RESUMO
To test for a role of the calcineurin-NFAT (nuclear factor of activated T cells) pathway in the regulation of fiber type-specific gene expression, slow and fast muscle-specific promoters were examined in C2C12 myotubes and in slow and fast muscle in the presence of calcineurin or NFAT2 expression plasmids. Overexpression of active calcineurin in myotubes induced both fast and slow muscle-specific promoters but not non-muscle-specific reporters. Overexpression of NFAT2 in myotubes did not activate muscle-specific promoters, although it strongly activated an NFAT reporter. Thus overexpression of active calcineurin activates transcription of muscle-specific promoters in vitro but likely not via the NFAT2 transcription factor. Slow myosin light chain 2 (MLC2) and fast sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1) reporter genes injected into rat soleus (slow) and extensor digitorum longus (EDL) (fast) muscles were not activated by coinjection of activated calcineurin or NFAT2 expression plasmids. However, an NFAT reporter was strongly activated by overexpression of NFAT2 in both muscle types. Calcineurin and NFAT protein expression and binding activity to NFAT oligonucleotides were different in slow vs. fast muscle. Taken together, these results indicate that neither calcineurin nor NFAT appear to have dominant roles in the induction and/or maintenance of slow or fast fiber type in adult skeletal muscle. Furthermore, different pathways may be involved in muscle-specific gene expression in vitro vs. in vivo.
Assuntos
Calcineurina/metabolismo , Miosinas Cardíacas , Proteínas de Ligação a DNA/metabolismo , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Músculo Esquelético/metabolismo , Proteínas Nucleares , Fatores de Transcrição/metabolismo , Animais , Northern Blotting , ATPases Transportadoras de Cálcio/biossíntese , ATPases Transportadoras de Cálcio/genética , Núcleo Celular/metabolismo , Células Cultivadas , Expressão Gênica/genética , Genes Reporter/genética , Camundongos , Músculo Esquelético/citologia , Cadeias Pesadas de Miosina/biossíntese , Cadeias Pesadas de Miosina/genética , Cadeias Leves de Miosina/genética , Cadeias Leves de Miosina/metabolismo , Fatores de Transcrição NFATC , Especificidade de Órgãos/genética , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/biossíntese , Ratos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Transdução de Sinais/genética , TransfecçãoRESUMO
The genes encoding the polyhydroxyalkanoate (PHA) biosynthetic pathway in Ralstonia eutropha (3-ketothiolase, phaA or bktB; acetoacetyl-CoA reductase, phaB; and PHA synthase, phaC) were engineered for plant plastid targeting and expressed using leaf (e35S) or seed-specific (7s or lesquerella hydroxylase) promoters in Arabidopsis and Brassica. PHA yields in homozygous transformants were 12-13% of the dry mass in homozygous Arabidopsis plants and approximately 7% of the seed weight in seeds from heterozygous canola plants. When a threonine deaminase was expressed in addition to bktB, phaB and phaC, a copolyester of 3-hydroxybutyrate and 3-hydroxyvalerate was produced in both Arabidopsis and Brassica.
Assuntos
Acetil-CoA C-Aciltransferase/metabolismo , Aciltransferases/metabolismo , Oxirredutases do Álcool/metabolismo , Arabidopsis/metabolismo , Cupriavidus necator/enzimologia , Poliésteres/metabolismo , Acetil-CoA C-Aciltransferase/genética , Aciltransferases/genética , Oxirredutases do Álcool/genética , Arabidopsis/genética , Cupriavidus necator/genética , Homozigoto , Modelos Moleculares , Estrutura Molecular , Folhas de Planta , Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteínas Recombinantes/metabolismo , SementesRESUMO
This paper reports an in-depth approach that identifies ascorbyl-2-sulfate (AS) in gastric, blood, liver, and muscle tissues of Atlantic salmon (Salmo salar). To insure the identify of the AS, the study utilized the latest high-performance liquid chromatography (HPLC) technology plus electron ionization mass spectrometry (EIMS). Just before saltwater adaptation stage, juvenile Atlantic salmon were force-fed AS, ascorbic acid (AA), and a molecular-equivalent combination of the two. After tissue analyses for AA and AS were performed by HPLC separation, the HPLC peaks were identified by EIMS. The data collected in this study indicate that Atlantic salmon can absorb AS through the gastric tissue when forced-fed AA and AS as described. The data also indicate that AS is transported through the blood to the liver. There is evidence to indicate that AS is converted to AA in the livers of these salmon. In addition, the muscle tissue contained a large portion of AA and AS.