Membrane permeation characteristics of abacavir in human erythrocytes and human T-lymphoblastoid CD4+ CEM cells: comparison with (-)-carbovir.

Abacavir, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol, is a novel purine carbocyclic nucleoside analogue that has been approved by the FDA for the treatment of HIV (as Ziagen trade mark [abacavir sulfate]). Chemically, abacavir and (-)-carbovir (CBV) differ only at the 6-position of the purine ring; abacavir contains a cyclopropylamino moiety in place of the 6-lactam functionality of CBV. Intracellularly both are ultimately metabolized to CBV triphosphate.

Induction of lipolysis in vitro and loss of body fat in vivo by zinc-alpha2-glycoprotein.

Loss of adipose tissue in cancer cachexia has been associated with tumour production of a lipid-mobilizing factor (LMF) which has been shown to be homologous with the plasma protein zinc-alpha(2)-glycoprotein (ZAG). The aim of this study was to compare the ability of human ZAG with LMF to stimulate lipolysis in vitro and induce loss of body fat in vivo, and to determine the mechanisms involved. ZAG was purified from human plasma using a combination of Q Sepharose and Superdex 75 chromatography, and was shown to stimulate glycerol release from isolated murine epididymal adipocytes in a dose-dependent manner.

A novel nonopioid action of enkephalins: competitive inhibition of the mammalian brain high affinity L-proline transporter.

The high affinity L-proline transporter (PROT) is a member of the family of Na+ (and Cl-)-dependent plasma membrane transport proteins that comprises transporters for several neurotransmitters, osmolytes, and metabolites. The brain-specific expression of PROT in a subset of putative glutamatergic pathways implies a specialized function for this novel transporter and its presumed natural substrate L-proline in excitatory synaptic transmission. However, definitive studies of the physiological role(s) of high affinity L-proline uptake have been precluded by the lack of specific uptake inhibitors.

Membrane permeation mechanisms of 2',3'-dideoxynucleosides.

The mechanism of membrane permeation of several 2',3'-dideoxynucleosides was investigated at 37 degrees with human erythrocytes using an "inhibitor-stop" assay. Transport (per 5 microL cells) via the nucleoside and nucleobase carriers was assessed by inhibition of influx with dilazep and adenine, respectively. Mechanisms of cellular entry were highly individualized: 2',3'-dideoxyadenosine and 3'-deoxythymidin-2'-ene via nonfacilitated diffusion, with high rates; 2',3'-dideoxyguanosine mainly via the nucleobase carrier (Km = 390 microM, Vmax = 32 pmol/sec); 2',3'-dideoxyinosine by both nucleobase (Km = 850 microM, Vmax = 2.

Transport of 5-fluorouracil and uracil into human erythrocytes.

The transport of 5-fluorouracil (5-FU) and uracil into human erythrocytes has been investigated under initial velocity conditions with an "inhibitor-stop" assay using a cold papaverine solution to terminate influx. At 37 degrees and pH 7.3, 5-FU influx was nonconcentrative; was partially inhibited by adenine, hypoxanthine, thymine, and uracil; and was insensitive to inhibition by nucleosides or inhibitors of nucleoside transport.

Inhibition of thymidine transport by 3'-azido-3'-deoxythymidine and its metabolites.

3'-Azido-3'-deoxythymidine (AZT) competitively inhibited the transport of thymidine (Km = 0.23 mM) into human erythrocytes with a Ki of 1.0 mM at 37 degrees C.

Enantiomeric selectivity of carbovir transport.

Carbovir (9-[4 alpha-(hydroxymethyl)cyclopent-2-ene-1 alpha-yl]guanine) (CBV) is a carbocyclic analogue of 2',3'-dideoxyguanosine that exhibits potent and selective in vitro activity against human immunodeficiency virus. Antiviral activity is associated with only the (-)-enantiomer. The transport characteristics of both (-)-CBV and (+)-CBV were investigated in human erythrocytes at 37 degrees C using a papaverine-stop assay.

Membrane permeation characteristics of 5'-modified thymidine analogs.

The membrane permeation characteristics of 5'-deoxythymidine (5'-ddThd) and 5'-azido-5'-deoxythymidine (5'-N3-5'-ddThd) were investigated in human erythrocytes, with an inhibitor-stop assay, at 20 degrees. Uptake of both nucleoside analogs occurred without metabolism, was nonconcentrative, and was partially inhibited by nucleosides or inhibitors of nucleoside transport at micromolar permeant concentrations. At higher permeant concentrations (greater than 1.

Desciclovir permeation of the human erythrocyte membrane by nonfacilitated diffusion.

The mechanism of transport of desciclovir (DCV)--a structural analogue and prodrug of acyclovir (ACV) which provides an improved oral bioavailability of ACV--was investigated in human erythrocytes with a "papaverine-stop" assay. DCV influx was nonconcentrative, linearly dependent on DCV concentration (0.9 microM to 15 mM), insensitive (less than or equal to 20% inhibition) to nucleobases, nucleosides, or potent inhibitors of nucleoside transport, and occurred without permeant metabolism.

Ganciclovir permeation of the human erythrocyte membrane.

The membrane permeation of ganciclovir (DHPG)--a structural analogue of acyclovir (ACV) with activity against cytomegalovirus--was investigated in human erythrocytes at 37 degrees with an "inhibitor-stop" assay. DHPG influx was nonconcentrative, occurred without permeant metabolism, and was rate-saturable. While substantial inhibition of the influx of 13 microM DHPG occurred only in the presence of permeants of the purine nucleobase carrier, nucleosides and inhibitors of nucleoside transport markedly inhibited DHPG influx at higher DHPG concentrations (greater than or equal to 200 microM).

2',3'-Dideoxythymidine permeation of the human erythrocyte membrane by nonfacilitated diffusion.

The influx of 2',3'-dideoxythymidine into human erythrocytes was characterized to gain insight into the molecular properties of 3'-azido-3'-deoxythymidine which allow this latter nucleoside analog to permeate cell membranes by nonfacilitated diffusion (J. Biol. Chem.

Acyclovir transport into human erythrocytes.

The mechanism of transport of the antiviral agent acyclovir (ACV) into human erythrocytes has been investigated. Initial velocities of ACV influx were determined with an "inhibitor-stop" assay that used papaverine to inhibit ACV influx rapidly and completely. ACV influx was nonconcentrative and appeared to be rate-saturable with a Km of 260 +/- 20 microM (n = 8).

Purine nucleobase transport in human erythrocytes. Reinvestigation with a novel "inhibitor-stop" assay.

A novel "inhibitor-stop" method for the determination of initial rates of purine nucleobase transport in human erythrocytes has been developed, based on the addition of seven assay volumes of cold 19 mM papaverine to terminate influx. In view of our finding that the initial velocities of adenine, guanine, and hypoxanthine influx into human erythrocytes were linear for only 4-6 s at 37 degrees C, the present method has been used to reexamine the kinetics of purine nucleobase transport in these cells. Initial influx rates of all three purine nucleobases were shown to be the result of concurrent facilitated and nonfacilitated diffusion.

Species-dependent expression and induction of homologues of rabbit cytochrome P-450 isozyme 5 in liver and lung.

The presence of homologues of rabbit cytochrome P-450 isozyme 5 in pulmonary and hepatic microsomal preparations from guinea pig, mouse, monkey, hamster, and rat was examined by immunoblotting and inhibition of metabolism of 2-aminofluorene with antibodies to isozyme 5. Homologues to isozyme 5 were detected in pulmonary preparations from all five species. However, only hepatic preparations from hamster, in addition to those from rabbit, contained detectable levels of this isozyme.

The cytochrome P-450 monooxygenase system of rabbit lung enzyme components, activities, and induction in the nonciliated bronchiolar epithelial (Clara) cell, alveolar type II cell, and alveolar macrophage.

Enzyme components and activities of the cytochrome P-450 monooxygenase system in microsomal preparations from the Clara cell, alveolar type II cell, and alveolar macrophage fractions isolated from lungs of untreated rabbits and rabbits treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin were examined. Results are compared to those obtained with microsomal preparations from whole lung. Concentrations of cytochrome P-450 isozymes 2 and 5 and NADPH-cytochrome P-450 reductase activities were higher in preparations from Clara cell fractions than in preparations from type II cell fractions or whole lung.

The effect of substrate on the expression of activity catalyzed by cytochrome P-450: metabolism mediated by rabbit isozyme 6 in pulmonary microsomal and reconstituted monooxygenase systems.

The content of cytochrome P-450, isozyme 6, in the rabbit pulmonary microsomal fraction was estimated by immunochemical methods to be 1 to 3% of the total cytochrome P-450. Following treatment of rabbits with 2,3,7,8-tetrachlorodibenzo-p-dioxin, the pulmonary microsomal concentration of isozyme 6 increased 16-fold. Isozyme 6 was also detected by immunochemical methods, but not by electrophoresis and staining for protein, in preparations of isozyme 5 isolated from the pulmonary microsomal fraction of untreated rabbits.

Correlation of placental microsomal activities with protein detected by antibodies to rabbit cytochrome P-450 isozyme 6 in preparations from humans exposed to polychlorinated biphenyls, quaterphenyls, and dibenzofurans.

Placental tissues were obtained from Chinese women in Taiwan who had been exposed to contaminated rice oils containing polychlorinated biphenyls and their thermal degradative products. Exposure via the diet occurred 4-5 years prior to pregnancy. Placental microsomal fractions from eight of the nine exposed subjects studied showed marked elevation of benzo(a)pyrene hydroxylation and 7-ethoxyresorufin O-deethylation activities related to control subjects.

Quantitation of rabbit cytochrome P-450, form 2, in microsomal preparations bound directly to nitrocellulose paper using a modified peroxidase-immunostaining procedure.

Rabbit hepatic microsomal suspensions were bound directly to nitrocellulose sheets using a "Hybridot" apparatus to ensure uniformity. Cytochrome P-450, form 2, was then detected by a modified immunochemical method wherein the nitrocellulose paper was incubated sequentially with antibody to form 2 for 1 h at 25 degrees C, rabbit anti-goat immunoglobulin G (IgG) at a 1:100 dilution for 15 min at 25 degrees C, goat peroxidase-antiperoxidase at a 1:2000 dilution for 15 min at 25 degrees C, and 3,3'-diaminobenzidine at 0.3 mg/ml plus 0.

Studies of amino-acid sequence in dihydrofolate reductase from a human methotrexate-resistant cell line KB/6b. Structural and kinetic comparison with mouse L1210 enzyme.

The partial amino acid sequence of dihydrofolate reductase (DHFR, EC 1.5.1.

Establishment of dihydrofolate reductase-increased human cell lines and relationship between dihydrofolate reductase levels and gene copy.

A series of methotrexate (MTX)-resistant human sublines developed by step increases in selected MTX concentrations have been cloned and examined for dihydrofolate reductase (DHFR) content, relative DNA copy number, and sensitivity to MTX. These cloned sublines had increased DHFR levels which were dependent on the presence of MTX in the medium. The increased levels of DHFR in the absence of MTX were stable in all the clones examined for over a year.

Quantitative correlation of homogeneously stained regions on chromosome 10 with dihydrofolate reductase enzyme in human cells.

Gene amplification may be visualized within a chromosome as a homogeneously stained region (HSR) and HSRs have rarely been reported in human tumor cells with identification of the amplified gene. A parental line and seven clones derived from KB cells resistant to methotrexate (MTX) contain dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase; EC 1.5.