Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 17 de 17
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Biomater Sci ; 9(19): 6574-6583, 2021 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-34582534

RESUMO

Porphyromonas gingivalis, the pathogen of periodontal disease, is thought to be involved in various diseases throughout the body via gingival tissue blood capillaries. However, the dynamic analysis of the infection mechanism, particularly the deep invasion process of the gingival tissue, has not yet been elucidated because of the lack of both in vivo and in vitro models. In this study, we developed a vascularized three-dimensional (3D) gingival model with an epithelial barrier expressing cell-cell junctions using collagen microfibers (CMFs) to enable the dynamic analysis of the P. gingivalis invasion process. Lipid raft disruption experiments in the gingival epithelial cell layer demonstrated that P. gingivalis migrates into the deeper epithelium via the intercellular pathway rather than intracellular routes. P. gingivalis was shown to invade the 3D gingival model, being found inside blood capillaries during two days of culture. Notably, the number of bacteria had increased greatly at least two days later, whereas the mutant P. gingivalis lacking the cysteine proteases, gingipains, showed a significantly lower number of survivors. The secretion of interleukin-6 (IL-6) from the gingival tissue decreased during the two days of infection with the wild type P. gingivalis, but the opposite was found for the mutant suggesting that P. gingivalis infection disturbs IL-6 secretion at an early stage. By allowing the dynamic observation of the P. gingivalis invasion from the epithelial cell layer into the blood capillaries for the first time, this model will be a powerful tool for the development of novel therapeutics against periodontal infection related diseases.


Assuntos
Capilares , Porphyromonas gingivalis , Células Cultivadas , Células Epiteliais , Gengiva , Humanos
2.
Nat Commun ; 12(1): 5059, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34429413

RESUMO

With the current interest in cultured meat, mammalian cell-based meat has mostly been unstructured. There is thus still a high demand for artificial steak-like meat. We demonstrate in vitro construction of engineered steak-like tissue assembled of three types of bovine cell fibers (muscle, fat, and vessel). Because actual meat is an aligned assembly of the fibers connected to the tendon for the actions of contraction and relaxation, tendon-gel integrated bioprinting was developed to construct tendon-like gels. In this study, a total of 72 fibers comprising 42 muscles, 28 adipose tissues, and 2 blood capillaries were constructed by tendon-gel integrated bioprinting and manually assembled to fabricate steak-like meat with a diameter of 5 mm and a length of 10 mm inspired by a meat cut. The developed tendon-gel integrated bioprinting here could be a promising technology for the fabrication of the desired types of steak-like cultured meats.


Assuntos
Bioimpressão/métodos , Géis , Carne , Tendões , Animais , Bovinos , Técnicas de Cultura de Células , Colágeno , Células Endoteliais , Músculos/citologia , Músculos/fisiologia , Impressão Tridimensional , Células-Tronco , Tendões/citologia , Engenharia Tecidual
3.
Neonatology ; 118(1): 28-36, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33326974

RESUMO

INTRODUCTION: Evidence on the reliability of using procalcitonin (PCT) and high-sensitivity C-reactive protein (hs-CRP) as diagnostic markers for early-onset neonatal bacterial infections is still insufficient because of their physiological elevation during the early neonatal period. This study aimed to assess the respiratory influence of serum PCT and hs-CRP levels and evaluate their predictive value for bacterial infections during the first 72 h of life in preterm neonates. METHODS: The preterm neonates enrolled in this single-center retrospective cohort study were categorized into 3 groups: reference, infection-unlikely respiratory failure, and probable bacterial infection; their serum PCT and hs-CRP levels were assessed. Subsequently, age-specific 95th percentile curves were plotted and the median and cutoff PCT and hs-CRP levels for predicting bacterial infections at birth and 7-18, 19-36, and 37-72 h after birth were determined. Moreover, the analysis of PCT and hs-CRP with a neonatal sequential organ failure assessment (nSOFA) score was performed in very low birth weight neonates. RESULTS: Serum PCT levels were influenced by respiratory failure. A significant difference was found in the median PCT and hs-CRP levels among the 3 groups at each time point. PCT sensitivities for predicting bacterial infection were slightly higher than those of hs-CRP in each time frame during the first 72 h of life. In both PCT and hs-CRP, there was no significant difference between infants with nSOFA scores of >4 and those with nSOFA scores of ≤4. DISCUSSION/CONCLUSION: Age-specific evaluation showed that PCT has better predictive value than hs-CRP for early-onset bacterial infections in preterm neonates.


Assuntos
Infecções Bacterianas , Proteína C-Reativa , Infecções Bacterianas/diagnóstico , Biomarcadores , Proteína C-Reativa/análise , Humanos , Lactente , Recém-Nascido , Pró-Calcitonina , Reprodutibilidade dos Testes , Estudos Retrospectivos
4.
Biology (Basel) ; 9(11)2020 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-33120912

RESUMO

Vascular invasion of cancer is a critical step in cancer progression, but no drug has been developed to inhibit vascular invasion. To achieve the eradication of cancer metastasis, elucidation of the mechanism for vascular invasion and the development of innovative treatment methods are required. Here, a simple and reproducible vascular invasion model is established using a vascular organoid culture in a fibrin gel with collagen microfibers. Using this model, it was possible to observe and evaluate the cell dynamics and histological positional relationship of invasive cancer cells in four dimensions. Cancer-derived exosomes promoted the vascular invasion of cancer cells and loosened tight junctions in the vascular endothelium. As a new evaluation method, research using this vascular invasion mimic model will be advanced, and applications to the evaluation of the vascular invasion suppression effect of a drug are expected.

5.
Curr Protoc Cell Biol ; 88(1): e112, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32776707

RESUMO

Construction of organized three-dimensional (3D) tissue with extracellular matrix (ECM) and multiple types of cells is important for tissue engineering to enable tissue function and enhance cellular function. However, the concentration of ECM and the thickness of the 3D tissue have been limited in previous methods due to a lack of permeability to nutrients and oxygen. Besides, it is difficult to use matured natural ECM as a cell scaffold without chemical modification due to its insolubility. In this article, we focus on multi-layered structure, which is commonly found in living tissue such as skin, blood vessels, and other organs. Here, we describe the preparation of a paper-like scaffold (ECM paper) from micro-fibered natural ECM and the construction of 3D multi-layered tissue composed of cell layers and ECM layers by stacking cell-seeded ECM papers. The thickness and components of the ECM layers are easily controllable by changing the composition of the ECM papers, and the fibrous structure of ECM paper shows high permeability and permits cell migration. Additionally, the ECM microfiber, which is physically defiberized from natural ECM, has a high ECM concentration equal to that of living tissue and high stability under physiological conditions. Therefore, this set of protocols enables construction of multi-layered 3D tissue composed of precisely controlled ECM layers and cell layers that may contribute to the assembly of tissue models. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Preparation of extracellular matrix paper Basic Protocol 2: Evaluation of cellular function of cells on extracellular matrix paper Basic Protocol 3: Construction of multi-layered 3D tissue.


Assuntos
Movimento Celular , Matriz Extracelular , Engenharia Tecidual , Tecidos Suporte , Animais , Células Cultivadas , Matriz Extracelular/química , Humanos , Modelos Biológicos , Engenharia Tecidual/métodos , Tecidos Suporte/química
6.
Adv Biosyst ; 4(5): e2000038, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32402125

RESUMO

Achieving vascularization of engineered tissues or structures is a major challenge in the field of tissue engineering. Hitherto, studies on vascularization have demonstrated limited control of vascular network geometry, such as vasculature direction and network density. An open vascular lumen is crucial to ensure that cells survive and that metabolic activity is fully functional in large-sized tissues. Herein, a method based on high water-dispersible collagen microfibers (CMF) to fabricate capillary orientation-controllable 3D tissue with an open vascular lumen using a dispensing machine is reported. A twenty micrometers-long CMF (CMF-20) with high dispersion property are shown to be more effective for dispensing a homogenous tissue and inducing formation of an interconnected capillary network than two hundred micrometers-long CMF (CMF-200). One of the advantages is the prevention of shrinkage on the z-axis of hydrogel-based tissue which acts as a microscaffold. The gaps between the fibers can support endothelial cell migration and maturation, thus forming a larger vascular lumen compared to CMF-free controls. Besides, shear forces produced by the dispensing process cause the collagen microfibers to align, and these microfibers guide cell alignment by integrin-induced adhesion. The findings based on CMF to allow blood capillary alignment and vascular lumen stabilization will be an important technology in tissue engineering.

7.
Brain Dev ; 42(7): 477-483, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32359890

RESUMO

OBJECTIVE: To evaluate the efficacy and safety of intravenous (i.v.) thiamylal in pediatric magnetic resonance imaging (MRI) sedation. METHODS: Infants and children from 1 month up to 8 years of age who underwent MRI in our hospital between April 2017 and March 2019 were included in this prospective observational study. Initial dose of 2 mg/kg thiamylal was given intravenously; however, additional doses were administered as needed. MRI was performed after adequate sedation was achieved. The primary endpoint was the success rate of MRI, while secondary endpoints were adverse events related to sedation, time to sedate, recovery time, and the dose of thiamylal. RESULTS: A total of 118 patients were included in the analysis with median age and weight of 31.5 months (14.0-56.8 months) and 12.6 kg (9.5-15.7 kg), respectively. The success rate of MRI was 96.6% (114/118), and the median dose of thiamylal per body weight was 3.6 (2.8-4.0) mg/kg. The median time from the first dose of thiamylal to MRI was 7 min (4-10 min) and that from the end of MRI scanning to the confirmation of emergence was 8 min (5-25 min). Adverse events encountered included respiratory arrests (n = 3) and reduction in oxygen saturation (SpO2; n = 9). There were no significant differences in the age, dose of thiamylal, sex, body weight, the American Society of Anesthesiologists physical status, and neurological abnormalities between the groups with and without respiratory complications. CONCLUSION: This study demonstrated an adequate efficacy and safety of i.v. thiamylal, with rapid sedation and patient recovery.


Assuntos
Hipnóticos e Sedativos/farmacologia , Imageamento por Ressonância Magnética/normas , Neuroimagem/normas , Tiamilal/farmacologia , Administração Intravenosa , Pré-Escolar , Feminino , Humanos , Hipnóticos e Sedativos/administração & dosagem , Hipnóticos e Sedativos/efeitos adversos , Lactente , Imageamento por Ressonância Magnética/métodos , Masculino , Neuroimagem/métodos , Estudos Prospectivos , Tiamilal/administração & dosagem , Tiamilal/efeitos adversos , Fatores de Tempo
8.
In Vivo ; 30(1): 17-25, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26709124

RESUMO

Periostin, a 90-kDa extracellular matrix protein, has been attracting attention as a novel biomarker of airway inflammatory diseases such as allergic rhinitis (AR) and asthma. Although oral administration of quercetin to patients with AR can favorably modify the clinical condition of this disease, the influence of quercetin on periostin functions is not well understood. The present study was, therefore, undertaken to examine the influence of quercetin on the production of both periostin and periostin-induced eosinophil chemoattractants from human nasal epithelial cells (HNEpC) in vitro. HNEpC were stimulated with 15.0 ng/ml interleukin (IL)-4 in the absence or presence of quercetin for 72 h. Periostin levels in the culture supernatants were measured using enzyme-linked immunosorbent assay (ELISA). Addition of 4.0 µM quercetin into cell cultures suppressed periostin production from HNEpC that was induced by IL-4 stimulation through inhibitation of signal transducer and activator of transcription 6 (STAT6) activation. We then examined whether quercetin could inhibit production of the periostin-induced eosinophil chemoattractants, regulated on activation, normal T-cell expressed and secreted (RANTES) and eotaxin, from HNEpC. HNEpC were stimulated with 2.0 ng/ml periostin in the absence or presence of quercetin for 72 h. RANTES and eotaxin levels in culture supernatants were examined using ELISA. Treatment of HNEpC with quercetin at a concentration of 4.0 µM suppressed the ability of cells to produce RANTES and eotaxin. This suppression was mediated through suppression of activation of the transcription factor nuclear factor-kappa B (NF-κB) p65, as measured using ELISA, and of chemokine mRNA expression, as measured using reverse transcriptase-polymerase chain reaction (RT-PCR). These results strongly suggest that quercetin suppresses the production of both periostin and periostin-induced eosinophil chemoattractants from HNEpC and results in improvement of the clinical condition of AR.


Assuntos
Moléculas de Adesão Celular/metabolismo , Quercetina/farmacologia , Células Cultivadas , Quimiocina CCL5/metabolismo , Eosinófilos/efeitos dos fármacos , Eosinófilos/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Interleucina-4/metabolismo , NF-kappa B/metabolismo , Fator de Transcrição STAT6/metabolismo , Fator de Transcrição RelA/metabolismo
9.
PLoS One ; 7(5): e35195, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22590500

RESUMO

BACKGROUND: The development of novel fertilization treatments, including in vitro fertilization and intracytoplasmic injection, has made pregnancy possible regardless of the level of activity of the spermatozoa; however, the etiology of male-factor infertility is poorly understood. Multiple studies, primarily through the use of transgenic animals, have contributed to a list of candidate genes that may affect male infertility in humans. We examined single nucleotide polymorphisms (SNPs) as a cause of male infertility in an analysis of spermatogenesis-specific genes. METHODS AND FINDING: We carried out the prevalence of SNPs in the coding region of phosphoglycerate mutase 4 (PGAM4) on the X chromosome by the direct sequencing of PCR-amplified DNA from male patients. Using RT-PCR and western blot analyses, we identified that PGAM4 is a functional retrogene that is expressed predominantly in the testes and is associated with male infertility. PGAM4 is expressed in post-meiotic stages, including spermatids and spermatozoa in the testes, and the principal piece of the flagellum and acrosome in ejaculated spermatozoa. A case-control study revealed that 4.5% of infertile patients carry the G75C polymorphism, which causes an amino acid substitution in the encoded protein. Furthermore, an assay for enzymatic activity demonstrated that this polymorphism decreases the enzyme's activity both in vitro and in vivo. CONCLUSION: These results suggest that PGAM4, an X-linked retrogene, is a fundamental gene in human male reproduction and may escape meiotic sex chromosome inactivation. These findings provide fresh insight into elucidating the mechanisms of male infertility.


Assuntos
Fertilidade/genética , Genes Ligados ao Cromossomo X , Doenças Genéticas Ligadas ao Cromossomo X/genética , Infertilidade Masculina/genética , Fosfoglicerato Mutase/genética , Polimorfismo de Nucleotídeo Único , Acrossomo/enzimologia , Feminino , Regulação da Expressão Gênica/genética , Doenças Genéticas Ligadas ao Cromossomo X/enzimologia , Humanos , Infertilidade Masculina/enzimologia , Masculino , Meiose/genética , Especificidade de Órgãos , Fosfoglicerato Mutase/biossíntese , Gravidez , Cauda do Espermatozoide/enzimologia , Espermátides/enzimologia , Testículo/enzimologia , Inativação do Cromossomo X/genética
10.
Proteomics ; 9(8): 2182-92, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19322776

RESUMO

To screen for glycoproteins showing aberrant sialylation patterns in sera of cancer patients and apply such information for biomarker identification, we performed SELDI-TOF MS analysis coupled with lectin-coupled ProteinChip arrays (Jacalin or SNA) using sera obtained from lung cancer patients and control individuals. Our approach consisted of three processes (i) removal of 14 abundant proteins in serum, (ii) enrichment of glycoproteins with lectin-coupled ProteinChip arrays, and (iii) SELDI-TOF MS analysis with acidic glycoprotein-compatible matrix. We identified 41 protein peaks showing significant differences (p<0.05) in the peak levels between the cancer and control groups using the Jacalin- and SNA-ProteinChips. Among them, we identified loss of Neu5Ac (alpha2,6) Gal/GalNAc structure in apolipoprotein C-III (apoC-III) in cancer patients through subsequent MALDI-QIT-TOF MS/MS. Furthermore, subsequent validation experiments using an additional set of 60 lung adenocarcinoma patients and 30 normal controls demonstrated that there is a higher frequency of serum apoC-III with loss of alpha2,6-linkage Neu5Ac residues in lung cancer patients compared to controls. Our results have demonstrated that lectin-coupled ProteinChip technology allows the high-throughput and specific recognition of cancer-associated aberrant glycosylations, and implied a possibility of its applicability to studies on other diseases.


Assuntos
Adenocarcinoma/sangue , Biomarcadores Tumorais/sangue , Neoplasias Pulmonares/sangue , Análise Serial de Proteínas/métodos , Processamento de Proteína Pós-Traducional , Idoso , Apolipoproteína C-III/sangue , Feminino , Glicosilação , Humanos , Lectinas/metabolismo , Masculino , Pessoa de Meia-Idade , Proteômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
11.
J Androl ; 30(4): 426-31, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19168450

RESUMO

To investigate the possible association between variations in the PRDM9 (MEISETZ) gene and impaired spermatogenesis in humans, we screened for mutations in the human PRDM9 gene using DNA from 217 sterile male patients and 162 proven fertile male volunteers. Two single-nucleotide polymorphisms (SNPs), 17353G>T (Gly433Val) and 18109C>G (Thr685Arg), were identified, as well as an intronic SNP, 15549G>T. These SNPs were identified in the heterozygous state in separate patients who demonstrated azoospermia. Neither variant was identified in fertile subjects. Our results suggest that mutations in PRDM9 may cause idiopathic infertility in human males.


Assuntos
Azoospermia/genética , Histona-Lisina N-Metiltransferase/genética , Infertilidade Masculina/genética , Sequência de Aminoácidos , Sequência de Bases , Humanos , Masculino , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único
12.
Gastroenterol Nurs ; 31(4): 263-72, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18708830

RESUMO

This study investigated the feelings of living donors about adult-to-adult liver transplantation. We interviewed 18 donors about their feelings before and after transplantation using semistructured interviews and then conducted a content analysis of their responses. Before transplantation, many donors reported that they wanted recipients to live for the donor or his or her family, and there was no one else to donate. Many donors were not anxious, did not feel coerced, and did not consider donation dangerous. Some reported being excited at facing a new experience. Some said they would not mind whatever happens. Others were anxious or unsure about the operation. Diagnostic testing and preoperative blood banking were painful. Donors experienced increasing stress just before the operation. After transplantation, some donors verbalized feeling more grateful to others and that they gained maturity. Throughout the process, donors were concerned about their recipients. Our results suggest that donors might act for themselves or their family. It is important to recognize the varied responses of donors' feelings toward liver transplant recipients.


Assuntos
Seleção do Doador/métodos , Transplante de Fígado/psicologia , Doadores Vivos/psicologia , Adaptação Fisiológica , Adaptação Psicológica , Adulto , Altruísmo , Estudos de Coortes , Emoções , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Nefrectomia/métodos , Cuidados Pós-Operatórios/métodos , Cuidados Pré-Operatórios/métodos , Qualidade de Vida , Inquéritos e Questionários , Fatores de Tempo
13.
J Biol Chem ; 283(27): 19039-48, 2008 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-18453535

RESUMO

Meichroacidin (MCA) is a highly hydrophilic protein that contains the membrane occupation and recognition nexus motif. MCA is expressed during the stages of spermatogenesis from pachytene spermatocytes to mature sperm development and is localized in the male meiotic metaphase chromosome and sperm flagellum. MCA sequences are highly conserved in Ciona intestinalis, Cyprinus carpio, and mammals. To investigate the physiological role of MCA, we generated MCA-disrupted mutant mice; homozygous MCA mutant males were infertile, but females were not. Sperm was rarely observed in the caput epididymidis of MCA mutant males. However, little to no difference was seen in testis mass between wild-type and mutant mice. During sperm morphogenesis, elongated spermatids had retarded flagellum formation and might increase phagocytosis by Sertoli cells. Immunohistochemical analysis revealed that MCA interacts with proteins located on the outer dense fibers of the flagellum. The testicular sperm of MCA mutant mice was capable of fertilizing eggs successfully via intracytoplasmic sperm injection and generated healthy progeny. Our results suggest that MCA is essential for sperm flagellum formation and the production of functional sperm.


Assuntos
Membrana Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fertilidade/fisiologia , Regulação da Expressão Gênica/fisiologia , Cauda do Espermatozoide/metabolismo , Espermatogênese/fisiologia , Motivos de Aminoácidos/genética , Animais , Carpas/genética , Carpas/metabolismo , Membrana Celular/genética , Cromossomos de Mamíferos/genética , Cromossomos de Mamíferos/metabolismo , Ciona intestinalis/genética , Ciona intestinalis/metabolismo , Proteínas de Ligação a DNA/genética , Epididimo/metabolismo , Masculino , Camundongos , Camundongos Mutantes , Fagocitose/fisiologia , Células de Sertoli/metabolismo , Espermátides/metabolismo , Cromossomo Y/genética , Cromossomo Y/metabolismo
14.
J Proteome Res ; 6(9): 3475-83, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17705522

RESUMO

The recent progress in various proteomic technologies allows us to screen serum biomarker including carbohydrate antigens. However, only a limited number of proteins could be detected by current conventional methods such as shotgun proteomics, primarily because of the enormous concentration distribution of serum proteins and peptides. To circumvent this difficulty and isolate potential cancer-specific biomarkers for diagnosis and treatment, we established a new screening system consisting of the sequential steps of (1) immunodepletion of 6 high-abundance proteins, (2) targeted enrichment of glycoproteins by lectin column chromatography, and (3) the quantitative proteome analysis using 12C6- or 13C6-NBS (2-nitrobenzenesulfenyl) stable isotope labeling followed by MALDI-QIT-TOF mass spectrometric analysis. Through this systematic analysis for five serum samples derived from patients with lung adenocarcinoma, we identified as candidate biomarkers 34 serum glycoproteins that revealed significant difference in alpha1,6-fucosylation level between lung cancer and healthy control, clearly demonstrating that the carbohydrate-focused proteomics could allow for the detection of serum components with cancer-specific features. In addition, we developed a more simplified and practical technique, mass spectrometry-based glycan structure analysis and lectin blotting, in order to validate glycan structure of candidate biomarkers that could be applicable in clinical use. Our new glycoproteomic strategy will provide highly sensitive and quantitative profiling of specific glycan structures on multiple proteins, which should be useful for serum biomarker discovery.


Assuntos
Biomarcadores Tumorais/química , Glicoproteínas/química , Neoplasias/sangue , Neoplasias/diagnóstico , Nitrobenzenos/química , Proteômica/métodos , Proteínas Sanguíneas/química , Regulação Neoplásica da Expressão Gênica , Glicosilação , Humanos , Imunoprecipitação , Lectinas/química , Espectrometria de Massas/métodos , Modelos Químicos , Polissacarídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
15.
J Virol ; 81(3): 1528-33, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17121798

RESUMO

Viral factors as well as host ones play major roles in the disease progression of human immunodeficiency virus type 1 (HIV-1) infection. We have examined cytotoxic T-lymphocyte activity and HIV-1 DNA PCR results of 312 high-risk seronegative drug users in northern Thailand and identified four seronegative cases positive for both assays. Furthermore, we have identified a synonymous mutation in nucleotide position 75 of the gag p17 gene (A426G) of HIV-1 that belongs to the CRF01_AE virus circulating in Thailand. The replication-competent HIV-1 clone containing the A426G mutation demonstrated a dramatic reduction of virion production and perturbation of viral morphogenesis without affecting viral protein synthesis in cells.


Assuntos
Genes gag/fisiologia , HIV-1/genética , Mutação , Vírion/fisiologia , DNA Viral/genética , Genes gag/genética , HIV-1/fisiologia , Humanos , Nucleotídeos
16.
Nucleic Acids Symp Ser (Oxf) ; (49): 99-100, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-17150652

RESUMO

We have identified a mutant human immunodeficiency virus type 1 (HIV-1) CRF01_AE that contains a single nucleotide mutation in gag gene from 4 HIV-1 seronegative drug users in Thailand. We found A to G mutation at the nucleotide position 75 of gag p17 gene (A75G) not changing the amino acid sequence. The mutant HIV-1 molecular clones were examined for their replication capability. Although the mutation dramatically reduced the level of virion production, it did not affect the amounts of viral protein synthesis within the transfected cells. In addition, this mutation did not affect the levels of Gag polyproteins. Furthermore, electron microscopic examinations have revealed a dramatic reduction of the virion production and perturbation of viral morphogenesis at the cytoplasmic membrane. These results indicate that the A75G mutation is attributable to the long-term sero-negativity of individuals at high risk of HIV-1 infection and suggest a novel mechanism that regulates HIV production.


Assuntos
Produtos do Gene gag/genética , Antígenos HIV/genética , Soronegatividade para HIV , HIV-1/genética , Mutação Puntual , Proteínas Virais/genética , Replicação Viral/genética , Produtos do Gene gag/classificação , Antígenos HIV/classificação , Infecções por HIV/epidemiologia , HIV-1/isolamento & purificação , HIV-1/fisiologia , Humanos , Filogenia , Transtornos Relacionados ao Uso de Substâncias/complicações , Proteínas Virais/classificação , Vírion/ultraestrutura , Produtos do Gene gag do Vírus da Imunodeficiência Humana
17.
J Biol Chem ; 277(16): 13973-82, 2002 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-11830582

RESUMO

Nerve growth factor (NGF) can induce apoptosis in neural cells via activation of the low affinity neurotrophin receptor p75NTR. NADE (p75NTR-associated cell death executor) is a p75NTR-associated protein that mediates apoptosis in response to NGF by interacting with the death domain of p75NTR in 293T, PC12, and nnr5 cells (Mukai, J., Hachiya, T., Shoji-Hoshino, S., Kimura, M. T., Nadano, D., Suvanto, P., Hanaoka, T., Li, Y., Irie, S., Greene, L. A., and Sato, T. A. (2000) J. Biol. Chem. 275, 17566-17570). We performed extensive mutational analysis on NADE, to better characterize its structural and functional features. Truncation of a minimal region, including amino acid residues 41-71 of NADE, was found to be sufficient to induce apoptosis. The designated regulatory region includes the C-terminal amino acid residues (72-112) and is essential for NGF-dependent regulation of NADE-induced apoptosis. Furthermore, the mutants with amino acid substitutions in the leucine-rich nuclear export signal (NES) sequence (residues 90-100) abolished the export of NADE from the nucleus to the cytoplasm. Mutation of the NES also abolished self-association of NADE, its interaction with p75NTR, and NGF-dependent apoptosis. Expression of a fragment of NADE (amino acid residues 81-124) blocked NGF-induced apoptosis in oligodendrocytes, suggesting that this region has a dominant negative effect on NGF/p75NTR-induced apoptosis. These studies identify distinct regions of NADE that are involved in regulating specific functions involved in p75NTR signal transduction.


Assuntos
Apoptose , Proteínas/química , Proteínas/fisiologia , Adenoviridae/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose , Linhagem Celular , Análise Mutacional de DNA , Técnicas de Transferência de Genes , Genes Dominantes , Proteínas de Fluorescência Verde , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Leucina/química , Proteínas Luminescentes/metabolismo , Camundongos , Microscopia de Fluorescência , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Células PC12 , Mutação Puntual , Conformação Proteica , Estrutura Terciária de Proteína , Ratos , Receptor de Fator de Crescimento Neural , Receptores de Fator de Crescimento Neural/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , Frações Subcelulares/metabolismo , Transfecção , Técnicas do Sistema de Duplo-Híbrido
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...