Alpha-fetoprotein-mediated uptake of fatty acids by human T lymphocytes.

The binding to resting and activated T lymphocytes of two radiolabelled fatty acids (oleic and arachidonic) was studied in the presence or in the absence of alpha-fetoprotein (AFP) as carrier protein. Fatty acid binding by resting and activated T lymphocytes was determined at 4 degrees C as a function of the concentration of fatty acid and AFP. Under the conditions employed, the following observations were made: (1) in the presence of AFP, fatty acids (oleic and arachidonic acid) are bound to cells by a two-component pathway; one is a saturable process, evidenced when the fatty acid to AFP (FA/AFP) molar ratio was fixed at 1 and the concentration of the fatty acid and the protein varied from 0.1 to 3.2 microM, and the second is a nonsaturable function of FA/AFP molar ratio and was linearly related to the unbound fatty acid concentration in the medium over the entire range studied; (2) in the absence of AFP, the nonsaturable process appears to be the only component of fatty acid binding; 3) at all tested concentrations of free (unbound) fatty acid in the medium, net fatty acid binding by either resting or activated T cells was considerably greater in the presence than in the absence of AFP; (4) in the presence of AFP, fatty acid binding was much higher in activated T cells than in resting T cells, whereas in the absence of AFP, nonsignificant differences were observed between activated and resting T cells; and (5) the time course of fatty acid and AFP binding at 4 degrees C revealed that, at equilibrium, the number of fatty acid molecules bound to the cell was much greater than that of AFP suggesting an accelerated dissociation of the fatty acid upon interaction of the AFP-fatty acid complex with putative cell receptors. It is concluded to the existence of an AFP/AFP-receptor pathway that facilitates the binding of fatty acids to T lymphocytes, particularly upon their blast transformation. This pathway may fulfill the increased requirement for fatty acids characteristic of proliferating cells and may serve to regulate the endocytosis of fatty acids with modulatory effects on lymphocyte function and to protect cells from their cytotoxic potential when internalized in excess.     

Transient responses of <Emphasis Type="Italic">Wickerhamia</Emphasis> sp. yeast continuous cultures to qualitative changes in carbon source supply: induction and catabolite repression of α-amylase synthesis

The purpose of this study was to evaluate the inductive effect of starch and maltose, and the repressive/inhibitory effect of glucose, on amy-1 gene expression and α-amylase production by Wickerhamia sp., using continuous culture under transient-state conditions at a dilution rate (D) of 0.083 h^?1. Induction and repression kinetics of α-amylase were studied by changing the medium feed from glucose to maltose or starch in the induction experiments and vice versa in the repression experiments. Expression levels of amy-1 gene were measured by RT-qPCR. Results showed that starch was a more efficient inducer of α-amylase synthesis compared to maltose, with maximum accumulation rate constants of 0.424 and 0.191 h^?1, respectively. In contrast, α-amylase synthesis in starch and maltose cultures was partially repressed by glucose as indicated by a specific activity close to basal levels and a decay constant rate (??0.065 and ??0.069 h^?1, respectively) higher than ??D. A linear dependence of the specific rate of α-amylase production on mRNA relative abundance of amy-1 gene was observed. An inhibitory effect of glucose was not observed even at a concentration of 30 g L^?1. In conclusion, the transient continuous culture is a useful tool to determine the qualitative and quantitative effects of maltose and starch on α-amylase induction and of glucose on enzyme repression, as well as to obtain a detailed understanding of the dynamic behavior of the yeast culture. Furthermore, results showed that amylaceous substrates can be very effective carbon sources for the production of α-amylase without being inhibited by glucose.     

Hexose keto-C-glycoside conjugates: design, synthesis, cytotoxicity, and evaluation of their affinity for the glucose transporter glut-1

The design, synthesis, cytotoxicity, and biological evaluation of carbohydrate/C-glycoside conjugates are described. The design concept is predicted on the idea that physiological barriers like the blood brain barrier could be crossed selectively by using glucose or glucose derivative/drug conjugates. The study demonstrates that, (1) carbohydrates and C-glycosides can be bonded at nonanomeric positions by the reaction of carbohydrate triflates with C-glycoside alkoxydes in the presence of DMPU; (2) there is a structure-activity relationship between the cytotoxicity of the conjugate and the nature of the carbohydrate residue; and (3) peracetylated hexose keto-C-glycoside conjugates are the most cytotoxic keto-C-glycosides.     

Practical Procedures for the Purification of Bacterial Viruses

The efficiencies of the various methods used for phage concentration have been compared. The two-phase concentration method (with polyethylene glycol and dextran sulfate) gave maximal recoveries of infectivity for coliphages of the T-even and T-odd series and for ribonucleic acid phages and single-stranded deoxyribonucleic acid phages. Precipitation of phages by acid gave high yields when applied to T2 and T4 phages but not with T3 and T7 coliphages. Differential centrifugation was efficient when sedimented phages were gently dispersed before repeating the centrifugation cycle. The efficiencies of the various methods have also been confirmed by electron microscope studies, which also show that the two-phase concentration method gave rise to intact phages. Zone centrifugations in sucrose gradients (12.5 to 52.5%) indicated that coliphages of the T-even series sediment faster than T-odd coliphages; they may thus be separated from each other and from empty ghosts by centrifugation at 100,000 x g for 40 min. Equilibrium centrifugation in preformed cesium chloride gradients was also useful for phage concentration and purification. This study also deals with some optical properties of purified phages; optical cross sections and absorbance ratios (at 260 and 280 nm) of the various preparations are given.     

Analyses of the interactions between retinoid-binding proteins and embryonal carcinoma cells

[3H]Retinoic acid (RA) and [3H]retinol bind in an unsaturable manner to isolated nuclei from Nulli-SCC1 and PCC4.aza1R embryonal carcinoma (EC) cells. When nuclei are challenged with the same labeled retinoids on their respective binding proteins (CRABP and CRBP), much less binding is observed and the binding is saturable. RA-CRABP does not compete with [3H]retinol-CRBP for binding to specific Nulli-SCC1 nuclear sites, whereas retinol-CRBP (but not apo-CRBP) actually potentiates the binding of [3H]RA-CRABP to these nuclei. The binding of [3H]RA-CRABP and [3H]retinol-CRBP is not dramatically affected by prior removal of the outer nuclear membrane with Triton X-100. However, treatment with the detergent after the binding reaction is complete removes about half of the bound [3H]RA-CRABP and almost all of the bound [3H]retinol-CRBP. We measured specific retinoid-binding activities in nucleoplasmic extracts of Nulli-SCC1 and PCC4.aza1R cells. The only readily detectable specific binding activity in nucleoplasmic extracts from untreated cells was for [3H]retinol in PCC4.aza1R preparations. Nucleoplasmic extracts from Nulli-SCC1 and PCC4.aza1R cells pretreated with RA had considerable levels of specific [3H]RA-binding activity with little or no increase in [3H]retinol binding. By contrast, similar extracts from Nulli-SCC1 cells treated with retinol bound large amounts of both [3H]retinol and [3H]RA. Under the same conditions, PCC4.aza1R extracts also contained [3H]RA-binding activity with no increase in [3H]retinol binding above the high endogenous levels. Although these results might reflect translocation of binding proteins from cytoplasm to nucleus, other interpretations must be considered since we often observed an increase, rather than the expected reduction, in cytoplasmic retinoid-binding protein levels.     

Turnover In Vivo of Lactosyl-ceramide and Other Glycosphingolipids in the Adult Rat Brain

The quantitative contribution of glucose to the biosynthesis of lactosyl-ceramide and other glycosphingolipids was studied in the adult rat brain in vivo using a semicompartmental model. Half-lives of glucose carbon in both the total carbon pool and the carbohydrate residue of the lipid were calculated. In all glycolipids the half-life of carbohydrate units was six to eight times shorter than the half-life of carbon in the total carbon pool of the same lipid. This carbohydrate half-life appears to be closely related to the turnover rate of the glycolipid. The shortest carbohydrate half-life (2.2 days) was obtained for lactosyl-ceramide followed by gangliosides. galactosyl-ceramides, and sulphatides. The results indicate that lactosyl-ceramide may serve as a branch point for the biosynthesis of cerebral gangliosides in vivo rather than occur as a breakdown product of more complex molecules.     

Morphological characterization of the pathway of endocytosis and intracellular processing of transferrin and alpha-fetoprotein in human T lymphocytes stimulated with phytohemagglutinin (PHA)

Covalent conjugates of transferrin (Tf) and alpha-fetoprotein (AFP) with horseradish peroxidase (HRP) have been used to follow, at the ultrastructural level, the uptake and the intracellular pathway of these proteins in peripheral blood human lymphocytes stimulated by phytohemagglutinin (PHA) to blast formation. Both proteins enter specifically the cells via vesicles (60-70 nm in diameter) and endosomes. They are then observed in multivesicular bodies and tubular vesicular elements in the Golgi region. AFP is thus found in the same subcellular compartments as Tf and is probably also recycled, as most of the 125I-labeled protein leaves the cells undegraded. Unstimulated lymphocytes do not internalize significantly AFP-HRP. The uptake of a noncovalent conjugate of AFP-HRP and [3H]-arachidonic acid [3H-(20:4)] is usually poor, at 37 degrees C, in unstimulated lymphocytes as well as, at 4 degrees C, in lymphocytes stimulated for 72 h. Stimulated lymphocytes incubated at 37 degrees C with the radioactive conjugate show a heavy labeling of cell organelles and more particularly of lipid droplets. AFP could regulate the intracellular delivery of fatty acid molecules.