Synthesis, biological properties and anti-HIV-1 activity of new pyrimidine P1,P2-dinucleotides.

New homo- and hetero-P(1),P(2)-dinucleotides were prepared with the use of multistep procedures starting from the monophosphates of 3'-fluoro-2-thiothymidine, 3'-fluoro-4-thiothymidine, AZT and 1-[(2-hydroxyethoxy)-methyl-5-propyl-6-phenylselenenyl]uracil. Anti-HIV properties of the synthesized P(1),P(2)-dinucleotides were evaluated against laboratory syncytia inducing strain HIV-1 in CEM-T4 cells. Anti-HIV activities were in the range of 5-45 nM, and therapeutic indexes were higher than 4666-14000. Interactions of the above mentioned compounds with recombinant HIV-1 reverse transcriptase were also investigated. The obtained results point to reverse transcriptase inhibition, with somewhat lower inhibitory activity than that of their parental nucleoside-5'-triphosphates. Compound 6 may be regarded as a potent anti-HIV/AIDS drug.

5'-O-ester prodrugs of potent and selective anti-HIV agent--2',3'-dideoxy-3'-fluoro-2-thiothymidine (S2FLT): synthesis and anti-HIV activity.

Novel synthesis of 2',3'-dideoxy-3'-fluoro-2-thiothymidine (SFLT) based on transformation of appropriately protected 1-beta-D-threo-ribofuranosylthymine is presented. The synthesis and evaluation of SFLT 5'-O-ester prodrugs enzymatic hydrolysis, as well as their anti-HIV activity, is also described.

The selection and evolution of viral quasispecies in HIV-1 infected children.

OBJECTIVES: To analyse the diversity and divergence of the viral populations in three mother-child pairs in longitudinally obtained samples for up to 7 years. METHODS: Peripheral blood mononuclear cells were obtained from three mothers at delivery and three to four samples were obtained from each of their children from 1.5 months up to 78 months of age. The V3 region of HIV-1 was amplified by polymerase chain reaction, cloned and sequenced. HIV-1 DNA sequence comparisons were performed by phylogenetic analysis. RESULTS: The viral population was initially homogenous in two children but highly heterogeneous in one child. Three patterns of vertical transmission seemed to have occurred: transmission of the most prevalent maternal strain, of a minor maternal strain and of multiple maternal strains. In one child, a possible reappearance of a maternal sequence was observed at 34 months of age. CONCLUSIONS: Children may become infected with the most prevalent maternal strain, a minor maternal variant or multiple maternal quasispecies. Maternal viral variants may reappear in children after several years of infection and could possibly be derived from a reservoir of founder quasispecies established during the children's primary HIV-1 infection.

Hepatitis C virus in lymphoid cells of patients coinfected with human immunodeficiency virus type 1: evidence of active replication in monocytes/macrophages and lymphocytes.

It has been reported that hepatitis C virus (HCV) may be lymphotropic in the setting of human immunodeficiency virus type 1 (HIV-1) coinfection. The present study was undertaken to determine the phenotype of lymphoid cells harboring replicating HCV in HIV-1-positive subjects. By means of highly strand-specific thermostable enzyme Tth-based reverse-transcriptase polymerase chain reaction, the presence of viral RNA-negative strand was sought in different subpopulations of peripheral blood mononuclear cells from 10 HIV-positive patients. HCV RNA-negative strand was most commonly present in monocytes/macrophages (4 cases), followed by CD8+ and CD4+ lymphocytes (2 cases) and CD19+ cells (1 case). In 2 cases that were further analyzed, viral-negative strand remained detectable in monocytes/macrophages cultured for 3 weeks. Moreover, monocyte/macrophage- and serum-derived viral sequences differed in the 5' untranslated region. These findings imply that, in HIV-infected subjects, HCV may replicate in the same cells as HIV-1, which raises the possibility of direct interactions between these pathogens.

Simultaneous detection of hepatitis C virus and human immunodeficiency virus RNA in serum using amplicor PCR tests.

Several tests are currently available to assist in the diagnosis of the hepatitis C virus (HCV) and human immunodeficiency virus (HIV). Tests that actually detect or quantify these viruses are based on the polymerase chain reaction (PCR) technique. However, the application of PCR is limited by the cost, labor, time-consumption, and potential for contamination. In this article we describe some procedures developed to reduce these limitations. We have developed and validated simultaneous detection methods for HIV RNA and HCV RNA in single serum samples using Amplicor PCR tests. The sensitivity and specificity of this method are comparable with the results obtained with commercial reverse transcription polymerase chain reaction (RT-PCR) techniques for HIV and HCV RNA detection. In addition we have modified the HIV Amplicor test for the RT-PCR procedure and the Chomczynski's method of RNA isolation. We hope that our method can find same applications in HIV and HCV coinfection research, blood screening, and medical diagnosis.

The role of surface and intracellular mucus in gastric mucosal protection against hydrogen ion. Compositional differences.

The role of surface and intracellular mucus in gastric mucosal protection against hydrogen ion was investigated. Gastric mucosa, prepared from dog and rat stomachs at various stages of mucus depletion, was mounted in the permeability chamber, and the diffusion of hydrogen ion from the luminal to the serosal side was measured. Removal of the surface mucus caused a 42.9% increase in the permeability of rat gastric mucosa and a 47.4% increase in the permeability of dog gastric mucosa. The permeability to hydrogen ion of gastric mucosa depleted of its surface and intracellular mucus increased 216% in the case of the dog and 280% with the rat. Compositional analysis showed that in both animals the intracellular mucus had a higher content of lipids, covalently bound fatty acids, and carbohydrates, whereas the protein content was higher in the surface mucus. The results suggest that, although both the surface and intracellular mucus participate in the retardation of hydrogen ion diffusion, the contribution of the latter appears to be greater.

Effect of ethanol on mucus glycoprotein fatty acyltransferase from gastric mucosa.

The enzyme activity that catalyzes the transfer of palmitic acid from palmitoyl coenzyme A to the deacylated intact or deglycosylated gastric mucus glycoprotein was demonstrated in the detergent extracts of the microsomal fraction of antral and body mucosa of the rat stomach. Both types of mucosa exhibited similar acyltransferase activities and acceptor specificities. A 10-14% decrease in the fatty acyltransferase activity was observed with the reduced and S-carboxymethylated mucus glycoprotein, but the proteolytically degraded glycoprotein showed no acceptor capacity. This indicated that the acylation of mucus glycoprotein with fatty acids occurs at its nonglycosylated polypeptide regions and that some of the fatty acids may be linked via thiol esters. Optimum enzyme activity was obtained at pH 7.4 with the detergent Triton X-100, NaF, and dithiothreitol. The apparent Km values for the intact and deglycosylated mucus glycoproteins were 0.45 and 0.89 microM, respectively. The acyltransferase activity of the microsomal enzyme was inhibited by ethanol. With both intact and deglycosylated glycoprotein substrates, the rate of inhibition was proportional to the ethanol concentration up to 0.4 M and was of the competitive type. The K1 values were 0.80 microM for the intact mucus glycoprotein and 1.82 microM for the deglycosylated glycoprotein. Preincubation of the microsomal enzyme with low concentrations of ethanol (up to 0.5 M) did not seem to exert any additional deterrent effect on acyltransferase activity. Higher concentrations of ethanol (1.0 M and above), however, caused substantial reduction in the transferase activity due to denaturation of the enzyme.

Identification of mucus glycoprotein fatty acyltransferase activity in human gastric mucosa.

The enzymatic activity which catalyzes the transfer of palmitic acid from palmitoyl coenzyme A to gastric mucus glycoprotein was demonstrated in antral and fundic mucosa of normal human stomach. Subcellular fractionation studies revealed that with both types of mucosa the enzyme activity was present in the microsomal fraction. The antral and fundic mucosa also exhibited similar enzyme activities, showed identical pH optimum, and required detergent, NaF and dithiothreitol. Optimum enzymatic activity for fatty acylation of mucus glycoprotein was obtained with 0.5% Triton X-100, 25 mM NaF, and 2 mM dithiothreitol at a pH of 7.4. The 14C-labeled product of the reaction comigrated on CsCl density gradient centrifugation with gastric mucus glycoprotein and contained the ester-bound palmitic acid.

Effect of bile acids on the glycoprotein constituent of gastric mucus.

The effect of bile acids on the glycoprotein constituent of gastric mucus was investigated. Ghosh-Lai rat stomachs were instilled in vivo for 1 h with buffered saline (control) followed by various bile acids in saline at pH 2.0-7.0. Following quantitative recovery, the instillates were used for the isolation of mucus glycoprotein. The results of analyses revealed that while taurocholic acid exerted essentially identical depletory effects throughout the entire pH range tested, the cholic acid had little depletory effect on the mucus below pH 5.0, and glycocholic acid below pH 4.0. The extent of mucus glycoprotein depletion by bile acids under optimal pH conditions was clearly dependent upon the bile acid concentration as well as its type. The maximum depletory effect with the lowest concentration of bile acid was achieved with taurocholate followed by glycocholate and cholate. Comparison of the depletory effects of mono-, di-, and trihydroxy conjugated and unconjugated bile acids revealed that, within each group, the highest degree of mucus glycoprotein depletion occurred with taurine conjugates. The number of hydroxyl groups on the bile acid molecule appears to exert a less evident effect on the depletory capacity. The results suggest that the in vivo depletion of gastric mucus of its glycoprotein constituent by bile acids depends on the pH of luminal exposure and on the bile acid composition.

Effects of colchicine on the Golgi complex and GERL of cultured rat peritoneal macrophages and epiphyseal chondrocytes.

Thioglycollate-elicited rat peritoneal macrophages and epiphyseal chondrocytes were cultured in vitro, treated with colchicine, and then studied by electron-microscopic and cytochemical techniques. Colchicine, but not lumicolchicine, caused disappearance of cytoplasmic microtubules and breakup of the Golgi complex with spreading of its dictyosomes from a well defined juxtanuclear area throughout the cytoplasm. There was also an altered distribution of lysosomes, which oriented themselves close to the dictyosomes both in control and colchicine-treated cells. Further, the structure of the individual dictyosomes was changed, especially in the chondrocytes. GERL equivalents were observed in control cells but were difficult to detect after exposure to colchicine. Reaction product for thiamine pyrophosphatase was found in narrow cisternae on the inner side of the dictyosomes in control cells but in vacuole-like structures in colchicine-treated cells. Reaction product for acid phosphatase was present in GERL equivalents and lysosomes in control cells but mainly in lysosomes in colchicine-treated cells. Nevertheless, the total specific activities of these enzymes as well as of 5'-nucleotidase, a plasma membrane marker, remained unaffected by the drug treatment. These observations show that cytoplasmic microtubules play an important and, in many respects similar, cytoskeletal role in two so functionally diverse cell types as macrophages and chondrocytes. They are particularly important for the structural integrity of the Golgi complex, which in both cells is normally organized in the area around the centrioles, from which numerous microtubules radiate into the cytoplasm. The observations further suggest that GERL is an integrated part of the Golgi complex in these cells.

Effects of colchicine on endocytosis of horseradish peroxidase by rat peritoneal macrophages.

Horseradish peroxidase (HRP) was used as an exogenous marker to study the effects of microtubule-disruptive drugs on endocytosis in cultures of thioglycollate-elicited rat peritoneal macrophages. Colchicine and vinblastine, but not lumicolchicine or cytochalasin B, reduced HRP uptake by about 30-40%. However, as determined by stereological measurements, the size of the HRP-containing compartment within the cells remained unaltered. In both control cells and cells treated with colchicine or vinblastine the HRP-reactive vesicles were preferentially located close to the dictyosomes (stacks of cisternae) despite the fact that the Golgi complex was disorganized in the treated cells. These results suggest that intact cytoplasmic complex was disorganized in the treated cells. These results suggest that intact cytoplasmic microtubules are required to maintain a normal rate of fluid phase endocytosis in macrophages. On the other hand, it seems as if microtubules are not necessary for the translocation of newly formed endocytic vesicles/lysosomes to the dictyosomes, from which they probably are supplied with digestive enzymes.

Effects of colchicine on endocytosis and cellular inactivation of horseradish peroxidase in cultured chondrocytes.

Horseradish peroxidase (HRP) was used as a marker to study the effects of microtubule-disruptive drugs on uptake and cellular inactivation of exogenous material in cultures of embryonic chick chondrocytes. HRP was ingested by fluid endocytosis, and intracellular enzyme activity subsequently diminished exponentially with time. Cytochemically, reaction product for HRP was found in vesicles often located close to the dictyosomes of the Golgi complex. Colchicine and vinblastine caused disappearance of cytoplasmic microtubules and disorganization of the Golgi complex with concomitant reduction in the cellular uptake of HRP to about half of that in the controls. Lumicolchicine, on the other hand, left cell fine structure and HRP uptake unaffected. These results indicate that microtubules are of considerable importance in the process of fluid endocytosis in cultured chondrocytes although the exact mechanism remains to be elucidated. The rate of intracellular inactivation of ingested HRP was not affected by colchicine or vinblastine. Double-labeling experiments with colloidal thorium dioxide and HRP likewise indicated that fusion of endocytic vesicles and lysosomes is not dependent on intact microtubules. The total specific activities of the three lysosomal enzymes examined were weakly or not at all changed by treatment of the cultures with colchicine or vinblastine. It therefore seems unlikely that microtubular organization plays an important role in the production or degradation of lysosomal enzymes in cultured chondrocytes.

Isolation and characterization of poly(glycosyl)ceramides (megaloglycolipids) with A, H and I blood-group activities.

Very complex glycosphingolipids with A, H and I blood-group activities were isolated from human erythrocyte membranes. The membranes were obtained from erythrocytes of blood group A, A2 and O respectively. A general formula for the antigens is: (Fuc)3-4(Gal)n(LlcNAc)n-2(Glc)1(Sphingosine)1(where Fus is fucose, Gal is galactose, GlcNAc is N-acetylglucosamine and Glc is glucose) with values of n ranging from 10-27. A-active preparations contain additionally 2-3 residues of N-acetylgalactosamine. In view of the unusual complexity of these compounds they were designated poly(glycosyl)ceramides (formerly megaloglycolipids). Individual poly(glycosyl)ceramide fractions were isolated from A erythrocytes and were found to differ by about 8 glycosyl residues per molecule forming a series of compounds with 22, 30, 38, 51 and 59 glycosyl residues per mole. Structural studies indicate that the main sequence of poly(glycosyl)ceramides consists of the residues of galactopyranose and 2-deoxy-2-acetamidoglucopyranose substituted at 3 and 4 position respectively. These residues are probably alternating. N-Acdtylglucosamine substituted at 3 position was not found in poly(glycosyl)ceramides. Brances of poly(glycosyl)ceramides originate from 3 and 6 position of galactopyranosyl residues. The number of branches is proportional to the degree of molecular complexity. In poly(glycosyl)ceramides isolated from A and A2 erythrocytes the branches are terminated with the following structures GalNAc alpha 1 leads to 3 [Fuc alpha 1 leads to 2] Gal; Fuc alpha 1 leads to 2 Gal and Gal (presumably Gal beta 1 leads to 4 GlcNAc). In poly(glycosyl)ceramides from A cells the total number of A and H-active structures per average molecule of 30-35 glycosyl residues amounts to 2.1 and 1.2 respectively while the number of terminal galactose structures is 1.8. For poly(glycosyl)ceramides from A2 erythrocytes the corresponding figures are 0.75, 3.5, and 2.1 respectively. Poly(glycosyl)ceramides from O cells comprise about 3.8 H-active structures and 1.8 terminal galactopyranosyl residues. In poly(glycosyl)ceramides with high "n" values the number of terminal galactose structures is increased. These fractions display high blood-group I activity. However, the removal of terminal galactose with beta-galactosidase affects I-activity only slightly.