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1.
Hemoglobin ; 43(1): 63-65, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31037981

RESUMO

Hb A'2 (or Hb B2) (HBD: c.49G>C) is the most frequent δ chain variant that has been described in Africa but not in Thailand. We report here a 10-month-old Thai infant with compound heterozygosity for ß0 codon 17 (A>T; HBB: c.52A>T) and ß+ IVS II-654 (C>T; HBB: c.316-197C>T). Under diagnosed ß-thalassemia (ß-thal) in her father, who carries Hb A'2 and a heterozygous ß0 codon 17 mutation, and the mother, who carries a heterozygous ß+ IVS II-654 mutation, was noted. Although Hb A'2 does not cause any problems, heterozygosity for Hb A'2 can lead to under diagnosis of ß-thal in Hb A'2 samples. This case highlights the importance of Hb A'2 in prenatal diagnosis (PND). Thus, molecular analysis for ß-thal mutations should be carried out when a small peak presents at the retention time (RT) of 4.71 min. on high performance liquid chromatography (HPLC) and the summation level of this peak and Hb A2 was equal or higher than 4.0%.


Assuntos
Hemoglobina A2/genética , Heterozigoto , Globinas beta/genética , Talassemia beta/diagnóstico , Talassemia beta/genética , Adulto , Cromatografia Líquida de Alta Pressão , Códon , Índices de Eritrócitos , Feminino , Genótipo , Hemoglobina A2/química , Hemoglobinas Anormais/química , Hemoglobinas Anormais/genética , Humanos , Lactente , Masculino , Mutação , Diagnóstico Pré-Natal , Globinas beta/química , Talassemia beta/sangue
2.
Cancer Biother Radiopharm ; 32(1): 1-8, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28118037

RESUMO

Overexpression of extracellular matrix metalloproteinase inducer (EMMPRIN) accelerates tumor invasion and metastasis via activation of matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator (uPA) expression. The authors were interested in whether the scFv-M6-1B9 intrabody against EMMPRIN that retains EMMPRIN in endoplasmic reticulum could be a potential tool to suppress cervical cancer invasion through inhibition of uPA. The chimeric adenoviral vector Ad5/F35-scFv-M6-1B9 was transferred into human cervical carcinoma HeLa cells to produce the scFv-M6-1B9 intrabody against EMMPRIN. Cell surface expression of EMMPRIN, the membrane-bound uPA, the enzymatic activity of secreted uPA, and the invasion ability were analyzed. The scFv-M6-1B9 intrabody successfully diminished the cell surface expression of EMMPRIN and the membrane-bound uPA on HeLa cells. uPA activity from tissue culture media of EMMPRIN-downregulated HeLa cells was decreased. The invasion ability of HeLa cells harboring scFv-M6-1B9 intrabody was also suppressed. These results suggested that the scFv-M6-1B9 intrabody might represent a potential approach for invasive cervical cancer treatment. The application of scFv-M6-1B9 intrabody in animal experiments and preclinical studies would be investigated further.


Assuntos
Basigina/genética , Terapia Genética/métodos , Anticorpos de Cadeia Única/genética , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/terapia , Adenoviridae , Regulação para Baixo , Feminino , Vetores Genéticos/farmacologia , Células HEK293 , Células HeLa , Humanos , Metaloproteinases da Matriz/metabolismo , Invasividade Neoplásica
3.
Appl Biochem Biotechnol ; 176(6): 1781-90, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26024713

RESUMO

This study was carried out to understand the effect of the recombinant multivalent extracellular matrix metalloproteinase inducer (EMMPRIN) extracellular domain, designated as rmEMMPRINex, on the apoptotic cell death of human leukemia U937 cells. Expression of monocarboxylate transporter 1 (MCT1) and caspase-9 in U937 treated with rmEMMPRINex was investigated in this study. Levels of membrane MCT1 and intracellular procaspase-9 were decreased in rmEMMPRINex-treated cells in comparison to controls. However, the expression of activated caspase-9 was undetectable. rmEMMPRINex also induced DNA fragmentation and apoptosis in U937 cells. Taken together, we concluded that interaction of rmEMMPRINex with U937 cells leads to inhibition of MCT1 membrane expression, intracellular activation of procaspase-9, followed by DNA fragmentation and apoptosis. This may contribute to the conceptual development of novel cancer drugs in the future.


Assuntos
Apoptose/efeitos dos fármacos , Basigina/farmacologia , Caspase 9/biossíntese , Regulação para Baixo/efeitos dos fármacos , Leucemia/metabolismo , Transportadores de Ácidos Monocarboxílicos/biossíntese , Proteínas de Neoplasias/biossíntese , Simportadores/biossíntese , Basigina/genética , Humanos , Leucemia/patologia , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Células U937
4.
J Cancer ; 6(3): 276-86, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25663946

RESUMO

Extracellular matrix metalloproteinase inducer (EMMPRIN) is a human leukocyte surface molecule that is enriched on the surface of many cancer cells, and it plays an important role in proliferation and metastasis. In this study, we utilized the chimeric adenoviral vector Ad5/F35 carrying gene encoding scFv against EMMPRIN (scFv-M6-1B9) to down-regulate EMMPRIN cell surface expression and investigated programmed cell death response in colorectal cancer (CRC) cell, Caco-2. The scFv-M6-1B9 intrabody exhibits robust activity in reducing EMMPRIN cell surface expression. This approach led to the inducing of apoptosis, which was relative to the increasing of apoptotic bodies in sub-G1 peak, phosphatidylserine externalization, as well as TUNEL-positive cells. In addition, real-time RT-PCR and western blotting analysis indicated that apoptosis was enhanced through the mitochondrial pathway, a marked reduction of Bcl-2, leading to the translocation of cytochrome c and also the dramatic activation of caspase-3. Moreover, carcinoembryonic antigen (CEA), a tumor marker for CRC, was found to have significantly diminished in both secreted protein and mRNA levels. In conclusion, these findings suggest that EMMPRIN down-regulation by scFv-M6-1B9 intrabody has great potential in enhancing the efficacy of apoptosis induction through the mitochondrial pathway and in effecting a decline in the CEA level. Thus, its benefits could be applied to project the future prospects for targeted gene therapy and therapeutic application in monitoring colorectal cancer.

5.
BMC Biotechnol ; 8: 5, 2008 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-18226275

RESUMO

BACKGROUND: Expression of intracellular antibodies (intrabodies) has become a broadly applicable technology for generation of phenotypic knockouts in vivo. The method uses surface depletion of cellular membrane proteins to examine their biological function. In this study, we used this strategy to block the transport of cell surface molecule CD147 to the cell membrane. Phage display technology was introduced to generate the functional antibody fragment to CD147, and we subsequently constructed a CD147-specific scFv that was expressed intracellularly and retained in the endoplasmic reticulum by adenoviral gene transfer. RESULTS: The recombinant antibody fragments, Fab and scFv, of the murine monoclonal antibody (clone M6-1B9) reacted specifically to CD147 by indirect enzyme-linked immunosorbent assays (ELISA) using a recombinant CD147-BCCP as a target. This indicated that the Fab- and scFv-M6-1B9 displaying on phage surfaces were correctly folded and functionally active. We subsequently constructed a CD147-specific scFv, scFv-M6-1B9-intrabody, in 293A cells. The expression of CD147 on 293A cell surface was monitored at 36 h after transduction by flow cytometry and demonstrated remarkable reduction. Colocalization of scFv-M6-1B9 intrabody with CD147 in the ER network was depicted using a 3D deconvolution microscopy system. CONCLUSION: The results suggest that our approach can generate antibody fragments suitable for decreasing the expression of CD147 on 293A cells. This study represents a step toward understanding the role of the cell surface protein, CD147.


Assuntos
Anticorpos Monoclonais/metabolismo , Membrana Celular/metabolismo , Fragmentos de Imunoglobulinas/metabolismo , Rim/metabolismo , Engenharia de Proteínas/métodos , Anticorpos Monoclonais/genética , Linhagem Celular , Humanos , Fragmentos de Imunoglobulinas/genética
6.
Immunobiology ; 214(6): 410-21, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-19264376

RESUMO

CD147, a multifunctional type I transmembrane glycoprotein, has been implicated in various physiological and pathological processes. It is involved in signal transduction pathways and also plays a crucial role in the invasive and metastatic activity of malignant tumor cells. Diminished expression of this molecule has been shown to be beneficial in suppression of tumor progression. In a previous study, we generated and characterized a recombinant antibody fragment, scFv, which reacted specifically to CD147. In the present study, we further investigated the biological properties, function and the effect of generated scFv on CD147 expression. The in vitro study showed that soluble scFv-M6-1B9 produced from E. coli HB2151 bound to CD147 surface molecule and inhibited OKT3-induced T cell proliferation. Furthermore, soluble lysate of scFv-M6-1B9 from 293A cells, transduced with a scFv-M6-1B9 expressing adenovirus vector, recognized both recombinant and native CD147. These results indicate that scFv-M6-1B9 binds with high efficiency and specificity. Importantly, scFv-M6-1B9 intrabody reduced the expression of CD147 on the cell surface of HeLa cells suggesting that scFv-M6-1B9 is biologically active. In conclusion, our present study demonstrated that scFv-M6-1B9 has a great potential to target both the intracellular and the extracellular CD147. The generated scFv-M6-1B9 may be an effective agent to clarify the cellular function of CD147 and may aid in efforts to develop a novel treatment in various human carcinomas.


Assuntos
Basigina/metabolismo , Muromonab-CD3/imunologia , Anticorpos de Cadeia Única/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologia , Membrana Celular/imunologia , Proliferação de Células , Gelatinases/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Anticorpos de Cadeia Única/genética , Linfócitos T/metabolismo
7.
Int Immunol ; 18(7): 1159-69, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16740601

RESUMO

CD147 is a broadly expressed cell-surface molecule and serves as a signaling receptor for extracellular cyclophilins. CD147 also appears to interact with immune cells, but its counter-receptor on these cells has not been clearly described. In the present report, we displayed multiple copies of the CD147 extracellular domain (CD147Ex) on VCSM13 phage to study the interaction of CD147 with its ligand. Recognition of phage containing fusion protein of CD147Ex and gpVIII (CD147Ex phage) by four different anti-CD147 mAbs indicated that at least parts of the CD147 are properly folded. Specific binding of CD147Ex phage to various cell types was demonstrated by flow cytometry. Morphological changes, however, were observed only in U937, a monocytic cell line, after 24 h incubation with multivalent CD147Ex phage. After 48 h, U937 cell propagation ceased. Staining with annexin V and the presence of cleaved caspase-3 indicated that many of the CD147Ex phage-treated cells had lost viability through apoptotic cell death. The above results suggest that CD147 induces apoptosis in U973 cells and that at least a portion of this cell death program involves a caspase-dependent pathway.


Assuntos
Apoptose/imunologia , Bacteriófagos/imunologia , Basigina/imunologia , Anexina A5/imunologia , Caspase 3 , Caspases/imunologia , Humanos , Células Jurkat , Células U937
8.
Protein Expr Purif ; 32(2): 323-31, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14965780

RESUMO

Production of VCSM13 phage displaying a high density of CD147 ectodomain (CD147Ex) was achieved when culturing conditions were modulated. A phagemid expressing CD147Ex was constructed and used to produce phage display CD147Ex gpVIII fusion protein in TG1 Escherichia coli. Displaying of CD147Ex via gpVIII was successfully increased when growing the transformed TG1 at 25 degrees C with IPTG-stimulation. In addition to temperature and IPTG-stimulation, the VCSM13 helper phage infection-period particularly affected the insertion of CD147Ex into phage progeny. By sandwich ELISA, incorporation of the CD147Ex into phage particle was confirmed. The correct size of the CD147Ex-gpVIII fusion protein at 28kDa was demonstrated by Western immunoblotting. Multivalent display of CD147Ex on phage particles will be valuable in discovering its ligand partner.


Assuntos
Antígenos CD , Antígenos de Neoplasias , Bacteriófago M13/genética , Isopropiltiogalactosídeo/farmacologia , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/química , Basigina , Western Blotting , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Plasmídeos/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Temperatura Ambiente , Fatores de Tempo
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