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1.
Animal ; 10(10): 1602-8, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26936422

RESUMO

Enterotoxigenic Escherichia coli (ETEC) is a type of pathogenic bacteria that cause diarrhea in piglets through colonizing pig small intestine epithelial cells by their surface fimbriae. Different fimbriae type of ETEC including F4, F18, K99 and F41 have been isolated from diarrheal pigs. In this study, we performed a genome-wide association study to map the loci associated with the susceptibility of pigs to ETEC F41 using 39454 single nucleotide polymorphisms (SNPs) in 667 F2 pigs from a White Duroc×Erhualian F2 cross. The most significant SNP (ALGA0022658, P=5.59×10-13) located at 6.95 Mb on chromosome 4. ALGA0022658 was in high linkage disequilibrium (r 2>0.5) with surrounding SNPs that span a 1.21 Mb interval. Within this 1.21 Mb region, we investigated ZFAT as a positional candidate gene. We re-sequenced cDNA of ZFAT in four pigs with different susceptibility phenotypes, and identified seven coding variants. We genotyped these seven variants in 287 unrelated pigs from 15 diverse breeds that were measured with ETEC F41 susceptibility phenotype. Five variants showed nominal significant association (P<0.05) with ETEC F41 susceptibility phenotype in International commercial pigs. This study provided refined region associated with susceptibility of pigs to ETEC F41 than that reported previously. Further works are needed to uncover the underlying causal mutation(s).


Assuntos
Escherichia coli Enterotoxigênica/patogenicidade , Infecções por Escherichia coli/veterinária , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla , Doenças dos Suínos/genética , Doenças dos Suínos/microbiologia , Suínos/genética , Suínos/microbiologia , Animais , Aderência Bacteriana , Cromossomos de Mamíferos/genética , Diarreia/genética , Diarreia/microbiologia , Diarreia/veterinária , Escherichia coli Enterotoxigênica/classificação , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Fímbrias Bacterianas/fisiologia , Desequilíbrio de Ligação , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Fatores de Transcrição/genética
2.
Oncogene ; 25(50): 6618-27, 2006 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-16702944

RESUMO

Phospholipid scramblase 1 (PLSCR1) is a multiply palmitoylated protein which is localized in either the cell membrane or nucleus depending on its palmitoylated state. The increasing evidence showed the biological roles of PLSCR1 in cell signaling, maturation and apoptosis. To investigate the functions of PLSCR1 in leukemic cells, we generated an inducible PLSCR1-expressing cell line using myeloid leukemic U937 cells. In this cell line, PLSCR1 was tightly regulated and induced upon tetracycline withdrawal. Our results showed that inducible PLSCR1 expression arrested the proliferation of U937 cells at G1 phase. Meanwhile, PLSCR1-overexpressing U937 cells also underwent granulocyte-like differentiation with increased sensitivity to etoposide-induced apoptosis. Furthermore, we also found that PLSCR1 induction increased cyclin-dependent kinase inhibitors p27(Kip1) and p21(Cip1) proteins, together with downregulation of S phase kinase-associated protein 2 (SKP2), an F-box subunit of the ubiquitin-ligase complex that targets proteins for degradation. Additionally, PLSCR1 induction significantly decreased c-Myc protein and antiapoptotic Bcl-2 protein. Although the exact mechanism by which PLSCR1 regulates these cellular events and gene expression remains unresolved, our results suggest that PLSCR1 plays the antagonistic role regarding leukemia development. These data will shed new insights into understanding the biochemical and biological functions of PLSCR1 protein.


Assuntos
Leucemia/genética , Proteínas de Transferência de Fosfolipídeos/fisiologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Ciclo Celular/genética , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células , Etoposídeo/farmacologia , Fase G1 , Regulação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia/metabolismo , Células Mieloides , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Transferência de Fosfolipídeos/metabolismo , Transfecção , Células Tumorais Cultivadas , Células U937
3.
Protein Expr Purif ; 19(1): 202-11, 2000 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10833408

RESUMO

alpha-N-Acetylglucosaminidase (EC 3.2.1.50) is a lysosomal enzyme that is deficient in the genetic disorder Sanfilippo syndrome type B. To study the human enzyme, we expressed its cDNA in Lec1 mutant Chinese hamster ovary (CHO) cells, which do not synthesize complex oligosaccharides. The enzyme was purified to apparent homogeneity from culture medium by chromatography on concanavalin A-Sepharose, Poros 20-heparin, and aminooctyl-agarose. The purified enzyme migrated as a single band of 83 kDa on SDS-PAGE and as two peaks corresponding to monomeric and dimeric forms on Sephacryl-300. It had an apparent K(m) of 0.22 mM toward 4-methylumbelliferyl-alpha-N-acetylglucosaminide and was competitively inhibited by two potential transition analogs, 2-acetamido-1,2-dideoxynojirimycin (K(i) = 0.45 microM) and 6-acetamido-6-deoxycastanospermine (K(i) = 0.087 microM). Activity was also inhibited by mercurials but not by N-ethylmaleimide or iodoacetamide, suggesting the presence of essential sulfhydryl residues that are buried. The purified enzyme preparation corrected the abnormal [(35)S]glycosaminoglycan catabolism of Sanfilippo B fibroblasts in a mannose 6-phosphate-inhibitable manner, but its effectiveness was surprisingly low. Metabolic labeling experiments showed that the recombinant alpha-N-acetylglucosaminidase secreted by CHO cells had only a trace of mannose 6-phosphate, probably derived from contaminating endogenous CHO enzyme. This contrasts with the presence of mannose 6-phosphate on naturally occurring alpha-N-acetylglucosaminidase secreted by diploid human fibroblasts and on recombinant human alpha-l-iduronidase secreted by the same CHO cells. Thus contrary to current belief, overexpressing CHO cells do not necessarily secrete recombinant lysosomal enzyme with the mannose 6-phosphate-targeting signal; this finding has implications for the preparation of such enzymes for therapeutic purposes.


Assuntos
Acetilglucosaminidase/isolamento & purificação , Acetilglucosaminidase/química , Acetilglucosaminidase/metabolismo , Animais , Células CHO , Células Cultivadas , Cromatografia em Agarose , Cricetinae , Meios de Cultivo Condicionados , Eletroforese em Gel de Poliacrilamida , Fibroblastos/metabolismo , Humanos , Manosefosfatos/metabolismo , Mucopolissacaridose III/metabolismo , Fosforilação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
4.
Mol Genet Metab ; 71(4): 573-80, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11136549

RESUMO

The Sanfilippo syndrome type B (MPS III B) is an autosomal recessive disease caused by deficiency of alpha-N-acetylglucosaminidase (EC 3. 2.1.50), one of the lysosomal enzymes required for the degradation of heparan sulfate. The disease is characterized by profound neurodegeneration but relatively mild somatic manifestations, and is usually fatal in the second decade. A mouse model had been generated by disruption of the Naglu gene in order to facilitate the study of pathogenesis and the development of therapy for this currently untreatable disease. Recombinant human alpha-N-acetylglucosaminidase (rhNAGLU) was prepared from secretions of Lec1 mutant Chinese hamster ovary cells. The enzyme, which has only unphosphorylated high-mannose carbohydrate chains, was endocytosed by mouse peritoneal macrophages via mannose receptors, with half-maximal uptake at ca. 10(-7) M. When administered intravenously to 3 month-old mice, rhNAGLU was taken up avidly by liver and spleen but marginally if at all by thymus, lung, kidney, heart, and brain (in order of diminishing uptake). The half-life of the enzyme was 2.5 days in liver and spleen. Immunohistochemistry and electron microscopy showed that only macrophages were involved in enzyme uptake and correction in these two organs, yet the storage of glycosaminoglycan was reduced to almost normal levels. The results show that the macrophage-targeted rhNAGLU can substantially reduce the body burden of glycosaminoglycan storage in the mouse model of Sanfilippo syndrome III B.


Assuntos
Acetilglucosaminidase/uso terapêutico , Modelos Animais de Doenças , Mucopolissacaridose III/enzimologia , Mucopolissacaridose III/terapia , Acetilglucosaminidase/deficiência , Acetilglucosaminidase/genética , Acetilglucosaminidase/farmacologia , Animais , Endocitose , Deleção de Genes , Glicosaminoglicanos/metabolismo , Meia-Vida , Heparitina Sulfato/metabolismo , Humanos , Imuno-Histoquímica , Injeções Intravenosas , Fígado/citologia , Fígado/enzimologia , Fígado/metabolismo , Fígado/ultraestrutura , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/enzimologia , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Mucopolissacaridose III/classificação , Mucopolissacaridose III/genética , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Baço/citologia , Baço/enzimologia , Baço/metabolismo
5.
J Biol Chem ; 272(36): 22758-65, 1997 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-9278435

RESUMO

alpha-L-Iduronidase is a lysosomal hydrolase that is deficient in Hurler syndrome and clinically milder variants. Recombinant human alpha-L-iduronidase, isolated from secretions of an overexpressing Chinese hamster ovary cell line, is potentially useful for replacement therapy of these disorders. Because of the importance of carbohydrate residues for endocytosis and lysosomal targeting, we examined the oligosaccharides of recombinant alpha-L-iduronidase at each of its six N-glycosylation sites. Biosynthetic radiolabeling showed that three or four of the six oligosaccharides of the secreted enzyme were cleaved by endo-beta-N-acetylglucosaminidase H, with phosphate present on the sensitive oligosaccharides and L-fucose on the resistant ones. For structural analysis, tryptic and chymotryptic glycopeptides were isolated by lectin binding and reverse phase high pressure liquid chromatography; their molecular mass was determined by matrix-assisted laser desorption-time of flight mass spectrometry before and after treatment with endo- or exoglycosidases or with alkaline phosphatase. Identification of the peptides was assisted by amino- or carboxyl-terminal sequence analysis. The major oligosaccharide structures found at each site were as follows: Asn-110, complex; Asn-190, complex; Asn-336, bisphosphorylated (P2Man7GlcNAc2); Asn-372, high mannose (mainly Man9GlcNAc2, some of which was monoglucosylated); Asn-415, mixed high mannose and complex; Asn-451, bisphosphorylated (P2Man7GlcNAc2). The Asn-451 glycopeptide was unexpectedly resistant to digestion by N-glycanase unless first dephosphorylated, but it was sensitive to endo-beta-N-acetylglucosaminidase H and to glycopeptidase A. The heterogeneity of carbohydrate structures must represent the accessibility of the glycosylation sites as well as the processing capability of the overexpressing Chinese hamster ovary cells.


Assuntos
Iduronidase/química , Sequência de Aminoácidos , Animais , Células CHO , Configuração de Carboidratos , Cromatografia Líquida de Alta Pressão , Cricetinae , Glicopeptídeos/química , Glicosilação , Humanos , Iduronidase/metabolismo , Radioisótopos do Iodo , Mapeamento de Peptídeos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tripsina/química
6.
Glycobiology ; 6(6): 619-26, 1996 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-8922957

RESUMO

The enzyme N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase (EC 3.1.4.45; uncovering enzyme) catalyzed the removal of N-acetylglucosamine from the N-acetylglucosamine-alpha-phospho-mannose portion of selected lysosomal enzyme oligosaccharide chains, thereby forming the mannose 6-phosphate signal which is responsible for the targeting of these lysosomal enzymes for transport into lysosomes. The uncovering enzyme has been purified approximately 7000-fold to electrophoretic homogeneity from Epstein-Barr virus-transformed human lymphoblast cells. The purification sequence involves solubilizing this membrane-bound enzyme with Tergitol NP-10, affinity chromatography on Lentil lectin-Sepharose 4B, ion-exchange chromatography on DEAE-Sephacel, chromatography on zinc(II)-IDA-Sepharose 6B, and preparative SDS-PAGE electrophoresis. The purified enzyme migrated as a single band of 114 kDa which was coincident with enzyme activity on analytical SDS-PAGE electrophoresis. Characterization studies of the purified enzyme demonstrated that catalytic activity was maximal at pH 6.95 and that the enzyme retained full activity following incubation for 10 min at 60 degrees C. No requirement was found for a divalent cation, but Zn2+, Hg2+, and Cu2+ were found to reduce the enzyme's activity by 30-40%. The highest catalytic efficiency was observed with N-acetylglucosamine-phospho-methylmannoside as a substrate while uridine diphosphate-N-acetylglucosamine, N-acetylglucosamine-phosphomannose-uteroferrin, and N-acetylglucosamine-phosphate were also cleaved by the enzyme with decreasing efficiency. Acetamino-deoxycastanospermine was a potent inhibitor of the human enzyme with a Ki of 0.35 microM, while N-acetylglucosamine phosphate (Ki 1.58 mM) and N-acetylglucosamine (Ki 5.1 mM) inhibited the enzyme to a lesser degree.


Assuntos
Linfócitos/enzimologia , Diester Fosfórico Hidrolases/isolamento & purificação , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Humanos , Diester Fosfórico Hidrolases/metabolismo , Especificidade por Substrato
7.
Zhongguo Zhong Xi Yi Jie He Za Zhi ; 13(5): 263-5, 259, 1993 May.
Artigo em Chinês | MEDLINE | ID: mdl-8219673

RESUMO

The extracts of Astragalus membranaceus have been further isolated by liquid chromatography. One of the fractions (Astragalan, M.W. 20,000-25,000) could enhance the secretion of tumor necrosis factor (TNF) from human peripheral blood mononuclear cells (PBMC) in vitro. After isolation of adherent and nonadherent mononuclear cells from PBMC, Astragalan increased the secretion of TNF-alpha and TNF-beta respectively. These results suggest further study of Astragalan would promote the application of Astragalan in cancer immunotherapy.


Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Leucócitos Mononucleares/imunologia , Polissacarídeos/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Adulto , Idoso , Astragalus propinquus , Feminino , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Linfotoxina-alfa/metabolismo , Masculino , Pessoa de Meia-Idade
8.
Glycobiology ; 2(2): 119-25, 1992 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-1318762

RESUMO

N-Acetylglucosamine-1-phosphotransferase (GlcNAcPTase) was solubilized with 2% Tergitol NP-10 from cultured human lymphoblast cells and purified 3840-fold with 14% recovery using lentil lectin-Sepharose 4B, DEAE-Sephacel and Sephacryl S-400 chromatographies. The partially purified enzyme requires the non-ionic detergent Tergitol NP-10 and a divalent cation, Mn2+ or Mg2+, for its activity and exhibits an optimal pH at 7.2-7.5 in Tris-maleate buffer. Kinetic studies demonstrated an apparent Km of 24 microM for the donor UDP-N-acetylglucosamine and of 117 mM for the artificial acceptor alpha-methylmannoside. The GlcNAcPTase is inhibited by UDP and UDP-glucose, and by negatively charged phospholipids including phosphatidylserine, phosphatidylglycerol and phosphatidic acid. The apparent mol. wt of the human lymphoblast GlcNAcPTase is approximately 1000 kDa, which is analogous to that reported for the partially purified enzyme from rat liver (Waheed et al., 1982).


Assuntos
Ativação Linfocitária , Linfócitos/enzimologia , Fosfotransferases/isolamento & purificação , Lectinas de Plantas , Transferases (Outros Grupos de Fosfato Substituídos) , Cromatografia de Afinidade , Cromatografia DEAE-Celulose , Cromatografia em Gel , Estabilidade Enzimática , Humanos , Lectinas , Fosfotransferases/antagonistas & inibidores , Fosfotransferases/química , Solubilidade , Especificidade por Substrato
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