RESUMO
SnoaL belongs to a family of small polyketide cyclases, which catalyse ring closure steps in the biosynthesis of polyketide antibiotics produced in Streptomyces. Several of these antibiotics are among the most used anti-cancer drugs currently in use. The crystal structure of SnoaL, involved in nogalamycin biosynthesis, with a bound product, has been determined to 1.35 A resolution. The fold of the subunit can be described as a distorted alpha+beta barrel, and the ligand is bound in the hydrophobic interior of the barrel. The 3D structure and site-directed mutagenesis experiments reveal that the mechanism of the intramolecular aldol condensation catalysed by SnoaL is different from that of the classical aldolases, which employ covalent Schiff base formation or a metal ion cofactor. The invariant residue Asp121 acts as an acid/base catalyst during the reaction. Stabilisation of the enol(ate) intermediate is mainly achieved by the delocalisation of the electron pair over the extended pi system of the substrate. These polyketide cyclases thus form of family of enzymes with a unique catalytic strategy for aldol condensation.
Assuntos
Aldeídos/metabolismo , Antibacterianos/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Isomerases/química , Isomerases/metabolismo , Modelos Moleculares , Nogalamicina/biossíntese , Streptomyces/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Biologia Computacional , Cristalografia por Raios X , Isomerases/genética , Espectrometria de Massas , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligonucleotídeos , Conformação Proteica , Alinhamento de SequênciaRESUMO
OBJECTIVE: To investigate the characterization of Notch gene involved in genetic regulatory networks in dental pulp stem cells. METHODS: The pulp tissue was separated from mouse teeth and digested by collagenase type I. Single-cell suspensions of dental pulp were seeded into 6-well plates with alpha modification of Eagle's medium supplemented with ES cell qualified Fetal Bovine Serum. Colony-forming efficiency was assessed in 14ds culture. Transcripts for Notch were detected by reverse transcription-PCR by using total RNA isolated from cells. RESULTS: There were clonogenic cells in dental pulp cell and the incidence of colony-forming cells derived from mouse dental pulp cells was 1.6-2.5 colonies/10(4) plate. Mouse-specific Notch mRNA expressed in colony-forming cells. CONCLUSION: Notch mRNA expressing in colony-forming cells provided a more detailed understanding of mouse dental pulp stem cell biology.
Assuntos
Polpa Dentária/citologia , Proteínas de Membrana/genética , Receptores de Superfície Celular/genética , Células-Tronco/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Polpa Dentária/metabolismo , Proteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Superfície Celular/biossíntese , Receptores Notch , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
Tax is a transcription activator encoded by human T-cell leukemia virus (HTLV)-1. Ribosomal protein L6 was also defined as Taxreb107 (Tax responsible element binding protein 107) for its activity of binding to the long terminal repeats of HTLV-1. To investigate the relationship between Tax and Taxreb107/RpL6, yeast two hybrid and GST pull-down assays were used. Results suggest that Tax can interact with Taxreb107/RpL6 directly and Taxreb107/RpL6 may regulate the function of Tax in HTLV-1 proliferation.
Assuntos
Produtos do Gene tax/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Proteínas Ribossômicas/metabolismo , Animais , DNA Complementar/genética , Produtos do Gene tax/genética , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Camundongos , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
To study the function of KyoT2 in vivo, two-hybrid yeast, purification of KyoT2 protein, preparation of antibody and GST-pulldown methods were used in the experiments. 42 clones were obtained after 5 x 10(6) clones were screened by four kinds of nutrition limitation and beta-galactosidase assay, 22 clones were obtained after restriction of positive clones. Finally, 13 genes were obtained by sequence assay. Two of these were RBP-Jk and PIAS1. After they and KyoT2 changed vectors, negative two-hybrid yeast was finished. The result was positive; Using KyoT2 protein and antibody GST-pulldown of KyoT2 and RBP-Jk, KyoT2 and PIAS1 were done, the result was also positive. Therefore, KyoT2 interacted with RBP-Jk and PIAS1.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/química , Proteínas Musculares , Proteínas Nucleares , Proteínas/metabolismo , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Clonagem Molecular/métodos , Proteínas de Ligação a DNA/genética , Biblioteca Gênica , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas com Domínio LIM , Proteínas Inibidoras de STAT Ativados , Proteínas/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , LevedurasRESUMO
The KyoT expression in the adult mouse was reported here for further investigation on the functions of KyoT in adult mouse. To study the expression of mRNA and protein of KyoT, Northern blot, RT-PCR, Immunohistochemical SABC methods and in situ hybridization methods were used in the experiments. Two kinds of KyoT were expressed at high levels in testis of adult mouse, and KyoT immunore activity was mainly located in Leydig's cells. The reactive substance was distributed in cytoplasm rather than in nuclei. KyoT mRNA hybridization signals were also detected in cytoplasm of Leydig's cells rather than in nuclei. The spermatogenic cells and negative controls showed negative results. These results suggest that KyoT was expressed in testis of adult mouse and mainly located in Leydig's cells.
RESUMO
A genetic screening system was established previously for the cloning of gene fragments encoding nuclear localization signals (NLS). In the current study, the system was improved and its ability to distinguish nuclear and cytoplasm-localized proteins was verified by inserting a known NLS-encoding gene. A mouse 10.5-d pc embryonic cDNA library was then inserted into the cloning site of the screening vector and 22 out of 10(4) clones were shown to be positive after screening using the system. The cDNA inserts of some of these clones were sequenced, and typical and untypical NLS were identified. The nuclear localizing activity of the cDNA inserts was further verified using the GFP fusion protein system. So, the system is useful in the cDNA library screening of genes encoding NLS-bearing proteins.
