Exposure of HIV-infected cells to phospholipid leads to membrane alterations and selective growth retardation.

The effect of exogenous phosphatidylcholine on structure and function of plasma membranes from HIV-1-producing cells and from their non-infected counterparts was determined. The membrane protein composition was not affected by phospholipid treatment. Membrane fluidity and Ca(2+)-permeability were increased in virus-producing cells and in control cells after lipid treatment. The triacylglycerol content of the plasma membranes was increased in virus-producing cells after lipid treatment, whereas the content of phospholipid and cholesterol was not changed. The increased triacylglycerol content was in accordance with a relatively higher rate of [14C]oleic acid incorporation into triacylglycerols of the virus-producing cells after lipid treatment as shown by metabolic labeling. The results suggest that a latent cytopathic effect of HIV-infection becomes manifest if the cells are exposed to exogenous phospholipid and this may open a way to preferentially eliminate HIV-producing cells.

Biosynthesis of tetrahydrobiopterin: a sensitive assay of 6-pyruvoyltetrahydropterin synthase using [2'-3H]dihydroneopterin 3'-triphosphate as substrate.

Pyruvoyltetrahydropterin synthase catalyzes the release of tritiated water from [2'-3H]dihydroneopterin 3'-triphosphate. The tritiated water formed during the enzymatic reaction is separated from substrate by adsorption of the latter to activated charcoal. The sensitivity and specificity of the assay allows the determination of the enzyme in crude cell extract.

Regulation of tetrahydrobiopterin synthesis during lectin stimulation of human peripheral blood lymphocytes.

Lectin stimulation of human T lymphocytes causes a 7-fold increase in the specific activities of GTP-cyclohydrolase and a 4-fold increase in the specific activities of sepiapterin reductase. GTP-cyclohydrolase activities are maximal after 48 h and subsequently decline, whereas sepiapterin reductase activities continue to increase during the 72 h period measured. The specific activities of 6-pyruvoyltetrahydropterin synthase remain unchanged upon stimulation. Tetrahydrobiopterin synthesis during blast transformation is thus directed by both GTP-cyclohydrolase and sepiapterin reductase.

Synthesis of tetrahydrobiopterin in Friend erythroleukemia cells and its modulator effect on cell proliferation.

The induction of the enzymes in the tetrahydrobiopterin pathway by dimethyl sulfoxide (DMSO) was investigated in subclones F4N and B8/3 of the proerythroblastoid Friend erythroleukemia cell line (MEL). GTP-cyclohydrolase, the initial enzyme in the biosynthetic pathway, is virtually absent in both clones, but expression increases during 3 days of DMSO treatment. The final enzyme levels show 12-fold (subclone B8/3) and 40-fold (subclone F4N) increases compared to initial values. Enhancement of 6-pyruvoyl tetrahydropterin synthase activity is detectable 6 h after exposure to DMSO and continues to increase in the 3-day time period to 2.4-fold and 1.8-fold levels in subclones B8/3 and F4N, respectively. Sepiapterin reductase is present in unstimulated F4N cells and absent in B8/3 cells. The enzyme activity is not affected by DMSO treatment in either cell line. This explains why DMSO treatment causes accumulation of tetrahydrobiopterin in the MEL subclone F4N, but not in subclone B8/3. MEL cells are devoid of phenylalanine hydroxylase for which tetrahydrobiopterin serves as cofactor. In F4N, but not in B8/3, tetrahydrobiopterin modulates the rate of [3H]thymidine incorporation, thus being functionally linked with cell proliferation rather than with differentiation. In contrast to T lymphocytes, periods of tetrahydrobiopterin synthesis and of modulator function are uncoupled in MEL cells.

Analysis of the tetrahydrobiopterin synthesizing system during maturation of murine reticulocytes.

The enzymes of tetrahydrobiopterin synthesis have been studied in murine bone marrow, in spleen, in erythrocytes, and in reticulocytes. Mice with chemically induced and with genetically conditioned reticulocytosis as found in the lactate dehydrogenase deficient strain (Ldh-1c/Ldh-1c) were used for analysis of reticulocytic enzyme activities. The activity of the biopterin synthesizing system is highest in bone marrow even though it amounts to only about 10% as compared with liver. The first enzyme of the biosynthetic pathway, GTP-cyclohydrolase, virtually disappears during the final maturation step of reticulocytes. In contrast, the activities of 6-pyruvoyltetrahydropterin synthase and of sepiapterin reductase of erythrocytes are only reduced to about one half of the reticulocyte level. The absence of biopterin in erythrocytes is therefore caused by the loss of the enzyme that initiates the pterin biosynthetic pathway.