Thymidine analog and multinucleoside resistance mutations are associated with decreased phenotypic susceptibility to stavudine in HIV type 1 isolated from zidovudine-naive patients experiencing viremia on stavudine-containing regimens.

Studies have demonstrated that HIV-1 isolated from subjects experiencing virologic failure on stavudine (d4T)-containing regimens often contains thymidine analog mutations (TAMs), consisting of reverse transcriptase (RT) mutations M41L, D67N, K70R, L210W, T215Y/F, and K219Q/E, previously associated only with zidovudine (ZDV) resistance. In clinical study NZT40012, HIV-1 was isolated from 86 ZDV-naive subjects experiencing viremia on d4T-based therapies (plasma HIV-1 RNA > or =1000 copies/ml) and analyzed to examine the association between RT mutations and phenotypic resistance to d4T. Resistance-associated mutations were analyzed from HIV-1 isolated from 85 subjects. Of these, 24 samples (28%) had TAMs, and 30 samples (35%) had either TAMs and/or the Q151M multinucleoside resistance (MNR) mutation. Phenotypic susceptibility to d4T was determined by two commercially available methods. Statistically significant increases (p < 0.001) in phenotypic fold resistance to d4T were observed in virus with at least one TAM or MNR mutation. However, the mean increases in phenotypic resistance were 4-fold for the Antivirogram assay and 3-fold for the Phenosense HIV assay, only slightly above the levels used to designate decreased susceptibility to d4T. Subjects can experience viremia on d4T-containing regimens with virus exhibiting only small increases in IC(50), suggesting that relatively small changes in viral susceptibility to d4T may influence drug efficacy.

Studies of Sulfate Utilization by Algae. 6. Adenosine-3'-Phosphate-5'-Phosphosulfate (PAPS) as an Intermediate in Thiosulfate Formation From Sulfate by Cell-Free Extracts of Chlorella.

When cell-free preparations of Chlorella pyrenoidosa Chick (Emerson strain 3) form thiosulfate from labeled sulfate, another radioactive compound also appears. This compound has been isolated in quantity and is shown to be identical with adenosine-3'-phosphate-5'-phosphosulfate (PAPS) on the basis of its chromatographic and electrophoretic behavior, chemical composition, sensitivity to selective degradative enzymes, and its ability to serve as a substrate for rat liver aryl sulphotransferase. In addition, as expected for PAPS, the compound on mild acid treatment yields all of its radioactive sulfur as sulfate, and is converted to a compound identical with adenosine-3',5'-diphosphate (PAP). Replacement of sulfate and ATP by this PAP(35)S in the usual incubation mixture yields the same product, thiosulfate, which can be isolated as such or detected as acid-volatile radioactivity. This conversion of PAP(35)S to thiosulfate still requires the addition of Mg(2+) and a reductant such as 2,3-dimercaptopropan-1-ol (BAL). The cause of our previous result that high concentrations of ATP inhibit thiosulfate formation from sulfate can be ascribed to a small amount of PAP contaminating the ATP preparations, since PAP proves to be an exceedingly effective inhibitor of the conversion of PAP(35)S to thiosulfate. Sulfate reduction to thiosulfate by Chlorella extracts is discussed and compared with similar systems from other organisms.

Studies of Sulfate Utilization by Algae. 7. In vivo Metabolism of Thiosulfate by Chlorella.

Chlorella pyrenoidosa Chick (Emerson strain 3) utilizes thiosulfate for growth as effectively as sulfate, and more effectively than a variety of organic sulfur compounds containing sulfur in various oxidation states. Thiosulfates, differentially labeled with (35)S in either the SH- or SO(3) - sulfur moieties, were used to follow the incorporation of thiosulfate-sulfur into constituents of the insoluble fraction and of the soluble pools. Labeled sulfate was also used for purposes of comparison. Label from both sulfur atoms of thiosulfate and from sulfate is incorporated into the cysteine, homocysteine, and glutathione of the soluble pools, and into the methionine and cystine of protein in the insoluble fraction. Label from SO(3)-sulfur of thiosulfate is incorporated more slowly into protein methionine and cystine than label from the SH-sulfur. Moreover, the SO(3)-sulfur of thiosulfate is recovered largely as sulfate in both the soluble pools and the insoluble fraction, while only a trace of SH-sulfur is recovered as sulfate in either case. Consistent with this, the metabolism of the SO(3)-sulfur of thiosulfate more closely resembles the metabolism of sulfate. Thus it would appear that exogenous thiosulfate undergoes early dismutation in which the SO(3)-sulfur is preferentially oxidized, and the SH-sulfur is preferentially incorporated in a reduced state. These results are discussed in relation to the conversion of sulfate to thiosulfate by cell-free extracts of Chlorella previously described.