RESUMO
Sugar-amino acid-nucleosides (SAAN) were synthesized to mimic glycosyl nucleotide donors based on the hypothesis that a basic amino acid may interact with carboxylate groups of the enzyme in a manner similar to the diphosphate metal ion complex. C-Glycoside analogues of the d-galactopyranose or l-arabinofuranose ring systems, and four amino acids (lysine, glutamine, tryptophan, and histidine), were chosen for this study. The targets were synthesized and tested against GlfT2, a galactofuranosyltransferase essential for cell wall galactan biosynthesis in Mycobacterium tuberculosis. The inhibition assay showed that analogues containing histidine and tryptophan are moderate inhibitors of GlfT2.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Glicosiltransferases/antagonistas & inibidores , Nucleosídeos/síntese química , Nucleosídeos/farmacologia , Aminoácidos/química , Carboidratos/química , Inibidores Enzimáticos/química , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , Nucleosídeos/químicaRESUMO
UDP-galactofuranose (UDP-Galf) is a substrate for two types of enzymes, UDP-galactopyranose mutase and galactofuranosyltransferases, which are present in many pathogenic organisms but absent from mammals. In particular, these enzymes are involved in the biosynthesis of cell wall galactan, a polymer essential for the survival of the causative agent of tuberculosis, Mycobacterium tuberculosis. We describe here the synthesis of derivatives of UDP-Galf modified at C-5 and C-6 using a chemoenzymatic route. In cell-free assays, these compounds prevented the formation of mycobacterial galactan, via the production of short "dead-end" intermediates resulting from their incorporation into the growing oligosaccharide chain. Modified UDP-furanoses thus constitute novel probes for the study of the two classes of enzymes involved in mycobacterial galactan assembly, and studies with these compounds may ultimately facilitate the future development of new therapeutic agents against tuberculosis.
Assuntos
Antituberculosos/química , Inibidores Enzimáticos/química , Galactanos/biossíntese , Galactose/análogos & derivados , Galactosiltransferases/antagonistas & inibidores , Difosfato de Uridina/análogos & derivados , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Galactanos/antagonistas & inibidores , Galactose/biossíntese , Galactose/química , Galactose/farmacologia , Galactosiltransferases/genética , Galactosiltransferases/metabolismo , Transferases Intramoleculares/antagonistas & inibidores , Transferases Intramoleculares/metabolismo , Klebsiella pneumoniae/enzimologia , Mycobacterium smegmatis/enzimologia , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Difosfato de Uridina/biossíntese , Difosfato de Uridina/química , Difosfato de Uridina/farmacologiaRESUMO
5'-AMP-activated protein kinase (AMPK) signaling initiates adaptive changes in skeletal muscle fibers that restore homeostatic energy balance. The purpose of this investigation was to examine, in rats, the fiber-type protein expression patterns of the alpha-catalytic subunit isoforms in various skeletal muscles, and changes in their respective contents within the tibialis anterior (TA) after chronic low-frequency electrical stimulation (CLFS; 10 Hz, 10 h daily), applied for 4 +/- 1.2 or 25 +/- 4.8 days. Immunocytochemical staining of soleus (SOL) and medial gastrocnemius (MG) showed that 86 +/- 4.1 to 97 +/- 1.4% of type IIA fibers stained for both the alpha1- and alpha2-isoforms progressively decreased to 63 +/- 12.2% of type IID/X and 9 +/- 2.4% of IIB fibers. 39 +/- 11.4% of IID/X and 83 +/- 7.9% of IIB fibers expressed only the alpha2 isoform in the MG, much of which was localized within nuclei. alpha1 and alpha2 contents, assessed by immunoblot, were lowest in the white gastrocnemius [WG; 80% myosin heavy chain (MHC) IIb; 20% MHCIId/x]. Compared with the WG, alpha1 content was 1.6 +/- 0.08 (P < 0.001) and 1.8 +/- 0.04 (P < 0.0001)-fold greater in the red gastrocnemius (RG: 13%, MHCIIa) and SOL (21%, MHCIIa), respectively, and increased in proportion to MHCIIa content. Similarly, alpha2 content was 1.4 +/- 0.10 (P < 0.02) and 1.5 +/- 0.07 (P < 0.001)-fold greater in RG and SOL compared with WG. CLFS induced 1.43 +/- 0.13 (P < 0.007) and 1.33 +/- 0.08 (P < 0.009)-fold increases in the alpha1 and alpha2 contents of the TA and coincided with the transition of faster type IIB and IID/X fibers toward IIA fibers. These findings indicate that fiber types differ with regard to their capacity for AMPK signaling and that this potential is increased by CLFS.
Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Complexos Multienzimáticos/metabolismo , Fibras Musculares Esqueléticas/enzimologia , Músculo Esquelético/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Ativadas por AMP , Animais , Western Blotting , Catálise , Núcleo Celular/metabolismo , Estimulação Elétrica , Eletroforese em Gel de Poliacrilamida , Imuno-Histoquímica , Isoenzimas/biossíntese , Isoenzimas/genética , Masculino , Complexos Multienzimáticos/biossíntese , Complexos Multienzimáticos/genética , Fibras Musculares de Contração Rápida/enzimologia , Fibras Musculares de Contração Rápida/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Fibras Musculares de Contração Lenta/enzimologia , Fibras Musculares de Contração Lenta/fisiologia , Músculo Esquelético/fisiologia , Cadeias Pesadas de Miosina/metabolismo , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Ratos , Ratos Sprague-Dawley , Regulação para Cima/fisiologiaRESUMO
The purpose of this investigation was to examine the temporal changes in uncoupling protein (UCP)-3 expression, as well as related adaptive changes in mitochondrial density and fast-to-slow fiber type transitions during chronically enhanced contractile activity. We examined the effects of 1-42 days of chronic low-frequency electrical stimulation (CLFS), applied to rat tibialis anterior (TA) for 10 h/day, on the expression of UCP-3 and concomitant changes in myosin heavy chain (MHC) protein expression and increases in oxidative capacity. UCP-3 protein content increased from 1 to 12 days, reaching 1.5-fold over control (P < 0.0005); it remained elevated for up to 42 days. In contrast, UCP-3 mRNA decreased in response to CLFS, reaching a level that was threefold lower than control (P < 0.0007). The activities of the mitochondrial reference enzymes citrate synthase (EC 4.1.3.7) and 3-hydroxyacyl-CoA-dehydrogenase (EC 1.1.1.35), which are known to increase in proportion to mitochondrial density, progressively increased up to an average of 2.3-fold (P < 0.00001). These changes were accompanied by fast-to-slow fiber type transitions, characterized by a shift in the pattern of MHC expression (P <0.0002): MHCI and MHCIIa expression increased by 1.7- and 4-fold, whereas MHCIIb displayed a 2.4-fold reduction. We conclude that absolute increases in UCP-3 protein content in the early adaptive phase were associated with the genesis of mitochondria containing a normal complement of UCP-3. However, during exposure to long-term CLFS, mitochondria were generated with a lower complement of UCP-3 and coincided with the emergence of a growing population of oxidative type IIA fibers.