AZT treatment dramatically alters the pattern of glycosphingolipid biosynthesis, nearly abolishing ganglioside synthesis at clinically relevant concentrations (1-5 M), 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. 3Ј-Azidothymidine (AZT)1 is one of the primary chemotherapeutic agents used in the treatment of HIV infection (1). This drug is effective because the triphosphate form of AZT, AZTTP, is a potent and somewhat selective inhibitor of HIV reverse transcriptase (2). Unfortunately, AZT therapy is often accompanied by side effects such as severe anemia and neutropenia due to inhibition of the maturation of blood stem cells, especially in the late stages of the disease (3).The current paradigm to explain AZT's hematologic toxicity focuses on DNA replication. AZT is proposed to impede growth or development of stem cells through incorporation of the analog into chromosomal DNA (3). This hypothesis is consistent with the rapid proliferation of blood stem cells and their general sensitivity toward inhibitors of DNA replication (for example cancer chemotherapeutics). However, AZTTP is a remarkably weak inhibitor of the three nuclear replicative DNA polymerases, ␣, ␦, and ⑀ (4, 5). Under physiological nucleotide concentrations, the amount of AZTTP needed to inhibit these enzymes is much higher than the concentrations that accumulate in treated cells (6) and raises the possibility that the myelosuppressive effects of AZT are not related to inhibition of chromosomal DNA replication.We recently demonstrated that the primary intracellular metabolite of AZT, AZTMP, is a potent competitive inhibitor of pyrimidine nucleotide sugar import into Golgi-enriched membrane fractions (7). Consequently, the glycosylation reactions that occur within the Golgi lumen were almost completely inhibited. Since AZTMP is known to accumulate to millimolar levels in several cell types (8), these observations suggested a novel mechanism for AZT toxicity, namely selective inhibition of lipid and protein glycosylation.Several lines of evidence indicate that inhibition of glycosylation could indeed lead to cytotoxicity. Small changes in glycosphingolipid synthesis can profoundly affect signal transduction, differentiation, and cell-cell interactions. Ganglioside synthesis varies in a characteristic manner during growth and differentiation (9, 10), and subtle changes in glycolipid composition dramatically alter the properties of many receptors and enzymes (10). For example, variations in ganglioside composition as small a...

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.

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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.