RESUMO
AIM: To report positive predictive values (PPVs) of mammographic findings (MFs) of a screening cohort in Taiwan with a view to providing radiologists around the world with adequate information for assessing MFs before recommending biopsy for Asian women. MATERIALS AND METHODS: Between January 2014 and June 2017, 18,449 women received screening mammography at Tri-Service General Hospital (TSGH). Of these women, 1,622 exhibited specific MFs, namely mass (n=518), microcalcification (n=668), focal asymmetry (FA; n=462), and architectural distortion (AD; n=117). The distribution and PPVs of each MF were calculated after stratification based on cancer type, age, and breast density. RESULTS: The age group with the highest proportion of women was 50-59 years (48.1%), and most women presented with dense breasts (68.6%). The most common MF in the recalled women was microcalcification (41.2%) and the least common was AD (7.2%). AD was the most predictive MF for overall breast cancers, invasive carcinomas, and carcinomas in situ. Microcalcification was the second most predictive MF among recalled women for predicting overall breast cancers; however, it was less predictive than mass and FA in women who received a biopsy recommendation or underwent biopsy. CONCLUSION: AD can indicate the likelihood of breast cancer development in Asian women with abnormal screening results. Benign breast diseases are more likely to occur in women recommended for or receiving breast biopsy owing to microcalcification than to mass or FA.
Assuntos
Neoplasias da Mama/diagnóstico por imagem , Carcinoma in Situ/diagnóstico por imagem , Mamografia/métodos , Adulto , Fatores Etários , Idoso , Ásia , Mama/diagnóstico por imagem , Doenças Mamárias/diagnóstico por imagem , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , TaiwanRESUMO
In animals, differentiation of germline from soma usually takes place during embryogenesis. Genes and their products that are preferentially expressed in the embryonic germ cells are regarded as candidates for maintaining germline fate or promoting germline identity. In Drosophila, for example, the protein encoded by the germline gene vasa is specifically restricted to the germ cells, while products of the gap gene hunchback (hb), a somatic gene, are preferentially expressed in the neuroblasts. In this study, we report the expression of both messenger RNA and protein encoded by Aphb, an hb orthologue in the asexual viviparous pea aphid Acyrthosiphon pisum, in germ cells as well as in neuroblasts. We infer that expression of Aphb messenger RNA in the germ cells during the formation of germaria is required for the anterior localization of Aphb in the protruding oocytes. Germarial expression and anterior localization of ApKrüppel was also identified but, unlike Aphb, its expression was not detected in the migrating germ cells. Very similar patterns of hb expression were also identified in the green peach aphid Myzus persicae, suggesting that germline expression of hb is conserved within the Aphididae. To date, this pattern of hb germline expression has not been reported in other insects.
Assuntos
Afídeos/metabolismo , Células Germinativas/metabolismo , Proteínas de Insetos/metabolismo , Animais , Afídeos/embriologia , Sequência de Bases , Proteínas de Ligação a DNA , Proteínas de Drosophila , Desenvolvimento Embrionário , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de TranscriçãoRESUMO
We report a design of low-loss THz Bragg fibers with a core size on the order of wavelength that operates near the cutoff frequency of its TE01 mode. We also propose a broadband Y-type mode converter based on branched rectangular metallic waveguides to facilitate coupling between the TE01 mode of the Bragg fiber and the TEM mode in free space with 60% efficiency. Our fiber holds strong promise to facilitate beam-wave interaction in gyrotron for high-efficiency THz generation.
RESUMO
Previously we identified anterior localization of hunchback (Aphb) mRNA in oocytes and early embryos of the parthenogenetic and viviparous pea aphid Acyrthosiphon pisum, suggesting that the breaking of anterior asymmetry in the oocytes leads to the formation of the anterior axis in embryos. In order to study posterior development in the asexual pea aphid, we cloned and analysed the developmental expression of caudal (Apcad), a posterior gene highly conserved in many animal phyla. We found that transcripts of Apcad were not detected in germaria, oocytes and embryos prior to the formation of the blastoderm in the asexual (viviparous) pea aphid. This unusual expression pattern differs from that of the existing insect models, including long- and short-germ insects, where maternal cad mRNA is passed to the early embryos and forms a posterior-anterior gradient. The first detectable Apcad expression occurred in the newly formed primordial germ cells and their adjacent blastodermal cells during late blastulation. From gastrulation onward, and as in other insects, Apcad mRNA is restricted to the posteriormost region of the germ band. Similarly, in the sexual (oviparous) oocytes we were able to identify anterior localization of Aphb mRNA but posterior localization of Apcad was not detected. This suggests that cad-driven posterior development is not conserved during early embryogenesis in asexual and sexual pea aphids.
Assuntos
Afídeos/embriologia , Desenvolvimento Embrionário , Proteínas de Homeodomínio/metabolismo , Proteínas de Insetos/metabolismo , Sequência de Aminoácidos , Animais , Afídeos/genética , Afídeos/metabolismo , Feminino , Proteínas de Homeodomínio/genética , Proteínas de Insetos/genética , Dados de Sequência Molecular , Ovário/metabolismo , RNA Mensageiro/metabolismo , Análise de Sequência de DNARESUMO
Specific receptor-mediated binding by Trichomonas vaginalis of human erythrocytes was demonstrated. The ability of live parasites to internalize erythrocytes was also documented. In vitro growth assays during lipid-free and iron-limiting conditions that do not support the survival of T. vaginalis organisms showed that purified erythrocyte lipids and hemoglobin were each able to provide lipids and/or hemoglobin iron for trichomonal growth and multiplication. Parasites bound hemoglobin in a highly specific receptor-mediated fashion, and only the homologous unlabeled hemoglobin, but not lactoferrin and transferrin, competed with iodinated hemoglobin binding. Two antibody-crossreactive surface proteins of the parasites were identified as adhesins, and antibody to the individual adhesins inhibited T. vaginalis recognition and binding of erythrocytes. Finally, patient sera possessed antibody to the adhesins, showing the immunogenic nature and in vivo relevance of the trichomonad proteins during infection.
Assuntos
Eritrócitos/parasitologia , Trichomonas vaginalis/metabolismo , Animais , Eletroforese em Gel de Poliacrilamida , Eritrócitos/metabolismo , Hemoglobinas/metabolismo , Humanos , Técnicas In Vitro , Ferro/metabolismo , Metabolismo dos Lipídeos , Proteínas de Protozoários/metabolismo , Trichomonas vaginalis/crescimento & desenvolvimentoRESUMO
Western blotting studies revealed that connexin43 (Cx43), one of the major gap junction proteins in human vascular endothelial cells, is posttranslationally modified during mitosis. This mitosis-specific modification results in a Cx43 species that migrates as a single protein band and was designated Cx43(m). Cx43(m) was shown to be the result of additional Ser/Thr phosphorylation as indicated by: (a) the increased gel mobility induced by both alkaline phosphatase and the Ser/ Thr-specific protein phosphatase-2A (PP2A) and (b) the removal of virtually all (32)P(i) from Cx43(m) by PP2A. Immunofluorescent confocal microscopy of mitotic cells revealed that Cx43 is intracellularly located, while in nonmitotic cells Cx43 is located at regions of cell-cell contact. Dye coupling studies revealed that mitotic endothelial cells were uncoupled from each other and from nonmitotic cells. After cytokinesis, sister cells resumed cell coupling independent of de novo protein synthesis. The mitosis-specific phosphorylation of Cx43 correlates with the transient loss of gap junction intercellular communication and redistribution of Cx43, suggesting that a protein kinase that regulates gap junctions is active in M-phase.
Assuntos
Conexina 43/análise , Conexina 43/metabolismo , Endotélio Vascular/metabolismo , Músculo Liso Vascular/metabolismo , Animais , Divisão Celular/fisiologia , Células Cultivadas/química , Células Cultivadas/citologia , Células Cultivadas/metabolismo , Conexina 43/química , Endotélio Vascular/química , Endotélio Vascular/citologia , Junções Comunicantes/química , Humanos , Mitose/fisiologia , Músculo Liso Vascular/química , Músculo Liso Vascular/citologia , Fosforilação , Ratos , Serina/metabolismo , Treonina/metabolismo , Veias Umbilicais/citologiaRESUMO
The DED1 gene, which encodes a putative RNA helicase, has been implicated in nuclear pre-messenger RNA splicing in the yeast Saccharomyces cerevisiae. It is shown here by genetic and biochemical analysis that translation, rather than splicing, is severely impaired in two newly isolated ded1 conditional mutants. Preliminary evidence suggests that the protein Ded1p may be required for the initiation step of translation, as is the distinct DEAD-box protein, eukaryotic initiation factor 4A (eIF4A). The DED1 gene could be functionally replaced by a mouse homolog, PL10, which suggests that the function of Ded1p in translation is evolutionarily conserved.
Assuntos
Biossíntese de Proteínas , RNA Nucleotidiltransferases/metabolismo , RNA Fúngico/genética , RNA Mensageiro/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Animais , Citoplasma/metabolismo , RNA Helicases DEAD-box , Fator de Iniciação 4A em Eucariotos , Genes Fúngicos , Camundongos , Mutação , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Fenótipo , RNA Helicases , RNA Nucleotidiltransferases/genética , Splicing de RNA , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/enzimologiaRESUMO
To determine if double-stranded RNA (dsRNA) viruses exist and are potential virulence factors in Trichomonas gallinae, virus purification via ultracentrifugation was attempted for 12 T. gallinae isolates recovered from wild birds. Following purification, virus-like particles were not observed by transmission electron microscopy, nor were dsRNA segments visualized in agarose gels after electrophoresis of extracted RNA from any of the 12 T. gallinae isolates. However, virus particles and dsRNA segments were detected from a previously determined virus-infected T. vaginalis isolate as a control using identical purification procedures. Subsequent reverse transcription-polymerase chain reaction analysis of the dsRNA of the virus in this isolate revealed a novel sequence of the RNA-dependent RNA polymerase gene of T. vaginalis viruses.
Assuntos
Vírus de RNA/genética , Vírus de RNA/isolamento & purificação , RNA de Cadeia Dupla/genética , Trichomonas/virologia , Animais , Aves/parasitologia , Análise por Conglomerados , Eletroforese em Gel de Ágar/métodos , Microscopia Eletrônica de Transmissão/métodos , Dados de Sequência Molecular , Filogenia , Vírus de RNA/classificação , RNA de Cadeia Dupla/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de Sequência , Trichomonas/isolamento & purificação , Ultracentrifugação/métodos , Vírion/ultraestruturaRESUMO
The purpose of this study is to investigate the clinical and histological features that may affect the survival of the patients and to evaluate the impact of post-operative adjuvant therapy on the outcomes of patients with stage IB and IIA carcinoma of the cervix. From August 1998 to January 2005, 140 patients with International Federation of Gynecology and Obstetrics stage IB and IIA cervical cancer were treated with radical hysterectomy and post-operative pelvic radiation therapy with or without chemotherapy. The median age was 55 years (range, 29-86 years). Seventy-six patients had stage IB and 64 patients had stage IIA disease. Tumour size was <4 cm in 96 patients and > or = 4 cm in 44 patients. One hundred and eleven patients had histology of squamous cell carcinoma, 12 patients has adenocarcinoma and 17 patients had other histologic types. Depth of stromal invasion was <2/3 in 20 patients and > or = 2/3 in 120 patients. Twenty-three patients had parametrial invasion and 117 patients had no parametrial invasion. Thirteen patients had lymphovascular space invasion and 127 had no lymphovascular space invasion. Nine patients had positive surgical margin and 131 patients had negative margin. Twenty-seven patients had pelvic lymph node metastasis and 113 patients had no pelvic lymph node metastasis. Seventy-five patients received concurrent chemoradiotherapy and 65 patients received radiotherapy alone. The 5-year overall survival (OAS) and disease-free survival were 83% and 72% respectively. In the log rank test, tumour size (P = 0.0235), pararmetrial invasion (P = 0.0121), pelvic lymph node metastasis (P < 0.0001) and adjuvant chemotherapy + radiotherapy (P = 0.0119) were significant prognostic factors for OAS, favouring tumour size <4 cm, absence of parametrial invasion and pelvic lymph node metastasis, and those who received adjuvant chemoradiotherapy. The patients who received radiation with concomitant chemotherapy had a 5-year OAS rate of 90% versus those who received radiotherapy alone, with a rate of 76%. For patients with high-risk early stage cervical cancer who underwent a radical hysterectomy and pelvic lymphadenectomy, adjuvant chemoradiotherapy resulted in better survival than radiotherapy alone. The addition of weekly cisplatin to radiotherapy is recommended. The treatment-related morbidity is tolerable.
Assuntos
Histerectomia , Neoplasias do Colo do Útero/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Quimioterapia Adjuvante/métodos , Intervalo Livre de Doença , Feminino , Humanos , Metástase Linfática , Pessoa de Meia-Idade , Cuidados Pós-Operatórios , Prognóstico , Radioterapia Adjuvante , Fatores de Risco , Neoplasias do Colo do Útero/mortalidade , Neoplasias do Colo do Útero/patologiaRESUMO
Aberrant protein glycosylation could be a distinct surface-marker of cancer cells that influences cancer progression and metastasis because glycosylation can regulate membrane protein folding which alters receptor activation and changes epitope exposure for antibody (Ab) recognition. Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), a glycophosphoinositol-anchored protein, is a heavily glycosylated tumor antigen. However, the clinical significance and biological effect of CEACAM6 glycosylation has not been addressed in cancers. We recently developed an anti-CEACAM6 Ab (TMU) from an immune llama library which can be engineered to a single-domain (sd)Ab or a heavy-chain (HC)Ab. The TMU HCAb specifically recognized glycosylated CEACAM6 compared to the conventional antibodies. Using the TMU HCAb, we found that glycosylated CEACAM6 was a tumor marker associated with recurrence in early-stage OSCC (oral squamous cell carcinoma) patients. CEACAM6 promoted OSCC cell invasion, migration, cytoskeletal rearrangement, and metastasis via interaction with epidermal growth factor (EGF) receptor (EGFR) and enhancing EGFR activation, clustering and intracellular signaling cascades. These functions were modulated by N-acetylglucosaminyltransferase 5 (MGAT5) which mediated N-glycosylation at Asn256 (N256) of CEACAM6. Finally, the TMU sdAb and HCAb treatment inhibited the migration, invasion and EGF-induced signaling in CEACAM6-overexpressing cells. In conclusion, the complex N-glycosylation of CEACAM6 is critical for EGFR signaling of OSCC invasion and metastasis. Targeting glycosylated CEACAM6 with the TMU sdAb or TMU HCAb could be a feasible therapy for OSCC.
Assuntos
Antígenos CD/metabolismo , Carcinoma de Células Escamosas/patologia , Moléculas de Adesão Celular/metabolismo , Receptores ErbB/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Bucais/patologia , Recidiva Local de Neoplasia/patologia , Adulto , Animais , Antígenos CD/genética , Asparagina/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/secundário , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular , Receptores ErbB/genética , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Glicosilação , Humanos , Neoplasias Pulmonares/secundário , Masculino , Camundongos , Camundongos SCID , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Invasividade Neoplásica/patologia , Estadiamento de Neoplasias , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
T cell proliferative responses to hepatitis B virus-encoded envelope antigen (S + preS2 + preS1), recombinant core antigen (HBcAg), and natural hepatitis B e antigen (HBeAg) were examined in 22 HBeAg-positive patients with chronic type B hepatitis and 17 healthy hepatitis B surface antigen (HBsAg) carriers. The results showed that HBeAg-positive patients had (a) higher levels of T cell responses to HBcAg/HBeAg than those of healthy HBsAg carriers (P less than 0.001 and P less than 0.01, respectively); (b) a further increase in these T cell responses during acute exacerbations (P less than 0.05 and P less than 0.05, respectively); (c) subsidence in the T cell responses to HBcAg/HBeAg after recovery from acute exacerbations and HBeAg seroconversion, whereas the responses would persist at high levels if the patients did not enter a clinical remission; and (d) low levels of T cell responses to S + preS2 + preS1 either before or after HBeAg seroconversion. The appearance of increasing T cell responses to HBcAg/HBeAg usually occurred in the early phase of acute exacerbations. These findings imply that HBcAg/HBeAg-specific T cells play an important role in the exacerbations of chronic hepatitis B and in HBeAg seroconversion. HBcAg/HBeAg-specific precursor T cell frequencies were serially studied in selected cases by limiting dilution assay. Elevation (two- to fourfold) of HBcAg/HBeAg-specific precursor T cell frequencies contributed to the increase of HBcAg/HBeAg-specific T cell proliferation during acute exacerbations.
Assuntos
Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Antígenos E da Hepatite B/imunologia , Hepatite B/imunologia , Hepatite Crônica/imunologia , Linfócitos T/imunologia , Adulto , Sequência de Bases , Feminino , Seguimentos , Humanos , Tolerância Imunológica/imunologia , Ativação Linfocitária/imunologia , Masculino , Dados de Sequência Molecular , Estudos Prospectivos , Fatores de TempoRESUMO
In vitro studies in patients with hepatitis B virus (HBV) infection have suggested that hepatocytolysis induced by CD8+ cytotoxic T lymphocytes (CTLs) is the most important effector pathway in eliminating infected cells. The recognition is implicated in the endogenously processed HBV antigens in the context of HLA class I molecules presented on the liver cell membrane. However, the naturally occurring HBV peptide antigens have not yet been demonstrated. We report here that a naturally processed peptide antigen P2 was isolated from HLA class I molecules of HBV-infected liver cell membrane. The P2 peptide exhibited the activity of sensitizing target cells for lysis by CD8+ CTLs. The P2 sequence (YVNVNMGLK) purified from liver tissue was in concordance with that encoded by the viral genome for the HBV nucleocapsid antigen or HBcAg 88-96. P2 peptide could also be isolated from the EBV-transformed B cells that were transfected by HBcAg-expressing vector. The P2 epitope, sharing the HLA-A11 binding motifs, was recognized by HLA-A11-restricted CD8+ CTLs. The data provided direct evidence that, in hepatitis B patients, antigenic peptides of HBV were processed by hepatocytes, presented with the class I MHC molecules, and recognized by CD8+ CTLs.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Antígenos da Hepatite B/imunologia , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Linfócitos T Citotóxicos/imunologia , Adulto , Sequência de Aminoácidos , Sequência de Bases , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/microbiologia , Epitopos , Antígenos HLA-A/imunologia , Antígeno HLA-A11 , Humanos , Masculino , Dados de Sequência Molecular , Peptídeos/imunologia , Processamento de Proteína Pós-Traducional , Subpopulações de Linfócitos T/imunologiaRESUMO
Homocysteine is an important and independent risk factor for arteriosclerosis. We showed previously that homocysteine stimulates vascular smooth muscle cell proliferation, a hallmark of arteriosclerosis. We show here that homocysteine and serum increased DNA synthesis synergistically in both human and rat aortic smooth muscle cells (RASMCs). Treatment of quiescent RASMCs with 1 mM homocysteine or 2% calf serum for 36 h increased cyclin A mRNA levels by 8- and 14-fold, respectively, whereas homocysteine plus serum increased cyclin A mRNA levels by 40-fold, indicating a synergistic induction of cyclin A mRNA. Homocysteine did not increase the half-life of cyclin A mRNA (2.9 h), but it did increase the transcriptional rate of the cyclin A gene in nuclear run-on experiments. The positive effect of homocysteine on cyclin A gene transcription was confirmed by our finding that homocysteine increased cyclin A promoter activity and ATF-binding protein levels in RASMCs. Finally, 1 mM homocysteine increased cyclin A protein levels and cyclin A-associated kinase activity by threefold. This homocysteine-induced expression lesions by promoting proliferation of vascular smooth muscle cells.
Assuntos
Ciclinas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Homocisteína/farmacologia , Músculo Liso Vascular/metabolismo , Transcrição Gênica/efeitos dos fármacos , Fatores Ativadores da Transcrição , Animais , Arteriosclerose/etiologia , Sequência de Bases , Becaplermina , Sangue , Proteínas Sanguíneas/análise , Bovinos , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/análise , Ciclinas/biossíntese , Ciclinas/metabolismo , DNA/biossíntese , Sinergismo Farmacológico , Humanos , Masculino , Dados de Sequência Molecular , Músculo Liso Vascular/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/farmacologia , Regiões Promotoras Genéticas , Protamina Quinase/metabolismo , Proteínas Proto-Oncogênicas c-sis , RNA Mensageiro/biossíntese , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição/análiseRESUMO
In Saccharomyces cerevisiae, ribosomal biogenesis takes place primarily in the nucleolus, in which a single 35S precursor rRNA (pre-rRNA) is first transcribed and sequentially processed into 25S, 5.8S, and 18S mature rRNAs, leading to the formation of the 40S and 60S ribosomal subunits. Although many components involved in this process have been identified, our understanding of this important cellular process remains limited. Here we report that one of the evolutionarily conserved DEAD-box protein genes in yeast, DBP3, is required for optimal ribosomal biogenesis. DBP3 encodes a putative RNA helicase, Dbp3p, of 523 amino acids in length, which bears a highly charged amino terminus consisting of 10 tandem lysine-lysine-X repeats ([KKX] repeats). Disruption of DBP3 is not lethal but yields a slow-growth phenotype. This genetic depletion of Dbp3p results in a deficiency of 60S ribosomal subunits and a delayed synthesis of the mature 25S rRNA, which is caused by a prominent kinetic delay in pre-rRNA processing at site A3 and to a lesser extent at sites A2 and A0. These data suggest that Dbp3p may directly or indirectly facilitate RNase MRP cleavage at site A3. The direct involvement of Dbp3p in ribosomal biogenesis is supported by the finding that Dbp3p is localized predominantly in the nucleolus. In addition, we show that the [KKX] repeats are dispensable for Dbp3p's function in ribosomal biogenesis but are required for its proper localization. The [KKX] repeats thus represent a novel signaling motif for nuclear localization and/or retention.
Assuntos
RNA Helicases , RNA Nucleotidiltransferases/genética , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA/fisiologia , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Nucléolo Celular/química , RNA Helicases DEAD-box , Dados de Sequência Molecular , RNA Nucleotidiltransferases/análise , RNA Nucleotidiltransferases/fisiologia , RNA Fúngico/metabolismo , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Ribossomos/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Deleção de SequênciaRESUMO
The yeast rna mutations (rna2 through rna10/11) are a set of temperature-sensitive mutations that result in the accumulation of pre-mRNAs at the nonpermissive temperature. Most of the yeast RNA gene products are involved in and essential for mRNA splicing in vitro, suggesting that they code for components of the splicing machinery. We tested this proposal by using an in vitro-synthesized RNA11 protein to complement the temperature-sensitive defect of the rna11 extract. During the in vitro complementation, the input RNA11 protein was associated with the 40S spliceosome and a 30S complex, suggesting that the RNA11 protein is indeed a component of the spliceosome. The formation of the RNA11-associated 30S complex did not require any exogenous RNA substrate, suggesting that this 30S particle is likely to be a preassembled complex involved in splicing. The RNA11-specific antibody inhibited the mRNA splicing in vitro, confirming the essential role of the RNA11 protein in mRNA splicing. Finally, using the anti-RNA11 antibody, we localized the RNA11 protein to the periphery of the yeast nucleus.
Assuntos
Núcleo Celular/análise , Proteínas Fúngicas/genética , Splicing de RNA , Proteínas de Saccharomyces cerevisiae , Leveduras/genética , Anticorpos Antifúngicos/imunologia , Sequência de Bases , Núcleo Celular/imunologia , Clonagem Molecular , Imunofluorescência , Proteínas Fúngicas/análise , Proteínas Fúngicas/imunologia , Teste de Complementação Genética , Dados de Sequência Molecular , Mutação , Proteínas de Ligação a RNA , Temperatura , Leveduras/imunologiaRESUMO
Typhula incarnata, the causal agent of gray snow mold, is an important winter pathogen of turfgrasses in the northern United States. The relative susceptibility of cultivars of three bent-grass species (creeping, colonial, and velvet bentgrass) to Typhula incarnata and the aggressiveness of 15 T. incarnata isolates obtained from infected turfgrasses on golf courses in Michigan, Minnesota, and Wisconsin were evaluated under controlled conditions. A hypersensitive type of resistance response to T. incarnata was not observed in any cultivar. Disease severity increased with higher inoculum concentration of T. incarnata. Colonization by gray snow mold gradually decreased with increasing plant age from 11 weeks after seeding in most cultivars tested, suggesting that age-related resistance was expressed over time. There were significant differences in disease severity among the three bentgrass species, particularly between tetraploid (creeping and colonial) and diploid (velvet) species, and among cultivars within each species, indicating varying levels of susceptibility to T. incarnata. All 15 isolates were pathogenic on bentgrass and were significantly different in aggressiveness, but aggressiveness was not related to geographic origin. Therefore, turfgrass breeders should be able to use one or a few virulent representative isolates of the pathogen to screen for resistance.
RESUMO
The dental follicle (DF), most often associated with unerupted teeth, is a condensation of ectomesenchymal cells that surrounds the tooth germ in early stages of tooth development. In the present study, we aim to isolate epithelial stem-like cells from the human DF and explore their potential differentiation into salivary gland (SG) cells. We demonstrated the expression of stem cell-related genes in the epithelial components of human DF tissues, and these epithelial progenitor cells could be isolated and ex vivo expanded in a reproducible manner. The human DF-derived epithelial cells possessed clonogenic and sphere-forming capabilities, as well as expressed a panel of epithelial stem cell-related genes, thus conferring stem cell properties (hDF-EpiSCs). When cultured under in vitro 3-dimensional induction conditions, hDF-EpiSCs were capable to differentiate into SG acinar and duct cells. Furthermore, transplantation of hDF-EpiSC-loaded native de-cellularized rat parotid gland scaffolds into the renal capsule of nude mice led to the differentiation of transplanted hDF-EpiSCs into salivary gland-like cells. These findings suggest that hDF-EpiSCs might be a promising source of epithelial stem cells for the development of stem cell-based therapy or bioengineering SG tissues to repair/regenerate SG dysfunction.
Assuntos
Saco Dentário/citologia , Células Epiteliais/citologia , Glândulas Salivares/citologia , Engenharia Tecidual/métodos , Animais , Western Blotting , Técnicas de Cultura de Células , Diferenciação Celular , Células Cultivadas , Feminino , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Nus , Microscopia Confocal , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Trichomonosis caused by Trichomonas vaginalis is the number one, non-viral sexually transmitted disease (STD) that affects more than 250 million people worldwide. Immunoglobulin A (IgA) has been implicated in resistance to mucosal infections by pathogens. No reports are available of IgA-reactive proteins and the role, if any, of this class of antibody in the control of this STD. The availability of an IgA monoclonal antibody (mAb) immunoreactive to trichomonads by whole cell (WC)-ELISA prompted us to characterize the IgA-reactive protein of T. vaginalis. RESULTS: An IgA mAb called 6B8 was isolated from a library of mAbs reactive to surface proteins of T. vaginalis. The 6B8 mAb recognized a 44-kDa protein (TV44) by immunoblot analysis, and a full-length cDNA clone encoded a protein of 438 amino acids. Southern analysis revealed the gene (tv44) of T. vaginalis to be single copy. The tv44 gene was down-regulated at both the transcriptional and translational levels in iron-depleted trichomonads as well as in parasites after contact with immortalized MS-74 vaginal epithelial cells (VECs). Immunofluorescence on non-permeabilized organisms confirmed surface localization of TV44, and the intensity of fluorescence was reduced after parasite adherence to VECs. Lastly, an identical protein and gene were present in Tritrichomonas foetus and Trichomonas tenax. CONCLUSION: This is the first report of a T. vaginalis gene (tv44) encoding a surface protein (TV44) reactive with an IgA mAb, and both gene and protein were conserved in human and bovine trichomonads. Further, TV44 is independently down-regulated in expression and surface placement by iron and contact with VECs. TV44 is another member of T. vaginalis genes that are regulated by at least two independent signaling mechanisms involving iron and contact with VECs.