RESUMO
Mg2+ is required at micromolar concentrations as a cofactor for ATP, enzymatic reactions, and other biological processes. We show that decreased extracellular Mg2+ reduced intracellular Mg2+ levels and impaired the Ca2+ flux, activation marker up-regulation, and proliferation after T cell receptor (TCR) stimulation. Reduced Mg2+ specifically impairs TCR signal transduction by IL-2-inducible T cell kinase (ITK) due to a requirement for a regulatory Mg2+ in the catalytic pocket of ITK. We also show that altered catalytic efficiency by millimolar changes in free basal Mg2+ is an unrecognized but conserved feature of other serine/threonine and tyrosine kinases, suggesting a Mg2+ regulatory paradigm of kinase function. Finally, a reduced serum Mg2+ concentration in mice causes an impaired CD8+ T cell response to influenza A virus infection, reduces T cell activation, and exacerbates morbidity. Thus, Mg2+ directly regulates the active site of specific kinases during T cell responses, and maintaining a high serum Mg2+ concentration is important for antiviral immunity in otherwise healthy animals.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Magnésio/farmacologia , Infecções por Orthomyxoviridae/imunologia , Proteínas Tirosina Quinases/metabolismo , Animais , Biocatálise/efeitos dos fármacos , Doadores de Sangue , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Cálcio/metabolismo , Domínio Catalítico/efeitos dos fármacos , Células Cultivadas , Humanos , Ativação Linfocitária/efeitos dos fármacos , Magnésio/sangue , Magnésio/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/sangue , Infecções por Orthomyxoviridae/virologia , Concentração Osmolar , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/química , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologiaRESUMO
Spliceosome-mediated RNA trans-splicing has emerged as an exciting mode of RNA therapy. Here we describe a novel trans-splicing strategy, which targets highly abundant pre-mRNAs, to produce therapeutic proteins in vivo. First, we used a pre-trans-splicing molecule (PTM) that mediated trans-splicing of human apolipoprotein A-I (hapoA-I) into the highly abundant mouse albumin exon 1. Hydrodynamic tail vein injection of the hapoA-I PTM plasmid in mice followed by analysis of the chimeric transcripts and protein, confirmed accurate and efficient trans-splicing into albumin pre-mRNA and production of hapoA-I protein. The versatility of this approach was demonstrated by producing functional human papillomavirus type-16 E7 (HPV16-E7) single-chain antibody in C57BL/6 mice and functional factor VIII (FVIII) and phenotypic correction in hemophilia A mice. Altogether, these studies demonstrate that trans-splicing to highly abundant albumin transcripts can be used as a general platform to produce therapeutic proteins in vivo.
Assuntos
Albuminas/genética , Trans-Splicing/genética , Animais , Apolipoproteína A-I/genética , Apolipoproteína A-I/fisiologia , Éxons/genética , Feminino , Terapia Genética/métodos , Vetores Genéticos/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Precursores de RNA/genética , Splicing de RNA/genética , Splicing de RNA/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Spliceossomos/genética , Spliceossomos/metabolismo , Trans-Splicing/fisiologiaRESUMO
<p><b>OBJECTIVE</b>To observe the changes of sperm chromatin in patients with oligo-astheno-teratozoospermia (OAT) syndrome after treated by integrated Chinese and Western medicine.</p><p><b>METHODS</b>Sixty patients with OAT syndrome were treated by integrated Chinese and Western medicine for 3 months. Their sperm samples were collected before and after the treatment, subjected to acridine orange staining and analyzed by fluorescent microscopy, flow cytometry and sperm routine detection.</p><p><b>RESULTS</b>Significant differences were shown in the master-group sperm signals (P < 0.01) and at and COMPalphat (P < 0.05) by flow cytometry, as well as in the green and the red groups (P < 0.05) by fluorescent microscopy before and after the treatment. Changes in sperm concentration, motility, vitality and deformity were noted after the treatment, with statistic difference between pre- and post-treatment (P < 0.05) except in forward sperm concentration.</p><p><b>CONCLUSION</b>Treatment by integrated Chinese and Western medicine can improve sperm chromatin in patients with OAT syndrome. Flow cytometry, along with fluorescent microscopy and sperm routine detection, plays an important role in the evaluation of male infertility therapy.</p>
