Lipid conjugates of antiretroviral agents. II. Disodium palmityl phosphonoformate: anti-HIV activity, physical properties, and interaction with plasma proteins

Disodium palmityl phosphonoformate, a novel lipid phosphoester of the anti HIV agent phosphonoformate (foscarnet), inhibits HIV replication in H9 cells and syncytia formation in MOLT-3 cells as effectively as foscarnet itself, as shown by dose-response data from assays for expression of p17 and p24 viral antigens and syncytia formation. Protein binding studies indicate that in serum, the derivative exists bound to albumin and the lipoproteins, and would therefore be likely to exhibit improved serum lifetime in vivo.     

Large scale purification and immunolocalization of bovine uroplakins I, II, and III. Molecular markers of urothelial differentiation

The differentiation of mammalian urothelium culminates in the formation of asymmetrical unit membrane (AUM). Using gradient centrifugation and detergent wash, we purified milligram quantities of AUMs which, interestingly, contained three major proteins (15, 27, and 47 kDa) that appeared to be identical to the three immunoaffinity purified, putatively AUM-associated proteins that we described earlier (Yu, J., Manabe, M., Wu, X.-R., Xu, C., Surya, B., and Sun, T.-T. (1990) J. Cell Biol., 111, 1207-1216). Peptide mapping and immunoblotting established that these three proteins were distinct molecules. Using monospecific antibodies to these three proteins, we showed that they were all restricted to the superficial urothelial cells and were AUM-associated. The 27- and 15-kDa proteins were detected exclusively on the luminal side of mature, apical AUMs. In contrast, epitopes of the 47-kDa protein were detected on both sides of apical AUMs suggesting a transmembranous configuration. These results (i) provide the strongest evidence thus far that AUM contains three major proteins (the 27-kDa uroplakin I, 15-kDa uroplakin II, and 47-kDa uroplakin III) which form an extremely insoluble complex, (ii) suggest that uroplakin II, like uroplakin I (Yu, J., Manabe, M., Wu, X.-R., Xu, C., Surya, B., and Sun, T.-T. (1990) J. Cell. Biol. 111, 1207-1216), translocates from one side of the membrane to another during AUM maturation, (iii) indicate that uroplakin III may play a different structural role than uroplakins I and II in AUM formation, and (iv) establish the three uroplakins as markers for an advanced stage of urothelial differentiation.     

A simple and efficient method for the separation and detection of small DNA fragments by electrophoresis in formamide containing agarose gels and Southern blotting to DBM-paper.

We show in this report that DNA fragments smaller than 300 bp are separated with high resolution by electrophoresis in concentrated (up to 7%) agarose gels containing 50% formamide. The separated DNA fragments can subsequently be quantitatively transferred to DBM-paper [Alwine, J.C. et al., Proc. Natl. Acad. Sci. USA (1977) 74, 5350-54] using the Southern technique [Southern, E.M., J. Mol. Biol. (1975) 98, 503-517], while preserving the sharpness of the original gel pattern. Since thin (0.2-0.4 mm) and thick (up to 5 mm) agarose slab gels can be easily handled in vertical or horizontal apparatus, this method should prove to be a very useful extention of the Southern technique, applicable to a variety of analytical and preparative purposes.     

Selective-Differential Medium for Isolation and Differentiation of Pectinatus from Other Brewery Microorganisms

An agar medium, LL-agar (lactate-lead acetate) was designed to selectively differentiate members of the genus Pectinatus (S. Y. Lee, M. S. Mabee, and N. O. Jangaard, Int. J. Syst. Bacteriol. 28:582-594, 1978; S. Y. Lee, M. S. Mabee, N. O. Jangaard, and E. K. Horiuchi, J. Inst. Brew. 86:28-30, 1980) from other brewery microorganisms. Selectivity was achieved by the use of sodium lactate as the sole source of carbon and phenylethyl alcohol as an inhibitor for aerobic gram-negative bacteria and yeast. Differentiation was established by the introduction of lead acetate into the medium, which reacted with the H₂S liberated by Pectinatus and resulted in a blackening of the Pectinatus colonies while the other brewery organisms, when present, remained white. In combination with the Lee tube (J. E. Ogg, S. Y. Lee, and B. J. Ogg, Can. J. Microbiol. 25:987-990, 1979) and this medium, isolation of Pectinatus organisms from beer samples was accomplished with convenience and simplicity.     

Degradation of Arachidonoyl-Labeled Phosphatidylinositols by Brain Synaptosomes

Abstract: Incubation of synaptosomes together with 1-acyl-2-[¹⁴C]arachi-donoyl-sn-glycerophosphoinositols (GPI) and sodium deoxycholate yielded diacylglycerols and free arachidonic acid. Diacylglycerol formation is attributed to hydrolysis by the diacyl-GPI-specific phospholipase C (EC 3.1.4.10), and this reaction requires sodium deoxycholate for optimal activity. The free arachidonic acid formed is attributed to hydrolysis of diacyl-GPI by phospholipase A (EC 3.1.1.5). Free fatty acid release was observed during incubation, even in the absence of bile salts, but this process was preferentially stimulated by sodium taurocholate. The release of fatty acids was not specific for diacyl-GPI, as similar release was obtained during incubation with other phosphoglycerides. In the presence of deoxycholate (2 mg/ml), the release of diacylglycerols was maximal at a diacyl-GPI concentration around 1.0 mM. However, the free fatty acid release was linear with respect to the substrate at least up to 1.4 mM. The rate of diacylglycerol release from diacyl-GPI was more rapid in the initial 30 min, whereas the free fatty acid release was linear with time up to 2 h. Under this incubation condition, calcium was found to stimulate both types of hydrolytic action, although the concentration needed to achieve this stimulation was rather high. This type of labeled precursor is potentially useful for studies of the different modes of diacyl-GPI degradation by enzymes in brain subcellular membranes.     

Arachidonate metabolism in the mouse thyroid implication of the lipoxygenase pathway in thyrotropin action

The present study of compares the effects of various inhibitors of arachidonate metabolism on mouse thyroid cyclo-oxygenase and lipoxygenase activities and thyrotropin-augmented cyclic-AMP accumulation. Mouse thyroid homogenate converts [1-14C]- arachidonate to several products of the cyclo-oxygenase pathway as well as one major product of the lipoxygenase pathway, 12-L-hydroxyeicosatetraenoic acid (12-Hete). Prostaglandin (PG) formation in thyroid homogenates is inhibited by 1-10 microM indomethacin and etya. 12-HETE accumulation is reduced by 91%, 83% and 20% by 5 microM ETYA, 15-HETE, and indomethacin, respectively. Thyrotropin-stimulated cyclic-AMP accumulation, measured in whole thyroid lobes by radioimmunoassay, is reduced by 45% and 73% by 50 microM and 100 microM ETYA, respectively; indomethacin is without effect at these concentrations. 15-HETE reduces thyrotropin-augmented cyclic-AMP accumulation by 57% and 100 microM. In product inhibition studies, 10 microM 12-HETE reduced the formation of radiolabeled 12-HETE by 20%. 10 microM PGE2, PGF2 alpha or PGD2 had no effect on [1-14C]-PG formation. 12-HETE, however, reduced PG synthesis by 76% at 10 microM. This is the first report implicating the arachidonate lipoxygenase pathway in thyrotropin action at the level of cyclic-AMP regulation. Additionally, our finding that 12-HETE inhibits prostaglandin synthesis suggests that the cyclo-oxygenase and lipoxygenase pathways in the mouse thyroid may be highly integrated.     

Preparation of two dinor-PGI2 metabolites from 6-keto-PGF1α by mycobacterium rhodochrous

The transformation of 6-keto-PGF_1α to two prostacyclin metabolites, 2,3-dinor-6-keto-PGF_1α (I) and 2,3-dinor-6,15-diketo-13,14-dihydro-PGF_1α (II) by $\text{Mycobacterium rhodochrous}$ UC-6176 is described. The finding that the bacterium oxidized 6-keto-PGF_1α to the 6,15-diketo metabolite II shows that it contains 15-hydroxy prostaglandin dehydrogenase and Δ¹³ reductase enzyme systems.     

Hepatic metabolism of prostacyclin (PGI2) in the rabbit: Formation of a potent novel inhibitor of platelet aggregation

Metabolism of [9-³H]-PGI₂ was studied in the isolated Tyrode's perfused rabbit liver. Five products, four radioactive and one non-radioactive, were identified in the perfusate: 19-hydroxy-6-keto-PGF_1α, 6-keto-PGF_1α, dinor-6-keto-PGF_1α, pentanor PGF_1α and a 6-keto-PGE₁-like substance. The first two, 19-hydroxy-6-keto-PGF_1α and 6-keto-PGF_1α, represented 5% and 45% respectively, of the total radioactivity; the last two accounted for 39%. The presence of dinor and pentanor derivatives of 6-keto-PGF_1α indicated that β -oxidation and oxidative-decarboxylation occurs in the liver as the major metabolic pathway of PGI₂. One non-radioactive metabolite which co-migrated with authentic 6-keto-PGE₁ was found to inhibit platelet aggregation, having a potency similar to authentic 6-keto-PGE₁, and its effect can be eliminated by boiling and by alkali treatment. This metabolite, having similar Rf value on TLC and biological behavior as 6-keto-PGE₁, may arise from oxidation of 6-keto-PGF_1α via the 9-hydroxyprostaglandin dehydrogenase pathway, as suggested by recovery of tritiated water in the aqueous phase of the perfusate. This material, a potent inhibitor of platelet aggregation, may arise from PGI₂ or its hydrolysis product, 6-keto-PGF_1α.     

Alveolar macrophage lipoxygenase products of arachidonic acid: isolation and recognition as the predominant constituents of the neutrophil chemotactic activity elaborated by alveolar macrophages

Human immune interferon preparations have anticellular activity on human cell lines (WISH and HEp-2). This anticellular activity copurified with the human immune interferon and appears to be a function of the immune interferon molecule. On the basis of a unit of antiviral activity, purified human immune interferon had about 20 and 100 times more anticellular activity than purified fibroblast or leukocyte interferon, respectively. The possible implications of this finding in the treatment of human neoplasia are discussed.