RESUMO
The specificity of the UDP-N-acetylglucosamine (UDP-GlcNAc) translocator for the binding of nucleoside monophosphates (NMPs) and nucleotide-sugars was examined in order to develop a quantitative understanding of how this enzyme recognizes its substrates and to provide a framework for development of novel drugs that target glycosylation. Competition studies reveal that tight binding requires a complete ribose ring and a 5'-phosphate. The enzyme is extremely tolerant to changes at the 3'-position, and the presence of 3'-F actually increases binding of the NMP to the enzyme. At the 2'-position, substitutions in the ribo configuration are well tolerated, although these same substitutions greatly diminish binding when present in the ara configuration. For the base, size appears to be the key feature for discrimination. The enzyme tolerates changing the C-4 oxygen of uridine to an amino group as well as substituting groups containing one or two carbons at C-5. However, substitution of groups containing three carbons at C-5, or exchange of the pyrimidine for a purine, greatly weakens binding to the translocator. Comparison of various UDP-sugars reveals that the UDP-GlcNAc translocator has lower affinity for UDP-N-acetylgalactosamine and UDP-glucose than for its cognate substrate and therefore indicates that this translocator requires both proper stereochemistry at C-4 and an aminoacetyl group at C-2. The impact of these observations on the design of more powerful nucleoside-based inhibitors of nucleotide-sugar import is discussed.
Assuntos
Proteínas de Transporte/antagonistas & inibidores , Complexo de Golgi/ultraestrutura , Membranas Intracelulares/metabolismo , Fígado/ultraestrutura , Nucleotídeos/farmacologia , Uridina Difosfato N-Acetilglicosamina/metabolismo , Animais , Ligação Competitiva , Transporte Biológico/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Glicosilação , Cinética , Nucleotídeos/química , Fosfatos/química , Fosfatos/farmacologia , Coelhos , Ribose/química , Relação Estrutura-Atividade , Uridina Difosfato N-Acetilglicosamina/farmacologia , Açúcares de Uridina Difosfato/metabolismo , Açúcares de Uridina Difosfato/farmacologiaRESUMO
The Zone model of pulmonary perfusion predicts that exercise reduces perfusion heterogeneity because increased vascular pressure redistributes flow to gravitationally nondependent lung, and causes dilation and recruitment of blood vessels. However, during exercise in animals, perfusion heterogeneity as measured by the relative dispersion (RD, SD/mean) is not significantly decreased. We evaluated the effect of exercise on pulmonary perfusion in six healthy supine humans using magnetic resonance imaging (MRI). Data were acquired at rest, while exercising (â¼27% of maximal oxygen consumption) using a MRI-compatible ergometer, and in recovery. Images were acquired in most of the right lung in the sagittal plane at functional residual capacity, using a 1.5-T MR scanner equipped with a torso coil. Perfusion was measured using arterial spin labeling (ASL-FAIRER) and regional proton density using a fast multiecho gradient-echo sequence. Perfusion images were corrected for coil-based signal heterogeneity, large conduit vessels removed and quantified (in ml·min(-1)·ml(-1)) (perfusion), and also normalized for density and quantified (in ml·min(-1)·g(-1)) (density-normalized perfusion, DNP) accounting for tissue redistribution. DNP increased during exercise (11.1 ± 3.5 rest, 18.8 ± 2.3 exercise, 13.2 ± 2.2 recovery, ml·min(-1)·g(-1), P < 0.0001), and the increase was largest in nondependent lung (110 ± 61% increase in nondependent, 63 ± 35% in mid, 70 ± 33% in dependent, P < 0.005). The RD of perfusion decreased with exercise (0.93 ± 0.21 rest, 0.73 ± 0.13 exercise, 0.94 ± 0.18 recovery, P < 0.005). The RD of DNP showed a similar trend (0.82 ± 0.14 rest, 0.75 ± 0.09 exercise, 0.81 ± 0.10 recovery, P = 0.13). In conclusion, in contrast to animal studies, in supine humans, mild exercise decreased perfusion heterogeneity, consistent with Zone model predictions.
Assuntos
Exercício Físico , Pulmão/irrigação sanguínea , Imageamento por Ressonância Magnética , Imagem de Perfusão/métodos , Circulação Pulmonar , Decúbito Dorsal , Adulto , Feminino , Humanos , Interpretação de Imagem Assistida por Computador , Medidas de Volume Pulmonar , Masculino , Consumo de Oxigênio , Valor Preditivo dos Testes , Fluxo Sanguíneo Regional , Fatores de TempoRESUMO
3'-Azido-3'-deoxythymidine (AZT) is one of the primary chemotherapeutic agents used in the treatment of human immunodeficiency virus (HIV) infection. Unfortunately, AZT therapy is accompanied by severe side effects. Using Golgi-enriched membrane fractions, we have determined that 3'-azido-3'-deoxythymidine monophosphate, the primary AZT metabolite in treated cells, potently inhibits protein glycosylation. This inhibition results from direct competition with several pyrimidine-sugars for transport into Golgi membranes. This potential mechanism of cytotoxicity does not involve 3'-azido-3'-deoxythymidine triphosphate, the AZT metabolite most likely responsible for its antiviral effects; thus, it may be possible to develop novel therapeutic strategies that prevent inhibition of glycosylation without affecting the anti-HIV properties of AZT.
Assuntos
Proteínas de Membrana/metabolismo , Açúcares de Nucleosídeo Difosfato/metabolismo , Zidovudina/toxicidade , Animais , Células CHO , Cricetinae , Ácido N-Acetilneuramínico do Monofosfato de Citidina/metabolismo , Didesoxinucleotídeos , Glicosilação/efeitos dos fármacos , Complexo de Golgi/metabolismo , HIV/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , Humanos , Membranas Intracelulares/metabolismo , Cinética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Nucleotídeos de Timina/toxicidade , Uridina Difosfato Galactose/metabolismo , Uridina Difosfato N-Acetilglicosamina/metabolismo , Zidovudina/análogos & derivados , Zidovudina/metabolismo , Zidovudina/uso terapêuticoRESUMO
The 1605 bp intron 4 of the Euglena gracilis chloroplast psbC gene was characterized as a group III twintron composed of an internal 1503 nt group III intron with an open reading frame of 1374 nt (ycf13, 458 amino acids), and an external group III intron of 102 nt. Twintron excision proceeds by a sequential splicing pathway. The splicing of the internal and external group III introns occurs via lariat intermediates. Branch sites were mapped by primer extension RNA sequencing. The unpaired adenosines in domains VI of the internal and external introns are covalently linked to the 5' nucleotide of the intron via 2'-5' phosphodiester bonds. This bond is susceptible to hydrolysis by the debranching activity of the HeLa nuclear S100 fraction. The internal intron and presumptive ycf13 mRNA accumulates primarily as a linear RNA, although a lariat precursor can also be detected. The ycf13 gene encodes a maturase-like protein that may be involved in group III intron metabolism.
Assuntos
Euglena gracilis/genética , Íntrons , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Animais , Sequência de Bases , Northern Blotting , Cloroplastos/química , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , RNA/química , Precursores de RNA/análise , Precursores de RNA/química , Splicing de RNA , RNA Mensageiro/análise , RNA Mensageiro/química , Ribonuclease H/metabolismo , Análise de SequênciaRESUMO
Recent in vitro work with Golgi-enriched membranes showed that 3'-azidothymidine-5'-monophosphate (AZTMP), the primary intracellular metabolite of 3'-azidothymidine (AZT), is a potent inhibitor of glycosylation reactions (Hall et al. (1994) J. Biol. Chem. 269, 14355-14358) and predicted that AZT treatment of whole cells should cause similar inhibition. In this report, we verify this prediction by showing that treatment of K562 cells with AZT inhibits lipid and protein glycosylation. AZT treatment dramatically alters the pattern of glycosphingolipid biosynthesis, nearly abolishing ganglioside synthesis at clinically relevant concentrations (1-5 microM), and suppresses the incorporation of both sialic acid and galactose into proteins. Control experiments demonstrate that these changes do not result from nonspecific effects on either the secretory apparatus or protein synthesis. On the other hand, studies using isolated nuclei as a model system for chromosomal DNA replication show that AZTTP is a very weak inhibitor of DNA synthesis. These observations strongly suggest that the myelosuppressive effects of AZT in vivo are due to inhibition of protein and/or lipid glycosylation and not to effects on chromosomal DNA replication.