cells showed a remarkable downregulation of IGFBP7 and resistance to IFN-alpha, compared with PLC-P. Parental PLC/PRF/5 cells transfected with short hairpin RNA against IGFBP7 showed a significant resistance to IFN-alpha relative to control cells (IC(50) fold increase=14.38 times). Insulin-like growth factor-binding protein 7 transfection into PLC-R restored sensitivity to IFN-alpha. In resected specimens, IGFBP7 expression significantly correlated with the response to IFN-alpha/5-FU therapy. CONCLUSION: IGFBP7 could be a useful predictor of the response to IFN-based therapy in advanced HCC.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Hepatocelular/genética , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Interferon-alfa/farmacologia , Neoplasias Renais/genética , Biomarcadores Tumorais/análise , Western Blotting , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/mortalidade , Linhagem Celular Tumoral , Fluoruracila/uso terapêutico , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Imunoprecipitação , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Estimativa de Kaplan-Meier , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/mortalidade , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Two scombropid fishes, Scombrops boops and Scombrops gilberti, are closely related and commercially important species in Japan. These species are often confused in commercial markets because of their morphological similarity. In this study, scombropid specimens collected from various Japanese coastal waters were subjected to polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and phylogenetic analysis of the 16S rRNA gene in mitochondrial DNA. These analyses showed that all the scombropid specimens collected from localities in the Sea of Japan were identified as S. boops, whereas those from the Pacific Ocean included two species, S. boops and S. gilberti. Almost all juvenile (<200 mm standard body length, S(L)) S. gilberti originated from the Pacific coastal waters of the northern Japan, whereas adults (>400 mm S(L)) were found only in deep water off the Izu Peninsula to the Izu Islands. This suggests that S. gilberti might migrate extensively during its life cycle. In addition, differences in the number of specimens and the distribution between the two species suggest that S. gilberti is less abundant than S. boops in Japanese waters.
Assuntos
Biodiversidade , Perciformes/classificação , Perciformes/genética , Animais , DNA Mitocondrial/genética , Demografia , Japão , Oceano Pacífico , Filogenia , RNA Ribossômico 16S/genética , Estações do AnoRESUMO
BACKGROUND: Hepatocellular carcinoma (HCC) with tumour thrombus (TT) in the inferior vena cava (IVC) or right atrium (RA) is a rare advanced disease state with a poor prognosis. The aim of this study was to examine survival after surgical resection. METHODS: Patients with HCC and TT of either the IVC or RA, who underwent liver resection between February 1997 and July 2017, were included. Their short- and long-term outcomes and surgical details were analysed retrospectively. RESULTS: Thirty-seven patients were included; 16 patients had TT in the IVC below the diaphragm, eight had TT in the IVC above the diaphragm, and 13 had TT entering the RA. Twelve patients had advanced portal vein TT (portal vein invasion (Vp) greater than Vp3 and Vp4), ten had bilobar disease, and 12 had extrahepatic disease. There were no in-hospital deaths, although two patients died within 90 days. Median survival did not differ between patients who had resection with curative intent (18·7 months) and those with residual tumour in the lung only (20·7 months), but survival was poor for patients with residual tumour in the liver (8·3 months). CONCLUSION: Liver resection with thrombectomy for advanced HCC with TT in the IVC or RA is safe and feasible, leading to moderate survival.
ANTECEDENTES: El carcinoma hepatocelular con trombo tumoral (TT) en la vena cava inferior (inferior vena cava, IVC) o en la aurícula derecha (right atrium, RA) es un estado avanzado de la enfermedad raro, con un pronóstico desfavorable. En este estudio analizamos la supervivencia después de la resección quirúrgica. MÉTODOS: Se incluyeron pacientes con carcinoma hepatocelular con TT en la IVC o en la RA, que se sometieron a resección hepática entre febrero de 1997 y julio de 2017. Los resultados a corto y a largo plazo de estos pacientes y los detalles quirúrgicos se analizaron retrospectivamente. RESULTADOS: Se incluyeron 37 pacientes. Entre estos pacientes, se identificaron 16 pacientes con TT en la IVC infradiafragmática, 8 pacientes con TT en la IVC supradiafragmática y 13 pacientes con TT entrando en la AR. Doce pacientes asociaron TT avanzado en la vena porta más allá de vp 3 y 4, 10 pacientes tenían enfermedad bilobar y 12 pacientes tenían enfermedad extrahepática. A pesar de que la tasa de mortalidad hospitalaria fue cero, dos pacientes fallecieron a los 90 días. Aunque la mediana del tiempo de supervivencia no fue diferente entre el grupo al que se le realizó resección con intención curativa (18,7 meses) y aquellos con tumor residual solo en el pulmón (20,7 meses), la supervivencia fue extremadamente pobre para los pacientes con tumor residual en el hígado (8,3 meses). CONCLUSIÓN: La resección hepática con trombectomía para el carcinoma hepatocelular avanzado con trombo tumoral en la vena cava inferior o en la aurícula derecha es segura y factible, asociándose a una supervivencia moderada.
Assuntos
Carcinoma Hepatocelular/cirurgia , Átrios do Coração/cirurgia , Hepatectomia/métodos , Neoplasias Hepáticas/cirurgia , Trombectomia/métodos , Veia Cava Inferior/cirurgia , Idoso , Carcinoma Hepatocelular/mortalidade , Feminino , Humanos , Japão , Neoplasias Hepáticas/mortalidade , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Análise de SobrevidaRESUMO
Type I IFN receptor type 2 (IFNAR2) expression correlates significantly with clinical response to interferon (IFN)-alpha/5-fluorouracil (5-FU) combination therapy for hepatocellular carcinoma (HCC). However, some IFNAR2-positive patients show no response to the therapy. This result suggests the possibility of other factors, which would be responsible for resistance to IFN-alpha/5-FU therapy. The aim of this study was to examine the mechanism of anti-proliferative effects of IFN-alpha/5-FU therapy and search for a biological marker of chemoresistance to such therapy. Gene expression profiling and molecular network analysis were used in the analysis of non-responders and responders with IFNAR2-positive HCC. The Wnt/beta-catenin signalling pathway contributed to resistance to IFN-alpha/5-FU therapy. Immunohistochemical analysis showed positive epithelial cell adhesion molecule (Ep-CAM) expression, the target molecule of Wnt/beta-catenin signalling, only in non-responders. In vitro studies showed that activation of Wnt/beta-catenin signalling by glycogen synthesis kinase-3 inhibitor (6-bromoindirubin-3'-oxime (BIO)) induced chemoresistance to IFN-alpha/5-FU. BrdU-based cell proliferation ELISA and cell cycle analysis showed that concurrent addition of BIO and IFN-alpha/5-FU significantly to hepatoma cell cultures reduced the inhibitory effects of the latter two on DNA synthesis and accumulation of cells in the S-phase. The results indicate that activation of Wnt/beta-catenin signalling pathway induces chemoresistance to IFN-alpha/5-FU therapy and suggest that Ep-CAM is a potentially useful marker for resistance to such therapy, especially in IFNAR2-positive cases.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Hepáticas/tratamento farmacológico , Proteínas Wnt/fisiologia , beta Catenina/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/fisiologia , Carcinoma Hepatocelular/genética , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/fisiologia , Linhagem Celular Tumoral , Molécula de Adesão da Célula Epitelial , Feminino , Fluoruracila/administração & dosagem , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Interferon-alfa/administração & dosagem , Neoplasias Hepáticas/genética , Masculino , Pessoa de Meia-Idade , Mutação/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Receptor de Interferon alfa e beta/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteínas Wnt/genética , beta Catenina/genéticaRESUMO
Intracisternal granules (ICG) develop in the rough ER of hyperstimulated thyrotrophs or thyroid hormone-secreting cells of the anterior pituitary gland. To determine the fate of these granules, we carried out morphological and immunocytochemical studies on pituitaries of thyroxine-treated, thyroidectomized rats. Under these conditions the ER of thyrotrophs is dramatically dilated and contains abundant ICG; the latter contain beta subunits of thyrotrophic hormone (TSH-beta). Based on purely morphologic criteria, intermediates were identified that appeared to represent stages in the transformation of a part rough/part smooth ER cisterna into a lysosome. Using immunocytochemical and cytochemical markers, two major types of intermediates were distinguished: type 1 lacked ribosomes but were labeled with antibodies against both ER markers (PDI, KDEL, ER membrane proteins) and a lysosomal membrane marker, lgp120. They also were reactive for the lysosomal enzyme, acid phosphatase, by enzyme cytochemistry. Type 2 intermediates were weakly reactive for ER markers and contained both lgp120 and lysosomal enzymes (cathepsin D, acid phosphatase). Taken together these results suggest that in hyperstimulated thyrotrophs part rough/part smooth ER elements containing ICG lose their ribosomes, their membrane is modified, and they sequentially acquire a lysosome-type membrane and lysosomal enzymes. The findings are compatible with the conclusion that a pathway exists by which under certain conditions, secretory proteins present in the ER as well as ER membrane and content proteins can be degraded by direct conversion of ER cisternae into lysosomes.
Assuntos
Fosfatase Ácida/metabolismo , Catepsina D/metabolismo , Retículo Endoplasmático/metabolismo , Lisossomos/metabolismo , Adeno-Hipófise/metabolismo , Tireotropina/metabolismo , Animais , Retículo Endoplasmático/ultraestrutura , Imunofluorescência , Isomerases/metabolismo , Microscopia Imunoeletrônica , Adeno-Hipófise/ultraestrutura , Isomerases de Dissulfetos de Proteínas , Ratos , Ratos Endogâmicos , TimectomiaRESUMO
Vps30p/Apg6p is required for both autophagy and sorting of carboxypeptidase Y (CPY). Although Vps30p is known to interact with Apg14p, its precise role remains unclear. We found that two proteins copurify with Vps30p. They were identified by mass spectrometry to be Vps38p and Vps34p, a phosphatidylinositol (PtdIns) 3-kinase. Vps34p, Vps38p, Apg14p, and Vps15p, an activator of Vps34p, were coimmunoprecipitated with Vps30p. These results indicate that Vps30p functions as a subunit of a Vps34 PtdIns 3-kinase complex(es). Phenotypic analyses indicated that Apg14p and Vps38p are each required for autophagy and CPY sorting, respectively, whereas Vps30p, Vps34p, and Vps15p are required for both processes. Coimmunoprecipitation using anti-Apg14p and anti-Vps38p antibodies and pull-down experiments showed that two distinct Vps34 PtdIns 3-kinase complexes exist: one, containing Vps15p, Vps30p, and Apg14p, functions in autophagy and the other containing Vps15p, Vps30p, and Vps38p functions in CPY sorting. The vps34 and vps15 mutants displayed additional phenotypes such as defects in transport of proteinase A and proteinase B, implying the existence of another PtdIns 3-kinase complex(es). We propose that multiple Vps34p-Vps15p complexes associated with specific regulatory proteins might fulfill their membrane trafficking events at different sites.
Assuntos
Autofagia , Carboxipeptidases/metabolismo , Proteínas Fúngicas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico/fisiologia , Saccharomyces cerevisiae/enzimologia , Catepsina A , Fracionamento Celular , Complexos Endossomais de Distribuição Requeridos para Transporte , Immunoblotting , Substâncias Macromoleculares , Modelos Biológicos , Mutação , Fenótipo , Plasmídeos , Testes de Precipitina , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae , Proteína VPS15 de Distribuição VacuolarRESUMO
For determination of the physiological role and mechanism of vacuolar proteolysis in the yeast Saccharomyces cerevisiae, mutant cells lacking proteinase A, B, and carboxypeptidase Y were transferred from a nutrient medium to a synthetic medium devoid of various nutrients and morphological changes of their vacuoles were investigated. After incubation for 1 h in nutrient-deficient media, a few spherical bodies appeared in the vacuoles and moved actively by Brownian movement. These bodies gradually increased in number and after 3 h they filled the vacuoles almost completely. During their accumulation, the volume of the vacuolar compartment also increased. Electron microscopic examination showed that these bodies were surrounded by a unit membrane which appeared thinner than any other intracellular membrane. The contents of the bodies were morphologically indistinguishable from the cytosol; these bodies contained cytoplasmic ribosomes, RER, mitochondria, lipid granules and glycogen granules, and the density of the cytoplasmic ribosomes in the bodies was almost the same as that of ribosomes in the cytosol. The diameter of the bodies ranged from 400 to 900 nm. Vacuoles that had accumulated these bodies were prepared by a modification of the method of Ohsumi and Anraku (Ohsumi, Y., and Y. Anraku. 1981. J. Biol. Chem. 256:2079-2082). The isolated vacuoles contained ribosomes and showed latent activity of the cytosolic enzyme glucose-6-phosphate dehydrogenase. These results suggest that these bodies sequestered the cytosol in the vacuoles. We named these spherical bodies "autophagic bodies." Accumulation of autophagic bodies in the vacuoles was induced not only by nitrogen starvation, but also by depletion of nutrients such as carbon and single amino acids that caused cessation of the cell cycle. Genetic analysis revealed that the accumulation of autophagic bodies in the vacuoles was the result of lack of the PRB1 product proteinase B, and disruption of the PRB1 gene confirmed this result. In the presence of PMSF, wild-type cells accumulated autophagic bodies in the vacuoles under nutrient-deficient conditions in the same manner as did multiple protease-deficient mutants or cells with a disrupted PRB1 gene. As the autophagic bodies disappeared rapidly after removal of PMSF from cultures of normal cells, they must be an intermediate in the normal autophagic process. This is the first report that nutrient-deficient conditions induce extensive autophagic degradation of cytosolic components in the vacuoles of yeast cells.
Assuntos
Autofagia/fisiologia , Macrolídeos , Saccharomyces cerevisiae/fisiologia , Vacúolos/fisiologia , Acetatos/farmacologia , Antibacterianos/farmacologia , Autofagia/efeitos dos fármacos , Calcimicina/farmacologia , Carbono/metabolismo , Cloranfenicol/farmacologia , Cicloeximida/farmacologia , Endopeptidases/genética , Endopeptidases/metabolismo , Etilmaleimida/farmacologia , Nitrogênio/metabolismo , Proteínas/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/ultraestrutura , Vacúolos/efeitos dos fármacos , Vacúolos/ultraestruturaRESUMO
Microtubule-associated protein 1B (MAP1B), one of the microtubule-associated proteins (MAPs), is a major component of the neuronal cytoskeleton. It is expressed at high levels in immature neurons during growth of their axons, which indicates that it plays a crucial role in neuronal morphogenesis and neurite extension. To better define the role of MAP1B in vivo, we have used gene targeting to disrupt the murine MAP1B gene. Heterozygotes of our MAP1B disruption exhibit no overt abnormalities in their development and behavior, while homozygotes showed a slightly decreased brain weight and delayed nervous system development. Our data indicate that while MAP1B is not essential for survival, it is essential for normal time course development of the murine nervous system. These conclusions are very different from those of a previous MAP1B gene-targeting study (Edelmann, W., M. Zervas, P. Costello, L. Roback, I. Fischer, A. Hammarback, N. Cowan, P. Davis, B. Wainer, and R. Kucherlapati. 1996. Proc. Natl. Acad. Sci. USA. 93: 1270-1275). In this previous effort, homozygotes died before reaching 8-d embryos, while heterozygotes showed severely abnormal phenotypes in their nervous systems. Because the gene targeting event in these mice produced a gene encoding a 571-amino acid truncated product of MAP1B, it seems likely that the phenotypes seen arise from the truncated MAP1B product acting in a dominant-negative fashion, rather than a loss of MAP1B function.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas Associadas aos Microtúbulos/genética , Sistema Nervoso/embriologia , Animais , Deleção de Genes , Marcação de Genes , Homozigoto , Camundongos , Camundongos MutantesRESUMO
Physiological roles of the members of the synaptophysin family, carrying four transmembrane segments and being basically distributed on intracellular membranes including synaptic vesicles, have not been established yet. Recently, mitsugumin29 (MG29) was identified as a novel member of the synaptophysin family from skeletal muscle. MG29 is expressed in the junctional membrane complex between the cell surface transverse (T) tubule and the sarcoplasmic reticulum (SR), called the triad junction, where the depolarization signal is converted to Ca(2+) release from the SR. In this study, we examined biological functions of MG29 by generating knockout mice. The MG29-deficient mice exhibited normal health and reproduction but were slightly reduced in body weight. Ultrastructural abnormalities of the membranes around the triad junction were detected in skeletal muscle from the mutant mice, i.e., swollen T tubules, irregular SR structures, and partial misformation of triad junctions. In the mutant muscle, apparently normal tetanus tension was observed, whereas twitch tension was significantly reduced. Moreover, the mutant muscle showed faster decrease of twitch tension under Ca(2+)-free conditions. The morphological and functional abnormalities of the mutant muscle seem to be related to each other and indicate that MG29 is essential for both refinement of the membrane structures and effective excitation-contraction coupling in the skeletal muscle triad junction. Our results further imply a role of MG29 as a synaptophysin family member in the accurate formation of junctional complexes between the cell surface and intracellular membranes.
Assuntos
Proteínas Musculares/deficiência , Proteínas Musculares/genética , Músculo Esquelético/anormalidades , Sinaptofisina/análogos & derivados , Sequência de Aminoácidos , Animais , Peso Corporal/genética , Membro Posterior/anormalidades , Membro Posterior/fisiopatologia , Membro Posterior/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Família Multigênica , Contração Muscular/genética , Proteínas Musculares/fisiologia , Músculo Esquelético/fisiopatologia , Músculo Esquelético/ultraestrutura , Sinaptofisina/deficiência , Sinaptofisina/genética , Sinaptofisina/fisiologiaRESUMO
Autophagy and the Cvt pathway are examples of nonclassical vesicular transport from the cytoplasm to the vacuole via double-membrane vesicles. Apg8/Aut7, which plays an important role in the formation of such vesicles, tends to bind to membranes in spite of its hydrophilic nature. We show here that the nature of the association of Apg8 with membranes changes depending on a series of modifications of the protein itself. First, the carboxy-terminal Arg residue of newly synthesized Apg8 is removed by Apg4/Aut2, a novel cysteine protease, and a Gly residue becomes the carboxy-terminal residue of the protein that is now designated Apg8FG. Subsequently, Apg8FG forms a conjugate with an unidentified molecule "X" and thereby binds tightly to membranes. This modification requires the carboxy-terminal Gly residue of Apg8FG and Apg7, a ubiquitin E1-like enzyme. Finally, the adduct Apg8FG-X is reversed to soluble or loosely membrane-bound Apg8FG by cleavage by Apg4. The mode of action of Apg4, which cleaves both newly synthesized Apg8 and modified Apg8FG, resembles that of deubiquitinating enzymes. A reaction similar to ubiquitination is probably involved in the second modification. The reversible modification of Apg8 appears to be coupled to the membrane dynamics of autophagy and the Cvt pathway.
Assuntos
Autofagia , Citoplasma/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Peroxissomos/metabolismo , Proteínas de Saccharomyces cerevisiae , Vacúolos/metabolismo , Sequência de Aminoácidos , Família da Proteína 8 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Transporte Biológico , Domínio Catalítico , Cisteína Endopeptidases/metabolismo , Ligases/metabolismo , Proteínas de Membrana/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Ligação Proteica , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas , Homologia de Sequência de Aminoácidos , Ubiquitina-Proteína Ligases , Ubiquitinas/metabolismoRESUMO
In nutrient-rich, vegetative conditions, the yeast Saccharomyces cerevisiae transports a resident protease, aminopeptidase I (API), to the vacuole by the cytoplasm to vacuole targeting (Cvt) pathway, thus contributing to the degradative capacity of this organelle. When cells subsequently encounter starvation conditions, the machinery that recruited precursor API (prAPI) also sequesters bulk cytosol for delivery, breakdown, and recycling in the vacuole by the autophagy pathway. Each of these overlapping alternative transport pathways is specifically mobilized depending on environmental cues. The basic mechanism of cargo packaging and delivery involves the formation of a double-membrane transport vesicle around prAPI and/or bulk cytosol. Upon completion, these Cvt and autophagic vesicles are targeted to the vacuole to allow delivery of their lumenal contents. Key questions remain regarding the origin and formation of the transport vesicle. In this study, we have cloned the APG9/CVT7 gene and characterized the gene product. Apg9p/Cvt7p is the first characterized integral membrane protein required for Cvt and autophagy transport. Biochemical and morphological analyses indicate that Apg9p/Cvt7p is localized to large perivacuolar punctate structures, but does not colocalize with typical endomembrane marker proteins. Finally, we have isolated a temperature conditional allele of APG9/CVT7 and demonstrate the direct role of Apg9p/Cvt7p in the formation of the Cvt and autophagic vesicles. From these results, we propose that Apg9p/Cvt7p may serve as a marker for a specialized compartment essential for these vesicle-mediated alternative targeting pathways.
Assuntos
Autofagia/fisiologia , Proteínas Fúngicas/metabolismo , Proteínas de Membrana/metabolismo , Animais , Transporte Biológico , Centrifugação com Gradiente de Concentração , Proteínas Fúngicas/genética , Proteínas de Membrana/genética , Coelhos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Frações Subcelulares , Vacúolos/metabolismoRESUMO
Synapsin I is one of the major synaptic vesicle-associated proteins. Previous experiments implicated its crucial role in synaptogenesis and transmitter release. To better define the role of synapsin I in vivo, we used gene targeting to disrupt the murine synapsin I gene. Mutant mice lacking synapsin I appeared to develop normally and did not have gross anatomical abnormalities. However, when we examined the presynaptic structure of the hippocampal CA3 field in detail, we found that the sizes of mossy fiber giant terminals were significantly smaller, the number of synaptic vesicles became reduced, and the presynaptic structures altered, although the mossy fiber long-term potentiation remained intact. These results suggest significant contribution of synapsin I to the formation and maintenance of the presynaptic structure.
Assuntos
Hipocampo/citologia , Neurônios/química , Terminações Pré-Sinápticas/química , Sinapsinas/deficiência , Animais , Cerebelo/citologia , Citoesqueleto/fisiologia , Feminino , Potenciação de Longa Duração/fisiologia , Masculino , Camundongos , Microscopia Eletrônica , Estrutura Molecular , Mutação/fisiologia , Neurônios/fisiologia , Neurônios/ultraestrutura , Terminações Pré-Sinápticas/fisiologia , Terminações Pré-Sinápticas/ultraestrutura , Sinapsinas/genética , Sinapsinas/fisiologia , Vesículas Sinápticas/química , Vesículas Sinápticas/genéticaRESUMO
We characterized Apg8/Aut7p essential for autophagy in yeast. Apg8p was transcriptionally upregulated in response to starvation and mostly existed as a protein bound to membrane under both growing and starvation conditions. Immunofluorescence microscopy revealed that the intracellular localization of Apg8p changed drastically after shift to starvation. Apg8p resided on unidentified tiny dot structures dispersed in the cytoplasm at growing phase. During starvation, it was localized on large punctate structures, some of which were confirmed to be autophagosomes and autophagic bodies by immuno-EM. Besides these structures, we found that Apg8p was enriched on isolation membranes and in electron less-dense regions, which should contain Apg8p-localized membrane- or lipid-containing structures. These structures would represent intermediate structures during autophagosome formation. Here, we also showed that microtubule does not play an essential role in the autophagy in yeast. The result does not match with the previously proposed role of Apg8/Aut7p, delivery of autophagosome to the vacuole along microtubule. Moreover, it is revealed that autophagosome formation is severely impaired in the apg8 null mutant. Apg8p would play an important role in the autophagosome formation.
Assuntos
Proteínas Associadas aos Microtúbulos/fisiologia , Fagossomos/fisiologia , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiologia , Família da Proteína 8 Relacionada à Autofagia , Proteínas Fúngicas/fisiologia , Microscopia Eletrônica , Fagocitose/fisiologia , Fagossomos/ultraestrutura , Saccharomyces cerevisiae/ultraestruturaRESUMO
Occludin is the only known integral membrane protein of tight junctions (TJs), and is now believed to be directly involved in the barrier and fence functions of TJs. Occludin-deficient embryonic stem (ES) cells were generated by targeted disruption of both alleles of the occludin gene. When these cells were subjected to suspension culture, they aggregated to form simple, and then cystic embryoid bodies (EBs) with the same time course as EB formation from wild-type ES cells. Immunofluorescence microscopy and ultrathin section electron microscopy revealed that polarized epithelial (visceral endoderm-like) cells were differentiated to delineate EBs not only from wild-type but also from occludin-deficient ES cells. Freeze fracture analyses indicated no significant differences in number or morphology of TJ strands between wild-type and occludin-deficient epithelial cells. Furthermore, zonula occludens (ZO)-1, a TJ-associated peripheral membrane protein, was still exclusively concentrated at TJ in occludin-deficient epithelial cells. In good agreement with these morphological observations, TJ in occludin-deficient epithelial cells functioned as a primary barrier to the diffusion of a low molecular mass tracer through the paracellular pathway. These findings indicate that there are as yet unidentified TJ integral membrane protein(s) which can form strand structures, recruit ZO-1, and function as a barrier without occludin.
Assuntos
Células Epiteliais/citologia , Proteínas de Membrana/fisiologia , Células-Tronco/citologia , Junções Íntimas/fisiologia , Animais , Biotina/análogos & derivados , Diferenciação Celular , Permeabilidade da Membrana Celular , Polaridade Celular , Células Cultivadas , Técnica de Fratura por Congelamento , Marcação de Genes , Proteínas de Membrana/análise , Camundongos , Ocludina , Fosfoproteínas/análise , Succinimidas , Junções Íntimas/química , Junções Íntimas/ultraestrutura , Proteína da Zônula de Oclusão-1RESUMO
The nerve axon is a good model system for studying the molecular mechanism of organelle transport in cells. Recently, the new kinesin superfamily proteins (KIFs) have been identified as candidate motor proteins involved in organelle transport. Among them KIF1A, a murine homologue of unc-104 gene of Caenorhabditis elegans, is a unique monomeric neuron- specific microtubule plus end-directed motor and has been proposed as a transporter of synaptic vesicle precursors (Okada, Y., H. Yamazaki, Y. Sekine-Aizawa, and N. Hirokawa. 1995. Cell. 81:769-780). To elucidate the function of KIF1A in vivo, we disrupted the KIF1A gene in mice. KIF1A mutants died mostly within a day after birth showing motor and sensory disturbances. In the nervous systems of these mutants, the transport of synaptic vesicle precursors showed a specific and significant decrease. Consequently, synaptic vesicle density decreased dramatically, and clusters of clear small vesicles accumulated in the cell bodies. Furthermore, marked neuronal degeneration and death occurred both in KIF1A mutant mice and in cultures of mutant neurons. The neuronal death in cultures was blocked by coculture with wild-type neurons or exposure to a low concentration of glutamate. These results in cultures suggested that the mutant neurons might not sufficiently receive afferent stimulation, such as neuronal contacts or neurotransmission, resulting in cell death. Thus, our results demonstrate that KIF1A transports a synaptic vesicle precursor and that KIF1A-mediated axonal transport plays a critical role in viability, maintenance, and function of neurons, particularly mature neurons.
Assuntos
Transporte Axonal/fisiologia , Proteínas de Ligação ao Cálcio , Morte Celular/fisiologia , Cinesinas/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios/citologia , Vesículas Sinápticas/metabolismo , Animais , Antígenos de Superfície/análise , Células Cultivadas , Sistema Nervoso Central/patologia , Técnicas de Cocultura , Ácido Glutâmico/farmacologia , Hipocampo/patologia , Cinesinas/genética , Glicoproteínas de Membrana/análise , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/genética , Malformações do Sistema Nervoso , Neurônios/química , Neurônios/metabolismo , Neurônios Aferentes , Dor , Nervo Isquiático , Vesículas Sinápticas/ultraestrutura , Sinaptofisina/análise , Sinaptotagminas , Sintaxina 1RESUMO
Familial adenomatous polyposis coli (FAP) is a disease characterized by the development of multiple colorectal adenomas, and affected individuals carry germline mutations in the APC gene. With the use of a conditional gene targeting system, a mouse model of FAP was created that circumvents the embryonic lethality of Apc deficiency and directs Apc inactivation specifically to the colorectal epithelium. loxP sites were inserted into the introns around Apc exon 14, and the resultant mutant allele (Apc580S) was introduced into the mouse germline. Mice homozygous for Apc580S were normal; however, upon infection of the colorectal region with an adenovirus encoding the Cre recombinase, the mice developed adenomas within 4 weeks. The adenomas showed deletion of Apc exon 14, indicating that the loss of Apc function was caused by Cre-loxP-mediated recombination.