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1.
Poult Sci ; 100(2): 452-460, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33518096

RESUMO

Ovomucoid is a major egg white protein which is considered as the most dominant allergen in chicken eggs. Owing to the difficulty of separating ovomucoid from egg whites, researchers have adopted genetic deletion for development of hypoallergenic eggs. Previously, we used CRISPR/Cas9 to establish chickens with ovomucoid gene (OVM) mutations, but it remained unknown whether such hens could produce eggs at maturity. Here, we have reported on eggs laid by OVM-targeted hens. Except for watery egg whites, the eggs had no evident abnormalities. Real-time PCR revealed alternative splicing of OVM mRNA in hens, but their expression was limited. Immunoblotting detected neither mature ovomucoid nor ovomucoid-truncated splicing variants in egg whites. Sixteen chicks hatched from 28 fertilized eggs laid by OVM-targeted hens, and fourteen of the sixteen chicks demonstrated healthy growth. Taken together, our results demonstrated that OVM knockout could almost completely eliminate ovomucoid from eggs, without abolishing fertility. Thus, the eggs developed in this study have potential as a hypoallergenic food source for most patients with egg allergies.


Assuntos
Galinhas/genética , Ovos/normas , Mutação , Ovomucina/genética , Alérgenos/genética , Animais , Galinhas/crescimento & desenvolvimento , Galinhas/fisiologia , Clara de Ovo/efeitos adversos , Clara de Ovo/química , Clara de Ovo/normas , Feminino , Deleção de Genes , Masculino , Oviposição/genética , Ovomucina/efeitos adversos , Óvulo
2.
Genes (Basel) ; 12(1)2020 12 30.
Artigo em Inglês | MEDLINE | ID: mdl-33396657

RESUMO

Increased commercial demand for monoclonal antibodies (mAbs) has resulted in the urgent need to establish efficient production systems. We previously developed a transgenic chicken bioreactor system that effectively produced human cytokines in egg whites using genome-edited transgenic chickens. Here, we describe the application of this system to mAb production. The genes encoding the heavy and light chains of humanized anti-HER2 mAb, linked by a 2A peptide sequence, were integrated into the chicken ovalbumin gene locus using a CRISPR/Cas9 protocol. The knock-in hens produced a fully assembled humanized mAb in their eggs. The mAb expression level in the egg white was 1.4-1.9 mg/mL, as determined by ELISA. Furthermore, the antigen binding affinity of the anti-HER2 mAb obtained was estimated to be equal to that of the therapeutic anti-HER2 mAb (trastuzumab). In addition, antigen-specific binding by the egg white mAb was demonstrated by immunofluorescence against HER2-positive and -negative cells. These results indicate that the chicken bioreactor system can efficiently produce mAbs with antigen binding capacity and can serve as an alternative production system for commercial mAbs.


Assuntos
Anticorpos Monoclonais/biossíntese , Sistemas CRISPR-Cas , Galinhas/genética , Clara de Ovo/química , Receptor ErbB-2/antagonistas & inibidores , Animais , Animais Geneticamente Modificados , Anticorpos Monoclonais/isolamento & purificação , Reatores Biológicos , Feminino , Edição de Genes/métodos , Humanos , Plasmídeos/química , Plasmídeos/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/imunologia , Trastuzumab/biossíntese , Trastuzumab/isolamento & purificação , Zigoto/química , Zigoto/metabolismo
3.
Sci Rep ; 8(1): 10203, 2018 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-29976933

RESUMO

Transgenic chickens could potentially serve as bioreactors for commercial production of recombinant proteins in egg white. Many transgenic chickens have been generated by randomly integrating viral vectors into their genomes, but transgene expression has proved insufficient and/or limited to the initial cohort. Herein, we demonstrate the feasibility of integrating human interferon beta (hIFN-ß) into the chicken ovalbumin locus and producing hIFN-ß in egg white. We knocked in hIFN-ß into primordial germ cells using a CRISPR/Cas9 protocol and then generated germline chimeric roosters by cell transplantation into recipient embryos. Two generation-zero founder roosters produced hIFN-ß knock-in offspring, and all knock-in female offspring produced abundant egg-white hIFN-ß (~3.5 mg/ml). Although female offspring of the first generation were sterile, their male counterparts were fertile and produced a second generation of knock-in hens, for which egg-white hIFN-ß production was comparable with that of the first generation. The hIFN-ß bioactivity represented only ~5% of total egg-white hIFN-ß, but unfolding and refolding of hIFN-ß in the egg white fully recovered the bioactivity. These results suggest that transgene insertion at the chicken ovalbumin locus can result in abundant and stable expression of an exogenous protein deposited into egg white and should be amenable to industrial applications.


Assuntos
Galinhas/genética , Clara de Ovo/química , Interferon beta/metabolismo , Ovalbumina/genética , Animais , Animais Geneticamente Modificados , Reatores Biológicos , Células Germinativas Embrionárias/citologia , Células Germinativas Embrionárias/metabolismo , Estudos de Viabilidade , Feminino , Técnicas de Introdução de Genes , Loci Gênicos , Humanos , Interferon beta/genética
4.
Sci Rep ; 6: 23980, 2016 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-27050479

RESUMO

The CRISPR/Cas9 system is a simple and powerful tool for genome editing in various organisms including livestock animals. However, the system has not been applied to poultry because of the difficulty in accessing their zygotes. Here we report the implementation of CRISPR/Cas9-mediated gene targeting in chickens. Two egg white genes, ovalbumin and ovomucoid, were efficiently (>90%) mutagenized in cultured chicken primordial germ cells (PGCs) by transfection of circular plasmids encoding Cas9, a single guide RNA, and a gene encoding drug resistance, followed by transient antibiotic selection. We transplanted CRISPR-induced mutant-ovomucoid PGCs into recipient chicken embryos and established three germline chimeric roosters (G0). All of the roosters had donor-derived mutant-ovomucoid spermatozoa, and the two with a high transmission rate of donor-derived gametes produced heterozygous mutant ovomucoid chickens as about half of their donor-derived offspring in the next generation (G1). Furthermore, we generated ovomucoid homozygous mutant offspring (G2) by crossing the G1 mutant chickens. Taken together, these results demonstrate that the CRISPR/Cas9 system is a simple and effective gene-targeting method in chickens.


Assuntos
Proteínas Aviárias/genética , Sistemas CRISPR-Cas , Galinhas/genética , Ovalbumina/genética , Ovomucina/genética , Animais , Células Cultivadas , Embrião de Galinha , Feminino , Marcação de Genes/métodos , Células Germinativas/citologia , Células Germinativas/metabolismo , Masculino , Mutagênese , Mutação , Reprodutibilidade dos Testes
5.
J Reprod Dev ; 62(2): 143-9, 2016 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-26727404

RESUMO

An in vitro culture system of chicken primordial germ cells (PGCs) has been recently developed, but the growth factor involved in the proliferation of PGCs is largely unknown. In the present study, we investigated the growth effects of chicken stem cell factor (chSCF) on the in vitro proliferation of chicken PGCs. We established two feeder cell lines (buffalo rat liver cells; BRL cells) that stably express the putative secreted form of chSCF (chSCF1-BRL) and membrane bound form of chSCF (chSCF2-BRL). Cultured PGC lines were incubated on chSCF1 or chSCF2-BRL feeder cells with fibroblast growth factor 2 (FGF2), and growth effects of each chSCF isoform were investigated. The in vitro proliferation rate of the PGCs cultured on chSCF2-BRL at 20 days of culture was more than threefold higher than those cultured on chSCF1-BRL cells and more than fivefold higher than those cultured on normal BRL cells. Thus, use of chSCF2-BRL feeder layer was effective for in vitro proliferation of chicken PGCs. However, the acceleration of PGC proliferation on chSCF2-BRL was not observed without FGF2, suggesting that chSCF2 would act as a proliferation co-factor of FGF2. We transferred the PGCs cultured on chSCF2-BRL cells to recipient embryos, generated germline chimeric chickens and assessed the germline competency of cultured PGCs by progeny test. Donor-derived progenies were obtained, and the frequency of germline transmission was 3.39%. The results of this study demonstrate that chSCF2 induces hyperproliferation of chicken PGCs retaining germline competency in vitro in cooperation with FGF2.


Assuntos
Proliferação de Células/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/metabolismo , Células Germinativas/citologia , Fator de Células-Tronco/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Galinhas , Criopreservação , Feminino , Humanos , Masculino , Proteínas Proto-Oncogênicas c-kit/metabolismo
6.
Proc Natl Acad Sci U S A ; 112(16): 5075-80, 2015 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-25848055

RESUMO

Limb skeletal elements originate from the limb progenitor cells, which undergo expansion and patterning to develop each skeletal element. Posterior-distal skeletal elements, such as the ulna/fibula and posterior digits develop in a Sonic hedgehog (Shh)-dependent manner. However, it is poorly understood how anterior-proximal elements, such as the humerus/femur, the radius/tibia and the anterior digits, are developed. Here we show that the zinc finger factors Sall4 and Gli3 cooperate for proper development of the anterior-proximal skeletal elements and also function upstream of Shh-dependent posterior skeletal element development. Conditional inactivation of Sall4 in the mesoderm before limb outgrowth caused severe defects in the anterior-proximal skeletal elements in the hindlimb. We found that Gli3 expression is reduced in Sall4 mutant hindlimbs, but not in forelimbs. This reduction caused posteriorization of nascent hindlimb buds, which is correlated with a loss of anterior digits. In proximal development, Sall4 integrates Gli3 and the Plzf-Hox system, in addition to proliferative expansion of cells in the mesenchymal core of nascent hindlimb buds. Whereas forelimbs developed normally in Sall4 mutants, further genetic analysis identified that the Sall4-Gli3 system is a common regulator of the early limb progenitor cells in both forelimbs and hindlimbs. The Sall4-Gli3 system also functions upstream of the Shh-expressing ZPA and the Fgf8-expressing AER in fore- and hindlimbs. Therefore, our study identified a critical role of the Sall4-Gli3 system at the early steps of limb development for proper development of the appendicular skeletal elements.


Assuntos
Osso e Ossos/embriologia , Proteínas de Ligação a DNA/metabolismo , Membro Anterior/embriologia , Membro Posterior/embriologia , Fatores de Transcrição Kruppel-Like/metabolismo , Botões de Extremidades/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição/metabolismo , Animais , Padronização Corporal , Osso e Ossos/metabolismo , Proliferação de Células , Proteínas de Ligação a DNA/genética , Epistasia Genética , Membro Anterior/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Membro Posterior/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Modelos Biológicos , Proteínas do Tecido Nervoso/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica , Transdução de Sinais , Fatores de Tempo , Fatores de Transcrição/genética , Proteína Gli3 com Dedos de Zinco
7.
Development ; 139(22): 4133-42, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23034636

RESUMO

Adult zebrafish possess a significant ability to regenerate injured heart tissue through proliferation of pre-existing cardiomyocytes, which contrasts with the inability of mammals to do so after the immediate postnatal period. Zebrafish therefore provide a model system in which to study how an injured heart can be repaired. However, it remains unknown what important processes cardiomyocytes are involved in other than partial de-differentiation and proliferation. Here we show that migration of cardiomyocytes to the injury site is essential for heart regeneration. Ventricular amputation induced expression of cxcl12a and cxcr4b, genes encoding a chemokine ligand and its receptor. We found that cxcl12a was expressed in the epicardial tissue and that Cxcr4 was expressed in cardiomyocytes. We show that pharmacological blocking of Cxcr4 function as well as genetic loss of cxcr4b function causes failure to regenerate the heart after ventricular resection. Cardiomyocyte proliferation was not affected but a large portion of proliferating cardiomyocytes remained localized outside the injury site. A photoconvertible fluorescent reporter-based cardiomyocyte-tracing assay demonstrates that cardiomyocytes migrated into the injury site in control hearts but that migration was inhibited in the Cxcr4-blocked hearts. By contrast, the epicardial cells and vascular endothelial cells were not affected by blocking Cxcr4 function. Our data show that the migration of cardiomyocytes into the injury site is regulated independently of proliferation, and that coordination of both processes is necessary for heart regeneration.


Assuntos
Quimiocina CXCL12/biossíntese , Coração/fisiologia , Miócitos Cardíacos/fisiologia , Receptores CXCR4/biossíntese , Regeneração , Proteínas de Peixe-Zebra/biossíntese , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Movimento Celular , Proliferação de Células , Quimiocina CXCL12/genética , Traumatismos Cardíacos/fisiopatologia , Ventrículos do Coração , Miocárdio/metabolismo , Receptores CXCR4/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologia , Proteínas de Peixe-Zebra/genética
9.
Dev Dyn ; 240(5): 1151-62, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21384471

RESUMO

The chromatin factors Hmgb1 and Hmgb2 have critical roles in cellular processes, including transcription and DNA modification. To identify the function of Hmgb genes in embryonic development, we generated double mutants of Hmgb1;Hmgb2 in mice. While double null embryos arrest at E9.5, Hmgb1(-/-) ; Hmgb2(+/-) embryos exhibit a loss of digit5, the most posterior digit, in the forelimb. We show that Hmgb1(-/-) ; Hmgb2(+/-) forelimbs have a reduced level of Shh signaling, as well as a clear downregulation of Wnt and BMP target genes in the posterior region. Moreover, we demonstrate that hmgb1 and hmgb2 in zebrafish embryos enhance Wnt signaling in a variety of tissues, and that double knockdown embryos have reduced Wnt signaling and shh expression in pectoral fin buds. Our data show that Hmgb1 and Hmgb2 function redundantly to enhance Wnt signaling in embryos, and further suggest that integrating Wnt, Shh, and BMP signaling regulates the development of digit5 in forelimbs.


Assuntos
Extremidades/embriologia , Proteínas HMGB/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Embrião de Mamíferos/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas HMGB/genética , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Proteína HMGB2/genética , Proteína HMGB2/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Mutantes , Modelos Biológicos , Gravidez , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Peixe-Zebra
10.
BMC Biotechnol ; 11: 5, 2011 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-21235743

RESUMO

BACKGROUND: A promoter capable of driving high-level transgene expression in oviduct cells is important for developing transgenic chickens capable of producing therapeutic proteins, including monoclonal antibodies (mAbs), in the whites of laid eggs. Ovalbumin promoters can be used as oviduct-specific regulatory sequences in transgenic chickens, but their promoter activities are not high, according to previous reports. RESULTS: In this study, while using a previously characterized ovalbumin promoter, we attempted to improve the expression level of mAbs using a Cre/loxP-mediated conditional excision system. We constructed a therapeutic mAb expression vector, pBS-DS-hIgG, driven by the CMV and CAG promoters, in which the expression of the heavy and light chains of humanized immunoglobulin G (hIgG) is preceded by two floxed stuffer reporter genes. In the presence of Cre, the stuffer genes were precisely excised and hIgG expression was induced in pBS-DS-hIgG-transfected 293T cells. In chicken oviduct primary culture cells, hIgG was expressed after transfection of pBS-DS-hIgG together with the ovalbumin promoter-driven Cre expression vector. The expression level of hIgG in these cells was increased 40-fold over that induced directly by the ovalbumin promoter. On the other hand, hIgG was not induced by the ovalbumin promoter-driven Cre in chicken embryonic fibroblast cells. CONCLUSIONS: The Cre/loxP-based system could significantly increase ovalbumin promoter-driven production of proteins of interest, specifically in oviduct cells. This expression system could be useful for producing therapeutic mAbs at high level using transgenic chickens as bioreactors.


Assuntos
Anticorpos Monoclonais/biossíntese , Ovalbumina/genética , Oviductos/fisiologia , Proteínas Recombinantes/biossíntese , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Linhagem Celular , Células Cultivadas , Galinhas , Feminino , Vetores Genéticos , Células HEK293 , Humanos , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Integrases/genética , Oviductos/citologia , Oviductos/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
11.
Biosci Biotechnol Biochem ; 74(12): 2426-30, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-21150102

RESUMO

Chicken primordial germ cells (PGCs) differentiate into germ cells in gonads. Because PGCs can be cloned and cultured maintaining germline competency, they are a good means of modifying the chicken genome, but the efficiency of plasmid transfection into PGCs is very low. In this study, I attempted to improve the efficiency of PGC transfection. Cultured PGCs were purified by Percoll density gradient centrifugation, and were then transfected with plasmid DNA. For transient transfection, the transfection efficiency increased more than 7-fold by the Percoll method. The efficiency of stable transfection of PGCs also increased significantly. The stable transfectants that were isolated by this method accumulated in the developing gonads after microinjection into bloodstream of chick embryos, indicating that gene transfection by Percoll purification did not alter the function of PGCs in vivo.


Assuntos
Centrifugação com Gradiente de Concentração/métodos , Galinhas , Células Germinativas/citologia , Células Germinativas/metabolismo , Povidona , Dióxido de Silício , Transfecção/métodos , Animais , Células Cultivadas , Embrião de Galinha , DNA/genética , Células Germinativas/transplante , Gônadas/citologia , Gônadas/crescimento & desenvolvimento , Ratos
12.
Dev Dyn ; 239(1): 1-15, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19530173

RESUMO

The Ror-family receptor tyrosine kinases (RTKs) play crucial roles in the development of various organs and tissues. In mammals, Ror2, a member of the Ror-family RTKs, has been shown to act as a receptor or coreceptor for Wnt5a to mediate noncanonical Wnt signaling. Ror2- and Wnt5a-deficient mice exhibit similar abnormalities during developmental morphogenesis, reflecting their defects in convergent extension movements and planar cell polarity, characteristic features mediated by noncanonical Wnt signaling. Furthermore, mutations within the human Ror2 gene are responsible for the genetic skeletal disorders dominant brachydactyly type B and recessive Robinow syndrome. Accumulating evidence demonstrate that Ror2 mediates noncanonical Wnt5a signaling by inhibiting the beta-catenin-TCF pathway and activating the Wnt/JNK pathway that results in polarized cell migration. In this article, we review recent progress in understanding the roles of noncanonical Wnt5a/Ror2 signaling in developmental morphogenesis and in human diseases, including heritable skeletal disorders and tumor invasion.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Doenças Genéticas Inatas/genética , Morfogênese/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Transdução de Sinais/fisiologia , Proteínas Wnt/metabolismo , Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Humanos , Modelos Biológicos , Proteína Wnt-5a
13.
AIDS Res Hum Retroviruses ; 24(12): 1561-4, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19102688

RESUMO

The genetic diversity of HIV-1 subtypes circulating in three districts of northern Kenya, i.e., Turkana, Mandera, and Moyale, was studied. DNA sequences encoding a portion of the env-C2-V3 region of the virus were amplified by PCR and sequenced directly. One hundred and fifty-nine samples were successfully sequenced in the env-C2-V3 region and analyzed. From the analysis, 57% were subtype A1, 27% were subtype C, 9% were subtype D, and the remaining 7% were unclassified. This study showed that HIV-1 subtype A1 was the dominant subtype in circulation in this region, though there was a significant percentage of HIV-1 subtype C in circulation there.


Assuntos
Variação Genética , Infecções por HIV/epidemiologia , Infecções por HIV/virologia , HIV-1/classificação , HIV-1/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Criança , Análise por Conglomerados , DNA Viral/química , DNA Viral/genética , Feminino , Genótipo , HIV-1/isolamento & purificação , Humanos , Quênia/epidemiologia , Epidemiologia Molecular , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Provírus/genética , Análise de Sequência de DNA
14.
Nat Genet ; 40(12): 1454-60, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19011629

RESUMO

It has been proposed that ciliated cells that produce a leftward fluid flow mediate left-right patterning in many vertebrate embryos. The cilia on these cells combine features of primary sensory and motile cilia, but how this cilia subtype is specified is unknown. We address this issue by analyzing the Xenopus and zebrafish homologs of Foxj1, a forkhead transcription factor necessary for ciliogenesis in multiciliated cells of the mouse. We show that the cilia that underlie left-right patterning on the Xenopus gastrocoel roof plate (GRP) and zebrafish Kupffer's vesicle are severely shortened or fail to form in Foxj1 morphants. We also show that misexpressing Foxj1 is sufficient to induce ectopic GRP-like cilia formation in frog embryos. Microarray analysis indicates that Xenopus Foxj1 induces the formation of cilia by upregulating the expression of motile cilia genes. These results indicate that Foxj1 is a critical determinant in the specification of cilia used in left-right patterning.


Assuntos
Cílios/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Padronização Corporal , Fatores de Transcrição Forkhead/genética , Xenopus/embriologia , Xenopus/genética , Proteínas de Xenopus/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra
15.
J Infect Chemother ; 14(1): 51-5, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18297450

RESUMO

By human immunodeficiency virus type 1 (HIV-1) antibody screening of people who visited sexually transmitted infection (STI)-related clinics (venereology, urology, and gynecology) and were considered to conduct high-risk sexual activities for HIV-1 infection in Osaka, Japan, during 1992 to 2004, a total of 54 HIV-1 infected individuals (51 Japanese males and 3 non-Japanese females) were identified. Based on the sequencing at env-C2V3 and pol regions, Japanese males were mostly of subtype B (50/51 cases), with the one remaining case being a recombinant circulating form, CRF01_AE, while 3/3 viruses in non-Japanese females were of CRF01_AE. Analysis of subtype B cases since 2001 showed that these viruses became wider in their genetic variation, including amino acid insertions and also deletions, than that of the cases before 2000. Thus, it was suggested that HIV-1 spreading in Osaka has been increasing in genetic variability. Although all these infected individuals were first recognized to be infected with HIV-1 by our screening, some of them were carriers of HIV-1 with drug-resistant pol sequences, indicating that they could be infected with drug-resistant HIV-1 mutants.


Assuntos
Infecções por HIV/virologia , HIV-1/genética , Instituições de Assistência Ambulatorial , Sequência de Aminoácidos , Clonagem Molecular , Farmacorresistência Viral , Feminino , Infecções por HIV/genética , Soropositividade para HIV/diagnóstico , HIV-1/classificação , Humanos , Japão , Masculino , Dados de Sequência Molecular , Mutagênese Insercional , Filogenia , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Análise de Sequência de DNA , Deleção de Sequência , Sexo sem Proteção , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene pol do Vírus da Imunodeficiência Humana/genética
16.
Nat Clin Pract Cardiovasc Med ; 4 Suppl 1: S77-82, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17230219

RESUMO

The migration of myocardial precursor cells towards the embryonic midline underlies the formation of the heart tube and is a key process of heart organogenesis. The zebrafish mutation miles-apart (mil), which affects the gene encoding a sphingosine-1-phosphate receptor, is characterized by defective migration of myocardial precursor cells and results in the formation of two laterally positioned hearts, a condition known as cardia bifida. The mechanism that disrupts myocardial migration in mil mutants remains largely unclear. To investigate how mil regulates this process, here we analyze the interactions between mil and other mediators of myocardial migration. We show that mil function is associated with the other known cardia bifida locus, natter/fibronectin (nat/fn), which encodes fibronectin, a major component of the extracellular matrix, in the control of myocardial migration. By using a primary culture system of embryonic zebrafish cells, we also show that signaling from the sphingosine-1-phosphate receptor regulates cell-fibronectin interactions in zebrafish. In addition, localized inhibition and activation of cell-fibronectin interactions during the stages of myocardial migration reveal that the temporal regulation of cell-fibronectin interaction by mil is required for proper myocardial migration. Our study reveals novel functional links between sphingosine-1-phosphate receptor signaling and cell-fibronectin interaction in the control of myocardial migration during zebrafish heart organogenesis.


Assuntos
Fibronectinas/fisiologia , Miócitos Cardíacos/fisiologia , Organogênese/fisiologia , Receptores de Lisoesfingolipídeo/genética , Animais , Comunicação Celular , Movimento Celular , Genótipo , Mutação , Organogênese/genética , Peixe-Zebra
17.
Nat Genet ; 38(11): 1316-22, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17013396

RESUMO

Primary cilia are microtubule-based organelles that project from the surface of nearly every animal cell. Although important functions of primary cilia in morphogenesis and tissue homeostasis have been identified, the mechanisms that control the formation of primary cilia are not understood. Here we characterize a zebrafish gene, termed duboraya (dub), that is essential for ciliogenesis. Knockdown of dub in zebrafish embryos results in both defects in primary cilia formation in Kupffer's vesicle and randomization of left-right organ asymmetries. We show that, at the molecular level, the function of dub in ciliogenesis is regulated by phosphorylation, which in turn depends on Frizzled-2-mediated noncanonical Wnt signaling. We also provide evidence that, at the cellular level, dub function is essential for actin organization in the cells lining Kupffer's vesicle. Taken together, our findings identify a molecular factor that links noncanonical Wnt signaling with the control of left-right axis specification, and provide an entry point for analyzing the mechanisms that regulate primary cilia formation.


Assuntos
Padronização Corporal/genética , Cílios/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas Wnt/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Padronização Corporal/fisiologia , Clonagem Molecular , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Organogênese/genética , Transdução de Sinais
18.
J Biol Chem ; 281(34): 24847-62, 2006 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-16798742

RESUMO

The anti-oncogenic Chk2 kinase plays a crucial role in DNA damage-induced cell cycle checkpoint regulation. Recently, we have shown that Chk2 associates with the oncogenic Wip1 (PPM1D) phosphatase and that Wip1 acts as a negative regulator of Chk2 during DNA damage response by dephosphorylating phosphorylated Thr-68 in activated Chk2 (Fujimoto, H., Onishi, N., Kato, N., Takekawa, M., Xu, X. Z., Kosugi, A., Kondo, T., Imamura, M., Oishi, I., Yoda, A., and Minami, Y. (2006) Cell Death Differ. 13, 1170-1180). Here, we performed structure-function analyses of Chk2 and Wip1 by using a series of deletion or amino acid-substituted mutant proteins of Chk2 and Wip1. We show that nuclear localization of both Chk2 and Wip1 is required for their association in cultured cells and that the serine-glutamine (SQ)/threonine-glutamine (TQ) domain of Chk2, containing Thr-68, and the N-terminal domain of Wip1, comprising about 100 amino acids, are necessary and sufficient for the association of both molecules. However, it was found that an intrinsic kinase activity of Chk2, but not phosphatase activity of Wip1, is required for the association of fulllength Chk2 and Wip1. Interestingly, we also show that the mutant Wip1 proteins, bearing the N-terminal domain of Wip1 alone or lacking an intrinsic phosphatase activity, exhibit dominant negative effects on the functions of the wild-type Wip1, i.e. ectopic expression of either of these Wip1 mutants inhibits dephosphorylation of Thr-68 in Chk2 by Wip1 and anti-apoptotic function of Wip1. These results provide a molecular basis for developing novel anti-cancer drugs, targeting oncogenic Wip1 phosphatase.


Assuntos
Fosfoproteínas Fosfatases , Proteínas Serina-Treonina Quinases , Animais , Linhagem Celular , Quinase do Ponto de Checagem 2 , Dano ao DNA , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Ligação Proteica , Proteína Fosfatase 2C , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato
19.
AIDS Res Hum Retroviruses ; 21(9): 810-4, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16218806

RESUMO

The genetic subtypes of HIV-1 circulating in northern Kenya have not been characterized. Here we report the partial sequencing and analysis of samples collected in the years 2003 and 2004 from 72 HIV-1-positive patients in northern Kenya, which borders Ethiopia, Somalia, and Sudan. From the analysis of partial env sequences, it was determined that 50% were subtype A, 39% subtype C, and 11% subtype D. This shows that in the northern border region of Kenya subtypes A and C are the dominant HIV-1 subtypes in circulation. Ethiopia is dominated mainly by HIV-1 subtype C, which incidentally is the dominant subtype in the town of Moyale, which borders Ethiopia. These results show that cross-border movements play an important role in the circulation of subtypes in Northern Kenya.


Assuntos
Infecções por HIV/epidemiologia , HIV-1/genética , Adolescente , Adulto , Criança , Pré-Escolar , Genes env/genética , Proteína gp41 do Envelope de HIV/genética , Humanos , Quênia/epidemiologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Filogenia , Especificidade da Espécie
20.
Genes Cells ; 9(12): 1227-38, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15569154

RESUMO

The brachydactylies are a group of inherited disorders of the hands characterized by shortened digits. Mutations in the tyrosine kinase receptor Ror2 cause brachydactyly type B (BDB). Mutations in GDF5, a member of the BMP/TGF-beta ligand family, cause brachydactyly type C (BDC) whereas mutations in the receptor for GDF5, BRI-b, cause brachydactyly type A2 (BDA2). There is considerable degree of phenotypic overlap between the subtypes BDB, BDC and BDA2. Here we demonstrate that all three components are involved in GDF5 induced regulation of chondrogenesis. We show that Ror2 (tyrosine kinase receptor) and BRI-b (serine/threonine kinase receptor) form a ligand independent heteromeric complex. The frizzled-like-CRD domain of Ror2 is required for this complex. Within that complex Ror2 gets transphosphorylated by BRI-b. We show that Ror2 modulates GDF5 signalling by inhibition of Smad1/5 signalling and by activating a Smad-independent pathway. Both pathways however, are needed for chondrogenic differentiation as demonstrated in ATDC5 cells. The functional interaction of Ror2 with GDF5 and BRI-b was genetically confirmed by the presence of epistatic effects in crosses of Ror2, BRI-b and Gdf5 deficient mice. These results indicate for the first time a direct interaction of Ser/Thr- and Tyr-Kinase receptors and provide evidence for modulation of the Smad-pathway and GDF5 triggered chondrogenesis.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/farmacologia , Células COS , Linhagem Celular , Condrogênese , Proteínas de Ligação a DNA/metabolismo , Genótipo , Glicosaminoglicanos/biossíntese , Fator 5 de Diferenciação de Crescimento , Humanos , Úmero/química , Ligantes , Camundongos , Mutação , Fosforilação , Receptores Proteína Tirosina Quinases/genética , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase , Receptores de Superfície Celular/genética , Transdução de Sinais , Proteínas Smad , Proteína Smad1 , Transativadores/metabolismo
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